Search results for: in vitro metabolites

Authors: Abubakar Bello Usman, Alhassan Muhammad Gani, Kolo Ibrahim

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The use of botanical raw materials to produce pharmaceuticals, herbal remedies, teas, spirits, cosmetics, sweets, dietary supplements, special industrial compounds and crude materials constitute an important global resource in terms of healthcare and economy. In Nigeria and other developing countries, the indigenous knowledge on the uses of plants lies with the older generation and the traditional healers. However, these custodians are decreasing in number due to death and other unforeseen occurrences. An Ethno-botanical survey was carried out to obtain information on the ethno medical values of wide range of plants used by the people of Gombe State, North-Eastern Nigeria, in the practice of healing and cure of typhoid (enteric) fever. Oral interviews were conducted so as to consider those with low literacy level who are involved in the practice of traditional medicine and thirty four (34) informants availed themselves for the interview and were consulted. All relevant information obtained from the respondents was recorded. A recent and valid nomenclature, along with local names, family names, part of the plant(s) used, methods of preparation and administration and fifty four (54) plant species belonging to 27 families as well as 7 unidentified species that are commonly used by the people of the state in ethnomedical treatment of the ailment were tabulated. Those interviewed included traditional practitioners, local herb sellers, traditional rulers, hunters, farmers and patients. Specific questions were asked and information supplied by informants was promptly documented. Results showed that the people of Gombe State are knowledgeable on herbal medicine in the treatment of diseases and ailments. Furthermore, the aqueous leaf extracts of Senna siamea, the plant species with the highest PPK (percentage of people who have knowledge about the use of a species for treating typhoid fever) in this ethnobotanical survey, was tested for its activity against clinical isolates of Salmonella typhi using the agar well diffusion method. The aqueous extracts showed some activity (zones of inhibition 11, 9, 7.5, 3.5, 1.3 mm) at 2000, 1800, 1600, 1400, 1200 µg/ml concentrations respectively. Preliminary phytochemical studies of the aqueous leaf extracts of the plant revealed the presence of secondary metabolites such as alkaloids, saponins, tannins, flavonoids and cardiac glycosides. Though a large number of traditionally used plants for the treatment of enteric fever were identified, further scientific validation of the traditional claims of anti-typhoid properties is imperative. This would establish their candidature for any possible future research for active principles and the possible development of new cheaper and more effective anti-typhoid drugs, as well as in the conservation of this rich diversity of medicinal plants.

Keywords: antimicrobial activities, ethnobotany, gombe state, north-eastern Nigeria, phytochemical screening, senna siamea, typhoid fever

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Authors: José M. Carmona, Diana Puigserver, Jofre Herrero

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Chlorinated solvents belong to the group of nonaqueous phase liquids (DNAPL) and have been involved in many contamination episodes. They are carcinogenic and recalcitrant pollutants that may be found in granular aquifers as: i) pools accumulated on low hydraulic conductivity layers; ii) immobile residual phase retained at the pore-scale by capillary forces; iii) dissolved phase in groundwater; iv) sorbed by particulate organic matter; and v) stored into the matrix of low hydraulic conductivity layers where they penetrated by molecular diffusion. The transition zone between granular aquifers and basal aquitards constitute the lowermost part of the aquifer and presents numerous fine-grained interbedded layers that give rise to significant textural contrasts. These layers condition the transport and fate of contaminants and lead to differences from the rest of the aquifer, given that: i) hydraulic conductivity of these layers is lower; ii) DNAPL tends to accumulate on them; iii) groundwater flow is slower in the transition zone and consequently pool dissolution is much slower; iv) sorbed concentrations are higher in the fine-grained layers because of their higher content in organic matter; v) a significant mass of pollutant penetrates into the matrix of these layers; and vi) this contaminant mass back-diffuses after remediation and the aquifer becomes contaminated again. Thus, contamination sources of chlorinated solvents are extremely more recalcitrant in transition zones, which has far-reaching implications for the environment. The aim of this study is to analyze the spatial and temporal differences in the evolution of biogeochemical processes in the transition zone and in the rest of the aquifer. For this, an unconfined aquifer with a transition zone in the lower part was selected at Vilafant (NE Spain). This aquifer was contaminated by perchloroethylene (PCE) in the 80’s. Distribution of PCE and other chloroethenes in groundwater and porewater was analyzed in: a) conventional piezometers along the plume and in two multilevel wells at the source of contamination; and b) porewater of fine grained materials from cores recovered when drilled the two multilevel wells. Currently, the highest concentrations continue to be recorded in the source area in the transition zone. By contrast, the lowest concentrations in this area correspond to the central part of the aquifer, where flow velocities are higher and a greater washing of the residual phase initially retained has occurred. The major findings of the study were: i) PCE metabolites were detected in the transition zone, where conditions were more reducing than in the rest of the aquifer; ii) however, reductive dechlorination was partial since only the formation of cis-dicholoroethylene (DCE) was reached; iii) In the central part of the aquifer, where conditions were predominantly oxidizing, the presence of nitrate significantly hindered the reductive declination of PCE. The remediation strategies to be implemented should be directed to enhance dissolution of the source, especially in the transition zone, where it is more recalcitrant. For example, by combining chemical and bioremediation methods, already tested at the laboratory scale with groundwater and sediments of this site.

Keywords: chlorinated solvents, chloroethenes, DNAPL, partial reductive dechlorination, PCE, transition zone to basal aquitard

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Authors: Shreya Sara Ittycheria, K. C. Sivakumar, Shijulal Nelson Sathi, Priya Srinivas

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hCG, a heterodimer composed of α and β subunits, is a peptide hormone having numerous biological functions. Although hCG is expressed by placenta during pregnancy, ectopic β-hCG secretion is observed in many non-trophoblastic tumors including that of breast. In-vitro and in-vivo studies done in the lab, have proved that BRCA1 defective cancers express β-hCG and when β-hCG is expressed or supplemented, it promotes tumor progression and exhibits resistance to carboplatin and ABT888, in such cancers but not in BRCA1 wild type cancers. In cancer cells, instead of binding to its regular receptor, LH-CGR, β-hCG binds with Transforming Growth Factor Receptor 2 (TGFβRII) and phosphorylates it resulting in faster tumor progression through the Smad signaling pathway. Targeting β-hCG could be a potential therapeutic strategy for managing BRCA1 defective cancers. Here, molecular docking and dynamic simulation studies were done to identify potential small molecule inhibitors against β-hCG as there are currently no such inhibitors reported. The binding sites of TGFβRII on β-hCG were identified from the top 10 predicted complexes from Z Dock. Virtual screening of selected commercially available small molecules from various libraries such as ZINC, NCI and Life Chemicals amounting to a total of 50,025 molecules were done. Four potential small molecule inhibitors were identified, RgcbPs-1, RgcbPs-2, RgcbPs-3 and RgcbPs-4 with binding affinities -60.778 kcal/mol, -45.447 kcal/mol, -65.2268 kcal/mol and -82.040 kcal/mol respectively. Further, 100ns Molecular Dynamics (MD) simulation showed that these molecules form stable complexes with β-hCG. RgcbPs-1 maintains hydrogen bonds with Q54, L52, Q46, C100, G36, C57, C38 residues, RgcbPs-2 maintains hydrogen bonds with A83 residue, RgcbPs-3 maintains hydrogen bonds with C57, Y58, R94, G101 residues and RgcbPs-4 maintains hydrogen bonds with G36, C38, T40, C57, D99, C100, G101 and L104 residues of β-hCG all of which coincide with the TGFβRII binding site on β-hCG. These results show that these two inhibitors could be used either singly or in combination for inhibiting β-hCG from binding to TGFβRII and thereby directly inhibiting the tumorigenesis pathway.

Keywords: β-hCG, breast cancer, dynamic simulations, molecular docking, small molecule inhibitors, virtual screening.

