Post-treatment with plant extracts used in Brazilian folk medicine caused a partial reversal of the antiproliferative effect of glyphosate in the<i> Allium cepa </i> test

Species of the genus Psychotria are used for multiple purposes in Brazilian folk medicine, either as water infusions, baths or poultices. This study was aimed to evaluate the genotoxic and antiproliferative effects of infusions of Psychotria brachypoda and P. birotula on the Allium cepa test. Exposure to distilled water was used as a negative control, while exposure to glyphosate was used as a positive control. The interaction of extracts (as a post-treatment) with the effects of glyphosate was also studied. Results showed that glyphosate and the extracts of both P. brachypoda and P. birotula reduced the mitotic index as compared with the negative control (distilled water). Surprisingly, however, both extracts from P. brachypoda and P. birotula caused a partial reversal of the antiproliferative effect of glyphosate when used as a post-treatment. Glyphosate also induced the highest number of cells with chromosomal alterations, which was followed by that of P. birotula extracts. However, the extracts from P. brachypoda did not show any signifi cant genotoxic effect. Post-treatment of glyphosate-treated samples with distilled water allowed a partial recovery of the genotoxic effect of glyphosate, and some of the Psychotria extracts also did so. Notably, post-treatment of glyphosatetreated samples with P. brachypoda extracts induced a statistically signifi cant apoptotic effect. It is concluded that P. brachypoda extracts show antiproliferative effects and are not genotoxic, while extracts of P. birotula show a less potent antiproliferative effect and may induce chromosomal abnormalities. The finding of a partial reversion of the effects of glyphosate by a post-treatment with extracts from both plants should be followed up.     

Alternative model to human skin organ culture: a preliminary study with Leibovitz L15 medium

Organ culture has been used to maintain the three-dimensional structure of the skin and the interaction between melanocytes and keratinocytes, which is essential for melanin production. In the current study we aimed to evaluate the general morphology, viability, and distribution of human melanocytes in a system that uses Leibovitz L15 medium at room temperature. By comparison with human skin explants maintained in Dulbecco's minimum Eagle's medium at 37 degrees C, we found that the skin was better preserved with Leibovitz L15 after 7 days in culture. The addition of 10% fetal bovine serum to this medium did not promote any change. Dividing cells labeled with Ki-67 were visualized at the basal and suprabasal epidermal layers. Retinoic acid was tested at 1 microg/mL and we recorded a reduction of the corneal layer after 48 hours and a complete detachment of the epidermis after 7 days, probably due to a toxic effect in the medium. Melanin and melanocytes were detected by ammoniacal silver and Dopa stainings under light microscopy. We observed that cells were viable throughout the culture period and melanin was distributed in melanocytes and keratinocytes. In conclusion, we suggest that the use of Leibovitz L15 medium at room temperature can be a viable alternative to the normal organ culture of human skin, which is an important system to study the activity and reaction of melanocytes to dermatological products and cosmetics.     

Genotoxic and mutagenic effects of permethrin in mice: Micronuclei analysis in peripheral blood erythrocytes

Pyrethroids such as permethrin are synthetic compounds widely used in the agriculture of many countries to combat plagues and in domestic products, such as acaricides. Not so long ago these chemicals were characterized as non‐toxic for non‐target organisms; however, recent studies have showed that these compounds could present toxic potential for many organisms. In this sense, this study presents genotoxic and mutagenic potential of permethrin administered intraperitoneally in mice under artificial conditions by the use of micronucleus assay in the peripheral blood of these animals. The mice were divided into five groups: group I = negative control (distilled water), group II = positive control (cyclophosphamide), group III = 30% of permethrin LD₅₀ (96 mg/kg), group IV = 50% of permethrin LD₅₀ (160 mg/kg), and group V = 80% of permethrin LD₅₀ (256 mg/kg). The peripheral blood was collected 24, 48, and 72 h after treatment. Results showed that all the tested permethrin dosages presented genotoxic and mutagenic effects 24 h after treatment, which would contradict the classification of this chemical product as moderately toxic, i.e., unable to cause damages to the cell DNA. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

Brief Note: Isolation,culture, characterization and optimization of human corneal stem cells

The effects of human versus mouse EGF on cell growth and culture duration were studied to optimize a human limbal stem cells culture method for therapeutical autologous transplantation. Limbal cells were obtained by trypsin digestion and transferred to a culture medium. The time needed to reach full confluence in culture was determined. Specific antibodies to corneal stem cell marker (P63) versus corneal epithelial differentiation marker (K3) were used for histochemical determinations. A high proportion of P63 positive cells (85± 4.6%), and a correspondingly low proportion K3 positive cells (15 ± 3.8%) indicated that most cultured cells remained undifferentiated and were considered as stem cells (mean ± SE, n=10). Cultures reached full confluency after 17.3 ± 1.2 days when the medium was supplemented with human EGF, while 21.7 ± 1.5 days were needed when the medium was supplemented with mouse EGF. The results showed that limbal stem cells proliferate more easily and reach to full confluency in a shorter time if the medium is supplemented with hEGF rather than with mEGF.     

