Bruno dos Santos Lima

Acute and sub-acute oral toxicity of Brazilian red propolis in rats

ETHNOPHARMACOLOGICAL RELEVANCE Propolis is a bee product widely used in folk medicine due to its numerous pharmacological properties. However, samples from different regions can differ in chemical composition, effectiveness, and side effects. Despite the widespread use of Brazilian red propolis, which is an isoflavone-rich variety, its toxicity has not been carefully studied. AIMS OF THE STUDY To assess the acute and sub-acute toxicity of the hydroethanolic extract of red propolis (HERP) administered orally to rats. MATERIALS AND METHODS HERP for the acute (300mg/kg) and sub-acute (10, 100 and 200mg/kg) toxicity studies was administered orally to rats according to OECD Guidelines 420 and 407, respectively. Clinical signs were identified, and hematological and biochemical analyses were performed. Water and food uptake as well as body and organ weights of animals were recorded. RESULTS AND CONCLUSIONS The acute study revealed no lethal effects at 300mg/kg of HERP, but toxic signs were observed, as HERP had an LD50 of more than 300mg/kg, indicating a warning. The most toxic signals in sub-acute studies were observed in males at a dose of 200mg/kg HERP. These results suggest estrogen-like activity, possibly from the isoflavones in HERP.

Gelatin-based membrane containing usnic acid-loaded liposome improves dermal burn healing in a porcine model.

There are a range of products available which claim to accelerate the healing of burns; these include topical agents, interactive dressings and biomembranes. The aim of this study was to assess the effect of a gelatin-based membrane containing usnic acid/liposomes on the healing of burns in comparison to silver sulfadiazine ointment and duoDerme® dressing, as well as examining its quantification by high performance liquid chromatography. The quantification of the usnic acid/liposomes was examined using high performance liquid chromatography (HPLC) by performing separate in vitro studies of the efficiency of the biomembranes in terms of encapsulation, drug release and transdermal absorption. Then, second-degree 5cm2 burn wounds were created on the dorsum of nine male pigs, assigned into three groups (n=3): SDZ - animals treated with silver sulfadiazine ointment; GDU - animals treated with duoDerme®; UAL - animals treated with a gelatin-based membrane containing usnic acid/liposomes. These groups were treated for 8, 18 and 30days. In the average rate of contraction, there was no difference among the groups (p>0.05). The results of the quantification showed that biomembranes containing usnic acid/liposomes were controlled released systems capable of transdermal absorption by skin layers. A macroscopic assay did not observe any clinical signs of secondary infections. Microscopy after 8days showed hydropic degeneration of the epithelium, with intense neutrophilic infiltration in all three groups. At 18days, although epidermal neo-formation was only partial in all three groups, it was most incipient in the SDZ group. Granulation tissue was more exuberant and cellularized in the UAL and GDU groups. At 30days, observed restricted granulation tissue in the region below the epithelium in the GDU and UAL groups was observed. In the analysis of collagen though picrosirius, the UAL group showed greater collagen density. Therefore, the UAL group displayed development and maturation of granulation tissue and scar repair that was comparable to that produced by duoDerme®, and better than that produced by treatment with sulfadiazine silver ointment In addition, the UAL group showed increased collagen deposition compared to the other two groups.

Effects of luteolin and quercetin 3-β-d-glucoside identified from Passiflora subpeltata leaves against acetaminophen induced hepatotoxicity in rats

Passiflora subpeltata has many beneficial effects in the treatment of various diseases including inflammation, pain and fever. This study was aimed to analyze the phytochemical compounds present in acetone extract of P. subpeltata leaves and to evaluate their performance against paracetamol induced hepatotoxicity activity. HPLC-DAD method was used to identify and quantify the phytochemical compounds. Hepatoprotective activity of acetone extract in the treatment of rat liver functions was monitored by the measurement of blood parameters and serum biochemical parameters such as SGOT, SGPT, ALP and in vivo antioxidant parameters viz. SOD, CAT and LPO. Further, liver tissues were also subjected to histopathological analysis. The HPLC-DAD results showed the luteolin and quercetin 3-β-d-glucoside as newly identified compounds in P. subpeltata species. Pre-treatment with acetone extract of P. subpeltata leaves at 200 and 400mg/kg doses significantly elevated the WBC, RBC and HB counts and retained the serum biochemical and enzymatic antioxidants levels to normal level. Based on this detailed study we conclude that acetone extract of P. subpeltata leaves offered better protection against hepatotoxicity induced by the acetaminophen.