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Authors: Marcin Dębowski, Marcin Zieliński, Mirosław Krzemieniewski

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As methane fermentation is a complex series of successive biochemical transformations, its subsequent stages are determined, to a various extent, by physical and chemical factors. A specific state of equilibrium is being settled in the functioning fermentation system between environmental conditions and the rate of biochemical reactions and products of successive transformations. In the case of physical factors that influence the effectiveness of methane fermentation transformations, the key significance is ascribed to temperature and intensity of biomass agitation. Among the chemical factors, significant are pH value, type, and availability of the culture medium (to put it simply: the C/N ratio) as well as the presence of toxic substances. One of the important elements which influence the effectiveness of methane fermentation is the pre-treatment of organic substrates and the mode in which the organic matter is made available to anaerobes. Out of all known and described methods for organic substrate pre-treatment before methane fermentation process, the ultrasound disintegration is one of the most interesting technologies. Investigations undertaken on the ultrasound field and the use of installations operating on the existing systems result principally from very wide and universal technological possibilities offered by the sonication process. This physical factor may induce deep physicochemical changes in ultrasonicated substrates that are highly beneficial from the viewpoint of methane fermentation processes. In this case, special role is ascribed to disintegration of biomass that is further subjected to methane fermentation. Once cell walls are damaged, cytoplasm and cellular enzymes are released. The released substances – either in dissolved or colloidal form – are immediately available to anaerobic bacteria for biodegradation. To ensure the maximal release of organic matter from dead biomass cells, disintegration processes are aimed to achieve particle size below 50 μm. It has been demonstrated in many research works and in systems operating in the technical scale that immediately after substrate supersonication the content of organic matter (characterized by COD, BOD5 and TOC indices) was increasing in the dissolved phase of sedimentation water. This phenomenon points to the immediate sonolysis of solid substances contained in the biomass and to the release of cell material, and consequently to the intensification of the hydrolytic phase of fermentation. It results in a significant reduction of fermentation time and increased effectiveness of production of gaseous metabolites of anaerobic bacteria. Because disintegration of Virginia fanpetals biomass via ultrasounds applied in order to intensify its conversion is a novel technique, it is often underestimated by exploiters of agri-biogas works. It has, however, many advantages that have a direct impact on its technological and economical superiority over thus far applied methods of biomass conversion. As for now, ultrasound disintegrators for biomass conversion are not produced on the mass-scale, but by specialized groups in scientific or R&D centers. Therefore, their quality and effectiveness are to a large extent determined by their manufacturers’ knowledge and skills in the fields of acoustics and electronic engineering.

Keywords: ultrasound disintegration, biomass, methane fermentation, biogas, Virginia fanpetals

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Authors: Razieh Zarei, Hassan zm, Abolghasem Ajami, Darush Moslemi, Narges Afsary, Amrollah Mostafa-zade

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Introduction: IL-23 is responsible for the differentiation and expansion of Th17/ThIL-17 cells from naive CD4+ T cells. Therefore, may be IL-23/IL17 axis involve in a variety of allergic and autoimmune diseases, such as RA, MS, inflammatory bowel disease (IBD), and asthma. TGF-β is also share for the differentiation Th17 producing IL-17 and CD4+CD25+Foxp3hiT regulatory cells from naïve CD4+ T cells which are involved in the regulation of immune response, maintaining immunological self-tolerance and immune homeostasis ,and the control of autoimmunity and cancer surveillance. Therefore, T regulatory cells play a key role in autoimmunity, allergy, cancer, infectious disease, and the induction of transplantation tolerance. Vitamin A and it's derivatives (retinoids) inhibit or reverse the carcinogenic process in some types of cancers in oral cavity,head and neck, breast, skin, liver, and blood cells. Shark is a murine organism and its cartilage has antitumor peptides to prevent angiogenesis, in vitro. Our purpose is whether simultaneous oral treatment vitamin A and shark cartilage can modulate IL-23/IL-17 and CD4CD25Foxp3 T regulatory cell/TGF-β pathways and Th1/Th2 immunity in patients with gastric cancer. Materials and Methods: First investigated an imbalanced supernatant of cytokines exist in patients with gastric cancer by ELISA. Associated with cytokines measuring such as IL-23,IL-17,TGF-β,IL-4 and γ-IFN, then flow cytometry was employed to determine whether the peripheral blood mononuclear cells such as CD4+CD25+Foxp3highT regulatory cells in patients with gastric cancer were changed correspondingly. Results: An imbalance between IL-17 secretion and TGF-β/Foxp3 t regulatory cell pathway and so, Th1 immunity (γ-IFN production) and TH2 immunity (IL-4 secretion) was not seen in patients with gastric cancer treated by vitamin A and shark cartilage. But, the simultaneously presented down-regulation of IL-23 indicated, at least cytokine level. Conclusion: Il-23, as a pro-angiogenesis cytokine, probably, help to tumor growth. Hence, suggested that down-regulation of IL-23, at least cytokine level, is useful for anti-tumor immune responses in patients with gastric cancer.

Keywords: IL-23/IL17 axis, TGF-β/CD4CD25Foxp3 T regulatory pathway, γ-IFN, IL-4, shark cartilage and gastric cancer

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Authors: Arief, Noovirman Jamarun, Benni Satria

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Background: The main problem in the development of livestock in Indonesia is feed both in terms of quality and quantity. On the other hand, conventional feed ingredients are expensive and difficult to obtain. Therefore, it is necessary to find alternative feed ingredients that have good quality, potential, and low cost. Feed ingredients that meet the above requirements are by-products of the palm oil industry, namely palm kernel cake (PKC). This study aims to obtain the best PKC composition for Etawa goat concentrate ration. Material and Methode : This research consists of 2 stages. Stage I is invitro study using Tilley and Terry method. The study used a Completely Randomized Design (CRD) with 4 treatments of rations and 4 replications. The treatment is the composition of the use of palm kernel cake (PKC) in the ration, namely, A). 10%, B). 20%, C). 30%, D). 40%. Other feed ingredients are corn, rice bran, tofu waste and minerals. The measured variables are the characteristics of the rumen fluid (pH, VFA and NH3). Stage II was done using the best ration of stage I (Ration C), followed by testing the use of Tithonia (Thitonia difersifolia) and agricultural waste of sweet potato leaves as a source of forage for livestock by in-vitro. The study used a Completely Randomized Design (CRD) with 3 treatments and 5 replications. The treatments were: Treatment A) Best Concentrate Ration Stage I + Titonia (Thitonia difersifolia), Treatment B) Best Concentrate Ration Stage I + Tithonia (Thitonia difersifolia) and Sweet potato Leaves, Treatment C) Best Concentrate Ration Stage I + Sweet potato leaves. The data obtained were analyzed using variance analysis while the differences between treatments were tested using the Duncant Multiple Range Test (DMRT) according to Steel and Torrie. Results of Stage II showed that the use of PKC in rations as concentrate feed combined with forage originating from Tithonia (Thitonia difersifolia) and sweet potato leaves produced pH, VFA and NH3-N which were still in normal conditions. The best treatment was obtained from diet B (P <0.05) with 6.9 pH, 116.29 mM VFA and 15mM NH3-N. Conclussion From the results of the study it can be concluded that PKC can be used as feed ingredients for dairy goat concentrate with a combination of forage from Tithonia (Tithonia difersifolia) and sweet potato leaves.

Keywords: palm oil by-product, palm kernel cake, concentrate, rumen fluid, Etawa goat

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Authors: Carmen Sica, Elena M. Scalisi, Sara Ignoto, Ludovica Palmeri, Martina Contino, Greta Ferruggia, Antonio Salvaggio, Santi C. Pavone, Gino Sorbello, Loreto Di Donato, Roberta Pecoraro, Maria V. Brundo

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Recently, a rise in the use of wireless internet technologies such as Wi-Fi and 5G routers/modems have been demonstrated. These devices emit a considerable amount of electromagnetic radiation (EMR), which could interact with the male reproductive system either by thermal or non-thermal mechanisms. The aim of this study was to investigate the direct in vitro influence of 5G radiation on sperm quality in Mytilus galloprovincialis, considered an excellent model for reproduction studies. The experiments at 27 GHz were conducted by using a no commercial high gain pyramidal horn antenna. To evaluate the specific absorption rate (SAR), a numerical simulation has been performed. The resulting incident power density was significantly lower than the power density limit of 10 mW/cm2 set by the international guidelines as a limit for nonthermal effects above 6 GHz. However, regarding temperature measurements of the aqueous sample, it has been verified an increase of 0.2°C, compared to the control samples. This very low-temperature increase couldn’t interfere with experiments. For experiments, sperm samples taken from sexually mature males of Mytilus galloprovincialis were placed in artificial seawater, salinity 30 + 1% and pH 8.3 filtered with a 0.2 m filter. After evaluating the number and quality of spermatozoa, sperm cells were exposed to electromagnetic fields a 27GHz. The effect of exposure on sperm motility and quality was evaluated after 10, 20, 30 and 40 minutes with a light microscope and also using the Eosin test to verify the vitality of the gametes. All the samples were performed in triplicate and statistical analysis was carried out using one-way analysis of variance (ANOVA) with Turkey test for multiple comparations of means to determine differences of sperm motility. A significant decrease (30%) in sperm motility was observed after 10 minutes of exposure and after 30 minutes, all sperms were immobile and not vital. Due to little literature data about this topic, these results could be useful for further studies concerning a great diffusion of these new technologies.

