Renato Ivan de Ávila

Campomanesia adamantium (Myrtaceae) fruits protect HEPG2 cells against carbon tetrachloride-induced toxicity

Campomanesia adamantium (Myrtaceae) is an antioxidant compounds-rich Brazilian fruit popularly known as gabiroba. In view of this, it was evaluated the hepatoprotective effects of pulp (GPE) or peel/seed (GPSE) hydroalcoholic extracts of gabiroba on injured liver-derived HepG2 cells by CCl4 (4 mM). The results showed the presence of total phenolic in GPSE was (60%) higher when compared to GPE, associated with interesting antioxidant activity using DPPH• assay. Additionally, HPLC chromatograms and thin layer chromatography of GPE and GPSE showed the presence of flavonoids. Pretreatment of HepG2 cells with GPE or GPSE (both at 800–1000 μg/mL) significantly (p < 0.0001) protected against cytotoxicity induced by CCl4. Additionally, the cells treated with both extracts (both at 1000 μg/mL) showed normal morphology (general and nuclear) contrasting with apoptotic characteristics in the cells only exposed to CCl4. In these experiments, GPSE also was more effective than GPE. In addition, CCl4 induced a marked increase in AST (p < 0.05) and ALT (p < 0.0001) levels, while GPE or GPSE significantly (p < 0.0001) reduced these levels, reaching values found in the control group. In conclusion, the results suggest that gabiroba fruits exert hepatoprotective effects on HepG2 cells against the CCl4-induced toxicity, probably, at least in part, associated with the presence of antioxidant compounds, especially flavonoids.

Mucoadhesive formulation of Bidens pilosa L. (Asteraceae) reduces intestinal injury from 5-fluorouracil-induced mucositis in mice

Gastrointestinal mucositis induced during cancer treatment is considered a serious dose-limiting side effect of chemotherapy and/or radiotherapy. Frequently, interruption of the cancer treatment due to this pathology leads to a reduction in cure rates, increase of treatment costs and decrease life quality of the patient. Natural products such as Bidens pilosa L. (Asteraceae), represent a potential alternative for the treatment of mucositis given its anti-inflammatory properties. In this study, B. pilosa glycolic extract was formulated (BPF) with poloxamer, a mucoadhesive copolymer, was used for treatment of 5-fluorouracil (5-FU)-induced mucositis in mice. As expected, animals only treated with 5-FU (200 mg/kg) presented marked weight loss, reduction of intestinal villi, crypts and muscular layer, which was associated with severe disruption of crypts, edema, inflammatory infiltrate and vacuolization in the intestinal tissue, as compared to the control group and healthy animals only treated with BPF. On the other hand, the treatment of intestinal mucositis-bearing mice with BPF (75, 100 or 125 mg/kg) managed to mitigate clinical and pathologic changes, noticeably at 100 mg/kg. This dose led to the restoration of intestinal proliferative activity through increasing Ki-67 levels; modulated the expression of Bax, Bcl2 and p53 apoptotic markers protecting intestinal cells from cell death. Moreover, this treatment regulated lipid peroxidation and inflammatory infiltration. No acute toxic effects were observed with this formulation. This work demonstrated that BPF was safe and effective against 5-FU-induced intestinal mucositis in mice. Additional studies are already in progress to further characterize the mechanisms involved in the protective effects of this technological formulation toward the development of a new medicine for the prevention and treatment of intestinal injury in patients undergoing chemotherapy/radiotherapy.

