Search results for: ABTS assay

Authors: Ruby Siwach, Manish Kumar, Raman Seth

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Extent of inactivation of pectin methylesterase (PME) in mosambi juice during thermal and thermosonication treatments was studied to obtain a minimally processed product. Effect of both treatments on cloud value, pH, titratable acidity, oBrix, and sensory attributes (flavour and taste) was studied. Thermal treatments (HT) were carried out at three temperatures 60, 70, and 80°C in a serological water bath for 5, 10, 15, and 20 min at each temperature. Thermosonication treatments (TS) were also given for same time-temperature combinations in water bath of a thermosonicator. Treated samples were stored in a deep freezer at 18°C for PME assay. PME activity of untreated sample was also assayed and residual PME activity and % loss in PME activity was calculated at each time-temperature combination. The extent of inactivation of PME increased with increase in treatment temperature and duration. Thermosonication treatments were found far more effective than thermal treatments of same time temperature combination in PME inactivation and retention of sensory attributes.

Keywords: pectin methylesterase, heat inactivation kinetics, thermosonication, thermal treatment

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Authors: Iman Farasat, Howard M. Salis

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Engineered genetic circuits reprogram cellular behavior to act as living computers with applications in detecting cancer, creating self-controlling artificial tissues, and dynamically regulating metabolic pathways. Phenemenological models are often used to simulate and design genetic circuit behavior towards a desired behavior. While such models assume that each circuit component’s function is modular and independent, even small changes in a circuit (e.g. a new promoter, a change in transcription factor expression level, or even a new media) can have significant effects on the circuit’s function. Here, we use statistical thermodynamics to account for the several factors that control transcriptional regulation in bacteria, and experimentally demonstrate the model’s accuracy across 825 measurements in several genetic contexts and hosts. We then employ our first principles model to design, experimentally construct, and characterize a family of signal amplifying genetic circuits (genetic OpAmps) that expand the dynamic range of cell sensors. To develop these models, we needed a new approach to measuring the in vivo binding free energies of transcription factors (TFs), a key ingredient of statistical thermodynamic models of gene regulation. We developed a new high-throughput assay to measure RNA polymerase and TF binding free energies, requiring the construction and characterization of only a few constructs and data analysis (Figure 1A). We experimentally verified the assay on 6 TetR-homolog repressors and a CRISPR/dCas9 guide RNA. We found that our binding free energy measurements quantitatively explains why changing TF expression levels alters circuit function. Altogether, by combining these measurements with our biophysical model of translation (the RBS Calculator) as well as other measurements (Figure 1B), our model can account for changes in TF binding sites, TF expression levels, circuit copy number, host genome size, and host growth rate (Figure 1C). Model predictions correctly accounted for how these 8 factors control a promoter’s transcription rate (Figure 1D). Using the model, we developed a design framework for engineering multi-promoter genetic circuits that greatly reduces the number of degrees of freedom (8 factors per promoter) to a single dimensionless unit. We propose the Ptashne (Pt) number to encapsulate the 8 co-dependent factors that control transcriptional regulation into a single number. Therefore, a single number controls a promoter’s output rather than these 8 co-dependent factors, and designing a genetic circuit with N promoters requires specification of only N Pt numbers. We demonstrate how to design genetic circuits in Pt number space by constructing and characterizing 15 2-repressor OpAmp circuits that act as signal amplifiers when within an optimal Pt region. We experimentally show that OpAmp circuits using different TFs and TF expression levels will only amplify the dynamic range of input signals when their corresponding Pt numbers are within the optimal region. Thus, the use of the Pt number greatly simplifies the genetic circuit design, particularly important as circuits employ more TFs to perform increasingly complex functions.

Keywords: transcription factor, synthetic biology, genetic circuit, biophysical model, binding energy measurement

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Authors: S. N. Harun, H. Ahmad

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A series of ruthenium(II) complexes, including two novel compounds [Ru(dppz)2(L)]2+ where dppz = dipyrido-[3,2-a:2’,3’-c]phenazine, and L = 2-phenylimidazo[4,5-f][1,10]phenanthroline (PIP) or 2-(4-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (p-HPIP) have been synthesized and characterized. The previously reported complexes [Ru(bpy)2L]2+ and [Ru(phen)2L]2+ were also prepared. All complexes were characterized by elemental analysis, 1H-NMR spectroscopy, ESI-Mass spectroscopy and FT-IR spectroscopy. The photophysical properties were analyzed by UV-Visible spectroscopy and fluorescence spectroscopy. [Ru(dppz)2(PIP)]2+ and [Ru(dppz)2(p-HPIP)]2+ displayed ‘molecular light-switch’ effect as they have high emission in acetonitrile but no emission in water. The cytotoxicity of all complexes against cancer cell lines Hela and MCF-7 were investigated through standard MTT assay. [Ru(dppz)2(PIP)]2+ showed moderate toxicity on both MCF-7 and Hela with IC50 of 37.64 µM and 28.02 µM, respectively. Interestingly, [Ru(dppz)2(p-HPIP)]2+ exhibited remarkable cytotoxicity results with IC50 of 13.52 µM on Hela and 11.63 µM on MCF-7 cell lines which are comparable to the infamous anti-cancer drug, cisplatin. The cytotoxicity of this complex series increased as the ligands size extended in order of [Ru(bpy)2(L)]2+ < [Ru(phen)2(L)]2+ < [Ru(dppz)2(L)]2+.