Keywords: mussel, spermatozoa, sperm motility, millimeter waves

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Authors: E. Kilicay, Z. Karahaliloglu, B. Hazer, E. B. Denkbas

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In this study, we have prepared concanavaline A (ConA) functionalized curcumin (CUR) loaded PHBHHx (poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)) nanoparticles as a novel and efficient drug delivery system. CUR is a promising anticancer agent for various cancer types. The aim of this study was to evaluate therapeutic potential of curcumin loaded PHBHHx nanoparticles (CUR-NPs) and concanavaline A conjugated curcumin loaded NPs (ConA-CUR NPs) for ovarian cancer treatment. ConA was covalently connected to the carboxylic group of nanoparticles by EDC/NHS activation method. In the ligand attachment experiment, the binding capacity of ConA on the surface of NPs was found about 90%. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis showed that the prepared nanoparticles were smooth and spherical in shape. The size and zeta potential of prepared NPs were about 228±5 nm and −21.3 mV respectively. ConA-CUR NPs were characterized by FT-IR spectroscopy which confirmed the existence of CUR and ConA in the nanoparticles. The entrapment and loading efficiencies of different polymer/drug weight ratios, 1/0.125 PHBHHx/CUR= 1.25CUR-NPs; 1/0.25 PHBHHx/CUR= 2.5CUR-NPs; 1/0.5 PHBHHx/CUR= 5CUR-NPs, ConA-1.25CUR NPs, ConA-2.5CUR NPs and ConA-5CUR NPs were found to be ≈ 68%-16.8%; 55%-17.7 %; 45%-33.6%; 70%-15.7%; 60%-17%; 51%-30.2% respectively. In vitro drug release showed that the sustained release of curcumin was observed from CUR-NPs and ConA-CUR NPs over a period of 19 days. After binding of ConA, the release rate was slightly increased due to the migration of curcumin to the surface of the nanoparticles and the matrix integrities was decreased because of the conjugation reaction. This functionalized nanoparticles demonstrated high drug loading capacity, sustained drug release profile, and high and long term anticancer efficacy in human cancer cell lines. Anticancer activity of ConA-CUR NPs was proved by MTT assay and reconfirmed by apoptosis and necrosis assay. The anticancer activity of ConA-CUR NPs was measured in ovarian cancer cells (SKOV-3) and the results revealed that the ConA-CUR NPs had better tumor cells decline activity than free curcumin. The nacked nanoparticles have no cytotoxicity against human ovarian carcinoma cells. Thus the developed functionalized nanoformulation could be a promising candidate in cancer therapy.

Keywords: curcumin, curcumin-PHBHHx nanoparticles, concanavalin A, concanavalin A-curcumin PHBHHx nanoparticles, PHBHHx nanoparticles, ovarian cancer cell

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Authors: Bhupendra Kumar Poudel, Sadhana Amatya, Tirtha Maiya Shrestha, Bharatmani Pokhrel, Mohan Prasad Amatya

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Background: H. salicifolia is a dense, branched, multipurpose, deciduous, nitrogen fixing, thorny willow-like small to moderate tree, restricted to the Himalaya. Among the two species of Nepal (Hippophae salicifolia and H. tibetana), it has been traditionally used as food additive, anticancer (bark), and treating toothache, tooth inflammation (anti-inflammatory) and radiation injury; while people of Western Nepal have largely undermined its veiled treasure by using it for fuel, wood and soil stabilization only. Therefore, the main objective of this study was to explore biological properties (analgesic, antidiabetic, cytotoxic and anti-inflammatory properties of this plant. Methodology: The transverse section of leaves and stems were viewed under microscope. Extracts obtained from soxhlation subjected to tests for phytochemical and biological studies. Rats (used to study antidiabetic and anti-inflammatory properties) and mice (used to study analgesic, CNS depressant, muscle relaxant and locomotor properties) were assumed to be normally distributed; then ANOVA and post hoc tukey test was used to find significance. The data obtained were analyzed by SPSS 17 and Excel 2007. Results and Conclusion: Pharmacognostical analysis revealed the presence of long stellate trichomes, double layered vascular bundle 5-6 in number and double layered compact sclerenchyma. The preliminary phytochemical screening of the extracts was found to exhibit the positive reaction tests for glycoside, steroid, tannin, flavonoid, saponin, coumarin and reducing sugar. The brine shrimp lethality bioassay tested in 1000, 100 and 10 ppm revealed cytotoxic activity inherent in methanol, water, chloroform and ethyl acetate extracts with LC50 (μg/ml) values of 61.42, 99.77, 292.72 and 277.84 respectively. The cytotoxic activity may be due to presence of tannins in the constituents. Antimicrobial screening of the extracts by cup diffusion method using Staphylococcus aereus, Escherichia coli and Pseudomonas aeruginosa against standard antibiotics (oxacillin, gentamycin and amikacin respectively) portrayed no activity against the microorganisms tested. The methanol extract of the stems and leaves showed various pharmacological properties: and antidiabetic, anti-inflammatory, analgesic [chemical writhing method], CNS depressant, muscle relaxant and locomotor activities in a dose-dependent fashion, indicating the possibility of the presence of different constituents in the stems and leaves responsible for these biological activities. All the effects when analyzed by post hoc tukey test were found to be significant at 95% confidence level. The antidiabetic activity was presumed to be due to flavonoids present in extract. Therefore, it can be concluded that this plant’s secondary metabolites possessed strong antidiabetic, anti-inflammatory and cytotoxic activity which could be isolated for further investigation.

Keywords: Hippophae salicifolia, constituents, antidiabetic, inflammatory, brine shrimp

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Authors: Afzal Hussain, Mohammad A. Altamimi, Syed Sarim Imam, Mudassar Shahid, Osamah Abdulrahman Alnemer

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Exhaustive application of sulfamethoxazole (SUX) became as a global threat for human health due to water contamination through diverse sources. The addressed combined application of Hansen solubility (HSPiP software) parameters and Quality by Design tool for developing various green nanoemulsions. HSPiP program assisted to screen suitable excipients based on Hansen solubility parameters and experimental solubility data. Various green nanoemulsions were prepared and characterized for globular size, size distribution, zeta potential, and removal efficiency. Design Expert (DoE) software further helped to identify critical factors responsible to have direct impact on percent removal efficiency, size, and viscosity. Morphological investigation was visualized under transmission electron microscopy (TEM). Finally, the treated was studied to negate the presence of the tested drug employing ICP-OES (inductively coupled plasma optical emission microscopy) technique and HPLC (high performance liquid chromatography). Results showed that HSPiP predicted biocompatible lipid, safe surfactant (lecithin), and propylene glycol (PG). Experimental solubility of the drug in the predicted excipients were quite convincing and vindicated. Various green nanoemulsions were fabricated, and these were evaluated for in vitro findings. Globular size (100-300 nm), PDI (0.1-0.5), zeta potential (~ 25 mV), and removal efficiency (%RE = 70-98%) were found to be in acceptable range for deciding input factors with level in DoE. Experimental design tool assisted to identify the most critical variables controlling %RE and optimized content of nanoemulsion under set constraints. Dispersion time was varied from 5-30 min. Finally, ICP-OES and HPLC techniques corroborated the absence of SUX in the treated water. Thus, the strategy is simple, economic, selective, and efficient.