Use of Bidens pilosa L. (Asteraceae) and Curcuma longa L. (Zingiberaceae) to treat intestinal mucositis in mice: Toxico-pharmacological evaluations

Introduction Several studies towards the development of an effective treatment for intestinal mucositis have been reported, since this condition represents a major problem in clinical oncology practice due to cytotoxic effects of chemotherapy. However standardized protocols and universally accepted treatment options are yet to be established. Objectives Given above, this study evaluated the protective effects of a mucoadhesive formulation containing both Bidens pilosa L. (Asteraceae) (BP) and curcuminoids from Curcuma longa L. (Zingiberaceae) (CL) on intestinal mucositis induced by 5-fluoruoacil (5-FU) in mice. Results As expected, animals only treated with 5-FU (200 mg/kg) showed a significant reduction of 60.3 and 42.4% in villi and crypts size, respectively, when compared to control. On the other hand, the proposed therapeutic/prophylactic treatment with mucoadhesive formulations managed to reduce histopathologic changes in mice bearing mucositis, especially at 125 mg/kg BP + 15 mg/kg CL dose. The formulation promoted an increase of 275.5% and 148.7% for villi and crypts size, respectively. Moreover, chemotherapy-related weight loss was reduced by 7.4% following the treatment. In addition, an increase of 10 and 30.5% in red and white blood cells was observed when compared to 5-FU group. Furthermore, treatments with the mucoadhesive formulation containing BP/CL up modulated Ki-67 and Bcl-2 expression while reduced pro-apoptotic regulator Bax. The formulation also modulated inflammatory response triggered by 5-FU through reduction of 68% of myeloperoxidase activity and a 4-fold increase in anti-inflammatory IL-10 levels. In parallel, the oxidative stress via lipid peroxidation was reduced as indicated by decrease of 63% of malondialdehyde concentrations. Additionally, the new formulation presented low acute oral systemic toxicity, being classified in the category 5 (2000 mg/kg < LD50 < 5000 mg/kg) of the Globally Harmonized Classification System. Conclusions This study showed an interesting potential of the mucoadhesive formulation of BP/CL for the treatment of 5-FU-induced intestinal mucositis. Given the perspectives for the development of a new medicine, clinical studies are in progress to better understand the protective effects of this innovative formulation in treating mucositis.

In vitro assessment of skin sensitization, photosensitization and phototoxicity potential of commercial glyphosate-containing formulations

This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.

In vitro safety and efficacy evaluations of a complex botanical mixture of Eugenia dysenterica DC. (Myrtaceae): Prospects for developing a new dermocosmetic product

In the context of developing a new natural product-based cosmetic, the in vitro efficacy and safety evaluations of a complex botanical mixture based on Eugenia dysenterica leaf hydroalcoholic extract (EDE) (2.5-1000μg/mL) were carried out. Chromatographic analysis demonstrated the presence of the tannin (ellagic acid) and flavonoids (quercetin and gallic acid) which characterize the EDE as a polyphenol-rich mixture. Using HFF-1 fibroblasts, it was shown that EDE promoted cell regeneration after UVA exposure. It also led to the inhibition of the collagenase, elastase and tyrosinase enzymes, which are involved in skin-related disorders. In terms of toxicological evaluation, the EDE was classified as non-phototoxic through the 3T3 Neutral Red Uptake Phototoxicity Test (OECD N° 432, 2004) and non-eye irritant by Bovine Corneal Opacity and Permeability (OECD N° 437, 2013) assay, in conjunction with corneal histomorphometric analysis. Furthermore, the EDE has no skin sensitization potential as demonstrated by a two-out-of-three prediction model [protein-binding/haptenization (OECD N° 442C, 2015), keratinocyte and dendritic cell activations]. In addition, it was shown that the EDE seems to be non-genotoxic through the cytokinesis-block micronucleus assay (OECD N° 487, 2014) using HepG2 cells. When considered together, these findings support the use of EDE botanical mixture in cosmetic/pharmaceutical products.

Toxicity evaluation of the photoprotective compound LQFM048: Eye irritation, skin toxicity and genotoxic endpoints