Keywords: ruthenium, cytotoxicity, molecular light-switch, anticancer

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Authors: A. H. Shaikat, M. B. Hossain, S. K. M. A. Islam, M. M. Hassan, S. A. Khan, A. K. M. Saifuddin, M. N. Islam, M. A. Hoque

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A total of eighteen (18) sex reversed chickens with unusual phenotypic characteristics of male birds were identified over 2000 Hyline layer chickens at Motaher Poultry Farm, Ramu, Cox’s Bazar. Chickens were subdivided into two groups (case = 18, control = 20) based on the appearance of sex-reversed secondary sexual characteristics. Phenotypic traits of studied chickens were measured with farm management details. Hormone assay using ELISA, autopsy followed by gross examination of viscera was performed. The study found higher body weight (gm) (1579.3; 95% CI: 1561.7-1596.8), comb length (cm) (12.2; 11.5-12.8), comb width (cm) (7.9; 7.7-8.2), wattle length (cm) (4.9; 4.8-5.1) distinct spur, and shortened pubic bones distance, suggesting decrease oviposition in sex-reversed chickens. Testosterone concentration (ng/ml) (8.5; 6.4-10.6) was significantly higher (p<0.001) along with decrease estrogen (pg/ml) (5.1; 4.9-5.5) and progesterone concentration (pg/ml) (310.9; 289.4-332.5) in sex-reversed chickens. Mass abdominal fat deposition with atrophied ovary was found upon exploration of viscera.

Keywords: ovary, phenotypic traits, sex hormone, sex reversal

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Authors: Ryeong-Hyeon Kim, Ah-Reum Lee, Seong-Soo Roh, Gyo-Nam Kim

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Appropriate regulation of melanogenesis is important for the management of skin pigmentation-related disease. Although several beneficial effects of fisetin (3,7,3’,4’-tetrahydroxyflavone) have been reported, the precise role and molecular mechanisms of fisetin in skin health both remain unclear. Here, we induced melanogenesis of HRM2 mice (n=7/group) by UVB irradiation for 20 days. UVB-induced HRM2 mice showed that the significantly increased melanin accumulation, however, fisetin treatment (25mg and 50mg/kg of body weight) dose-dependently and significantly inhibits UVB-induced melanogenesis. In line with this, fisetin treatment effectively down-regulated m RNA and expression levels of tyrosinase, TRP2, and MITF. In addition, our inhibitor assay revealed the down-regulated melanogenic marker genes by fisetin treatment were mediated with connective tissue growth factor (CCN2)/TGF-β signaling pathway. Useful information is provided for development of functional foods using fisetin for skin health.

Keywords: connective tissue growth factor, fisetin, melanogenesis, skin, TGF-beta

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Authors: K. Suresh, R. Arunkumar

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The objective of this study is to investigate the preparation of gramine nano suspension and protective effect of Gramine on the apoptosis of laryngeal cancer cells cell line (HEp-2 and KB). The growth inhibition rate of Hep-2 and KB cells in vitro were measured by MTT assay and apoptosis by, levels of reactive oxygen species, mitochondrial membrane potential, morphological changes and flowcytometry. Based on the results, we determined the effective doses of gramine as 127.23µm/ml for 24 hr and 119.81 µm/ml for 48hr in hep-2 cell line and 147.58 µm ml for 24 hr and 123.74µm µm/ml for 48hr in KB cell line. cytotoxicity effects of gramine were confirmed by treatment of HEp-2 cell and KB cell with IC50 concentration of gramine resulted in sequences of events marked by the enhance the apoptosis accompanied by loss of cell viability, modulation of reactive oxygen species and cell cycle arrest through the induction of G0/G1 phase arrest on HEp-2 cells. Our study suggests that the nanosuspension of gramine possesses the more cytotoxic effect of cancer cells and a novel candidate for cancer chemoprevention.

Keywords: apoptosis, HEp-2 cell line, KB cell line mitochondria, gramine, nanosuspension

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Authors: Nermin Isik, Ozlem Derinbay Ekici, Oguzhan Avci

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The aim of this study was to determination of role infection in neonatal calves in Central Anatolian, Turkey. A total 300 fecal samples were collected from diarrheic neonatal calves, aged between 0–90 days from Konya, Karaman, and Aksaray from January to April 2014. Fecal specimens from calves with clinically diarrheic symptoms were examined for the presence of Bovine Coronavirus, Bovine Rotavirus, Cryptosporidium sp., and E. coli by commercially available capture direct enzyme linked immunosorbent assay (ELISA) kit and Modified Ziehl Neelsen method (MZN). Calves were grouped according to their age as follows: 1-14, 15-29, and 30-90 days. Cryptosporidium sp. infection was detected in 52.8%, 58.8%, and 39.2% by ELISA and 33.9%, 47%, 26.7% by MZN in the respective age groups. The seroprevalance of Rotavirus (12.5 %, 40 %, 12.5 %), Coronavirus (2.5%, 0%, 3.5%) and E. coli (5%, 4.7%, 8.9%) infections were determined according to the age groups respectively. Cryptosporidium sp. was the most detected enteropathogen (52 %) of calves and coronavirus was the least detected (2 %). The detection rate of the mixed enfection was 12.3%. In conclusion, it must be evaluated by mix infections in calves with diarrhea. These results will provide an important contribution against the factors that cause diarrhea