A new bioreactor for the controlled application of complex mechanical stimuli for cartilage tissue engineering

Mechanical stimuli have been shown to enhance chondrogenesis on both animal and human chondrocytes cultured in vitro. Different mechanical stimuli act simultaneously in vivo in cartilage tissue and their effects have been extensively studied in vitro, although often in a separated manner. A new bioreactor is described where different mechanical stimuli, i.e. shear stress and hydrostatic pressure, can be combined in different ways to study the mechanobiology of tissue engineered cartilage. Shear stress is imposed on cells by forcing the culture medium through the scaffolds, whereas a high hydrostatic pressure up to 15 MPa is generated by pressurizing the culture medium. Fluid-dynamic experimental tests have been performed and successful validation of the bioreactor has been carried out by dynamic culture of tissue-engineered cartilage constructs. The bioreactor system allows the investigation of the combined effects of different mechanical stimuli on the development of engineered cartilage, as well as other possible three-dimensional tissue-engineered constructs.     

Reactive oxygen species in bovine embryo in vitro production.

Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39 degrees C in 5% CO2: 95% humidified air. In vitro fertilization was carried out in IVF-mSOF with frozen-thawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90% N2: 5% CO2: 5% O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2',7'-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P < 0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time.     

In situ cultured preantral follicles is a useful model to evaluate the effect of anticancer drugs on caprine folliculogenesis

Despite the increase in the incidence of cancer, the number of women who survive cancer treatment is growing. However, one of the principal results of chemotherapy is premature ovarian failure (POF). The aim of this study was to use the in situ culture preantral follicles as an in vitro model to evaluate the toxicity of two anticancer drugs, doxorubicin (DXR) and paclitaxel (PTX), on the integrity and development of ovarian follicles. Fragments of the ovarian cortex of goats were cultured in vitro for 1 or 7 days in α‐MEM⁺ supplemented with different concentrations of DXR (0.003, 0.03, or 0.3 µg/mL) and PTX (0.001, 0.01, or 0.1 µg/mL). Analyses were performed before and after culture to evaluate tissue integrity by classical histology, apoptosis by TUNEL assay, DNA laddering kit and the detection of activated caspase 3, and DNA damage by the immune detection of phosphorylated histone H2A.x (H2AXph139). Both DXR and PTX reduced the number of morphologically normal primordial and developing follicles. Positive staining for TUNEL and active caspase 3 was detected in all the samples (P < 0.05). Therefore, we propose the in situ culture of caprine preantral follicles as a useful experimental model for assessing the toxic effects of the chemotherapeutic agents on ovarian folliculogenesis. Microsc. Res. Tech. 79:773–781, 2016. © 2016 Wiley Periodicals, Inc.     

Role of LIBS in Elemental Analysis of Psidium guajava Responsible for Glycemic Potential

Abstract

The present study deals with the detection of elements responsible for glycemic potential of ripe and unripe fruit peel aqueous extracts of Psidium guajava (P. guajava). Treatment with the aqueous extract of unripe fruit peel showed a significant fall of 17.5% (p<0.001) in blood glucose levels (BGLs) of normal rats during fasting blood glucose (FBG) test with a dose of 400 mg/kg bw. In sub‐diabetic rats, a fall of 19.8% (p<0.001) was observed with the same dose during a glucose tolerance test (GTT). The significant fall observed in FBG, post prandial glucose (PPG) and urine sugar levels of severely diabetic rats was 20.7%, 17.5% (p<0.05), and 66.6% (p<0.01), respectively. On the contrary, the effect of ripe fruit peel aqueous extract showed a regular rise of 24.4% (p<0.01) in BGL of normal rats and of 90.0% (p<0.001) in sub‐diabetic rats. Laser Induced Breakdown Spectroscopy (LIBS) has been used for the identification of elements responsible for the glycemic potential of fruit peel aqueous extracts of P. guajava. Concentration of Mg was found higher in unripe fruit peel aqueous extract than in the ripe fruit peel aqueous extract whereas the concentration of K was found lower in the extract of unripe fruit peel than in the extract of ripe fruit peel. Thus, the LIBS results help in defining the role of Mg and K in diabetes management. However, the concentrations of other minerals, like Na, N, O, and C, are nearly the same in both of the extracts.     