A new molecule, LQFM048, originally designed through molecular hybridization using green chemistry approach, is in development as a photoprotective agent. Eye irritation, skin toxicity and genotoxicity evaluations are mandatory for predicting health risks. In this context, the purpose of this study was to investigate the eye irritation potential of LQFM048 by combining Short Time Exposure (STE), Bovine Corneal Opacity and Permeability (BCOP) associated with corneal histomorphometry and Hens Egg Test-Chorioallantoic Membrane (HET-CAM). Additionally, skin toxicity was evaluated by interleukin-18 production in the HaCaT keratinocyte, Local Lymph Node Assay (LLNA:BrdU-ELISA) method, 3T3 Neutral red uptake (NRU) assay and in vivo phototoxicity test. Genotoxic potential of LQFM048 was also analyzed by cytokinesis-block micronucleus assay (MNvit test-cytoB) in HepG2 cells. Our results showed that LQFM048 did not induce eye irritation and it was classified as UN GHS No Category for both STE and BCOP assays and non-irritating for HET-CAM test. LQFM048 showed non-potential skin sensitization with stimulation index (SI=0.7) in the LLNA:BrdU-ELISA method. Corroborating in vivo tests, it did not promote significant cytotoxicity in HaCaT cells and it showed similar levels of IL-18 when compared to control. Furthermore, LQFM048 induced non-phototoxic potential with photo-irritation factor (PIF) and mean photo effect (MPE) of 1 and -0.138, respectively, for 3T3 cells. Similarly, it was not phototoxic for in vivo testing with or without exposure to UVA, showing SI values of 1 and 1.2, respectively. The micronucleus test showed that LQFM048 was not genotoxic, under the conditions tested.In conclusion, LQFM048, a heterocyclic compound obtained through an environmentally acceptable simple synthetic route, seems to be safe for human use, especially for the development of a new sunscreen product, since it is neither an eye irritant, nor a contact allergen, nor mutagenic and nor phototoxic.

Eugenia dysenterica DC. (Myrtaceae) exerts chemopreventive effects against hexavalent chromium-induced damage in vitro and in vivo

Abstract Context: Eugenia dysenterica DC. (Myrtaceae) has been widely used in the folk medicine and it presents phytochemicals constituents associated to antioxidant properties. Objective: The objective of this study was to investigate the protective effects of E. dysenterica leaf hydroalcoholic extract (EDE) in vitro and in vivo using AMJ2-C11 cells and Swiss mice exposed to hexavalent chromium [Cr(VI)], respectively. Materials and methods: AMJ2-C11 cells were pretreated with EDE and exposed to Cr(VI) to evaluate cytotoxicity and the pathways involved in the chemopreventive effects of the extract. Mice were daily pretreated with EDE and then exposed to Cr(VI). Survival analysis, histopathological examination and determination of Cr levels in biological tissues were carried out. Results: In vitro studies showed that pretreatment of the AMJ2-C11 cells with EDE protected against the cytotoxicity and oxidative stress induced by Cr(VI). Consequently, the pretreatment with EDE reduced reactive oxygen species and apoptosis triggered by Cr(VI), probably by a marked antioxidant and chelating activities demonstrated by EDE. Regarding in vivo studies, pretreatment for 10 days with EDE increased survival of the mice exposed to Cr(VI). In addition, EDE prevented liver and kidney pathological damages, in parallel with reduction in chromium levels found in these organs and plasma. EDE also showed a marked antioxidant potential associated with the presence of polyphenols, especially flavonoids and tannins, as confirmed by HPLC-PDA. Conclusion: The study showed that EDE protects against Cr(VI)-induced damage in vitro and in vivo supporting further studies for the development of therapeutic products applied to prevent the damage induced by toxic metals, especially Cr(VI).

Punica granatum L. protects mice against hexavalent chromium-induced genotoxicity

This study investigated the chemoprotective effects of Punica granatum L. (Punicaceae) fruits alcoholic extract (PGE) on mice exposed to hexavalent chromium [Cr(VI)]. Animals were pretreated with PGE (25, 50 or 75 mg/kg/day) for 10 days and subsequently exposed to a sub-lethal dose of Cr(VI) (30 mg/kg). The frequency of micronucleated polychromatic erythrocytes in the bone marrow was investigated and the Cr(VI) levels were measured in the kidneys, liver and plasm. For the survival analysis, mice were previously treated with PGE for 10 days and exposed to a single lethal dose of Cr(VI) (50 mg/kg). Exposure to a sub-lethal dose of Cr(VI) induced a significant increase in the frequency of micronucleated cells. However, the prophylactic treatment with PGE led to a reduction of 44.5% (25 mg/kg), 86.3% (50 mg/kg) and 64.2% (75 mg/kg) in the incidence of micronuclei. In addition, the 50 mg/kg dose of PGE produced a higher chemoprotective effect, since the survival rate was 90%, when compared to that of the non-treated group. In these animals, reduced amounts of chromium were detected in the biological materials, in comparison with the other groups. Taken together, the results demonstrated that PGE exerts a protective effect against Cr(VI)-induced genotoxicity.

