Stephalagine, an aporphine alkaloid from Annona crassiflora fruit peel, induces antinociceptive effects by TRPA1 and TRPV1 channels modulation in mice.

Pain relief represents a critical unresolved medical need. Consequently, the search for new analgesic agents is intensively studied. Annona crassiflora, a native species of the Brazilian Savanna, represents a potential source for painful treatment. This study aimed to investigate the antinociceptive potential of A. crassiflora fruit peel, focusing on its major alkaloid, stephalagine, in animal models of pain evoked by the activation of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels. Male C57BL/6/J mice were submitted to formalin-, cinnamaldehyde-, and capsaicin-induced nociception tests to assess nociceptive behavior, and to the open-field and rotarod tests for motor performance analyses. Moreover, the stephalagine's effect was tested on capsaicin- and cinnamaldehyde-induced Ca2+ influx in spinal cord synaptosomes. In silico assessments of the absorption, distribution, metabolism and central nervous system permeability of stephalagine were carried out. The ethanol extract and alkaloidal fraction reduced the nociception induced by formalin. When administered by oral route (1 mg/kg), stephalagine reduced the spontaneous nociception and paw edema induced by TRPV1 agonist, capsaicin, and by TRPA1 agonists, cinnamaldehyde- and formalin, without altering the animals' locomotor activity. The prediction of in silico pharmacokinetic properties of stephalagine suggests its capacity to cross the blood-brain barrier. Furthermore, this alkaloid reduces the capsaicin- and cinnamaldehyde-mediated Ca2+ influx, indicating a possible modulation of TRPV1 and TRPA1 channels, respectively. Together, our results support the antinociceptive and anti-edematogenic effects of the A. crassiflora fruit peel and suggest that these effects are triggered, at least in part, by TRPV1 and TRPA1 modulation by stephalagine.

Protective effects of a polyphenol-enriched fraction of the fruit peel of Annona crassiflora Mart. on acute and persistent inflammatory pain.

Different parts of Annona crassiflora Mart., a native species from Brazilian savanna, were traditionally used for the treatment of a wide variety of ailments including arthritis. Thus, this study aimed to investigate the possible antinociceptive and anti-inflammatory properties of a polyphenol-enriched fraction of the fruit peel of A. crassiflora, named here as EtOAc, in mice. Pro-inflammatory cytokines and nitric oxide (NO) production were evaluated in LPS-activated macrophages. Then, EtOAc fraction was administered by oral route in male C57BL/6/J mice, and the animals were submitted to glutamate-induced nociception and complete Freund's adjuvant (CFA)-induced monoarthritis tests to assess nociception (mechanical, spontaneous and cold pain) and inflammation (edema and neutrophil infiltration), and to the open-field and rotarod tests for motor performance analysis. EtOAc fraction inhibited the production of IL-6 and NO in the LPS-induced macrophages, and reduced spontaneous nociception induced by glutamate, without altering the animals' locomotor activity. In addition, the polyphenol-enriched fraction was able to revert the early and late hyperalgesia induced by CFA, as well as edema at the acute phase. Reduction of myeloperoxidase activity and inflammatory cell infiltration was observed in the paw tissue of mice injected with CFA and treated with EtOAc fraction. Together, our results support the antinociceptive and anti-inflammatory effects of the polyphenol-enriched fraction of A. crassiflora fruit peel and suggest that these effects are triggered, at least in part, by suppressing pro-inflammatory cytokines and neutrophils infiltration.

Pro-Fibrogenic and Anti-Inflammatory Potential of a Polyphenol-Enriched Fraction from Annona crassiflora in Skin Repair.

A polyphenol-enriched fraction from Annona crassiflora fruit peel (PEF-Ac) containing chlorogenic acid, epi-catechin, procyanidins B2 and C1, quercetin-glucoside, kaempferol, and caffeoyl-glucoside was investigated for its anti-inflammatory, pro-angiogenic, and profibrogenic potential in the healing of cutaneous wounds. Four wounds were performed on the back of C57 mice and the lesions were treated with the vehicle (Vaseline and lanolin) and PEF-Ac at concentrations of 2%, 4%, and 6% for 4 and 7 d. Neutrophils and macrophages activities were evaluated indirectly by the activity of myeloperoxidase and N-acetyl-ß-D-glycosaminidase, angiogenesis was evaluated by hemoglobin dosing and vessel count in histological sections, and collagen deposition was assessed from histological sections stained with picrosirius red. PEF-Ac demonstrated anti-inflammatory activity, with reduced activities of neutrophil and macrophage in the cutaneous wounds. In addition, there was an increase in the synthesis of types I and III collagen, as well as in the percentage of wound closure, mainly after 4 d of treatment. On the other hand, PEF-Ac did not present an effective pro-angiogenic activity. A. crassiflora fruit peel showed anti-inflammatory and profibrogenic properties, indicating a promising natural source of bioactive molecules for treatment of cutaneous wounds.