Impact of pituitary FSH purification on <i>in vitro</i> early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol^® and Folltropin^®) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol^® (50 ng/mL) or Folltropin^® (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, only Stimufol^® maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin^® at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin^® than Stimufol^®. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol^® was better to preserve follicular morphology while Folltropin^® was more efficient to promote follicular growth.     

尿和血浆中劳拉西泮的气相色谱法研究

建立一种灵敏的检测人尿和血浆中劳拉西泮的气相色谱方法。酶水解的尿和血浆样品在pH =9用乙醚提取 ,提取物用MTBDMS衍生化 ,衍生物用HP - 5毛细柱进行色谱分离 ,用电子捕获检测器检测。尿和血浆中提取率分别为 84. 4 %和 81. 3% ,检测限分别为 4. 2ng/mL和 2. 4ng/mL。方法已用于口服 2mg劳拉西泮人的尿和血浆分析 ,分析结果表明本方法适合于麻醉抢劫案中药物分析。本方法已与非衍生化、TMS衍生化气相色谱分析方法比较 ,本方法是最灵敏的方法。     

Extract of white button mushroom affects skin healing and angiogenesis

White button mushroom extract was examined in this study on (1) its potential effect on angiogenesis in chorioallantoic culture and (2) its recovering effect on the skin after injury in the ICR mice. Methods used included TUNEL assay on apoptosis, immunohistochemistry for vascular endothelial growth factor (VEGF), proliferative cell nuclear antigen (PCNA), epidermal growth factor (EGF), transforming growth factor β (TGF‐β), and immune factor CD4 and western blotting. The results of chorioallantoic culture showed that the mushroom treatment led to significant increase in densities of VEGF sites. In the skin injury, ICR mice model increased EGF, PCNA, and collagen fibers, along with decrease of TUNEL positive apoptotic cells and limited reaction of TGF‐β and CD4 indicated that white button mushroom extract appeared to have beneficial effects on skin in regeneration and after injury. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

枸杞子提取物中多糖含量测定方法研究

采用苯酚-硫酸法衍生,紫外-可见分光光度法测定枸杞子提取物中枸杞子多糖含量。对样品制备及含量测定方法进行充分验证,最大吸收波长490nm,葡萄糖线性范围为2.65～13.3μg/mL,r=0.99939,平均回收率为101.32%,RSD为2.39%。所建立方法准确、可靠,可用于枸杞子提取物中多糖的含量测定。     

带 Thom盘的 Flettner转子的 CFD模拟研究

呈现了在8800雷诺数下,三维流动掠过转动比为3．0及6．0的带盘子圆柱转子的数值模拟。为了评估大尺寸盘子对流体动力的影响,雷诺数及几何模型与Thom在1934年的试验报告保持一致。参考往年文献,模拟得出了相反的结论。     

Key morphologic changes and DNA strand breaks in human lymphoid cells: Discriminating apoptosis from necrosis

Apoptosis is an important form of physiologic cell death displayed by an enormous variety of tissues under divergent conditions. The recent attention toward apoptosis in virtually all aspects of modern biology indicates that rapid and accurate differentiation between apoptosis and necrotic death is of considerable interest. Apoptosis is distinguishable from necrosis on the basis of several criteria. In this study, we undertook to examine the effects of mild hyperthermia (43°C leading to apoptotic death) and severe hyperthermia (50°C leading to necrotic killing) on associated DNA fragmentation. Using laser scanning and fluorescent microscopic evaluation of DNA “comets” in the single cell gel assay, we compared necrotic and apoptotic DNA damage in a variety of human leukemia and lymphoma cell lines at the level of the individual cell. We show that necrotic cells do display detectable DNA damage. We confirm our preliminary report that comet “tail moment” is sufficient to distinguish between necrotic and apoptotic DNA damage, while comet tail length may confuse the two forms. We report that a recovery period is necessary for expression of increasing apoptotic but not necrotic DNA damage. We show that apoptosis increases with prolonged hyperthermia and confirm that the mode of death changes from apoptosis to necrosis with higher heat loads, producing a greater fraction of cells showing damage. In addition, we show that for necrotic cells, DNA tail moment reflects sensitivity to prolonged exposure without a concomitant change in tail length.     