Keywords: cryptosporidium sp., bovine coronavirus, bovine rotavirus, E.coli, calve, ELISA

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Authors: Liza Meutia Sari, Gus Permana Subita, Elza Ibrahim Auerkari

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Background: Many herbs have been discovered to be potential sources of anticancer drugs. Biji Pinang or areca nut (Areca catechu Linn.) has a high content of phenolics and flavonoids, and which is related to antioxidant activity. However, data on its effects on oral squamous cell carcinoma is not available. Objectives: Identification of the cytotoxicity and apoptosis activity in HSC-2 and HSC-3. Methods: The areca nut was extracted by ethanol 96%, MTS assay and apoptosis activity with flow cytometry. Results: The extract of areca nut showed higher toxicity on HSC-3 cell compared to HSC-2. The IC₅₀ of HSC-3 was 164.06 μg/ml vs. 629.50 μg/ml in HSC-2. There was an increase in late apoptosis percentage after 24 and 48 hours in HSC-2. There was a significant increase in early apoptosis percentage after 24 hours and late in 48 hours in HSC-3. Conclusion: The antioxidant activity of the extract of areca nut might be associated with the selective cytotoxicity on HSC-2 and HSC-3. Apoptosis is the major cell death mechanism involved. The areca nut may play an important role in anticancer herb medicine.

Keywords: areca nut, cytotoxicity, apoptosis, oral carcinoma

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Authors: Salmin K. Alshalmani, Nada H. Zobi, Ismaeel H. Bozakouk

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Marine organisms are potentially prolific sources of highly bio active secondary metabolites that might represent useful leads in the development of new pharmaceutical agents. The Libyan marine biodiversity including macroalgae remains partially unexplored in term of their potential bio activities. The phytochemical analysis of the alcoholic extracts of some commonly occurring seaweed Cystoseira compressa, enteromorpha intestinals, corallina, and Ulva lactuca and their evaluated for antibacterial activity by well diffusion assay were studied. Four different solvents namely water, ethanol 99 %, methanol 99 %, and methylated spirit 95 % were used for extraction. The phytochemical analysis revealed the presence of carbohydrates, steroids, tannin & phenols, saponins, proteins, and glycosides. The extracts were subjected for study of antibacterial activity. The zone of inhibition ranged between 8 to 16 mm in aqueous extract and up to 16 mm in methanol extract. The maximum activity (16 mm) was recorded from methanol extract of Ulva lactuca against Staphylococcus aureus and, minimum activity (8mm) recorded by Cystoseira compressa against S. aureus.

Keywords: macroalgae, phytochemicals, antibacterial activity, methanolic extract

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Authors: A. M. Gadanya, B. A. Ahmad, U. Maigatari

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Effect of oral administration of Gadagi tea on superoxide dismutase activity was assessed on twenty (20) male subjects (aged 21-40years). Ten (10) male non Gadagi tea consumers (aged 20-26 years), were used as control. Blood samples were collected from the subjects and analysed for serum superoxide dismutase activity using R&D Enzyme Linked Immunosorbent Assay method (ELISA). The subjects were grouped into four based on age i.e group I (21-25 years), group II (26-30 years), and also based on duration of the tea consumption, i.e group A (5-9 years) , group B (10-14 years), group C (15-19 years) and group D (20-24 years). The subjects in group I (0.12 U mg-l +0.05), group II (0.11 U mg-l +0.01), group III (0.14 U mg-l +0.08) and group IV (0.17 U mg-l +0.11) showed increased activity of serum superoxide dismutase when compared with the control subjects (0.88 U mg-l +0.02) (P<0.05). There was no statistical significant difference in superoxide dismutase activity within the case groups (P<0.05), based on age and duration of consumption of the tea. Thus, Gadagi tea consumption could increase serum superoxide dismutase activity in humans.

Keywords: “Gadagi” tea, Serum, Superoxide dismutase, Humans.

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Authors: Adrian Garrido Sanchis, Richard Pashley

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The MS2 (ATCC15597-B1) virus was used as a surrogate to estimate the inactivation rates for enteric viruses when using a hot air bubble column evaporator (HBCE) system in the treatment of household wastewater. In this study, we have combined MS2 virus surface charging properties with thermal inactivation rates, using an improved double layer plaque assay technique, in order to assess the efficiency of the HBCE process for virus removal in water. When bubbling a continuous flow of dry air, at 200°C, only heats the aqueous solution in the bubble column to about 50°C. Viruses are not inactivated by this solution temperature, as confirmed separately from water bath heating experiments. Hence, the efficiency of the HBCE process for virus removal in water appeared to be caused entirely by collisions between the hot air bubbles and the virus organisms. This new energy efficient treatment for water reuse applications can reduce the thermal energy required to only 25% (about 113.7 kJ/L) of that required for boiling (about 450 kJ/L).