Keywords: quality by design, sulfamethoxazole, green nanoemulsion, water treatment, icp-oes, hansen program (hspip software

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Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc

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Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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Authors: Amanda C. Q. M. Vieira, Cybelly M. Melo, Camila B. M. Figueirêdo, Giovanna C. R. M. Schver, Salvana P. M. Costa, Magaly A. M. de Lyra, Ping I. Lee, José L. Soares-Sobrinho, Pedro J. Rolim-Neto, Mônica F. R. Soares

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Epiisopiloturine (EPI) is a drug candidate that is extracted from Pilocarpus microphyllus and isolated from the waste of Pilocarpine. EPI has demonstrated promising schistosomicidal, leishmanicide, anti-inflammatory and antinociceptive activities, according to in vitro studies that have been carried out since 2009. However, this molecule shows poor aqueous solubility, which represents a problem for the release of the drug candidate and its absorption by the organism. The purpose of the present study is to investigate the extent of enhancement of kinetic solubility of a solid solution (SS) of EPI in hipromellose phthalate HP-55 (HPMCP), an enteric polymer carrier. SS was obtained by the solvent evaporation methodology, using acetone/methanol (60:40) as solvent system. Both EPI and polymer (drug loading 10%) were dissolved in this solvent until a clear solution was obtained, and then dried in oven at 60ºC during 12 hours, followed by drying in a vacuum oven for 4 h. The results show a considerable modification in the crystalline structure of the drug candidate. For instance, X-ray diffraction (XRD) shows a crystalline behavior for the EPI, which becomes amorphous for the SS. Polarized light microscopy, a more sensitive technique than XRD, also shows completely absence of crystals in SS sample. Differential Scanning Calorimetric (DSC) curves show no signal of EPI melting point in SS curve, indicating, once more, no presence of crystal in this system. Interaction between the drug candidate and the polymer were found in Infrared microscopy, which shows a carbonyl 43.3 cm-1 band shift, indicating a moderate-strong interaction between them, probably one of the reasons to the SS formation. Under sink conditions (pH 6.8), EPI SS had its dissolution performance increased in 2.8 times when compared with the isolated drug candidate. EPI SS sample provided a release of more than 95% of the drug candidate in 15 min, whereas only 45% of EPI (alone) could be dissolved in 15 min and 70% in 90 min. Thus, HPMCP demonstrates to have a good potential to enhance the kinetic solubility profile of EPI. Future studies to evaluate the stability of SS are required to conclude the benefits of this system.

Keywords: epiisopiloturine, hipromellose phthalate HP-55, pharmaceuticaltechnology, solubility

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Authors: Jienny Lee, In-Soo Cho, Sang-Ho Cha

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Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.

Keywords: mesenchymal stem cells, cryopreservation, stemness, senescence

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Authors: Vijaya Madhuri Devraj, Swarnalatha Guditi, Kiran Kumar Bokara, Gangadhar Taduri

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Cell-based strategies may open therapeutic approaches that promote tolerance through manipulation of macrophages to increase long-term transplant survival rates and minimize side effects of the current immune suppressive regimens. The aim of the present study was, therefore, to test and compare the therapeutic potential of MSC and Tregs on macrophage polarization to develop an alternate cell-based treatment option in kidney transplantation. In the current protocol, macrophages from kidney transplant recipients with graft dysfunction were co-cultured with MSCs and Treg cells with and without cell-cell contact on transwell plates, further to quantitatively assess macrophage polarization in response to MSC and Treg treatment over time, M1 and M2 cell surface markers were used. Additionally, multiple soluble analytes were analyzed in cell supernatant by using bead-based immunoassays. Furthermore, to confirm our findings, gene expression analysis was done. MSCs induced the formation of M2 macrophages more than Tregs when macrophages M0 were cultured in transwell without cell contact. From this, we deduced the mechanism that soluble factors present in the MSCs condition media are involved in skewing of macrophages towards type 2 macrophages; similarly, in co-culture with cell-cell contact, MSCs resulted in more M2 type macrophages than Tregs. And an important finding of this study is the combination of both MSC-Treg showed significantly effective and consistent results in both with and without cell contact setups. Hence, it is suggestive to prefer MSCs over Tregs for secretome-based therapy and a combination of both for either therapy for effective transplantation outcomes. Our findings underline a key role of Tregs and MSCs in promoting macrophage polarization towards anti-inflammatory type. The study has great importance in prolongation of allograft and patient survival without any rejection by cell-based therapy, which induce self-tolerance and controlling infection.

Keywords: graft rejection, graft tolerance, macrophage polarization, mesenchymal stem cells, regulatory T cells, transplant immunology

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Authors: Asif Bilal, Fouzia Tanvir, Sibtain Ahmad

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Estrogen receptor alpha, also known as estrogen receptor-1, is highly involved in risk of mammary carcinoma. The objectives of this study were to identify non-synonymous SNPs of estrogen receptor and their association with breast cancer and to identify the chemotherapeutic responses of phytochemicals against it via in-silico study design. For this purpose, different online tools. to identify pathogenic SNPs the tools were SIFT, Polyphen, Polyphen-2, fuNTRp, SNAP2, for finding disease associated SNPs the tools SNP&GO, PhD-SNP, PredictSNP, MAPP, SNAP, MetaSNP, PANTHER, and to check protein stability Mu-Pro, I-Mutant, and CONSURF were used. Post-translational modifications (PTMs) were detected by Musitedeep, Protein secondary structure by SOPMA, protein to protein interaction by STRING, molecular docking by PyRx. Seven SNPs having rsIDs (rs760766066, rs779180038, rs956399300, rs773683317, rs397509428, rs755020320, and rs1131692059) showing mutations on I229T, R243C, Y246H, P336R, Q375H, R394S, and R394H, respectively found to be completely deleterious. The PTMs found were 96 times Glycosylation; 30 times Ubiquitination, a single time Acetylation; and no Hydroxylation and Phosphorylation were found. The protein secondary structure consisted of Alpha helix (Hh) is (28%), Extended strand (Ee) is (21%), Beta turn (Tt) is 7.89% and Random coil (Cc) is (44.11%). Protein-protein interaction analysis revealed that it has strong interaction with Myeloperoxidase, Xanthine dehydrogenase, carboxylesterase 1, Glutathione S-transferase Mu 1, and with estrogen receptors. For molecular docking we used Asiaticoside, Ilekudinuside, Robustoflavone, Irinoticane, Withanolides, and 9-amin0-5 as ligands that extract from phytochemicals and docked with this protein. We found that there was great interaction (from -8.6 to -9.7) of these ligands of phytochemicals at ESR1 wild and two mutants (I229T and R394S). It is concluded that these SNPs found in ESR1 are involved in breast cancer and given phytochemicals are highly helpful against breast cancer as chemotherapeutic agents. Further in vitro and in vivo analysis should be performed to conduct these interactions.

Keywords: breast cancer, ESR1, phytochemicals, molecular docking

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Authors: Birgul Hizlar, Sibel Karakaya

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Nowadays, consumers are more and more aware of the benefits beyond basic nutrition provided by food and food compounds. Between these, carotenoids have been demonstrated to exhibit multiple health benefits (for example, some types of cancer, cardiovascular diseases, eye disorders, among others). However, carotenoid bioaccessibility and bioavailability is generally rather low due to their specific localization in plant tissue and lipophilic nature. This situation is worldwide issue, since both developed and developing countries have their interest and benefits in increasing the uptake of carotenoids from the human diet. Recently, a new class of foods designed to improve the bioaccessibility/bioavailability of orally administered bioactive compounds is introduced: excipient foods. Excipient foods are specially designed foods which are prepared depending on the physicochemical properties of target bioactive compounds and increasing the bioavailability or bioaccessibility of bioactive compound. In this study, effects of food matrix (greating, boiling and mashing) and different excipient foods (olive oil, lemon juice, whey curd and dried artichoke leaf powder) on bioaccessibility of β-carotene in carrot were investigated by means of simulating in vitro gastrointestinal (GI) digestion. β-carotene contents of grated, boiled and mashed (after boiling process) carrots were 79.28, 147.63 and 151.19 μg/g respectively. No significant differences among boiled and mashed samples indicated that mashing process had no effect on the release of β-carotene from the food matrix (p > 0.05). On the contrary, mashing causes significant increase in the β-carotene bioaccessibility (p < 0.05). The highest β-carotene content was found in the mashed carrots incorporated with olive oil and lemon juice (C2). However, no significant differences between that sample and C1 (mashed carrot with lemon juice, olive oil, dried artichoke leaf powder), C3 (mashed carrot with addition of olive oil, lemon juice, whey curd) and). Similarly, the highest β-carotene bioaccessibility (50.26%) was found mashed C3 sample (p < 0.05). The increase in the bioaccessibility was approximately 5 fold and 50 fold when compared to grated and mashed samples containing olive oil, lemon juice and whey curd. The results demonstrate that both, food matrix and excipient foods, are able to increase the bioaccessibility of β-carotene.

