Phosphate-buffered Saline Extracts Research Articles

Allergenic Potential of Common Hops (Humulus lupulus L.) in the Context of Cross-Reactions with Pollen Allergens.

Common hops (Humulus lupulus L.) play a key role in brewing, providing the bitterness, flavor, and aroma of beer, and are widely used in supplements for their antibacterial, anti-inflammatory, and antioxidant properties. However, despite their broad applications, the allergenic potential of common hops remains underexplored, particularly when compared to the closely related Humulus japonicus. This preliminary study aimed to investigate the allergenic potential of common hops and their potential cross-reactivity with common pollen allergens. The immunoreactivity of hop stalks, leaves, and cones was assessed using antibodies against major allergens from birch (Bet v1a), mugwort (Art v1), and timothy grass (Phl p5b), as well as three sera from pollen-allergic patients. Slot Blot analysis was performed using phosphate-buffered saline extracts from the stalks, leaves, and cones of three hop cultivars, while Western Blotting followed SDS-PAGE protein separation. The results revealed significant immunoreactivity in native hop proteins, with diminished reactivity observed in denatured proteins. Cross-reactivity between hop proteins and major pollen allergens was confirmed, indicating that hop proteins may contribute to allergic sensitization in pollen-sensitive individuals. These findings underscore the potential allergenic risks associated with the consumption or exposure to hop-containing products.

In Silico Analysis of Binding Sites for a Novel ssDNA Aptamer Specific to Verrucarin A and Detection in Dust Extracts.

An aptamer is a single-stranded oligonucleotide that serves as a chemical antibody with a high specificity and binding affinity that can recognize a wide range of molecules. Effective modification and truncation of aptamers can enhance their binding affinities to particular targets while also broadening their application for uses, such as biosensors. However, a conventional trial-and-error methodology hinders this process. Herein, we demonstrate an in silico method to elucidate the binding site of single-stranded DNA aptamer specific to verrucarin A, a mycotoxin produced by molds in indoor buildings that causes adverse effects in living organisms. The novel ssDNA aptamer exhibited a binding affinity of 29.5 nM, demonstrating a relatively strong affinity compared to those of previously reported typical aptamers for small molecules. Furthermore, the selected aptamer was highly specific toward verrucarin A among structurally related mycotoxins (i.e., verrucarol and zearalenone). The specific binding site of the aptamer predicted via molecular dynamics and molecular docking simulations was highly consistent with the results observed via truncation, single base mutation, and circular dichroism experiments. The fluorescence assay revealed limits of detection and quantification of 4.1 and 12 nM for the aptamer, respectively. Comparing our developed aptasensor with LC-MS/MS methodology revealed that it could detect verrucarin A levels in phosphate-buffered saline and dust extracts with robust precision and consistency. Our findings provide insight for future studies exploring interaction mechanisms with intended targets and practical sensing applications, such as point-of-care detection of verrucarin A.

CAPN5 attenuates cigarette smoke extract-induced apoptosis and inflammation in BEAS-2B cells.

Apoptosis and chronic inflammation are the main phenotypes in chronic obstructive pulmonary disease (COPD) pathogenesis. Cigarette smoke exposure is the leading risk factor for COPD, which causes aberrant airway epithelial structure and function. As a non-classical calpain, the molecular function of calpain5 (CAPN5) in COPD remains unclear. This study investigated the role of CAPN5 in mediating cigarette smoke extract (CSE)-induced apoptosis and inflammation. Immunohistochemistry (IHC) and Western blotting (WB) were performed to detect the location and expression of CAPN5. In vitro, BEAS-2B cells were transfected with CAPN5 siRNA or CAPN5 plasmid, followed by phosphate-buffered saline (PBS) or cigarette smoke extract (CSE) treatment. The protein expression levels of CAPN5, NF-κB p65, p-p65, IκBα, p-IκBα and apoptosis proteins (BCL-2, BAX) were measured by WB. Flow cytometry (FCM) was performed to analyze the cell apoptosis index. CAPN5 was mainly expressed in the airway epithelium and significantly decreased in the COPD-smoker and emphysema-mouse groups. Silencing CAPN5 significantly decreased the protein expression of BCL-2, IκBα, and increased p-p65 and BAX protein expression. Additionally, an increased apoptosis index was detected after silencing CAPN5. Moreover, overexpression of CAPN5 partly inhibited IκBα degradation and p65 activation, and reduced CSE-induced inflammation and apoptosis. These combined results indicate that CAPN5 could protect against CSE-induced apoptosis and inflammation, which may provide a potential therapeutic target for smoking-related COPD.