Immune and Hormonal Response to High-intensity Exercise During Orienteering.

Orienteering is an endurance sport that combines physical and cognitive activity, during which the athlete must complete a course with several points distributed over unknown terrain in the shortest possible time. A number of studies have investigated the body's physiological adaptations to the stress caused during competition, but not the immunological changes. To that end, the present study evaluated the immunological, physiological and pathological responses in athletes performing high-intensity physical exercise during an orienteering race. The 30 athletes tested belonged to the elite orienteering category and participated in the regional championship. Cortisol levels were determined before and after the competition to assess stress response, as were the cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17 to evaluate the immune response. Cortisol levels increased after the competition, indicating a stress condition. IFN-γ, IL-6 and IL-10 levels also rose post competition. The results indicate that orienteers are exposed to high stress levels, and that this condition affects their immune and endocrine systems, triggering a predominantly anti-inflammatory response, likely an athlete's mechanism of adaptation to the stress imposed by high-intensity physical exercise.

Effect of saliva collection methods and oral hygiene on salivary biomarkers.

The aim of this study was to evaluate the influence of unstimulated and stimulated saliva collection methods, as well as tooth brushing, on the secretion rate of salivary total protein, nitrite, total antioxidant capacity and alpha-amylase. Saliva of 14 healthy individuals were collected with stimulation using Salivette®, Parafilm® and chewing gum and without stimulation from spit with and without fluid accumulation, before and after oral hygiene. Total protein, nitrite, total antioxidant capacity and alpha-amylase concentration (sAA) were evaluated. The collection of saliva stimulated with Parafilm® and chewing gum increased the salivary flow (1.5 ± 0.4 and 3.4 ± 0.7 mL/min, respectively) and the secretion rate of salivary total protein (1.0 ± 0.2 and 2.3 ± 0.5 mg/min, respectively). Also, chewing gum increases the salivary nitrite secretion (213 ± 58 nmol/min) and total antioxidant capacity (410 ± 47 nmol trolox eq/min). Interestingly, the unstimulated method without saliva accumulation prior to collection resulted in low sAA levels (23,531 ± 7979 pixel density). Furthermore, oral hygiene decreased salivary flow (1.3 ± 0.5 to 1.0 ± 0.4 mL/min), reduced the secretion rate of total protein (1.0 ± 0.5 to 0.6 ± 0.2 mg/min, p < .05) and increased sAA (13,159 ± 7114 to 20,075 ± 25,656 pixel density, p < .05). The type of stimulation can activate autonomous receptors responsible for the secretion and composition of saliva. Therefore, the evaluation of saliva collection methods and oral hygiene on salivary biomarkers is important for understanding and standardizing variations in salivary composition to strengthen the use of saliva as a diagnostic fluid.

Phytochemical composition of aerial parts and roots of Pfaffia glomerata (Spreng.) Pedersen and anticholinesterase, antioxidant, and antiglycation activities.

The Pfaffia glomerata, a plant popularly called Brazilian ginseng, is widely used in Brazil for the treatment of various pathologies, including those associated with the Central Nervous System. 20-hydroxyecdysone (20E), a phytosteroid present in this plant, can promote adaptogenic effects in the organism, providing greater body resistance to stressors. This study aimed to evaluate the phytochemical composition and the anticholinesterase, antioxidant, and antiglycation effects of extracts and fractions of aerial parts and roots of P. glomerata, also analyzing their possible cytotoxic effects. The fractions were obtained by partitioning methanol extracts from the aerial part and roots of P. glomerata with hexane, dichloromethane, ethyl acetate, n-butanol, and water. The samples were initially tested in anticholinesterase, antioxidant, and antiglycation assays, and the most promising samples were submitted for cytotoxicity and chromatographic analyses. Mass spectrometry and chromatography methods revealed that 20E was the main compound in the dichloromethane fractions, there being 35% more 20E in the aerial part (APD) than in the roots (RD). Added to the higher concentration of 20E, the APD fraction also presented more promising results than the RD fraction in anticholinesterase and antioxidant analyses, indicating that their effects may be related to the concentration of 20E. These same fractions showed no hemolytic effects but were cytotoxic in high concentrations. These new findings contribute to scientific information about P. glomerata and open more perspectives for the understanding of its therapeutic properties, allowing the association of biological activity with the presence of 20E.

