Comparative sperm recovery rate after density gradient centrifugation with two media for in vitro fertilization.

OBJECTIVE: To compare the efficacy of two density gradient centrifugation media for retrieving spermatozoa from semen samples by evaluating the total motile sperm count (TMSC) and the percentage recovery. METHODS: Twenty-two men with different sperm counts participated in the study. The samples were divided into two equal aliquots and processed using the commercial ISolate Sperm Separation Medium (Irvine Scientific, United States) and the GV Gradiente (IngáMed, Brazil). After separation, samples were counted and evaluated for motile sperm recovery. RESULTS: The mean TMSC in the fresh sample was 19.65±21.08 million/mL. After the ISolate separation the TMSC was 6.71±7.29 million/mL, and for the GV Gradiente it was 6.27±6.82 million/mL. The percentage of motile spermatozoa recovered was 36.47%±21.61 for ISolate and 35.22%±21.24 for the GV Gradiente (p>0.05). The samples from 6 oligospermic patients (27%) were evaluated separately and the TMSC for ISolate was 4.83±2.92 million/mL, and for the GV Gradiente, it was 4.16±3.12 million/mL (p=0.54). When evaluating only normospermic patient samples, the TMSC for ISolate was 9.05±7.29 million/mL, and for the GV Gradiente, it was 8.47±6.79 million/mL (p=0.83). CONCLUSIONS: There was no statistical difference in retrieving motile sperm using the GV Gradiente and the ISolate Separation Medium.

Manufacturing Different Types of Solid Dispersions of BCS Class IV Polyphenol (Daidzein) by Spray Drying: Formulation and Bioavailability.

Daidzein (DZ) is a polyphenolic compound belonging to Biopharmaceutical Classification System class IV, which shows that it may have limited therapeutic effects due to its low solubility and poor bioavailability. This study aimed to obtain high-purity DZ and prepare and characterize different types of solid dispersions (SDs) in order to enhance aqueous solubility and bioavailability. Excipients were investigated in order to manufacture different types of solid dispersions (SDs). Second-generation solid dispersions (SG), third-generation solid dispersions (TG), and second- and third-generation pH-modulated solid dispersions (SD and TG pHM-SD) were produced via spray drying. The SDs were characterized and tested for in vitro DZ release and oral bioavailability. SDs have shown increased aqueous solubility and in vitro release rate. Solid-state characterization showed that DZ was in an amorphous state in most of the formulations. The enhanced aqueous solubility of TG-pHM SD was reflected by an increase in oral bioavailability, which significantly increased the maximum plasma concentration approximately 20-fold and decreased the time to reach the maximum plasma concentration. The production of pHM SDs that contain DZ via spray drying is a simple and effective approach for oral drug delivery, which has the potential to greatly reduce the dose and enhance therapeutics effects.

In vitro antioxidant potential and in vivo effects of Schinus terebinthifolia Raddi leaf extract in diabetic rats and determination of chemical composition by HPLC-ESI-MS/MS.

The present study investigated the in vitro and in vivo antioxidant potential and phytochemical composition of Schinus terebinthifolia, which is widely used in folk medicine for various therapeutic purposes. The in vitro analyses indicated that the hydroethanolic extract (HE) had 312.50 ± 0.50 mg GAE/g of total phenols. It also presented anti-DPPH• and anti-ABTS•+ activity, reduced phosphomolybden and metal ions and blocked the bleaching of ß-carotene. The HE at concentrations of 3.0 and 2.0 µg/mL had TRAP values of 2.223 ± 0.018 and 1.894 ± 0.026 µM Trolox, respectively. The HE increased the availability of antioxidants in plasma in treated animals in vivo. HPLC-ESI-MS/MS indicated the presence of 11 phenols: cumaric acid, (+)-catechin, myricetin-3-O-glicuronide, kaempferol-3-O-glucoside, myricetin, myricitrin, quercetin, gallic acid, methyl galate, pentagalloyl glucose and ethyl galate. Thus, S. terebinthifolia has potential for the prevention or treatment of diseases that are related to oxidative stress, such as diabetes mellitus.

Flavan-3-ols and proanthocyanidins from Limonium brasiliense inhibit the adhesion of Porphyromonas gingivalis to epithelial host cells by interaction with gingipains.

Porphyromonas gingivalis is a pathogen strongly involved in chronic and aggressive forms of periodontitis. Natural products, mainly polyphenols, have been described for advanced treatment of periodontitis by inhibition of the bacterial adhesion of P. gingivalis to the epithelial host cells. An acetone:water extract (LBE) from the rhizomes of Limonium brasiliense (Boiss.) Kuntze was tested under in vitro conditions for potential antiadhesive effects against P. gingivalis to human KB cells and for inhibition of the proteolytic activity of gingipains, the main virulence factor of P. gingivalis. LBE≤100µg/mL had no cytotoxicity against the bacteria and did not influence the cell physiology of human epithelial KB cells. At 100µg/mL LBE reduced the adhesion of P. gingivalis to KB cells significantly by about 80%. LBE at 20µg/mL reduced the proteolytic activity of the arginin-specific Rgp gingipain by about 75%. Chemical profiling of LBE indicated the presence of gallic acid, epigallocatechin-3-O-gallate and samarangenins A and B as lead compounds. UHPLC by using MS and UV detection displays a suitable method for quality control of the extract for identification and quantification of the lead compounds.

Guaraná (Paullinia cupana) seeds: Selective supercritical extraction of phenolic compounds.