Alzheimer proteopathic tau seeds are biochemically a forme fruste of mature paired helical filaments.

Aggregation prone molecules, such as tau, form both historically well characterized fibrillar deposits (neurofibrillary tangles) and recently identified phosphate-buffered saline (PBS) extract species called proteopathic seeds. Both can cause normal endogenous tau to undergo templated misfolding. The relationship of these seeds to the fibrils that define tau-related diseases is unknown. We characterized the aqueous extractable and sarkosyl insoluble fibrillar tau species derived from human Alzheimer brain using mass spectrometry and in vitro bioassays. Post-translational modifications (PTMs) including phosphorylation, acetylation and ubiquitination are identified in both preparations. PBS extract seed competent tau can be distinguished from sarkosyl insoluble tau by the presence of overlapping, but less abundant, PTMs and an absence of some PTMs unique to the latter. The presence of ubiquitin and other PTMs on the PBS-extracted tau species correlates with the amount of tau in the seed competent size exclusion fractions, with the bioactivity and with the aggressiveness of clinical disease. These results demonstrate that the PTMs present on bioactive, seed competent PBS extract tau species are closely related to, but distinct from, the PTMs of mature paired helical filaments, consistent with the idea that they are a forme fruste of tau species that ultimately form fibrils.

Hemagglutinin from the Root Tuber of Dioscorea preussii Pax.

In this study, Dioscorea preussii root tuber hemagglutinin was purified and its physicochemical properties were determined. The antioxidative and anti-hemolytic activities of the hemagglutinin were also investigated. The hemagglutinating assay was used to detect the presence of lectin in the phosphate-buffered saline extract of the D. preussii root tuber. The lectin was purified using ammonium sulfate fractionation and molecular sieve chromatography. The optimum pH and temperature were determined. In addition, antioxidant activity was assessed using 2,2 diphenylpicrylhydrazyl (DPPH) radical scavenging, metal chelating, ferric reducing antioxidant power (FRAP), and lipid peroxidation inhibition assays. Red blood cells subjected to oxidative damage caused by H2O2 were employed to evaluate their antihemolytic ability. Starch inhibited hemagglutinin activity. Dioscorea preussii hemagglutinin (DPH) maintained full hemagglutinating activity from 30 °C to 60 °C and pH 5-13. Ethylene diamine tetraacetic acid did not affect the hemagglutinating activity of hemagglutinin. All denaturing agents (Guanidine-HCl, urea, and β-mercaptoethanol) reduced the hemagglutinating activity of the hemagglutinin to different degrees. The hemagglutinin scavenged the DPPH radical and chelated iron metal with half maximum inhibitory concentration (IC50) of 0.727 ± 0.035 mg/mL and 0.583 ± 0.078 mg/mL, respectively, while the FRAP assay showed that it contained 76 mg of ascorbic acid equivalent per gram of the purified hemagglutinin. In the absence of hemolytic agents and at lower concentration tested, hemagglutinin showed positive membrane integrity protection. This study provides information on the antioxidant properties of D. preussii root tuber hemagglutinin as well as its cell membrane protective ability. The lectin is a starch-binding, which makes it a novel lectin.