Bioactive compounds from the leaves of Maytenus ilicifolia Mart. ex Reissek: Inhibition of LDL oxidation, glycation, lipid peroxidation, target enzymes, and microbial growth.

ETHNOPHARMACOLOGICAL RELEVANCE: Maytenus ilicifolia Mart. ex Reissek, a medicinal plant used for treating gastritis, ulcers, and gastric disorders, possesses therapeutic properties attributed to diverse leaf compounds-terpenoids, alkaloids, flavonoids, phenols, and tannins, reflecting the ethnopharmacological knowledge of traditional users. AIMS OF THE STUDY: We aimed to assess the antioxidant and antiglycant capacities of Maytenus ilicifolia's ethanolic extract and organic fractions, identify bioactive compounds through HPLC-MS/MS analysis, and conduct phytochemical assessments. We also assessed their potential to inhibit digestive and cholinesterase enzymes, mitigate oxidation of human LDL and rat hepatic tissue, and examine their antimicrobial and cytotoxic properties. MATERIALS AND METHODS: Organic fractions (hexane - HF-Mi, dichloromethane - DMF-Mi, ethyl acetate - EAF-Mi, n-butanol - BF-Mi, and hydromethanolic - HMF-Mi) were obtained via liquid-liquid partitioning. Antioxidant (DPPH, FRAP, ORAC) and antiglycant (BSA/FRU, BSA/MGO, ARG/MGO/LDL/MGO models) capacities were tested. Phytochemical analysis employed HPLC-MS/MS. We also studied the inhibitory effects on α-amylase, acetylcholinesterase, butyrylcholinesterase, human LDL and rat hepatic tissue oxidation, antimicrobial activity, and cytotoxicity against RAW 264.7 macrophages. RESULTS: HPLC-ESI-MS/MS identified antioxidant compounds such as catechin, quercetin, and kaempferol derivatives. Ethanolic extract (EE-Mi) and organic fractions demonstrated robust antioxidant and antiglycant activity. EAF-Mi and BF-Mi inhibited α-amylase (2.42 µg/mL and 7.95 µg/mL) compared to acarbose (0.144 µg/mL). Most organic fractions exhibited ∼50% inhibition of acetylcholinesterase and butyrylcholinesterase, rivaling galantamine and rivastigmine. EAF-Mi, BF-Mi, and EE-Mi excelled in inhibiting lipid peroxidation. All fractions, except HMF-Mi, effectively countered LDL oxidation, evidenced by the area under the curve. These fractions protected LDL against lipid peroxidation. CONCLUSION: This study unveils Maytenus ilicifolia's ethanolic extract and organic fractions properties. Through rigorous analysis, we identify bioactive compounds and highlight their antioxidant, antiglycant, enzyme inhibition, and protective properties against oxidative damage. These findings underline its significance in modern pharmacology and its potential applications in healthcare.

Exploring the composition and properties of Centella asiatica metabolites and investigating their impact on BSA glycation, LDL oxidation and α-amylase inhibition.