The photoprotective and anti-inflammatory activity of red propolis extract in rats

The negative effects triggered by ultraviolet radiation, such as premature aging and carcinogenesis, have motivated several studies on photoprotection. Recent strategies for photoprotection have included the incorporation of natural antioxidant and anti-inflammatory compounds, such as flavonoids, into sunscreens and the oral administration of natural antioxidant extracts. Brazilian Red propolis extract contains isoflavonoids with antioxidant and anti-inflammatory activities. Here, we investigate the photoprotective effects of orally- or topically-administered formulations containing hydroalcoholic extract of red propolis (HERP) in a rodent model. HERP showed markers identified as: daidzein (4.68 μg/mL), formononetin (31.81 μg/mL) and biochanin A (9.58 μg/mL). A fourth peak was found in the chromatogram but was not identified. The antioxidant activity of HERP was calculated to be 3.07 mmol Trolox/g and 2.13 mmol Trolox/g, respectively. Topical HERP exerted a protective action against UVB radiation, which was similar to that exerted by oxybenzone filter. Oral HERP as an adjuvant treatment did not increase sunburn protection. However, the oral administration of HERP presented chemoprotective and anti-inflammatory activity (p < 0.05) similar or better than Polypodium leucotomos oral treatment (positive control). In conclusion, topical administration of HERP has photoprotective activity in a murine model and the mechanisms of protection can be related to the antioxidant and anti-inflammatory characteristics of HERP compounds.

Hydroalcoholic extract of Brazilian red propolis exerts protective effects on acetic acid-induced ulcerative colitis in a rodent model

Ulcerative colitis (UC) is a common intestinal inflammatory disease with an etiology that is not well understood. Although the anti-inflammatory and anti-oxidant effects of the hydroalcoholic extract of Brazilian red propolis (HERP) have been reported in various experimental models, its protective effect in models of UC have not been evaluated. The purpose of this study was to investigate the chemopreventive effect of hydroalcoholic extract of Brazilian red propolis (HERP) in acetic acid-induced colitis (AAIC) using a rodent model. The HERP was chemically characterised by HPLC/DAD analyses. Male rats were randomly assigned into four groups: sham, vehicle (with AAIC, treated with vehicle), P10 (with AAIC, treated with 10mg/kg HERP), and P100 (with AAIC, treated with 100mg/kg HERP). Treatments were performed for 7days, and colitis was induced on day seven. Animals were euthanized 24h after colitis induction and body weight, colon length, gross and histological scores, malondialdehyde (MDA) and myeloperoxidase (MPO) concentrations in colon tissue, and the immunohistochemical expression of inducible nitric oxide synthase (iNOS) were assessed. The major compounds found in HERP were liquiritigenin (68.8mg/g), formononetin (54.29mg/g), biochanin A (30.97mg/g), and daidzein (19.90mg/g). Rats treated with 10mg/kg HERP demonstrated significant decreases in MPO concentrations, gross and histological scores of tissue damage, and iNOS expression (p<0.05). Similarly, rats treated with 100mg/kg HERP demonstrated significant decreases in MPO levels (p<0.05) and histological scores of tissue damage (p<0.05). The results of this study indicate that oral administration of HERP attenuates AAIC in rats, which may be due to anti-inflammatory effects related to iNOS inhibition.

Aqueous extract of Chrysobalanus icaco leaves, in lower doses, prevent fat gain in obese high-fat fed mice.

ETHNOPHARMACOLOGICAL RELEVANCE Due to the rise in obesity, the necessity for resources and treatments that could reduce the morbidity and mortality associated to this pandemia has emerged. The development of new anti-obesity drugs through herbal sources has been increasing in the past decades which are being used not only as medicine but also as food supplements. Previous studies with the aqueous extract of Chrysobalanus icaco L (AECI) have demonstrated activity on lowering blood glucose levels and body weight. AIM OF THE STUDY Investigate C. icaco effects in overall adiposity and glycemic homeostasis. MATERIAL AND METHODS C57BL/6J mice were randomly assigned to standard chow (SC) or high-fat diet (HFD) and treated with AECI in 0.35mg/mL or 0.7mg/mL concentrations ad libitum. Food intake, feed efficiency, metabolic efficiency, body, fat pads and gastrocnemius weight, adiposity index, serum lipids, fecal lipid excretion, locomotor activity in the open field test and insulin and glucose tolerance tests were analyzed and compared. The major components of the extract were demonstrated through HPLC and its antioxidant activity analyzed through DPPH and lipid peroxidation. RESULTS The AECI in the 0.35mg/mL concentration did not affect food intake or body weight. However, it promoted lower adipose tissue gain, TG levels, and fecal lipid excretion, increased locomotor activity and lean mass weight, and normalized insulin sensitivity and glucose tolerance. Moreover, AECI showed the presence of myricetin 3-O-glucuronide, rutin, quercitrin and myricitrin and demonstrated high-antioxidant activity. CONCLUSIONS AECI in lower concentrations can prevent fat storage or enhance fat utilization through the increase of locomotor activity. Also, this reinforces its ability to maintain glucose homeostasis through the normalization of insulin sensitivity and glucose tolerance despite the high-fat diet intake. These activities could be associated to the extracts polyphenol content.