Approximately 70% of the Brazilian production of guaraná (Paullinia cupana) seeds is absorbed by the beverage industries. Guaraná has several pharmacological properties: energy stimulant, antimicrobial, chemoprophylactic, antigenotoxic, antidepressive, anxiolytic, and anti-amnesic effects. Supercritical carbon dioxide extraction of bioactive compounds from guaraná seeds was carried out and optimized by an orthogonal array design (OA9(3(4))). The factors/levels studied were: modifier(s) (ethanol and/or methanol), extraction time (20, 40, and 60min), temperature (40, 50, and 60°C), and pressure (100, 200, and 300bar). The statistical design was repeated with increasing proportions of modifiers. The percentage of modifier used was proportional to the amount of polar compounds extracted. The best conditions for the supercritical extraction, based on the content of polyphenols, epicatechin/catechin quantification, yield and operating cost, proved to be: 40% ethanol:methanol during 40min, under 40°C, and 100bar. The temperature had a significant effect on the total phenolic content.

Stryphnodendron adstringens: Clarifying Wound Healing in Streptozotocin-Induced Diabetic Rats.

Diabetes mellitus is a serious public health problem in which a major complication is impaired wound healing. Among the strategies developed to foster tissue repair is the use of medicinal plants. The bark of Stryphnodendron adstringens, which is popularly used as an aid in wound healing, has a documented effect on wound repair in normal rats. This study evaluated the healing action of the crude extract of S. adstringens in diabetic rats, and its chemical content. Compounds present in the crude extract were characterized by mass spectrometry. In diabetic rats (streptozotocin 35 mg/kg), two wounds made in the skin were treated daily for 4, 7, 10, and 14 days with gel containing 1 % crude extract or with base gel. Histological analyses involved the measurement of the length and thickness of the re-epithelialized surface, quantification of the number of cells in mitosis, and types I and III collagen fibers. Also, cutaneous permeation by photoacoustic spectroscopy, and the expression of cyclooxygenase-2 and vascular endothelial growth factor by Western blot were assessed. The crude extract fingerprint showed masses indicating proanthocyanidins. The crude extract mainly stimulated cell migration and proliferation of keratinocytes at the beginning of the treatment in addition to stimulating the replacement of type III collagen fibers by type I collagen fibers at 10 and 14 days. The photoacoustic spectroscopy technique showed that the gel containing 1 % of crude extract permeated through the skin to the dermis, where the crude extract was found. Vascular endothelial growth factor was stimulated after 7 days of treatment with the crude extract and cyclooxygenase-2 at 4, 7, and 10 days. The crude extract of S. adstringens acted in tissue repair in wounds in diabetic rats by stimulating the production of collagen fibers at the wound site. The crude extract favored the formation of a more organized extracellular matrix and filled the entire extent of the wound, and also fostered the upregulation of cyclooxygenase-2 and vascular endothelial growth factor, which are essential to this process. These crude extract actions in diabetic wounds are probably due to the presence of proanthocyanidins.

Hydrolyzable tannins from hydroalcoholic extract from Poincianella pluviosa stem bark and its wound-healing properties: phytochemical investigations and influence on in vitro cell physiology of human keratinocytes and dermal fibroblasts.

Extracts from Poincianella pluviosa stem bark are used in traditional medicine of South America for its wound healing properties. For validation of this traditional use and for rationalizing a potential pharmaceutical development towards standardized preparations bioassay-guided fractionation of EtOH-water (1:1v/v) extract (crude extract, CE) of P. pluviosa bark was performed. HaCaT keratinocytes cell line and human primary dermal fibroblasts (pNHDF) were used as in vitro systems. Significant stimulation of mitochondrial activity was found for CE on both cell types, which caused a strong increase of cell proliferation of keratinocytes. Fractionation of CE over Sephadex LH20 revealed two inactive fractions (FA and FB) and an active fraction FC, which was further fractionated by MPLC into 4 subfractions. Subfraction FC1 increased mitochondrial activity and proliferation of keratinocytes and dermal fibroblasts in a dose dependent manner (10 to 100 µg/mL) and did not show necrotic cytotoxicity on keratinocytes (LDH release assay). FC1 was investigated by ESI-MS/MS and solid-state (13)C NMR which confirmed the presence of various polyphenols and hydrolyzable tannins. MS studies suggest the presence of pyrogallol (1), gallic acid (2), gallic acid methyl ester (3), ellagic acid (4), corilagin (5), 1,4,6-tri-O-galloyl-glucose (6), tellimagrandin I (7), 1,2,3,6-tetra-O-galloyl-glucose (8), mallotinic acid (9), tellimagrandin II (10), 1,2,3,4,6-penta-O-galloyl-glucose (11), geraniin (12), and mallotusinic acid (13).

Preparation of Spray-Dried Soy Isoflavone-Loaded Gelatin Microspheres for Enhancement of Dissolution: Formulation, Characterization and in Vitro Evaluation.

The most bioactive soy isoflavones (SI), daidzein (DAI) and genistein (GEN) have poor water solubility, which reduces their bioavailability and health benefits and limits their use in industry. The goal of this study was to develop and characterize a new gelatin matrix to microencapsulate DAI and GEN from soy extract (SE) by spray drying, in order to obtain solid dispersions to overcome solubility problems and to allow controlled release. The influences of 1:2 (MP2) and 1:3 (MP3) SE/polymer ratios on the solid state, yield, morphology, encapsulation efficiency, particle size distribution, release kinetics and cumulative release were evaluated. Analyses showed integral microparticles and high drug content. MP3 and MP2 yield were 43.6% and 55.9%, respectively, with similar mean size (p > 0.05), respectively. X-ray diffraction revealed the amorphous solid state of SE. In vitro release tests showed that dissolution was drastically increased. The results indicated that SE microencapsulation might offer a good system to control SI release, as an alternative to improve bioavailability and industrial applications.