Keywords: bioaccessibility, carotenoids, carrot, β-carotene

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Authors: Ariel Vilchez, Francisca Acevedo, Rodrigo Navia

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The wound healing process can be accelerated and improved by the action of antioxidants such as curcumin (Cur) over the tissues; however, the efficacy of curcumin used through the digestive system is not enough to exploit its benefits. Electrospinning presents an alternative to carry curcumin directly to the wounds, and polyhydroxybutyrate (PHB) is proposed as the matrix to load curcumin owing to its biodegradable and biocompatible properties. PHB is among 150 types of Polyhydroxyalkanoates (PHAs) identified, it is a natural thermoplastic polyester produced by microbial fermentation obtained from microorganisms. The proposed objective is to develop electrospun bacterial PHB-based microfibers containing curcumin for possible biomedical applications. Commercial PHB was solved in Chloroform: Dimethylformamide (4:1) to a final concentration of 7% m/V. Curcumin was added to the polymeric solution at 1%, and 7% m/m regarding PHB. The electrospinning equipment (NEU-BM, China) with a rotary collector was used to obtain Cur-PHB fibers at different voltages and flow rate of the polymeric solution considering a distance of 20 cm from the needle to the collector. Scanning electron microscopy (SEM) was used to determine the diameter and morphology of the obtained fibers. Thermal stability was obtained from Thermogravimetric (TGA) analysis, and Fourier Transform Infrared Spectroscopy (FT-IR) was carried out in order to study the chemical bonds and interactions. A preliminary curcumin release to Phosphate Buffer Saline (PBS) pH = 7.4 was obtained in vitro and measured by spectrophotometry. PHB fibers presented an intact chemical composition regarding the original condition (dust) according to FTIR spectra, the diameter fluctuates between 0.761 ± 0.123 and 2.157 ± 0.882 μm, with different qualities according to their morphology. The best fibers in terms of quality and diameter resulted in sample 2 and sample 6, obtained at 0-10kV and 0.5 mL/hr, and 0-10kV and 1.5 mL/hr, respectively. The melting temperature resulted near 178 °C, according to the bibliography. The crystallinity of fibers decreases while curcumin concentration increases for the studied interval. The curcumin release reaches near 14% at 37 °C at 54h in PBS adjusted to a quasi-Fickian Diffusion. We conclude that it is possible to load curcumin in PHB to obtain continuous, homogeneous, and solvent-free microfibers by electrospinning. Between 0% and 7% of curcumin, the crystallinity of fibers decreases as the concentration of curcumin increases. Thus, curcumin enhances the flexibility of the obtained material. HPLC should be used in further analysis of curcumin release.

Keywords: antioxidant, curcumin, polyhydroxybutyrate, wound healing

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Authors: Ikram Elsiddig, Yacouba Djamila, Amna Hamad

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Abstract—The worldwide increasing of using herbs in form of medicine with or without prescription medications potentiates the interactions between herbal products and conventional medicines; due to more research for herb-drug interactions are needed. for a long time hyperglycemia had been treated with several medicinal plants. A. sativum, belonging to the Liliaceae family is well known for its medicinal uses in African traditional medicine, it used for treating of many human diseases mainly diabetes, high cholesterol and high blood pressure. The purpose of this study is to determine the interaction effect between A. sativum bulb extracts and metformin drug used in diabetes treatment. The in vitro and in vivo evaluation were conducted by glucose reuptake using isolated rats hemidiaphgrams tissue and by estimate glucose tolerance in glucose-loaded wistar albino rats. The results showed that, petroleum ether, chloroform and ethyl acetate extracts were found to have activity of glucose uptake in isolated rats hemidiaphgrams of 24.11 mg/g, 19.07 mg/g and 15.66 mg/g compared to metformin drug of 17 mg/g. These activity were reducded to 17.8 mg/g, 13.59 mg/g and 14.46 mg/g after combination with metformin, metformin itself reduced to 13.59 mg/g, 14.46 mg/g and 12.71 mg/g in comination with chloroform and ethyl acetate. These decrease in activity could be due to herbal–drug interaction between the extracts of A. sativum bulb and metformin drug. The interaction between A. sativum extract and metformin was also shown by in vivo study on the induced hyperglycemic rats. The glucose level after administered of 200 mg/kg was found to be increase with 47.2 % and 17.7% at first and second hour compared to the increase of blood glucose in the control group of 82.6% and76.7%.. At fourth hour the glucose level was became less than normal with 3.4% compared to control which continue to increase with 68.2%. Dose of 400 mg/kg at first hour showed increase in blood glucose of 31.5 %, at second and fourth hours the glucose level was became less than normal with decrease of 3.2 % and 30.4%. After combination the activity was found to be less than that of extract at both high and low dose, whereas, at first and second hour, the glucose level was found to be increase with 50.4% and 21.2%, at fourth hour the glucose level was became less than normal with 14%. Therefore A. sativum could be a potential source for anti-diabetic when it used alone, and it is significant important to use the garlic extract alone instead of combined with Metformin drug in diabetes- treatment.

Keywords: Antagonistic, Garlic, Metformin, Synergistic

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Authors: Junichi Hosoi

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Epidermal Langerhans cells reside in upper layer of epidermis and play a role in immune surveillance. The finding of the close association of nerve endings to Langerhans cells triggered the research on systemic regulation of Langerhans cells. They disappear from epidermis after exposure to environmental and internal stimuli and reappear about a week later. Myeloid progenitor cells are assumed to be one of the sources of Langerhans cells. We examined the effects of cortisol on the reappearance of Langerhans cells in vitro. Cord-blood derived CD34-positive cells were cultured in the medium supplemented with stem cell factor/Flt3 ligand/granulocyte macrophage-colony stimulating factor/tumor necrosis factor alpha/bone morphologic protein 7/transforming growth factor beta in the presence or absence of cortisol. Cells were analyzed by flow cytometry for CD1a (cluster differentiation 1a), a marker of Langerhans cells and dermal dendritic cells, and CD39 (cluster differentiation factor 39), extracellular adenosine triphosphatase. Both CD1a-positive cells and CD39-positive cells were decreased by treatment with cortisol (suppression by 35% and 22% compared to no stress hormone, respectively). Differentiated Langerhans cells are attracted to epidermis by chemokines that are secreted from keratinocytes. Epidermal keratinocytes were cultured in the presence or absence of cortisol and analyzed for the expression of CCL2 (C-C motif chemokine ligand 2) and CCL20 (C-C motif chemokine ligand 20), which are typical attractants of Langerhans cells, by quantitative reverse transcriptase polymerase chain reaction. The expression of both chemokines, CCL2 and CCL20, were suppressed by treatment with cortisol (suppression by 38% and 48% compared to no stress hormone, respectively). We examined the possible regulation of the suppression by cortisol with plant extracts. The extracts of Ganoderma lucidum and Iris protected the suppression of the differentiation to CD39-positive cells and also the suppression of the gene expression of LC-chemoattractants. These results suggest that cortisol, which is either systemic or locally produced, blocks the supply of epidermal Langerhans cells at 2 steps, differentiation from the precursor and attraction to epidermis. The suppression is possibly blocked by some plant extracts.