Antimicrobial activities of three seaweeds extract against some human viral and bacterial pathogens

Microbial infections cause complicated health influences along with bad economic impacts. In the present investigation, three dominant seaweeds namely, Amphiroa anceps, Corallina officinalis and Sargassum filipendula were collected from different Egyptian sites at the Red Sea and Mediterranean Sea during autumn 2019. Organic extracts of the three algae were screened for their antibacterial activity against three pathogenic bacteria Salmonella typhiimurium, Staphylococcus aureus and Escherichia coli, in addition to in vitro antiviral activity against Rotavirus (RV), and Coxsackie virus B3 (CVB3) that cause severe diseases in human. Organic extract of A. anceps, C. officinalis and S. filipendula inhibit E. coli cells by 57.1%, 85.7%, and 91.4%, respectively. The highest level of concentration of the C. officinalis extract (100 µg/mL) inhibits 100% of Staphylococcus aureus cells followed by S. filipendula and A. anceps extract which inhibit 82.5% and 75% of S. aureus. Similarly, the highest concentration of C. officinalis extract inhibits S. typhiimurium by 80%. The extract of A. anceps exhibited a high antiviral effect against RV infection with TI = 22 and virus titers lessened by 2.75 log TCID₅₀ followed by extractions of C. officinalis with TI = 18.3 and virus titers reduced by 2.5 log TCID₅₀. Against CVB3 infection, the extract of A. anceps causes the highest antiviral activity with TI = 15 and reduce the viral titers by 2.5 log TCID₅₀, followed by extractions of C. officinalis with TI = 8.8 and inhibition of virus titers by 1.75 log TCID₅₀. Extract of S. filipendula displayed the lowest antiviral effects against RV and CVB3 infection with TI = 2.4 and 1.4, respectively. The obtained results clarified that the extract of three marine seaweeds maintains a potent antimicrobial activity, making them a future promising source of new antimicrobial drugs.

     

毛细管气相色谱法测定面包中脱氢乙酸含量

建立了面包中脱氢乙酸含量的毛细管气相色谱测定方法。该法采用WBI进样口,毛细管色谱柱J＆WDB1701（中等极性30m×0.53mm×1μm）,FID检测器进行检测。进样口温度：250℃,载气流速：10mL/min,柱室温度：150℃恒温,检测器温度：250℃,样品用硫酸溶液酸化无水乙醇定容后直接进样测定。方法简单、快速、重现性好,平均回收率大于95%,RSD小于3.O%,线性范围为20μg/mL一400μg/mL。     

Optimization of medicinal plant extraction methods and their encapsulation through extrusion technology

In this study, aqueous extracts of Plectranthus ecklonii, Plectranthus grandidentatus, Plectranthus ornatus, Plectranthus porcatus, and Plectranthus saccatus were prepared using infusion, decoction, microwave and ultrasonic methods. Rosmarinic and caffeic acids were their major components while chlorogenic acid was for the first time found in a Lamiaceae extract (P. saccatus), being the acetylcholinesterase (AChE) inhibition and the antioxidant activities furthermore evaluated. P. ecklonii microwave extract with a high yield (22.9 ± 0.3 mg of dry plant extract/mL), the best AChE inhibition activity (59.14 ± 4.97%) and a strong antioxidant activity (25.47 ± 1.1%) at 1 mg/mL, was then successfully encapsulated into alginate beads (98.64–100.0%). The high rosmarinic and caffeic acids content as well the high AChE inhibition and antioxidant properties after the encapsulation strongly indicate that this strategy could potentiate the action of the plant extract by promoting a protective effect and a sustained release of active constituents and, ultimately, improve their pharmacological effects.     

小新月菱形藻固定化培养初步研究

用褐藻酸钙对小新月菱形藻进行固定化培养实验,测定不同胶粒大小、胶珠密度、CaCl2浓度及不同接种量对该藻的影响,比较了自由化生长与固定化细胞的生长曲线。小新月菱形藻在褐藻酸钙凝胶中仍具有呼吸和光合作用的能力。球径为3.5mm,CaCl2浓度为2%时,细胞生长快,每50mL培养液中加入200个胶珠时细胞生长量大,接种量不能低于104个细胞/mL。与游离的小新月菱形藻相比,固定化小新月菱形藻生长慢,但生长周期长。