Hesperetin-loaded lipid-core nanocapsules in polyamide: a new textile formulation for topical drug delivery

Chronic venous insufficiency is characterized by chronic reflux disorder of blood from the peripheral to the central vein, with subsequent venous hypertension and resulting changes in the skin. Traditionally, nonsurgical treatments relied on the use of compression therapy, and more recently a variety of flavonoids have been shown to have positive effects. There have also been developments of more effective drug delivery systems using various textiles and nanotechnology to provide new therapeutic options. Our objective was to use nanotechnology to develop a new formulation containing hesperetin (Hst), a substance not previously used in the treatment of chronic venous insufficiency, impregnated into textile fibers as a possible alternative treatment of venous diseases. We prepared the nanocapsules using the interfacial deposition of preformed polymer method with an Hst concentration of 0.5 mg/mL and then characterized the size and distribution of particles. To quantify the Hst in the samples, we developed an analytical method using high-performance liquid chromatography. Studies of encapsulation efficiency (98.81%±0.28%), microscopy, drug release (free-Hst: 104.96%±12.83%; lipid-core nanocapsule-Hst: 69.90%±1.33%), penetration/permeation, drug content (0.46±0.01 mg/mL) and the effect of washing the textile after drug impregnation were performed as part of the study. The results showed that nanoparticles of a suitable size and distribution with controlled release of the drug and penetration/permeation into the skin layers were achieved. Furthermore, it was established that polyamide was able to hold more of the drug, with a 2.54 times higher content than the cotton fiber; after one wash and after five washes, this relation was 2.80 times higher. In conclusion, this is a promising therapeutic alternative to be further studied in clinical trials.

Polyphenols rich Passiflora leschenaultii leaves modulating Farnesoid X Receptor and Pregnane X Receptor against paracetamol-induced hepatotoxicity in rats

The hepatoprotective effect of P. leschenaultii. (DC) leaves was investigated in rats under paracetamol induced oxidative stress. Leaf acetone extract (200 and 400mg/kg) were administered daily via gavage for 14days before paracetamol (2000mg/kg, p.o.) treatment. After the experiment, the levels of serum biochemical parameters and enzymatic antioxidant levels were determined. Furthermore, liver tissues were analyzed histopathologically. Additionally, the molecular docking studies of the identified compounds against PXR and FXR proteins were also performed. The assessment revealed that the acetone extract significantly reduced the elevated levels of SGPT, SGOT and ALP in serum. Moreover, the enzymatic antioxidants such as SOD, CAT and LPO were also retained normally by the plant extract. From histopathological analysis, it was clearly evident that the cellular architecture of plant extract treated rat liver tissues were not affected by the paracetamol induction at the higher dose. The results of docking studies also revealed that the identified compounds showed steric interactions (between nonpolar atoms) with amino acid groups. Collectively, the present study suggests that P. leschenaultii leaves extract protects the liver from paracetamol induced hepatic damage.

UHPLC-QqQ-MS/MS identification, quantification of polyphenols from Passiflora subpeltata fruit pulp and determination of nutritional, antioxidant, α-amylase and α-glucosidase key enzymes inhibition properties.

The diabetic key enzymes inhibition, nutritional, antioxidant activity and bioactive compounds identification of Passiflora subpeltata fruit pulp were investigated. Fifteen polyphenolic compounds including protocatechuic acid, ferulic acid, vanillic acid, epicatechin, p-coumaric acid, cinnamic acid, eriodictyol and quercetin-3-glucoside were identified in the pulp of this species by using UHPLC-QqQ-MS/MS analysis. The total carbohydrates and crude protein contents in fruit pulp were 2.62 mg glucose equivalent/g sample fruit pulp and 8.80 mg BSA equivalent/g sample fruit pulp, respectively. The fresh fruit pulp of P. subpeltata contained high total phenolic (724.76 mg GAE/g sample) content and it revealed very high DPPH• (IC50 of 5.667 μg/mL) and ABTS+• (6794.96 μM trolox equivalent/g sample) scavenging activities. In the key enzymes assays useful for diabetic inhibition the fresh fruit pulp characterized maximum inhibition of α-amylase and α-glucosidase IC50 of 18.69 and 32.63 μg/mL, respectively. Thus, these results lead to conclude that this fruit specie could be very useful source in nutraceutical products preparations for Type 2 diabetic suffering humans.

Inclusion complex between β-cyclodextrin and hecogenin acetate produces superior analgesic effect in animal models for orofacial pain

Hecogenin acetate (HA) is a steroidal sapogenin-acetylated with pharmacological properties which have already been described in the literature such as, anti-inflammatory, anti-hyperalgesic and antinociceptive, but it has low solubility in aqueous media. Therefore, in an attempt to overcome this, we set out to create inclusion complexes between HA and b-cyclodextrin (b-CD) and evaluate the antinociceptive effects in the orofacial nociception in mice. The complexes were prepared using different methods in the molar ratios 1:1 and 1:2 and characterized physicochemically. The results of the physicochemical characterization elucidated inclusion complexes formation between b-CD and HA by freeze drying method in the molar ratio 1:2, which obtained a complexation efficiency of 92% and produced superior analgesic effect in animal models for orofacial pain at a lower dose when compared to HA alone.