Centella asiatica (L.) Urb. is a small herbaceous plant belonging to the Apiaceae family that is rich in triterpenes, such as asiaticoside and madecassoside. Centella asiatica finds broad application in promoting wound healing, addressing skin disorders, and boosting both memory and cognitive function. Given its extensive therapeutic potential, this study aimed not only to investigate the Centella asiatica ethanolic extract but also to analyze the biological properties of its organic fractions, such as antioxidant antiglycation capacity, which are little explored. We also identified the main bioactive compounds through spectrometry analysis. The ethanolic extract (EE) was obtained through a static maceration for seven days, while organic fractions (HF: hexane fraction; DF: dichloromethane fraction; EAF: ethyl acetate fraction; BF: n-butanol fraction and HMF: hydromethanolic fraction) were obtained via liquid-liquid fractionation. The concentration of phenolic compounds, flavonoids, and tannins in each sample was quantified. Additionally, the antiglycation (BSA/FRU, BSA/MGO, and ARG/MGO models) and antioxidant (FRAP, ORAC, and DPPH) properties, as well as the ability to inhibit LDL oxidation and hepatic tissue peroxidation were evaluated. The inhibition of enzyme activity was also analyzed (α-amylase, α-glycosidase, acetylcholinesterase, and butyrylcholinesterase). We also evaluated the antimicrobial and cytotoxicity against RAW 264.7 macrophages. The main compounds present in the most bioactive fractions were elucidated through ESI FT-ICR MS and HPLC-ESI-MS/MS analysis. In the assessment of antioxidant capacity (FRAP, ORAC, and DPPH), the EAF and BF fractions exhibited notable results, and as they are the phenolic compounds richest fractions, they also inhibited LDL oxidation, protected the hepatic tissue from peroxidation and inhibited α-amylase activity. Regarding glycation models, the EE, EAF, BF, and HMF fractions demonstrated substantial activity in the BSA/FRU model. However, BF was the only fraction that presented non-cytotoxic activity in RAW 264.7 macrophages at all tested concentrations. In conclusion, this study provides valuable insights into the antioxidant, antiglycation, and enzymatic inhibition capacities of the ethanolic extract and organic fractions of Centella asiatica. The findings suggest that further in vivo studies, particularly focusing on the butanol fraction (BF), may be promising routes for future research and potential therapeutic applications.

Annona crassiflora Mart. Fruit Peel Polyphenols Preserve Cardiac Antioxidant Defense and Reduce Oxidative Damage in Hyperlipidemic Mice.

Dyslipidemia and oxidative stress are directly related to the pathogenesis of cardiovascular diseases. Annona crassiflora Mart. (ACM) has been traditionally used in folk medicine to alleviate inflammation and pain. This plant is rich in polyphenols, which exhibit high antioxidant capacity. The present study aimed to elucidate the antioxidant properties of ACM in the heart of hyperlipidemic mice. The animals were orally administered either a crude ethanol extract (CEAc) or a polyphenols-rich fraction (PFAc) obtained from ACM fruit peel. There were correlations between blood and fecal biochemical data with cardiac oxidative stress biomarkers. Here, the pre-treatment with CEAc for 12 d led to an increase in glutathione content (GSH) and a reduction in the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase. Moreover, PFAc was found to enhance the total antioxidant capacity as well as GSH, SOD and CAT activities, which were reduced by Triton WR-1339-induced hyperlipidemia. Moreover, the administration of PFAc before the treatment resulted in a decrease in protein carbonylation and lipid peroxidation levels, as well as a reduction in the activities of glutathione reductase and glucose-6-phosphate dehydrogenase. ACM fruit peel showed improvement in the glutathione system, mainly its polyphenols-rich fraction, indicating a potential cardioprotective antioxidant usage of this plant extract.

Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oils.

This study addresses the current trend of essential oils in alternative medicine using the non-chordate model Drosophila melanogaster. Following the three R's principles, it proposes non-chordate models to fill knowledge gaps on essential oil toxicity. Copaiba, lavender, and ginger essential oils are evaluated for effects on D. melanogaster lifespan, climbing ability, and brain structure, while their anti-inflammatory properties are also analyzed. Results show dose-related differences: higher concentrations (0.25% v/v) cause brain deterioration and impaired climbing, while lower concentrations (0.0625% v/v for copaiba and ginger; 0.125% for lavender) have no effect on climbing or brain structure. Lavender oil significantly extends lifespan and maintains anti-inflammatory activity when ingested, underscoring its therapeutic potential. These findings highlight the importance of D. melanogaster as a model for studying essential oil properties, potentially replacing chordate models. In addition, this research advances alternative remedies for currently incurable diseases, with lavender oil emerging as a promising candidate for drug discovery.

Fructose and methylglyoxal-induced glycation alters structural and functional properties of salivary proteins, albumin and lysozyme.