UHPLC-MS/MS method for the simultaneous determination of nicotine and tobacco-specific nitrosamines NNN and NNK for use in preclinical studies

A new method was developed and validated for the simultaneous determination of nicotine and tobacco-specific nitrosamines (TSNAs) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in two different tests matrices: porcine buccal epithelium tissue and phosphate buffered saline (PBS) extracts of smokeless tobacco products. The novelty of this work is in the development of a liquid chromatography tandem mass spectrometry method that can provide simultaneous quantification of trace levels of TSNAs and high concentrations of nicotine in biological media. Precision, accuracy, and stability were evaluated during method validation to ensure the method was fit for purpose. Several sample preparation and extraction methods were evaluated to minimize matrix effects and maximize analyte recoveries. The method was accurate in the range of 81.1% – 117%; repeatability was estimated in the range of 1.5% – 13.6% across multiple concentrations. The linear regression correlation coefficient (R2) was greater than 0.9959 for all analytes, and the limit of detection (LOD) was determined for nicotine, NNK, and NNN at 1 ng/mL 0.005 ng/mL, and 0.006 ng/ mL, respectively. Our method was found to be appropriate for the analysis of nicotine, NNN, and NNK in the porcine buccal epithelium and PBS extracts of smokeless tobacco products.Graphical abstract

In-vitro Evaluation of Acetylcholinesterase and Butyrylcholinesterase Inhibitory Activities of Antlion Larvae Extracts

Antlion is used in traditional medicine by natives of the southern part of Nigeria particularly the Yorubas for memory enhancement. The progress made so far in the use of this organism as a memory booster lead to investigating the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) (the key enzymes in the pathogenesis of Alzheimer's disease) inhibitory activities of methanolic and phosphate buffered saline (PBS) extracts. The activities of these enzymes were investigated using Ellman’s method. The kinetics of the inhibition patterns was also studied using eserine as the standard inhibitor. The concentration of the extract required for 50% inhibition (IC50) of the AChE was 49.00 ± 1.20 and 271.40 ± 0.10 µg/mL, for the PBS and methanolic extracts respectively, compared to Eserine with IC50 of 2.25 x 10-2 ± 0.15 x 10-2 µg/mL. Similarly, the IC50 for the BuChE was 66.30 ± 0.40 and 216.70 ± 1.10 µg/mL respectively, for the methanol and PBS extracts, compared to Eserine with IC50 of 1.10 ± 0.30 µg/mL. The pattern of inhibition of the BuChE in the presence of the extracts was non-competitive, while AChE exhibited non-competitive and competitive inhibitions for the methanolic and PBS extracts respectively. It is therefore evident that extracts of the antlion larvae contained cholinesterase inhibitors which might be binding to AChE and BuChE; with the PBS extract inhibitory activity towards AChE being more potent than the methanolic extract. Suggesting a beneficial effect in cognitive deficit and related dementia associated with Alzheimer’s disease.

Treatment with Uncaria tomentosa Promotes Apoptosis in B16-BL6 Mouse Melanoma Cells and Inhibits the Growth of B16-BL6 Tumours.

Uncaria tomentosa is a medicinal plant native to Peru that has been traditionally used in the treatment of various inflammatory disorders. In this study, the effectiveness of U. tomentosa as an anti-cancer agent was assessed using the growth and survival of B16-BL6 mouse melanoma cells. B16-BL6 cell cultures treated with both ethanol and phosphate-buffered saline (PBS) extracts of U. tomentosa displayed up to 80% lower levels of growth and increased apoptosis compared to vehicle controls. Treatment with ethanolic extracts of Uncaria tomentosa were much more effective than treatment with aqueous extracts. U. tomentosa was also shown to inhibit B16-BL6 cell growth in C57/bl mice in vivo. Mice injected with both the ethanolic and aqueous extracts of U. tomentosa showed a 59 ± 13% decrease in B16-BL6 tumour weight and a 40 ± 9% decrease in tumour size. Histochemical analysis of the B16-BL6 tumours showed a strong reduction in the Ki-67 cell proliferation marker in U. tomentosa-treated mice and a small, but insignificant increase in terminal transferase dUTP nick labelling (TUNEL) staining. Furthermore, U. tomentosa extracts reduced angiogenic markers and reduced the infiltration of T cells into the tumours. Collectively, the results in this study concluded that U. tomentosa has potent anti-cancer activity that significantly inhibited cancer cells in vitro and in vivo.