Keywords: Langerhans cell, stress, CD39, chemokine

Procedia PDF Downloads 160

Authors: A. Dashti, M. Eskandari, L. Farahmand, P. Parvin, A. Jafargholi

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Breast Cancer is one of the most considerable diseases in the United States and other countries and is the second leading cause of death in women. Common breast cancer treatments would lead to adverse side effects such as loss of hair, nausea, and weakness. These complications arise because these cancer treatments damage some healthy cells while eliminating the cancer cells. In an effort to address these complications, laser radiation was utilized and tested as a targeted cancer treatment for breast cancer. In this regard, tissue engineering approaches are being employed by using an electrospun scaffold in order to facilitate the growth of breast cancer cells. Polycaprolacton (PCL) was used as a material for scaffold fabricating because of its biocompatibility, biodegradability, and supporting cell growth. The specific breast cancer cells have the ability to create a three-dimensional cell cluster due to the spontaneous accumulation of cells in the porosity of the scaffold under some specific conditions. Therefore, we are looking for a higher density of porosity and larger pore size. Fibers showed uniform diameter distribution and final scaffold had optimum characteristics with approximately 40% porosity. The images were taken by SEM and the density and the size of the porosity were determined with the Image. After scaffold preparation, it has cross-linked by glutaraldehyde. Then, it has been washed with glycine and phosphate buffer saline (PBS), in order to neutralize the residual glutaraldehyde. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) results have represented approximately 91.13% viability of the scaffolds for cancer cells. In order to create a cluster, Michigan Cancer Foundation-7 (MCF-7, breast cancer cell line) and Michigan Cancer Foundation-10A (MCF-10A, human mammary epithelial cell line) cells were cultured on the scaffold in 24 well plate for five days. Then, we have exposed the cluster to the laser diode 808 nm radiation to investigate the effect of laser on the tumor with different power and time. Under the same conditions, cancer cells lost their viability more than the healthy ones. In conclusion, laser therapy is a viable method to destroy the target cells and has a minimum effect on the healthy tissues and cells and it can improve the other method of cancer treatments limitations.

Keywords: breast cancer, electrospun scaffold, polycaprolacton, laser diode, cancer treatment

Procedia PDF Downloads 123

Authors: Rawia M. Khalil, Ghada Abd-Elbary, Mona Basha, Ghada E. A. Awad, Hadeer A. Elhashemy

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Background: Bacterial infections of the eye are the clinical conditions responsible for ocular morbidity and blindness. Conjunctivitis is an inflammation of the conjunctiva, due to Staphylococcus aureus. Lomefloxacin HCl (LXN) is a third generation flouroquinolone antibiotic with a broad spectrum against wide range of bacteria and very effective against Staph infections especially in conjunctiva (conjunctivitis). The present study aims to develop and evaluate novel ocular proniosomal gels of Lomefloxacin Hcl (LXN); in order to improve its ocular bioavailability for the management of bacterial conjunctivitis. Materials and methods: Proniosomes were prepared by coacervation phase separation method using different types of nonionic surfactants (Span 60,40,20,Tween 20,40,60,80,Brij 35,98,72) solely and as mixtures with Span® 60. The formed gels were characterized for entrapment efficiency, vesicle size and in vitro drug release. The optimum proniosomal gel; P-LXN 7 were characterized for pH measurement, transmission electron microscopy (TEM) and differential scanning calorimetry (DSC) as well as Stability study and microbiological evaluation .The results revealed that only Span 60 was able to form stable LXN proniosomal gel when used individually while the other nonionic surfactants formed gels only in combination with Span 60 at different ratios. The optimum proniosomal gel; P-LXN 7 (Span60:Tween60, 9:1) appeared as spherical shaped vesicles having high entrapment efficiency (>80 %), appropriate vesicle size (187 nm) as well as controlled drug release over 12h. DSC confirmed the amorphous nature and the uniformity of LXN inclusion within the vesicles. Physical stability study did not show any significant changes in appearance or entrapment efficiency or vesicle size after storage for 3 months at 4°C. Ocular irritancy test revealed that P-LXN 7 was safe, well tolerable and suitable for ocular delivery. In vivo antibacterial activity of P-LXN 7 evaluated using the susceptibility test and topical therapy of induced ocular conjunctivitis confirmed the enhanced antibacterial therapeutic efficacy of the LXN-proniosomal gel compared to the commercially available LXN eye drops; Orchacin®. Conclusions: Our results suggest that proniosomal gels could provide a promising carrier of LXN for efficient ocular treatment of bacterial conjunctivitis.

Keywords: bacterial conjunctivitis, lomefloxacin HCl, ocular drug delivery, proniosomes

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Authors: Pavel Bulai, Taras Pitlik, Tatsiana Kulahava, Timofei Lipski

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The method of Electrocardiography (ECG) interpretation for post-exercise recovery tracking was developed. Metabolic indices (aerobic and anaerobic) were designed using ECG-derived features. This study reports the associations between aerobic and anaerobic indices and classical parameters of the person’s physiological state, including blood biochemistry, glycogen concentration and VO2max changes. During the study 9 participants, healthy, physically active medium trained men and women, which trained 2-4 times per week for at least 9 weeks, fulfilled (i) ECG monitoring using Apple Watch Series 4 (AWS4); (ii) blood biochemical analysis; (iii) maximal oxygen consumption (VO2max) test, (iv) bioimpedance analysis (BIA). ECG signals from a single-lead wrist-wearable device were processed with detection of QRS-complex. Aerobic index (AI) was derived as the normalized slope of QR segment. Anaerobic index (ANI) was derived as the normalized slope of SJ segment. Biochemical parameters, glycogen content and VO2max were evaluated eight times within 3-60 hours after training. ECGs were recorded 5 times per day, plus before and after training, cycloergometry and BIA. The negative correlation between AI and blood markers of the muscles functional status including creatine phosphokinase (r=-0.238, p < 0.008), aspartate aminotransferase (r=-0.249, p < 0.004) and uric acid (r = -0.293, p<0.004) were observed. ANI was also correlated with creatine phosphokinase (r= -0.265, p < 0.003), aspartate aminotransferase (r = -0.292, p < 0.001), lactate dehydrogenase (LDH) (r = -0.190, p < 0.050). So, when the level of muscular enzymes increases during post-exercise fatigue, AI and ANI decrease. During recovery, the level of metabolites is restored, and metabolic indices rising is registered. It can be concluded that AI and ANI adequately reflect the physiology of the muscles during recovery. One of the markers of an athlete’s physiological state is the ratio between testosterone and cortisol (TCR). TCR provides a relative indication of anabolic-catabolic balance and is considered to be more sensitive to training stress than measuring testosterone and cortisol separately. AI shows a strong negative correlation with TCR (r=-0.437, p < 0.001) and correctly represents post-exercise physiology. In order to reveal the relation between the ECG-derived metabolic indices and the state of the cardiorespiratory system, direct measurements of VO2max were carried out at various time points after training sessions. The negative correlation between AI and VO2max (r = -0.342, p < 0.001) was obtained. These data testifying VO2max rising during fatigue are controversial. However, some studies have revealed increased stroke volume after training, that agrees with findings. It is important to note that post-exercise increase in VO2max does not mean an athlete’s readiness for the next training session, because the recovery of the cardiovascular system occurs over a substantially longer period. Negative correlations registered for ANI with glycogen (r = -0.303, p < 0.001), albumin (r = -0.205, p < 0.021) and creatinine (r = -0.268, p < 0.002) reflect the dehydration status of participants after training. Correlations between designed metabolic indices and physiological parameters revealed in this study can be considered as the sufficient evidence to use these indices for assessing the state of person’s aerobic and anaerobic metabolic systems after training during fatigue, recovery and supercompensation.

Keywords: aerobic index, anaerobic index, electrocardiography, supercompensation

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Authors: Jesryl B. Paulite, Irene Alcantara-Papa, Teofila O. Zulaybar, Jocelyn T. Zarate, Virgie Ugay

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Banana is one of the major fruits in the Philippines in terms of volume of production and export earnings. The Philippines export of fresh Cavendish banana ranked No.1 with 22% share. One major threat to the industry is Fusarium wilt caused by Fusarium oxysporum f. sp. cubense. It tops as a major concern today affecting the Philippine banana industry since 2002 up to the present in Mindanao. Because of environmental and health issues concerning the use of chemical pesticides in the control of diseases, utilization of microorganisms has been significant in recent years as a promising alternative. This study aims to evaluate the potential of actinomycetes to control Fusarium wilt in Cavendish banana. The in-vitro experiments was carried out in Complete Randomized Design (CRD) while field experiment was laid out in a Randomized Complete Block Design (RCBD) with three treatments and three replications. Actinomycetes were isolated from mangrove soils in areas in Quezon and Bataan, Philippines. A total of 199 actinomycetes were isolated and 82 actinomycetes showed activity against the local Fusarium oxysporum (Foc) by agar plug assay. The test for antagonisms (AQ6, AQ30, and AQ121) of three best isolates Foc to were selected inhibiting Foc by 21.0mm, 22.0mm and 20.5mm, respectively. The same actinomycetes inhibited well Foc Tropical Race 4 showing 24.6 mm, 20.2mm and 19.0 mm zones of inhibition by agar plug assay, respectively. Combinations of the three isolates yielded an inhibition of 13.5 mm by cup cylinder assay. These findings led to the formulation of the mixed actinomycetes as biocontrol agents against Foc. A field experiment to evaluate the formulated mixed actinomycetes against Foc in a Foc infested field in Kinamayan, Sto Tomas, Davao Del Norte, Philippines. was conducted. Results showed that preventive method of application of the mixed actinomycetes against Foc showed promising results. A 56.66% mortality was observed in control set-up (no biocontrol agent added) compared to 33.33% mortality in preventive method. Further validation of the effectiveness of the mixed actinomycetes as biocontrol agent is presently being conducted in Asuncion, Davao Del Norte, Philippines.