Glycation process refers to reactions between reduction sugars and amino acids that can lead to formation of advanced glycation end products (AGEs) which are related to changes in chemical and functional properties of biological structures that accumulate during aging and diseases. The aim of this study was to perform and analyze in vitro glycation by fructose and methylglyoxal (MGO) using salivary fluid, albumin, lysozyme, and salivary α-amylase (sAA). Glycation effect was analyzed by biochemical and spectroscopic methods. The results were obtained by fluorescence analysis, infrared spectroscopy (total attenuated reflection-Fourier transform, ATR-FTIR) followed by multivariate analysis of principal components (PCA), protein profile, immunodetection, enzymatic activity and oxidative damage to proteins. Fluorescence increased in all glycated samples, except in saliva with fructose. The ATR-FTIR spectra and PCA analysis showed structural changes related to the vibrational mode of glycation of albumin, lysozyme, and salivary proteins. Glycation increased the relative molecular mass (Mr) in protein profile of albumin and lysozyme. Saliva showed a decrease in band intensity when glycated. The analysis of sAA immunoblotting indicated a relative reduction in intensity of its correspondent Mr after sAA glycation; and a decrease in its enzymatic activity was observed. Carbonylation levels increased in all glycated samples, except for saliva with fructose. Thiol content decreased only for glycated lysozyme and saliva with MGO. Therefore, glycation of salivary fluid and sAA may have the potential to identify products derived by glycation process. This opens perspectives for further studies on the use of saliva, an easy and non-invasive collection fluid, to monitor glycated proteins in the aging process and evolution of diseases.

Polyphenols-Rich Fraction from Annona muricata Linn. Leaves Attenuates Oxidative and Inflammatory Responses in Neutrophils, Macrophages, and Experimental Lung Injury.

Annona muricata Linn. is a common plant found in the warmest regions of South and Central America and its use in traditional medicine has been reported for the treatment of various illnesses. In the current study, we investigate the antioxidant and anti-inflammatory activities of crude extract and fractions from A. muricata L. leaves in isolated murine phagocytic immune cells as well as experimental LPS-induced acute lung injury (ALI). In a luminol-dependent chemiluminescence assay, we showed that ethyl acetate (EtOAc.f) and n-butanol (BuOH.f) fractions-both rich in polyphenols-reduced the generation of reactive oxygen species (ROS) by neutrophils stimulated with opsonized zymosan; similar results were found in culture of bone marrow-derived macrophages (BMDMs). By evaluating anti-inflammatory activity in BMDMs, EtOAc.f and BuOH.f reduced secretion of IL-6 and expression of the co-stimulatory molecule CD40. Furthermore, in LPS-induced ALI, oral administration of EtOAc.f reduced myeloperoxidase (MPO) activity in lung tissue. In addition, on a mechanism dependent on glutathione levels, the oxidative damage was also attenuated. These findings revealed direct antioxidant and anti-inflammatory activities of polyphenols-rich fractions of A. muricata L. leaves on neutrophils and macrophages. Moreover, the reduced oxidative damage and levels of inflammatory markers in experimental ALI suggest that these fractions might be explored for the development of new therapies for inflammatory conditions.

Flavonoids and proanthocyanidins-rich fractions from Eugenia dysenterica fruits and leaves inhibit the formation of advanced glycation end-products and the activities of α-amylase and α-glucosidase.

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Eugenia dysenterica have been popularly used in Brazil for treating diabetes mellitus and its complications. The present study aimed to screen extracts from E. dysenterica fruit pulp, peel, seed and leaf for carbohydrate digestive enzymes inhibitors with antioxidant and anti-glycation capacities. MATERIALS AND METHODS: Ethanol extracts of E. dysenterica were subjected to a liquid-liquid fractionation and the fractions were used to evaluate their antioxidant properties and inhibitory potential against the formation of advanced glycation end-products (AGEs) and α-amylase and α-glucosidase. RESULTS: The ethyl acetate fraction (EtOAcF) from seed and the dichloromethane fraction (CH2Cl2F) and EtOAcF from leaf had high antioxidant capacities (ORAC >5500 µmol trolox eq g-1, FRAP >1500 µmol trolox eq g-1 and DPPH IC50 < 35 µg mL-1) and showed exceptional inhibitory activities against AGEs formation (glycation inhibition above 80% at 10 µg mL-1) and α-amylase and α-glucosidase (inhibition above 50% at 10 µg mL-1). The gallated B-types proanthocyanidins were the most active ingredients found in the leaf of E. dysenterica (CH2Cl2 and EtOAcF), being responsible for the notorious inhibitory effects against glycation and glycoside hydrolases due to their ortho-hydroxyl groups, which play role in scavenge and quench free radicals and glycated products, and may occupy the enzymes' substrate binding pocket. Furthermore, gallic acid, quercetin and its glycoside derivatives were detected by the first time in the E. dysenterica fruit seed (EtOAcF). CONCLUSIONS: The results strongly contribute to the understanding of the antidiabetic potential of seeds and leaves from E. dysenterica, a species from a global biodiversity hotspot, which appears to be linked to the prevention of oxidative stress, AGEs production and postprandial hyperglycemia.