Keywords: MS2 virus inactivation, water reuse, hot bubble column evaporator, water treatment

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Authors: Forough Ataollahi, Sumit Pramanik, Belinda Pingguan-Murphy, Wan Abu Bakar Bin Wan Abas, Noor Azuan Bin Abu Osman

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Endothelium proliferation is an important process in cardiovascular homeostasis and can be regulated by extracellular environment, as cells can actively sense mechanical environment. In this study, we evaluated endothelial cell proliferation on PDMS/alumina (Al2O3) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 5% and 10% Al2O3 at curing temperature 50˚C for 4 h and then characterized by mechanical, structural and morphological analyses. Higher stiffness was found in the composites compared to the pure PDMS substrate. Cell proliferation of the cultured bovine aortic endothelial cells on substrate materials were evaluated via Resazurin assay and 1, 1’-Dioctadecyl-1, 3, 3, 3’, 3’-Tetramethylindocarbocyanine Perchlorate-Acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The results revealed that stiffer substrates promote more endothelial cells proliferation to the less stiff substrates. Therefore, this study firmly hypothesizes that the stiffness elevates endothelial cells proliferation.

Keywords: stiffness, proliferation, bovine aortic endothelial cells, extra cellular matrix, vascular

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Authors: D. Zujur, J. Moret, D. Rodriguez, L. Cruz, J. Lira, L. Gil, E. Dominguez, J. F. Alvarez-Barreto

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In this work, chitosan (CH) has been used to produce a novel coating for Ti6Al4V, the most widely used alloy in orthopedic implants, so as to improve the biological tissue response at the metallic surface. The Ti6Al4V surface was sandblasted with alumina particles and observed by SEM. Chitosan was chemically modified, via crodiimide chemistry, with lactobionic and 4-azidebenzoic acid to make it soluble at physiological pH and photo-crosslinkable, respectively. The reaction was verified by FTIR, NMR, and UV/vis spectroscopy. Ti6Al4V surfaces were coated with solutions of the modified CH and exposed to UV light, causing the polymer crosslinking, and formation of a hydrogel on the surface. The crosslinking reaction was monitored by FTIR at different exposure times. Coating morphology was observed by SEM. The coating´s cytocompatibility was determined in vitro through the culture of rat bone marrow´s mesenchymal stem cells, using an MTT assay. The results show that the developed coating is cytocompatible, easy to apply and could be used for further studies in the encapsulation of bioactive molecules to improve osteogenic potential at the tissue-implant interface.

Keywords: chitosan, photo-crosslinking, Ti6Al4V, bioactive coating, hydrogel

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Authors: Meiling Yu, Nadia Rouatbi, Khuloud T. Al-Jamal

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Background: Treatment of neurological disorders with modern medical and surgical approaches remains difficult. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. The treatment of brain diseases with gene therapy requires the gene-editing tool to be delivered efficiently to the central nervous system. In this study, we explored the efficiency of different delivery routes, namely intravenous (i.v.), intra-cranial (i.c.), and intra-nasal (i.n.), to deliver stable nucleic acid-lipid particles (SNALPs) containing gene-editing tools namely Cas9 mRNA and sgRNA encoding for GFP as a reporter protein. We hypothesise that SNALPs can reach the brain and perform gene-editing to different extents depending on the administration route. Intranasal administration (i.n.) offers an attractive and non-invasive way to access the brain circumventing the blood–brain barrier. Successful delivery of gene-editing tools to the brain offers a great opportunity for therapeutic target validation and nucleic acids therapeutics delivery to improve treatment options for a range of neurodegenerative diseases. In this study, we utilised Rosa26-Cas9 knock-in mice, expressing GFP, to study brain distribution and gene-editing efficiency of SNALPs after i.v.; i.c. and i.n. routes of administration. Methods: Single guide RNA (sgRNA) against GFP has been designed and validated by in vitro nuclease assay. SNALPs were formulated and characterised using dynamic light scattering. The encapsulation efficiency of nucleic acids (NA) was measured by RiboGreen™ assay. SNALPs were incubated in serum to assess their ability to protect NA from degradation. Rosa26-Cas9 knock-in mice were i.v., i.n., or i.c. administered with SNALPs to test in vivo gene-editing (GFP knockout) efficiency. SNALPs were given as three doses of 0.64 mg/kg sgGFP following i.v. and i.n. or a single dose of 0.25 mg/kg sgGFP following i.c.. knockout efficiency was assessed after seven days using Sanger Sequencing and Inference of CRISPR Edits (ICE) analysis. In vivo, the biodistribution of DiR labelled SNALPs (SNALPs-DiR) was assessed at 24h post-administration using IVIS Lumina Series III. Results: Serum-stable SNALPs produced were 130-140 nm in diameter with ~90% nucleic acid loading efficiency. SNALPs could reach and stay in the brain for up to 24h following i.v.; i.n. and i.c. administration. Decreasing GFP expression (around 50% after i.v. and i.c. and 20% following i.n.) was confirmed by optical imaging. Despite the small number of mice used, ICE analysis confirmed GFP knockout in mice brains. Additional studies are currently taking place to increase mice numbers. Conclusion: Results confirmed efficient gene knockout achieved by SNALPs in Rosa26-Cas9 knock-in mice expressing GFP following different routes of administrations in the following order i.v.= i.c.> i.n. Each of the administration routes has its pros and cons. The next stages of the project involve assessing gene-editing efficiency in wild-type mice and replacing GFP as a model target with therapeutic target genes implicated in Motor Neuron Disease pathology.