Values of antioxidant activities (ABTS and DPPH) and ferric reducing and chelating powers of gamma-irradiated rosemary extract

Abstract This study was carried out to evaluate the effect of the various gamma dose irradiations on the antioxidant activity of rosemary PBS (phosphate-buffered saline) extract. The PBS extract of rosemary was irradiated with gamma rays at the doses of 10, 20, 30, 40 and 50 kGy, and their antioxidant activities were investigated by DPPH (1,1-diphenyl-2-picrylhydrazyl), radical cation ABTS•+ (2,2-Azino-Bis (3-Ethylbenzthiazoline-6-Sulfonic Acid), Diammonium Salt), ferric reducing antioxidant power assay (FRAP) and ferric chelating capacity using ferrozine. The scavenging activities of gamma irradiated rosemary were observed to be significantly higher at 30 and 40 kGy using both methods, DPPH and ABST. Also, the reducing power values of 30 kGy gamma irradiated rosemary were higher than that of non-irradiated (P < 0.05) and the other doses of irradiation could not make any significant difference in reducing power of non-irradiated rosemary. The gamma irradiation of rosemary (at 10–50 kGy), significantly increased the Fe2+ chelating activity compared to non-irradiated rosemary while increasing the absorbed doses reduced Fe2+ chelating activity in rosemary (P < 0.05). There is no significant difference in the rosemary irradiated with 10 kGy and 30 kGy. The gamma irradiated rosemary showed higher antioxidant activities at doses of 30 kGy (79.5 %) and 40 kGy (80 %) with DPPH method, while slightly lower activities were recorded at doses of 30 kGy (34.8 %) and 40 kGy (34.2 %) with ABTS test. Also, results indicate that the 30 kGy might be an effective dose with maximum enhancement in the antioxidant properties of the gamma irradiated rosemary.

Preparation of an anti-isoprocarb monoclonal antibody and its application in developing an immunochromatographic strip assay.

In this study, a carboxyl group was introduced into the isoprocarb molecule to obtain an isoprocarb hapten, which was then coupled with a protein to obtain an artificial antigen. Three monoclonal antibody cell lines, 1D11, 6E6 and 1B5, were finally obtained by mouse immunization, cell fusion and subcloning, and the antibody produced by cell line 1B5 had the best affinity and sensitivity. The monoclonal antibody was highly sensitive and specific for isoprocarb, with an IC50 of 2.09 ng/ml and a cross-reactivity rate of <0.21%. By optimizing the indirect competitive (ic)-ELISA, the optimal conditions were determined to be pH 7.4, 0% methanol and 0.8% NaCl, the limit of detection value was 0.23 ng/ml, and the linear range of the ic-ELISA was 0.46-9.62 ng/ml. The recovery rate of the isoprocarb cucumber sample was 97-99% for the ic-ELISA method. In addition, we successfully developed an immunochromatographic test strip for the detection of isoprocarb residues. The cutoff values in phosphate-buffered saline and cucumber extract were 10 and 25 ng/ml, respectively. Both methods met the requirements for isoprocarb residue detection in agricultural products, and can be used for semiquantitative and qualitative analysis of isoprocarb in vegetables.