Increased selenium concentration in the synthesis of CdSe magic-sized quantum dots affects how the brain responds to oxidative stress.

CdSe magic-sized quantum dots (MSQDs) have been widely used as fluorescent probes in biological systems due to their excellent optical properties with a broader fluorescence spectrum and stable luminescence in biological media. However, they can be cytotoxic and alter the redox balance depending on the amounts of Cd2+ adsorbed on their surface. Thus, the present study aimed to evaluate whether increases in selenium concentration in the synthesis of CdSe-MSQDs decrease the oxidative stress caused by Cd2+ -based quantum dots. CdSe-MSQDs synthesized with different concentrations of selenium were investigated against oxidative stress in the brain of chicken embryos by examining total antioxidant capacity, lipid peroxidation, thiol, and glutathione contents, as well as the activities of glutathione peroxidase, superoxide dismutase (SOD), catalase (CAT), and glutathione reductase. In addition, the vascularization of the chorioallantoic membrane (CAM) analysis was performed. Higher selenium concentrations alter the surface defect levels (decrease free Cd2+ ) and controlled the oxidative effects of CdSe-MSQDs by reducing the lipid peroxidation, restoring the glutathione defense system and the antioxidant enzymes SOD and CAT, and maintaining the vascular density of the CAM. The current findings reinforce the study of the effects of the presence of Cd2+ ions on the surface of quantum dots, changing toxicity, and aiming interesting strategies of nanomaterials in biological systems.

Topic use of Annona crassiflora Mart. contributes to wound healing due to the antioxidant and proliferative effects of fibroblasts.

Annona crassiflora Mart. is a species native to the Cerrado biome, whose fruit is known as araticum or marolo. Plant parts are widely used in folk medicine to treat inflammation and pain associated with rheumatism, wounds, venereal diseases, snakebites, and microbial infections. Thus, we investigated a fraction rich in phenolic compounds (PCAc) obtained from the crude extract of the peel of these fruits on non-cytotoxic, anti-inflammatory, antioxidant, and collagen biosynthesis properties in the healing of wounds induced on the back of BALB/c mice. For the control group, the induced wounds were not treated and for the others, wounds were treated topically with vehicle or vehicle plus PCAc. Both fractions contained in PCAc demonstrated effective protection on fibroblasts. We highlight the effect of the ethyl acetate fraction which, in addition to the protective effect, has a proliferative activity on these cells. In addition, PCAc caused improvement in healing after 7 days of treatment and in the longest period of treatment with PCAc (7, 14, and 21 days) there was a greater contraction of the wound, accompanied by resolution of the inflammatory process, antioxidant defense, increasing collagen synthesis, and modulation of metalloproteinases. PCAc demonstrated better re-epithelialization and organization of the dermis at the end of treatment. The changes promoted by the phenolic compounds of A. crassiflora were important in the healing process, especially in activities related to inflammation, oxidative stress, and fibrogenesis.

Stephalagine, an aporphinic alkaloid with therapeutic effects in acute gout arthritis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gout is an inflammatory disease characterized by the accumulation of monosodium urate crystals (MSU) in the joints, leading to severe pain and inflammation. Stephalagine is a Brazilian Savanna aporphine alkaloid isolated from Annona crassiflora Mart. Fruit peel, that has been popularly used to treat rheumatism and have been described with antinociceptive properties. However, no studies evaluated the possible therapeutic properties of stephalagine in arthritic pain. AIM OF THE STUDY: To evaluate the possible antinociceptive and anti-inflammatory effects of stephalagine in an acute gout attack in mice. MATERIALS AND METHODS: Adult male wild type C57BL/6/J/UFU mice (20-25 g) were used (process number 018/17). The treated group received stephalagine (1 mg/kg, by gavage) and the vehicle group received saline (10 mL/kg, by gavage), both 1 h before the MSU crystals (100 µg/ankle joint) administration. All groups were analyzed for mechanical allodynia, thermal hyperalgesia, overt pain-like behaviors, and edema development at 2, 4, 6 and 24 h after injections. Synovial fluid and the ankle articulation from the injected joint were collected 4 h after administrations for myeloperoxidase enzyme activity, IL-1ß measurement, and histological analysis. RESULTS: Stephalagine had a significant antinociceptive effect on mechanical allodynia, when compared to vehicle group at 2-24 h after intra-articular injection of MSU and 2 h for spontaneous and cold thermal sensitivity. Stephalagine was also able to significantly reduce the articular edema (45 ± 1%), the activity of the myeloperoxidase enzyme (37 ± 6%), and IL-1ß levels (43 ± 3%). The histological analysis confirms that stephalagine dramatically reduced the number of infiltrating inflammatory cells (75 ± 6%) in MSU injected animals. Also, stephalagine treatment did not alter the uric acid levels, xanthine oxidase activity, AST and ALT activities, urea and creatinine levels, neither cause any macroscopic changes in the mice's weight, deformations, changes in the coat, or feces. CONCLUSION: Stephalagine may be an alternative for the management of gout, once it was able to induce antinociceptive and anti-inflammatory effects without causing adverse effects on the evaluated parameters.