Keywords: CRISPR, nanoparticles, brain diseases, administration routes

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Authors: Piyaporn Buaklang, Narisa Kengtrong Bordeerat

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The use of nanoparticles is increasing worldwide and there are many nanotech-based daily products available in the market. The toxicity of nanoparticles results from their extremely small size which can be transported easily into the blood stream and other organs. We aimed to study the genotoxicity of two nanoparticles, Titanium dioxide (TiO2-NPs) and Zinc oxide (ZnO-NPs), in TK6 cells by micronucleus assay. The cells were tested at 8, 24, and 48 hours after exposed to 0.10, 0.25, 0.50 and 1.00 µg/mL of TiO2-NPs particles size < 25 nm and < 100 nm and to ZnO-NPs at 1, 10, 50, and 100 µg/mL, particles size < 50 nm and < 100 nm. At 24 hours of incubation transmission electron microscope (TEM) revealed that the nanoparticles TiO2-NPs at 1.00 µg/mL and ZnO-NPs at 10 µg/mL were able to be taken into the cells and induced the production of increasing amount of micronucleus in dose-dependent manner. The effect of the two nanoparticles on chromosome aberration indicated that TiO2-NPs and ZnO-NPs are genotoxic. In addition, the toxicity of TiO2-NPs was found to be 10 times more toxic than ZnO-NPs after 24 hours exposure. Analysis showed that the TiO2-NPs induced formation of micronucleus was both time and dose dependent, whereas the genotoxicity of ZnO-NPs was only dose dependent. In conclusion, TiO2-NPs and ZnO-NPs were able to transport through the cells membrane and directly genotoxic to TK6 cells in dose-dependent manner.

Keywords: nanoparticles, genotoxicity, human lymphoblast cells (TK6), micronucleus

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Authors: Rekia. Cherbi, Mokhtar. Saidi, Mohamed. Yousfi, Zhor. Rahmani

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Lawsonia alba (Henna) is widely used in folkloric medicinal for a treatment of various skin diseases such as Eczema (atopic dermatitis), boils and sores. The aim of the present study is to determine the antioxidant activity, total phenolics, flavonoids, and condensed tannins content of extracts from the seeds of Lawsonia. alba grown in Algeria and selected from three different regions (Adrar, Biskra, and Ouargla). Total phenolics content ranged from 68,42 ± 0,54 to 88,31 ± 0,78mg gallic acid equivalents (GAE)/g dry weight, the flavonoids content varied from 1,13 ± 0,0035 to 1,367 ± 0,002mg quercetin equivalents (Q)/ g dry weight and condensed tannins (14,47 ± 0,138 to 25,50 ± 0,076 mg catechin equivalents (CE)/g dry weight). The antioxidant activities of the extracts were evaluated by DPPH assay. The results showed that all extracts from the seeds of Lawsonia. alba seem to be good trappers of radicals, the IC50 values of the extracts ranged between 0,00826 and 0,01 g/l.

Keywords: antioxidant activity, Lawsonia. alba, phenolic compounds, seeds

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Authors: Pallavi Moudgil, J. S. Bedi, R. S. Aulakh, J. P. S. Gill

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Mycotoxins in milk are of major public health concern. The present study was envisaged with an aim to monitor the occurrence of aflatoxin M1 and ochratoxin A in raw milk samples collected from individual animals from dairy farms located in Punjab (India). A total of 168 raw milk samples were collected and analysed using competitive ELISA kits. Out of these, 9 (5.4%) samples were found positive for aflatoxin M1 with the mean concentration of 0.006-0.13 ng/ml and 2 (1.2%) samples exceeded the established maximum residue limit of 0.05 ng/ml established by the European Union. For ochratoxin A, 2 (0.1%) samples were found positive with the mean concentration of 0.61-0.83 ng/ml with both the samples below the established maximum residue limit of 2 ng/ml. The results showed that the milk of dairy cattle is safe with respect to ochratoxin A contamination but occurrence of aflatoxin M1 above maximum residue limit suggested that feed contaminated with mycotoxins might have been offered to dairy cattle that can pose serious health risks to consumers.

Keywords: Aflatoxin M1, health risks, maximum residue limit, milk, Ochratoxin A

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Authors: A. Gramza-Michałowska, J. Skręty, M. Antczak, J. Kobus-Cisowska, D. Kmiecik, J. Korczak, Kulczyński Bartosz

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Background: Modern consumer highly appreciates the correlation between eating habits and health. Intensified research showed many proofs confirming that food besides its basic nutritional function, possess also significant prophylactic and therapeutic potential. Preventive potential of selected food is commonly used as improvement factor of patients life standard. World Health Organization indicates that diabetes (Diabetes mellitus) and obesity are two of the most common and dangerous diseases. Diet therapy is an element of diabetes education program and a part of healing process, allowing maintaining and remaining the optimal metabolic state of the system. It must be remembered that diabetes treatment should be individualized to each patient. One of highly recommended vegetable for diabetes is asparagus (Asparagus officinalis L.), low calories common plant, growing in European countries. Objective: To propose the technology of unsweetened muesli production with addition of new components, we investigated the effects of selected vegetable addition on antioxidative capacity and consumer’s acceptance of muesli as representative of breakfast product. Methods: Muesli was formulated from a composition of oat flakes, flaxseed, bran, carrots, broccoli and asparagus. Basic composition of muesli was evaluated as content of protein, lipids, fatty acid composition, ash, selected minerals and caloricity. Antioxidant capacity of muesli was evaluated with use radical scavenging methods (DPPH, ABTS), ORAC value and PCL - photochemiluminescence antiradical potential. Proposed muesli as new product was also characterized with sensory analysis, which included color, scent, taste, consistency and overall acceptance of a product. Results: Results showed that addition of freeze-dried asparagus into muesli allowed to lower the fat content and caloricity of a product according to the base product. No significant loss in antioxidant potential was evaluated, also the sensory value of a product was not negative. Conclusion: Designed muesli would be an answer for obese people looking for healthy snack during the daytime. Results showed that product with asparagus addition would be accepted by the consumers and because of its antidiabetic potential could be a n important factor in prevention of diabetes or obesity. Financial support by the UE Project no PO IG 01.01.00.00-061/09