Proteolytic Activity of Edible Spruce Morchella esculenta

Abstract This study focused on the determination of non-specific proteolytic activity of edible spruce Morchella esculenta in water extract, phosphate-buffered saline (PBS) solution (pH = 7.5) extract and a suspension prepared from 200 mg DW (dry weight) of edible spruce in PBS solution (pH 7.5). A clear casein solution was used as a substrate. The absorbances were measured in quartz cuvettes at the wavelength of 280 nm against a blank with zero concentration of trypsin. Non-specific proteolytic activity was expressed as trypsin equivalents per kilogram of mushroom dry weight (mg.kg−1 DW). All of the extracts demonstrated non-specific enzymatic activity. The highest activity was observed in the PBS suspension and the lowest enzymatic activity was measured in the water extract of the Morchella esculenta fungi. The non-specific proteolytic activity decreased in the following order: PBS suspension extract (pH 7.5; 22.9 mg.kg−1 DW), followed by PBS extract (pH 7.5; 13.6 mg.kg−1 DW) and finally the water extract (10.94 mg.kg−1 DW).

IgE-mediated allergic responses associated to Ailanthus altissima pollen using an animal model

BackgroundMurine models have been widely used in the study of allergy as sensitized mice can produce IgE and/or IgG1in response after the injection of an antigen/adjuvant combination. Ailanthus altissima pollen (AAP) has been recently reported as an emerging aeroallergen in Iran. So far, several AAP candidate allergens by the screening of allergen-specific IgE in the sera from AAP sensitized patients in Iran. ObjectiveThe aim of the present study was to detect and compare the allergens eliciting an IgE response in a mouse model, and in human, using pollen extract of A. altissima and an immunoproteomics based approach. MethodsThe pollen proteins were extracted in phosphate-buffered saline (PBS). Thirty male BALB/c mice were randomly divided into two groups of AP extract sensitized and sham that respectively received AAP PBS extract and a PBS control by intraperitoneal injections at regular intervals. The optimized AAP protein extracts were analyzed using 2D-gel electrophoresis and were subsequently confronted to pooled sera of sensitized mice. ResultsTwo-D gel electrophoresis of AAP extract allowed the separation of 125 protein spots distributed in a wide range of pI and molecular masses. Two-DE immunoblotting using pooled sera of sensitized mice led to the detection of 14 IgE reactive spots with molecular masses ranging from 12 to 40–42kDa. ConclusionThe results do not correlate with our previous analyses using human AAP-sensitized sera. These findings reflect some differences in the sIgE reactivity to allergenic proteins in animal models.

Isolation of lactic acid bacteria bound to the porcine intestinal mucosa and an analysis of their moonlighting adhesins.

The adhesion of lactic acid bacteria (LAB) to the intestinal mucosa is one of the criteria in selecting for probiotics. Eighteen LAB were isolated from porcine intestinal mucin (PIM): ten strains of Lactobacillus, six strains of Weissella, and two strains of Streptococcus. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for phosphate-buffered saline (PBS) extracts from the LAB, many bands were detected in half of the samples, while a few and/or no clear bands were detected in the other half. All six of the selected LAB showed adhesion to PIM. L. johnsonii MYU 214 and MYU 221 showed adhesion at more than 10%. W. viridescens MYU 208, L. reuteri MYU 213, L. mucosae MYU 225, and L. agilis MYU 227 showed medium levels of adhesion at 5.9–8.3%. In a comprehensive analysis for the adhesins in the PBS extracts using a receptor overlay analysis, many moonlighting proteins were detected and identified as candidates for adhesins: GroEL, enolase, and elongation factor Tu in MYU 208; peptidase C1, enolase, formyl-CoA transferase, phosphoglyceromutase, triosephosphate isomerase, and phosphofructokinase in MYU 221; and DnaK, enolase, and phosphoglycerate kinase in MYU 227. These proteins in the PBS extracts, which included such things as molecular chaperones and glycolytic enzymes, may play important roles as adhesins.

Selenium added unripe carica papaya pulp extracts enhance wound repair through TGF-β1 and VEGF-a signalling pathway.