Pharmacological evaluation and molecular docking of new di-tert-butylphenol compound, LQFM-091, a new dual 5-LOX/COX inhibitor

Abstract Dual 5‐LOX/COX inhibitors are potential new dual drugs to treat inflammatory conditions. This research aimed to design, synthesis and to evaluate the anti‐inflammatory and antinociceptive effects of the new compound, which is derived from nimesulide and darbufelone lead compounds. The new dual inhibitor 5‐LOX/COX has the possible advantage of gastrointestinal safety. A voltammetric experiment was conducted to observe the drugs antioxidative effect. A formalin test, a hot plate test and carrageenan‐induced mechanical hyperalgesia were employed to evaluate the analgesic nature of LQFM‐091. To evaluate anti‐inflammatory activity, we measured edema, leukocyte count, myeloperoxidase activity and cytokines levels in carrageenan‐induced inflammation tests. We elucidated the underlying mechanisms by assessing the interaction the with COXs and LOX enzymes by colorimetric screening assay and molecular docking. The lethal dose (LD50) was estimated using 3T3 Neutral Red Uptake assay. Our results indicate that the LQFM‐091 prototype is a powerful antioxidant, as well as able to inhibit COX‐1, COX‐2 and LOX activities. LQFM091 was classified in GHS category 4 (300 < LD50 < 2000 mg/Kg). This prototype showed analgesic activity in the formalin test and decreased carrageenan‐induced mechanical hyperalgesia. Furthermore, LQFM‐091 reduced the paw edema induced by carrageenan and reduced the leukocyte count, myeloperoxidase activity, TNF‐&agr; and IL‐1&bgr; levels in the pleural exudate. Another interesting finding was the absence of gastrointestinal lesions. These data indicate that LQFM‐091 produced antinociceptive and anti‐inflammatory effects while maintaining gastrointestinal safety. Furthermore, this compound presented a safe toxicological profile. Blocked COXs and LOX enzymes are important targets for manipulating the mechanism of this compound. Graphical abstract Figure. No Caption available.

Photoprotective effect and acute oral systemic toxicity evaluation of the novel heterocyclic compound LQFM048

The new heterocyclic derivative LQFM048 (3) (2,4,6-tris ((E)-ethyl 2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylate)-1,3,5-triazine) was originally designed through the molecular hybridization strategy from Uvinul® T 150 (1) and (E)-ethyl 2-cyano-3-(4hydroxy-3-methoxyphenyl)acrylate (2) sunscreens, using green chemistry approach. This compound was obtained in global yields (80%) and showed an interesting redox potential. In addition, it is thermally stable up to temperatures around 250°C. It was observed that LQFM048 (3) showed a low degradation after 150min of sunlight exposure at 39°C, whereas the extreme radiation conditions induced a considerable photodegradation of the LQFM048 (3), especially when irradiated by VIS and VIS+UVA. During the determination of sun protection factor, LQFM048 (3) showed interesting results, specially as in association with other photoprotective compounds and commercial sunscreen. Additionally, the compound (3) did not promote cytotoxicity for 3T3 fibroblasts. Moreover, it was not able to trigger acute oral systemic toxicity in mice, being classified as a compound with low acute toxicity hazard (2.000mg/kg>LD50<5.000mg/kg). Therefore, this compound synthesized using green chemistry approach is promising showing potential to development of a new sunscreen product with advantage of presenting redox potential, indicating antioxidant properties.