Keywords: muesli, vegetables, asparagus, antioxidant potential, lipids

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Authors: Abubakar Umar Birnin-yauri

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Plastic wastes arising from Polyethylene (PE)-based materials are increasingly becoming environmental problem, this is owed to the fact that these PE waste materials will only decompose over hundreds, or even thousands of years, during which they cause serious environmental problems. In this research, Polymer blends prepared from PE, Cashewnut flour (CNF) and Gum Arabic (GA) were studied in order to assay their biodegradation potentials via composting method. Different sample formulations were made i.e., X1= (70% PE, 25% CNF and 5% GA, X2= (70% PE, 20% CNF and 10% GA), X3= (70% PE, 15% CNF and 15% GA), X4 = (70% PE, 10% CNF and 20% GA) and X5 = (70% PE, 5% CNF and 25% GA) respectively. The results obtained showed that X1 recorded weight loss of 9.89% of its original weight after the first 20 days and 37.45% after 100 day, and X2 lost 12.67 % after the first 20 days and 42.56% after 100day, sample X5 experienced the greatest weight lost in the two methods adopted which are 52.9% and 57.89%. Instrumental analysis such as Fourier Transform Infrared Spectroscopy, Thermogravimetric analysis and Scanning electron microscopy were performed on the polymer blends before and after biodegradation. The study revealed that the biodegradation of the polymer blends is influenced by the contents of both the CNF and GA added into the blends.

Keywords: polyethylene, cashewnut, gum Arabic, biodegradation, blend, environment

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Authors: A. Jamsheera, F. Arjmand, D. K. Mohapatra

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Small molecules binding to specific sites along DNA molecule are considered as potential chemotherapeutic agents. Their role as mediators of key biological functions and their unique intrinsic properties make them particularly attractive therapeutic agents. Keeping in view, novel dipeptide complexes Cu(II)-Val-Pro (1), Zn(II)-Val-Pro (2), Cu(II)-Ala-Pro (3) and Zn(II)-Ala-Pro (4) were synthesized and thoroughly characterized using different spectroscopic techniques including elemental analyses, IR, NMR, ESI–MS and molar conductance measurements. The solution stability study carried out by UV–vis absorption titration over a broad range of pH proved the stability of the complexes in solution. In vitro DNA binding studies of complexes 1–4 carried out employing absorption, fluorescence, circular dichroism and viscometric studies revealed the binding of complexes to DNA via groove binding. UV–vis titrations of 1–4 with mononucleotides of interest viz., 5´-GMP and 5´-TMP were also carried out. The DNA cleavage activity of the complexes 1 and 2 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents and the cleavage mechanism involved a hydrolytic pathway. Furthermore, in vitro antitumor activity of complex 1 was screened against human cancer cell lines of different histological origin.

Keywords: dipeptide Cu(II) and Zn(II) complexes, DNA binding profile, pBR322 DNA cleavage, in vitro anticancer activity

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Authors: E. Carraro, Sa. Bonetta, Si. Bonetta, E. Ceretti, G. C. V. Viola, C. Pignata, S. Levorato, T. Salvatori, S. Vannini, V. Romanazzi, A. Carducci, G. Donzelli, T. Schilirò, A. De Donno, T. Grassi, S. Bonizzoni, A. Bonetti, G. Gilli, U. Gelatti

Abstract:

In 2013, air pollution and particulate matter were classified as carcinogenic to human by the IARC. At present, PM is Europe's most problematic pollutant in terms of harm to health, as reported by European Environmental Agency (EEA) in the EEA Technical Report on Air quality in Europe, 2015. A percentage between 17-30 of the EU urban population lives in areas where the EU air quality 24-hour limit value for PM10 is exceeded. Many studies have found a consistent association between exposure to PM and the incidence and mortality for some chronic diseases (i.e. lung cancer, cardiovascular diseases). Among the mechanisms responsible for these adverse effects, genotoxic damage is of particular concern. Children are a high-risk group in terms of the health effects of air pollution and early exposure during childhood can increase the risk of developing chronic diseases in adulthood. The MAPEC_LIFE (Monitoring Air Pollution Effects on Children for supporting public health policy) is a project founded by EU Life+ Programme (LIFE12 ENV/IT/000614) which intends to evaluate the associations between air pollution and early biological effects in children and to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in children. This work is focused on the micronuclei frequency in child buccal cells in association with airborne PM levels taking into account the influence of other factors associated with the lifestyle of children. The micronucleus test was performed in exfoliated buccal cells of 6–8 years old children from 5 Italian towns with different air pollution levels. Data on air quality during the study period were obtained from the Regional Agency for Environmental Protection. A questionnaire administered to children’s parents was used to obtain details on family socio-economic status, children health condition, exposures to other indoor and outdoor pollutants (i.e. passive smoke) and life-style, with particular reference to eating habits. During the first sampling campaign (winter 2014-15) 1315 children were recruited and sampled for Micronuclei test in buccal cells. In the sampling period the levels of the main pollutants and PM10 were, as expected, higher in the North of Italy (PM10 mean values 62 μg/m3 in Torino and 40 μg/m3 in Brescia) than in the other towns (Pisa, Perugia, Lecce). A higher Micronucleus frequency in buccal cells of children was found in Brescia (0.6/1000 cells) than in the other towns (range 0.3-0.5/1000 cells). The statistical analysis underlines a relation of the micronuclei frequency with PM concentrations, traffic level near child residence, and level of education of parents. The results suggest that, in addition to air pollution exposure, some other factors, related to lifestyle or further exposures, may influence micronucleus frequency and cellular response to air pollutants.