BackgroundIncreased wound healing efficiency by Se2+ added Carica papaya L. (Caricaceae) fruit extract was linked to increased antioxidant and anti-inflammatory responses during healing.We investigated the impact of Se2+ or Zn2+ added papaya water (WE) and phosphate-buffered saline (PE) extracts on cells recruitment and bio-molecular alterations on days 4 and 10 post wounding in an in vivo excision wound.MethodsExcision wounds were created on the dorsum of Sprague Dawley rats and treated topically twice/day with 20 μL of PE and WE (5 mg extract/mL), 0.5 μgSe2+ added PE and WE (PES and WES), or 100 μMZn2+ added PE and WE (PEZ and WEZ). Deionised water (negative) and Solcoseryl (positive) were applied on the control groups. Histochemical and biochemical assays were used to evaluate cellular and bio-molecular changes in the wound.ResultsPES (PE + 0.5 μg Se2+) only increased significantly (p < 0.05) wound total protein content (95.14 ± 1.15 mg/g tissue vs positive control; 80.42 ± 0.86 mg/g tissue) on day 10 post wounding. PES increased significantly (p < 0.05) the number of fibroblasts/high power field (HPF) (75.60 ± 9.66) but decreased significantly (p < 0.05) the number of polymorphonuclear leukocytes/HPF (59.20 ± 12.64) in the wound compared to positive control (50.60 ± 12.58 fibroblasts/HPF, 101.00 ± 27.99 polymorphonuclear leukocytes/HPF) on day 4. Similar results were recorded for WES. PES demonstrated increased neovascularization, TGF-β1 and VEGFA expressions at day 4 and increased collagen at day 10.ConclusionPapaya extract improved wound repair by increasing fibroblasts recruitment and reducing polymorphonuclear leukocytes infiltration through early transient expressions of TGF-β1 and VEGFA at the wound area. The processes were amplified with Se2+ addition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0900-4) contains supplementary material, which is available to authorized users.

Anti-inflammatory and antioxidant properties of unripe papaya extract in an excision wound model

Context: Carica papaya L. (Caricaceae) fruit was shown to exhibit wound healing properties.Objectives: We investigated anti-inflammatory and antioxidant potential of papaya fruit phosphate-buffered saline extract (PE) during wound healing and enhancement of the potentials due to trace ions addition.Materials and methods: Rat excision wounds were topically treated twice/day with 20 µL of PE (5 mg extract/mL), 0.5 µg Se2+ added PE (PES), or 100 µM Zn2+ added PE (PEZ). Control groups were treated with deionized water (negative) and deproteinized calf blood extract ointment (Solcoseryl®, positive). Lipid peroxidation (LPX), antioxidant, proinflammatory, and arginine metabolic enzymes were estimated in the wound excised on days 4 and 10 post wounding.Results: PE (5 mg/mL; 9.80 ± 0.33 d) and PES (PE + 0.5 µg Se2+; 8.90 ± 0.23 d) significantly (p < 0.05) reduced the average time for complete wound closure compared with the negative (13.00 ± 0.37 d) and positive (9.80 ± 0.33 d) controls, respectively. Biochemical evaluations of LPX product (malondialdehyde), antioxidant (catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx)), and pro-inflammatory (cyclooxygenase-2 and myeloperoxidase (MPO)) enzyme activities and metabolites (nitrite and urea), on days 4 and 10 post wounding, confirmed the anti-inflammatory and antioxidant properties of PE and PES in this study.Discussion and conclusion: Treatment of excision wounds with papaya extract, especially with the addition of selenium for 10 d, reduced inflammation associated oxidative damage apparently via cyclooxygenase specific inhibition, arginine metabolism, and up-regulation of antioxidant enzymes.