Inhibitors of angiotensin I converting enzyme potentiate fibromyalgia-like pain symptoms via kinin receptors in mice.

Fibromyalgia is a potentially disabling chronic disease, characterized by widespread pain and a range of comorbidities such as hypertension. Among the mechanisms involved in fibromyalgia-like pain symptoms are kinins and their B1 and B2 receptors. Moreover, angiotensin I converting enzyme (ACE) inhibitors, commonly used as antihypertensive drugs, can enhance pain by blocking the degradation of peptides such as substance P and bradykinin, besides enhancing kinin receptors signalling. We investigated the effect of ACE inhibitors on reserpine-induced fibromyalgia-like pain symptoms and the involvement of kinins in this effect in mice. Nociceptive parameters (mechanical and cold allodynia and overt nociception) were evaluated after ACE inhibitors administration in mice previously treated with reserpine. The role of kinin B1 and B2 receptors was investigated using pharmacological antagonism. Additionally, bradykinin levels, as well as the activity of ACE and kininase I, were measured in the sciatic nerve, spinal cord and cerebral cortex of the mice. The ACE inhibitors enalapril and captopril enhanced reserpine-induced mechanical allodynia, and this increase was prevented by kinin B1 and B2 receptor antagonists. Substance P and bradykinin caused overt nociception and increased mechanical allodynia in animals treated with reserpine. Reserpine plus ACE inhibitors increased bradykinin-related peptide levels and inhibited ACE activity in pain modulation structures. Since hypertension is a frequent comorbidity affecting fibromyalgia patients, hypertension treatment with ACE inhibitors in these patients should be reviewed once this could enhance fibromyalgia-like pain symptoms. Thus, the treatment of hypertensive patients with fibromyalgia could include other classes of antihypertensive drugs, different from ACE inhibitors.

A 20-hydroxyecdysone-enriched fraction from Pfaffia glomerata (Spreng.) pedersen roots alleviates stress, anxiety, and depression in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Pfaffia glomerata roots are widely used in Brazil to treat various pathological conditions, particularly psychological disorders. 20-hydroxyecdysone, a phytosteroid present in the plant, can promote greater body resistance against exogenous and endogenous stressors. The objective of this study was to evaluate the possible neuroprotective effect of a 20-hydroxyecdysone-enriched fraction (20E-EF), obtained from P. glomerata roots, in an acute murine stress model. MATERIAL AND METHODS: The 20E-EF was obtained by partitioning the methanol extract from P. glomerata roots with dichloromethane. Mice were treated by gavage with three doses of 20E-EF (3, 10, and 30 mg/kg) and parameters of stress, anxiety, and depression were evaluated. Biomarkers of oxidative stress (enzymes, antioxidant profile, and oxidized molecules) were evaluated in the cortex, striatum (basal ganglia), and hippocampus of animals treated with 30 mg/kg of 20E-EF. RESULTS: Mass spectrometry revealed that 20E was the main compound in the dichloromethane fraction. At a dose of 30 mg/kg, 20E-EF reduced stress, anxiety, and depression, while stimulating antioxidant enzymes (catalase, superoxide dismutase, and glutathione peroxidase), promoting antioxidant activity (antioxidant capacity, sulfhydryl groups, and reduced glutathione), and reducing oxidative markers (lipid peroxidation). In addition, 20E increased the concentration of NO in the striatum, possibly improving memory function and antioxidant activity. CONCLUSION: A 30 mg/kg dose of 20E-EF was able to reduce stress, anxiety, and depression, in addition to maintaining antioxidant defenses of the cortex and striatum. These findings open new perspectives for understanding the therapeutic properties of P. glomerata and the underlying mechanism(s).