Keywords: air pollution, buccal cells, children, micronucleus cytome assay

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Authors: Maomao Cao

Abstract:

Objective: To test the performance of ctDNA methylation for the detection of liver cancer. Methods: A total of 1233 individuals have been recruited in 2017. 15 male and 15 female samples (including 10 cases of liver cancer) were randomly selected in the present study. CfDNA was extracted by MagPure Circulating DNA Maxi Kit. The concentration of cfDNA was obtained by Qubit™ dsDNA HS Assay Kit. A pre-constructed predictive model was used to analyze methylation data and to give a predictive score for each cfDNA sample. Individuals with a predictive score greater than or equal to 80 were classified as having liver cancer. CT tests were considered the gold standard. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the diagnosis of liver cancer were calculated. Results: 9 patients were diagnosed with liver cancer according to the prediction model (with high sensitivity and threshold of 80 points), with scores of 99.2, 91.9, 96.6, 92.4, 91.3, 92.5, 96.8, 91.1, and 92.2, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of ctDNA methylation for the diagnosis of liver cancer were 0.70, 0.90, 0.78, and 0.86, respectively. Conclusions: ctDNA methylation could be an acceptable diagnostic modality for the detection of liver cancer.

Keywords: liver cancer, ctDNA methylation, detection, diagnostic performance

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Authors: Manal E. A Elhalwagy, Nadia Amin Abdulmajeed, Hanan S. Alnahdi, Enas N. Danial

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Baron is herbicide includes (48% glyphosate) widely used in Egypt. The present study assesses the cytotoxic and genotoxic effect of baron on rats liver. Two groups of rats were treated orally with 1/10 LD 50, (275.49 mg kg -1) and 1/40 LD 50, (68.86 mg kg-1) glyphosate for 28 days compared with control group. Serum and liver tissues were taken at 14 and 28 days of treatment. An inhibition in Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were recorded at both treatment periods and reduction in total serum protein (TP) and albumin (ALB). However, non-significant changes in serum acetylcholinesterase (AChE). Elevation in oxidative stress biomarker malondyaldehyde (MDA) and the decline in detoxification biomarker total reduced glutathione (GSH), Glutathione S-transferase (GST) and superoxide dismutase (SOD) in liver tissues led to increase in percentage of DNA damage. Destruction in liver tissue architecture was observed . Although, Baron was classified in the safe category pesticides repeated exposure to small doses has great danger effect.

Keywords: glyphosate, liver toxicity, oxidative stress, DNA damage, commet assay

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Authors: Abdolreza Esmaeilzadeh, Mehri Mirzaei

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Objective: Rheumatic disease is a chronic disease that causes pain, stiffness, swelling and limited motion and function of many joints. RA is the most common form of autoimmune arthritis, affecting more than 1.3 million Americans. Of these, about 75% are women. Materials and Methods: This study was formed due to the misconception about ANA test, which is frequently performed with methods based upon solid phase as ELISA. This experiment was conducted on 430 patients, with clinical symptoms that are likely affected with rheumatic diseases, simultaneously by means of ANA and FANA. Results: 36 cases (8.37%) of patients, despite positive ANA, have demonstrated negative results via Indirect Immunofluorescence Assay (IIFA), (false positive). 116 cases (27%) have demonstrated negative ANA results, by means of the ELISA technique, although they had positive IIFA results. Conclusion: Other advantages of IIFA are antibody titration and specific pattern detection that have the capability of distinguishing positive dsDNA results. According to the restrictions and false negative cases, in patients, IIFA test is highly recommended for these disease's diagnosis.