English

A galactose-specific lectin was purified from the skin mucus of African catfish (Clarias gariepinus) and the physicochemical properties determined. Phosphate buffered saline extract of the skin mucus of African catfish specifically agglutinated erythrocytes of rabbit and human blood group B, but did not agglutinate bat, rat, hen and human blood A and O erythrocytes. The haemagglutinating activity of the lectin was completely inhibited by lactose while galactose and melibiose inhibited the activity to some extent and was calcium-independent. The purified lectin has a native and subunit molecular weight of 63, 000 and 20, 000 Daltons, respectively suggesting a homotrimeric structure for the protein. The protein contained 126 amino acid residues per subunit. This was characterized by large amount of polar amino acids that constituted about 60% of the total amino acids. The lectin showed maximum activity over the pH range 6 – 9 and was heat stable up to 50°C. Ethylenediaminetetraacetic acid (EDTA) had no inhibitory effect on its haemagglutinating activity. Periodic acid–Schiff (PAS) staining showed that the lectin was not a glycoprotein. Chemical modifications of serine and arginine residues of the protein did not affect its haemagglutination activity while modifications of cysteine, tryptophan and histidine residues led to total loss of its activity. The study concluded that African catfish skin mucus lectin exhibited similar physicochemical properties with lectins from other fish skin mucus. Key words: African catfish, Lectin, haemagglutination, skin mucus, galactose-specific, Clarias gariepinus

Immunomodulatory activities of Gelidium amansii gel extracts on murine RAW 264.7 macrophages

The objective of this study was to investigate the effects of Gelidium amansii, a red algae cultivated in the northeastern coast of Taiwan, on immune regulatory activities in RAW 264.7 macrophages. G. amansii gel was prepared by boiling and filtering the dried G. amansii algae. Phosphate-buffered saline (PBS) and ethanol extracts of the freeze-dried gel were then used to treat the cells. The results showed that the PBS extracts of G. amansii gel activated the macrophage by increasing cell proliferation and by enhancing the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6; the ethanol extracts did not show such effects. By contrast, neither PBS nor ethanol extracts of G. amansii gel affected NO production in lipopolysaccharide (LPS)-stimulated macrophages, although the ethanol extracts suppressed LPS-stimulated TNF-α, IL-1β, and IL-6 production. In conclusion, the PBS extracts of G. amansii gel activated the macrophage by enhancing the production of immune mediators, whereas the ethanol extracts showed anti-inflammatory activities by suppressing the cytokine production in LPS-stimulated RAW 264.7 macrophages.

Addition of Selenium to &lt;i&gt;Carica papaya&lt;/i&gt; Linn Pulp Extract Enhances Dermal Wound Healing Activity

Purpose: To investigate the combined effect of Carica papaya extract and mineral elements wound healing.Methods: Phosphate-buffered saline (PBS) and water extracts of unripe Carica papaya. papaya were analyzed for mineral element composition (Ca, calcium; Mg, magnesium; Na, sodium; Fe, iron; Mn, manganese; Zn, zinc; Ag, silver; and Se, selenium) using a microwave-assisted digestion procedure. Atomic absorption spectrophotometry was used for the analysis of the elements. Phytochemical analysis was conducted using standard protocols. Mice weighing 25 - 30 g were anesthetized, shaved and inflicted with wound at the dorsal region using a biopsy punch. The mice were treated topically twice daily with or without 5 mg/ml PBS or water extracts containing 100 or 200 ìM Zn2+ or 1.0 or 0.5 ìg/g Se2+. The standard (positive control) group was treated with solcoseryl and the negative controlwith deionised H2O. Wound area was monitored with a camera and evaluated by software.Results: PBS extract formulated with Se2+. (0.5 or 1.0 mg/g) took approximately 9 days for complete wound healing while negative control took 14 days and other treatment groups least 10 days. PBS extract contained 9 % protein but no tannin while water extract contained 0.58 % protein and tannins. Calcium (137.98 mg/100 g extract) constituted the most abundant macro-element while Se (32.14 mg/100 g extract) was the most abundant microelement in C. papaya pulp.Conclusion: Addition of selenium to C. papaya extracts augments the wound healing activity of the extracts.Keywords: Papaya, Zn, Selenium, dermal wound healing