Keywords: actinomycetes, biocontrol agents, cavendish banana, Fusarium oxysporum f. sp. cubense

Procedia PDF Downloads 559

Authors: Umara Nissar Rafiqi, Irum Gul, Nazima Nasrullah, Monica Saifi, Malik Z. Abdin

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Background: Stevia rebaudiana, a member of Asteraceae family is an important medicinal plant and produces a commercially used non-caloric natural sweetener, which is also an alternate herbal cure for diabetes. Steviol glycosides are the main sweetening compounds present in these plants. Secondary metabolites are crucial to the adaption of plants to the environment and its overcoming stress conditions. In agricultural procedures, the abiotic stresses like salinity, high metal toxicity and drought, in particular, are responsible for the majority of the reduction that differentiates yield potential from harvestable yield. Salt stress and heavy metal toxicity lead to increased production of reactive oxygen species (ROS). To avoid oxidative damage due to ROS and osmotic stress, plants have a system of anti-oxidant enzymes along with several stress induced enzymes. This helps in scavenging the ROS and relieve the osmotic stress in different cell compartments. However, whether stress induced toxicity modulates the activity of these enzymes in Stevia rebaudiana is poorly understood. Aim: The present study focussed on the effect of salinity, heavy metal toxicity (lead and mercury) on physiological traits and transcriptional profiling of Stevia rebaudiana. Method: Stevia rebaudiana plants were collected from the Central Institute of Medicinal and Aromatic plants (CIMAP), Patnagar, India and maintained under controlled conditions in a greenhouse at Hamdard University, Delhi, India. The plants were subjected to different concentrations of salt (0, 25, 50 and 75 mM respectively) and heavy metals, lead and mercury (0, 100, 200 and 300 µM respectively). The physiological traits such as shoot length, root numbers, leaf growth were evaluated. The samples were collected at different developmental stages and analysed for transcription profiling by RT-PCR. Transcriptional studies in stevia rebaudiana involves important antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), cytochrome P450 monooxygenase (CYP) and stress induced aquaporin (AQU), auxin repressed protein (ARP-1), Ndhc gene. The data was analysed using GraphPad Prism and expressed as mean ± SD. Result: Low salinity and lower metal toxicity did not affect the fresh weight of the plant. However, this was substantially decreased by 55% at high salinity and heavy metal treatment. With increasing salinity and heavy metal toxicity, the values of all studied physiological traits were significantly decreased. Chlorosis in treated plants was also observed which could be due to changes in Fe:Zn ratio. At low concentrations (upto 25 mM) of NaCl and heavy metals, we did not observe any significant difference in the gene expressions of treated plants compared to control plants. Interestingly, at high salt concentration and high metal toxicity, a significant increase in the expression profile of stress induced genes was observed in treated plants compared to control (p < 0.005). Conclusion: Stevia rebaudiana is tolerant to lower salt and heavy metal concentration. This study also suggests that with the increase in concentrations of salt and heavy metals, harvest yield of S. rebaudiana was hampered.

Keywords: Stevia rebaudiana, natural sweetener, salinity, heavy metal toxicity

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Authors: Neslihan Demirci, Serdar Durdağı

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Mycobacterium tuberculosis is still a worldwide disease-causing agent that, according to WHO, led to the death of 1.5 million people from tuberculosis (TB) in 2020. The bacteria reside in macrophages located specifically in the lung. There is a known quadruple drug therapy regimen for TB consisting of isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB). Over the past 60 years, there have been great contributions to treatment options, such as recently approved delamanid (OPC67683) and bedaquiline (TMC207/R207910), targeting mycolic acid and ATP synthesis, respectively. Also, there are natural compounds that can block the tryptophanyl-tRNA synthetase (TrpRS) enzyme, chuangxinmycin, and indolmycin. Yet, already the drug resistance is reported for those agents. In this study, the newly released TrpRS enzyme structure is investigated for potential inhibitor drugs from already synthesized molecules to help the treatment of resistant cases and to propose an alternative drug for the quadruple drug therapy of tuberculosis. Maestro, Schrodinger is used for docking and molecular dynamic simulations. In-house library containing ~8000 compounds among FDA-approved indole-containing compounds, a total of 57 obtained from the ChemBL were used for both ATP and tryptophan binding pocket docking. Best of indole-containing 57 compounds were subjected to hit expansion and compared later with virtual screening workflow (VSW) results. After docking, VSW was done. Glide-XP docking algorithm was chosen. When compared, VSW alone performed better than the hit expansion module. Best scored compounds were kept for ten ns molecular dynamic simulations by Desmond. Further, 100 ns molecular dynamic simulation was performed for elected molecules according to Z-score. The top three MMGBSA-scored compounds were subjected to steered molecular dynamic (SMD) simulations by Gromacs. While SMD simulations are still being conducted, ponesimod (for multiple sclerosis), vilanterol (β₂ adrenoreceptor agonist), and silodosin (for benign prostatic hyperplasia) were found to have a significant affinity for tuberculosis TrpRS, which is the propulsive force for the urge to expand the research with in vitro studies. Interestingly, top-scored ponesimod has been reported to have a side effect that makes the patient prone to upper respiratory tract infections.

Keywords: drug repurposing, molecular dynamics, tryptophanyl-tRNA synthetase, tuberculosis

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Authors: Gökçe Erdemir, İlhan Yaylım, Serap Erdem-Kuruca, Musa Mutlu Can

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It has been determined that the main reason for the death of cancer disease is caused by metastases rather than the primary tumor. The cells that leave the primary tumor and enter the circulation and cause metastasis in the secondary organs are called "circulating tumor cells" (CTCs). The presence and number of circulating tumor cells has been associated with poor prognosis in many major types of cancer, including breast, prostate, and colorectal cancer. It is thought that knowledge of circulating tumor cells, which are seen as the main cause of cancer-related deaths due to metastasis, plays a key role in the diagnosis and treatment of cancer. The fact that tissue biopsies used in cancer diagnosis and follow-up are an invasive method and are insufficient in understanding the risk of metastasis and the progression of the disease have led to new searches. Liquid biopsy tests performed with a small amount of blood sample taken from the patient for the detection of CTCs are easy and reliable, as well as allowing more than one sample to be taken over time to follow the prognosis. However, since these cells are found in very small amounts in the blood, it is very difficult to capture them and specially designed analytical techniques and devices are required. Methods based on the biological and physical properties of the cells are used to capture these cells in the blood. Early diagnosis is very important in following the prognosis of tumors of epithelial origin such as breast, lung, colon and prostate. Molecules such as EpCAM, vimentin, and cytokeratins are expressed on the surface of cells that pass into the circulation from very few primary tumors and reach secondary organs from the circulation, and are used in the diagnosis of cancer in the early stage. For example, increased EpCAM expression in breast and prostate cancer has been associated with prognosis. These molecules can be determined in some blood or body fluids to be taken from patients. However, more sensitive methods are required to be able to determine when they are at a low level according to the course of the disease. The aim is to detect these molecules found in very few cancer cells with the help of sensitive, fast-sensing biosensors, first in breast cancer cells reproduced in vitro and then in blood samples taken from breast cancer patients. In this way, cancer cells can be diagnosed early and easily and effectively treated.