Antioxidant compounds from Annona crassiflora fruit peel reduce lipid levels and oxidative damage and maintain the glutathione defense in hepatic tissue of Triton WR-1339-induced hyperlipidemic mice.

Dyslipidemia is a risk factor for the pathogenesis of several diseases, such as obesity, hypertension, atherosclerosis and cardiovascular diseases. In addition to interfering with serum concentrations of cholesterol and triglycerides, hyperlipidemia is involved in oxidative stress increase and reduction of the endogenous antioxidant defenses. The fruit peel of Annona crassiflora crude extract (CEAc) and its polyphenols-rich fraction (PFAc) were investigated against hypertriglyceridemia, hypercholesterolemia and hepatic oxidative stress in Triton WR-1339-induced hyperlipidemic mice. Lipid parameters in serum, feces and liver, as well as hepatic oxidative status, and enzymatic and non-enzymatic antioxidant defense systems were analyzed. Pre-treatment with CEAc for 12 days decreased hepatic triglycerides and total cholesterol, and similar to PFAc, increased the high-density lipoprotein level. There were reductions in lipid peroxidation and protein carbonylation, as well as restoration of the glutathione defense system and total thiol content in the liver of the hyperlipidemic mice treated with PFAc. The fruit peel of A. crassiflora, a promising natural source of bioactive molecules, showed a potential lipid-lowering action and hepatoprotective activities triggered by reduction of oxidative damage and maintenance of the enzymatic and non-enzymatic antioxidant systems impaired by the hyperlipidemic state.

Anti-parasitic activity of Annona muricata L. leaf ethanolic extract and its fractions against Toxoplasma gondii in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Sulfadiazine and pyrimethamine are the two drugs used as part of the standard therapy for toxoplasmosis, however; they may cause adverse side effects and fail to prevent relapse in many patients, rendering infected individuals at risk of reactivation upon becoming immunocompromised. Extracts from various parts of Annona muricata have been widely used medicinally for the management, control and/or treatment of several human diseases, acting against parasites that cause diseases in humans. AIM OF THE STUDY: This study was performed to investigate the action of the ethanolic extract of A. muricata (EtOHAm) and its fractions in the control of the apicomplexan parasite Toxoplasma gondii in vitro and in vivo, and the effect of EtOHAm on the inflammatory response and lipid profile alteration induced by in vivo T. gondii infection. MATERIALS AND METHODS: The cytotoxicity of EtOHAm and its fractions ethyl acetate (EtOAcAm), n-butanol (BuOHAm), aqueous (H2OAm), hexane (HexAm) and dichloromethane (CH2Cl2Am) was evaluated in NIH/3T3 fibroblasts using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cells were infected with T. gondii, treated with the extracts, and parasite proliferation was analyzed. For the in vivo experiments, C57BL/6 mice were orally infected with T. gondii and, treated with different concentrations of extract fractions that were effective in vitro (EtOHAm, EtOAcAm, HexAm and CH2Cl2Am). Tissue parasitism, histological alterations, systemic cytokine and lipid profile were investigated. RESULTS: EtOHAm, EtOAcAm, BuOHAm, H2OAm presented low cytotoxicity until doses of 200 µg/mL, while HexAm and CH2Cl2Am presented toxicity from doses of 100µg/mL. EtOHAm, HexAm and CH2Cl2Am decreased the parasitism in vitro, presenting a therapeutic index of 2.62, 2.44, and 2.96, respectively. In vivo, EtOHAm, HexAm and CH2Cl2Am improved the survival rate of infected animals, however, only EtOHAm was able to decrease the parasitism in the small intestine and lung. Additionally, EtOHAm decreased the systemic interferon (IFN)-γ and tumor necrosis factor (TNF) systemically in infected mice, and was able to maintain the triglycerides and very-low-density lipoprotein (VLDL) lipid fractions at similar levels to uninfected animals. Although treatment with EtOHAm could not control the inflammation induced by oral infection in the tissues analyzed, it was able to preserve the number of goblet cells in the small intestine. CONCLUSIONS: Ethanolic A. muricata leaf extract could be considered as a good candidate for the development of a complementary/alternative therapy against toxoplasmosis, and also as an anti-inflammatory alternative for decreasing TNF and IFN-γ concentrations and lipid fractions in specific diseases.