Extraction Of House Dust Mite Allergen, Der P1, From Dust

A recent article in this journal found that the type of extraction buffer did not affect measured levels of the major group one allergen (Der p 1) from the house dust mite, Dermatophagoides pteronyssinus, in household dust samples (1). This differed from a previous study where about twice the levels of Der p 1 were found in borate-buffered saline extracts, compared to ammonium bicarbonate buffer or phosphate-buffered saline extracts, irrespective of extraction temperature or time of extraction (2). In discussing our previous study (2), the authors state that we did not use Tween-20 in our extraction buffers. Although our article did not make a mention of Tween-20, we in fact did use it in our extraction media. It was an oversight of us in that we did not mention this in our article. We further explored temperature and buffer effects on Der p 1 extraction in an external quality control program where six dust samples were sent to 23 laboratories worldwide (3). Results from this study again showed that type of extraction buffer and extraction temperature influenced Der p 1 measurements. The majority of the participant laboratories did use Tween-20 as a wetting agent in their extraction buffers. As pointed out by the authors, adding Tween-20 to extraction media results in higher endotoxin levels and is recommended by the European Committee for Standardisation. The authors call for further investigation on the effects of Tween-20 on extraction effi ciency of all indoor allergens from dust. However, the majority of indoor allergen analysis laboratories use ELISA or MARIATM kits from Indoor Biotechnologies where the recommended extraction medium is phosphate-buffered saline containing 0.02 % Tween-20. It would take much effort to study this systematically to obtain results with suffi cient internal and external validity.

Development of an optimized method for the recovery of infectious F-RNA coliphage MS2 from meat

F-RNA coliphages, part of the gut flora and likely to be deposited on meat along with other enteric organisms during carcass dressing and processing, may be regarded as an indicator and/or surrogate for potential zoonotic enteric viruses. There is no recommended sampling method for viruses on meats and there is a lack of information on the attachment of enteric viruses or F-RNA coliphages to gauze swabs, cellulose sponges and muscle and fat tissue. The objective of this work was to optimize the recovery of MS2 from muscle and fat tissue of meat by comparing phosphate buffered saline (PBS), 10% beef extract pH 7.2, and tryptose phosphate (2.9%) glycine (6%) broth pH 9.5 as eluants. The sampling techniques of excision, swabbing with gauze or cellulose sponges were compared with homogenizing the inoculated entire muscle or fat surface area. The recovery of MS2 from cellulose sponges using beef extract was significantly higher (P=0.001) than tryptose phosphate glycine broth which was significantly higher (P=0.0001) than PBS. There was no significant difference in the recovery between tryptose phosphate glycine broth and beef extract (P=0.92) and there was no significant difference between PBS and beef extract (P=0.10) when MS2 was recovered from gauze. No significant differences were observed between the different eluants with muscle tissue (P=0.91). When MS2 was recovered from muscle tissue with beef extract significant differences were observed (P=0.002); the sampling techniques of homogenizing the entire sample (56%) was equal to excision (43%) (P=0.23) and swabbing with a cellulose sponge (38%) (P=0.06) which were significantly higher than swabbing with gauze (28%), a second grouping of means indicated that homogenizing the entire sample was significantly higher than the other three sampling techniques. When MS2 was recovered from fat, significant differences were observed (P=0.000); homogenizing the entire sample (78%) was equal to excision (74%), which were significantly higher than swabbing with gauze (49%) or cellulose sponge (29%). The recovery of MS2 from meat is affected by the sampling technique. When choosing a nondestructive sampling method such as a cellulose sponge, a higher recovery can be obtained with beef extract as an eluant.