Keywords: electrochemical biosensors, breast cancer, circulating tumor cells, EpCAM, Vimentin, Cytokeratins

Procedia PDF Downloads 240

Authors: M. R. Aher, R. B. Laware, G. S. Asane, B. S. Kuchekar

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Objective: Studies have shown that a new combination therapy with Artemisinin derivatives and curcumin is unique, with potential advantages over known ACTs. In present study an attempt was made to prepare microparticulate drug delivery system of Curcumin-Zn complex and evaluate it in combination with artemether for antimalarial activity. Material and method: Curcumin Zn complex was prepared and encapsulated using sodium alginate. Microparticles thus obtained are further coated with various enteric polymers at different coating thickness to control the release. Microparticles are evaluated for encapsulation efficiency, drug loading and in vitro drug release. Roentgenographic Studies was conducted in rabbits with BaSO 4 tagged formulation. Optimized formulation was screened for antimalarial activity using P. berghei-infected mice survival test and % paracetemia inhibition, alone (three oral dose of 5mg/day) and in combination with arthemether (i.p. 500, 1000 and 1500µg). Curcumin-Zn(II) was estimated in serum after oral administration to rats by using spectroflurometry. Result: Microparticles coated with Cellulose acetate phthalate showed most satisfactory and controlled release with 479 min time for 60% drug release. X-ray images taken at different time intervals confirmed the retention of formulation in GI tract. Estimation of curcumin in serum by spectroflurometry showed that drug concentration is maintained in the blood for longer time with tmax of 6 hours. The survival time (40 days post treatment) of mice infected with P. berghei was compared to survival after treatment with either Curcumin-Zn(II) microparticles artemether combination, curcumin-Zn complex and artemether. Oral administration of Curcumin-Zn(II)-artemether prolonged the survival of P.berghei-infected mice. All the mice treated with Curcumin-Zn(II) microparticles (5mg/day) artemether (1000µg) survived for more than 40 days and recovered with no detectable parasitemia. Administration of Curcumin-Zn(II) artemether combination reduced the parasitemia in mice by more than 90% compared to that in control mice for the first 3 days after treatment. Conclusion: Antimalarial activity of the curcumin Zn-artemether combination was more pronounced than mono therapy. A single dose of 1000µg of artemether in curcumin-Zn combination gives complete protection in P. berghei-infected mice. This may reduce the chances of drug resistance in malaria management.

Keywords: formulation, microparticulate drug delivery, antimalarial, pharmaceutics

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Authors: Gulnaz Khan, Meha F. Aftab, Munazza Murtaza, Rizwana S. Waraich

Abstract:

Advanced glycation end products (AGEs) precursor and its abnormal accumulation cause damage to various tissues and organs. AGEs have pathogenic implication in several diseases including diabetes. Existing AGEs inhibitors are not in clinical use, and there is a need for development of novel inhibitors. The present investigation aimed at identifying the novel AGEs inhibitors and assessing their mechanism of action for treating insulin resistance in mice model of diabetes. Novel derivatives of benzylidene of indan-1,3-dione were synthesized. The compounds were selected to study their action mechanism in improving insulin resistance, in vitro, in human hepatocytes and murine adipocytes and then, in vivo, in mice genetic model of diabetes (db/db). Mice were treated with novel derivatives of benzylidene of indane 1,3-dione. AGEs mediated ROS production was measured by dihydroethidium fluorescence assay. AGEs level in the serum of treated mice was observed by ELISA. Gene expression of receptor for AGEs (RAGE), PPAR-gamma, TNF-alpha and GLUT-4 was evaluated by RT-PCR. Glucose uptake was measured by fluorescent method. Microscopy was used to analyze glycogen synthesis in muscle. Among several derivatives of benzylidene of indan-1,3-dione, IDD-24, demonstrated highest inhibition of AGESs. IDD-24 significantly reduced AGEs formation and expression of receptor for advanced glycation end products (RAGE) in fat, liver of db/db mice. Suppression of AGEs mediated ROS production was also observed in hepatocytes and fat cell, after treatment with IDD-24. Glycogen synthesis was increased in muscle tissue of mice treated with IDD-24. In adipocytes, IDD-24 prevented AGEs induced reduced glucose uptake. Mice treated with IDD-24 exhibited increased glucose tolerance, serum adiponectin levels and decreased insulin resistance. The result of present study suggested that IDD-24 can be a possible treatment target to address glycotoxins induced insulin resistance.

Keywords: advance glycation end product, hyperglycemia, indan-1, 3-dione, insulin resistance

Procedia PDF Downloads 139

Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

Abstract:

Klebsiella pneumoniae causes a wide range of infections, including urinary tract infections, sepsis, bacteremia, pneumonia, and liver abscesses. The emergence of multi-drug resistance in this bacterium led to a major setback for clinical management. WHO also endorsed a need for finding alternative therapy to antibiotics for the treatment of these infections. Development of vaccines and passive antibody therapy has been proven as a potent alternative to antibiotics in the case of MDR, XDR, and PDR Klebsiella infections. Siderophore receptors have been demonstrated to be overexpressed for the internalization of iron siderophore complexes during infections in most Gram-negative bacteria. For the present study, immune response to siderophore receptors to establish this protein as a potential immunogen for the development of therapeutic leads was explored. Clinical strains of Klebsiella pneumoniae were grown in iron-deficient conditions, and the iron-regulated outer membrane proteins were extracted and characterized through mass spectrometry for specific identification. The gene for identified protein was cloned in pET- 28a vector and expressed in E. coli. The native protein and the recombinant protein were isolated and purified and used as antigens for the generation of immune response in BALB/c mice. The native protein of Klebsiella pneumoniae grown in iron-deficient conditions was identified as FepA (Ferrienterobactin receptor) and other siderophore receptors. This 80 kDa protein generated an immune response in BALB/c mice. The antiserum from mice after subsequent booster doses was collected and showed binding with FepA protein in western blot and phagocytic uptake of the K. pneumoniae in the presence antiserum from immunized mice also observed from the animal studies after bacterial challenge post immunisation in mice have shown bacterial clearance. The antiserum from mice showed binding and clearance of the Klebsiella pneumoniae bacteria in vitro and in vivo. These antigens used for generating an active immune response in mice can further be used for therapeutic monoclonal antibody development against Klebsiella pneumoniae infections.

Keywords: antiserum, FepA, Klebsiella pneumoniae, multi drug resistance, siderophore receptor

Procedia PDF Downloads 79

Authors: Jamboor K. Vishwanatha

Abstract:

Among the potent anti-cancer agents, curcumin has been found to be very efficacious against various cancer cells. Despite multiple medicinal benefits of curcumin, poor water solubility, poor physiochemical properties and low bioavailability continue to pose major challenges in developing a formulation for clinical efficacy. To improve its potential application in the clinical area, we formulated poly lactic-co-glycolic acid (PLGA) nanoparticles. The PLGA nanoparticles were formulated using solid-oil/water emulsion solvent evaporation method and then characterized for percent yield, encapsulation efficiency, surface morphology, particle size, drug distribution within nanoparticles and drug polymer interaction. Our studies showed the successful formation of smooth and spherical curcumin loaded PLGA nanoparticles with a high percent yield of about 92.01±0.13% and an encapsulation efficiency of 90.88±0.14%. The mean particle size of the nanoparticles was found to be 145nm. The in vitro drug release profile showed 55-60% drug release from the nanoparticles over a period of 24 hours with continued sustained release over a period of 8 days. Exposure to curcumin loaded nanoparticles resulted in reduced cell viability of cancer cells compared to normal cells. We used a novel non-covalent insertion of a homo-bifunctional spacer for targeted delivery of curcumin to various cancer cells. Functionalized nanoparticles for antibody/targeting agent conjugation was prepared using a cross-linking ligand, bis(sulfosuccinimidyl) suberate (BS3), which has reactive carboxyl group to conjugate efficiently to the primary amino groups of the targeting agents. In our studies, we demonstrated successful conjugation of antibodies, Annexin A2 or prostate specific membrane antigen (PSMA), to curcumin loaded PLGA nanoparticles for targeting to prostate and breast cancer cells. The percent antibody attachment to PLGA nanoparticles was found to be 92.8%. Efficient intra-cellular uptake of the targeted nanoparticles was observed in the cancer cells. These results have emphasized the potential of our multifunctional curcumin nanoparticles to improve the clinical efficacy of curcumin therapy in patients with cancer.

Keywords: polymeric nanoparticles, cancer therapy, sustained release, curcumin

Procedia PDF Downloads 306