Keywords: autoimmune disease, IIFA, EIA, rheumatic disease

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Authors: Prajna Paramita Naik, Sujit Kumar Bhutia

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Background: Regardless of the development multimodal treatment strategies, oral squamous cell carcinoma (OSCC) is often associated with a high rate of recurrence, metastasis and chemo- and radio- resistance. The present study inspected the relevance of CD44, ABCB1 and ADAM17 expression as a putative stem cell compartment in oral squamous cell carcinoma (OSCC) and deciphered the role of autophagy in regulating the expression of aforementioned proteins, stemness and chemoresistance. Methods: A retrospective analysis of CD44, ABCB1 and ADAM17 expression with respect to the various clinicopathological factors of sixty OSCC patients were determined via immunohistochemistry. The correlation among CD44, ABCB1 and ADAM17 expression was established. Sphere formation assay, flow cytometry and fluorescence microscopy were conducted to elucidate the stemness and chemoresistance nature of established cisplatin-resistant oral cancer cells (FaDu). The pattern of expression of CD44, ABCB1 and ADAM17 in parental (FaDu-P) and resistant FaDu cells (FaDu-CDDP-R) were investigated through fluorescence microscopy. Western blot analysis of autophagy marker proteins was performed to compare the status of autophagy in parental and resistant FaDu cell. To investigate the role of autophagy in chemoresistance and stemness, sphere formation assay, immunofluorescence and Western blot analysis was performed post transfection with siATG14 and the level of expression of autophagic proteins, mitochondrial protein and stemness-associated proteins were analyzed. The statistical analysis was performed by GraphPad Prism 4.0 software. p-value was defined as follows: not significant (n.s.): p > 0.05;*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001; ****: p ≤ 0.0001 were considered statistically significant. Results: In OSCC, high CD44, ABCB1 and ADAM17 expression were significantly correlated with higher tumor grades and poor differentiation. However, the expression of these proteins was not related to the age and sex of OSCC patients. Moreover, the expression of CD44, ABCB1 and ADAM17 were positively correlated with each other. In vitro and OSCC tissue double labeling experiment data showed that CD44+ cells were highly associated with ABCB1 and ADAM17 expression. Further, FaDu-CDDP-R cells showed higher sphere forming capacity along with increased fraction of the CD44+ population and β-catenin expression FaDu-CDDP-R cells also showed accelerated expression of CD44, ABCB1 and ADAM17. A comparatively higher autophagic flux was observed in FaDu-CDDP-R against FaDu-P cells. The expression of mitochondrial proteins was noticeably reduced in resistant cells as compared to parental cells indicating the occurrence of autophagy-mediated mitochondrial degradation in oral cancer. Moreover, inhibition of autophagy was coupled with the decreased formation of orospheres suggesting autophagy-mediated stemness in oral cancer. Blockade of autophagy was also found to induce the restoration of mitochondrial proteins in FaDu-CDDP-R cells indicating the involvement of mitophagy in chemoresistance. Furthermore, a reduced expression of CD44, ABCB1 and ADAM17 was also observed in ATG14 deficient cells FaDu-P and FaDu-CDDP-R cells. Conclusion: The CD44+ ⁄ABCB1+ ⁄ADAM17+ expression in OSCC might be associated with chemoresistance and a putative CSC compartment. Further, the present study highlights the contribution of mitophagy in chemoresistance and confirms the potential involvement of autophagic regulation in acquisition of stem-like characteristics in OSCC.

Keywords: ABCB1, ADAM17, autophagy, CD44, chemoresistance, mitophagy, OSCC, stemness

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Authors: Josselyn Mata Calidonio, Arianna I. Maddox, Kimberly Hamad-Schifferli

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Rapid diagnostics are critical infectious disease tools that are designed to detect a known biomarker using antibodies specific to that biomarker. However, a way to detect unknown viruses has not yet been achieved in a paper test format. We describe here a route to make an adaptable paper immunoassay that can detect an unknown biomarker, demonstrating it on SARS-CoV-2 variants. The immunoassay repurposes cross-reactive antibodies raised against the alpha variant. Gold nanoparticles of two different colors conjugated to two different antibodies create a colorimetric signal, and machine learning of the resulting colorimetric pattern is used to train the assay to discriminate between variants of alpha and Omicron BA.5. By using principal component analysis, the colorimetric test patterns can pick up and discriminate an unknown that it has not encountered before, Omicron BA.1. The test has an accuracy of 100% and a potential calculated discriminatory power of 900. We show that it can be used adaptively and that it can be used to pick up emerging variants without the need to raise new antibodies.

Keywords: adaptive immunoassay, detecting unknown viruses, gold nanoparticles, paper immunoassay, repurposing antibodies

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Authors: Abdelsabour G. A. Khaled, Ahmed W. A. Abdalla And Sabry Y. M. Mahmoud

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Banana plants showing typical mosaic and yellow stripes on leaves as symptoms were collected from Assiut Governorate in Egypt. The causal agent was identified as Cucumber mosaic virus (CMV) on the basis of symptoms, transmission, serology, transmission electron microscopy and reverse transcription polymerase chain reaction (RT-PCR). Coat protein (CP) gene was amplified using gene specific primers for coat protein (CP), followed by cloning into desired cloning vector for sequencing. In this study the CMV was transmitted into propagation host either by aphid or mechanically. The transmission was confirmed through Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA). Analysis of the 120 deduced amino acid sequence of the coat protein gene revealed that the EG-A strain of CMV shared from 97.50 to 98.33% with those strains belonging to subgroup IA. The cluster analysis grouped the Egyptian isolate with strains Fny and Ri8 belonging sub-group IA. It appears that there occurs a high incidence of CMV infecting banana belonging to IA subgroup in most parts of Egypt.

Keywords: banana, CMV, transmission, CP gene, RT-PCR

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Authors: Chijindu Okpalaoka, Charles Chinedu Onuselogu

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COVID-19, humanity's coronavirus disease caused by SARS-CoV-2, was first detected in late 2019 in Wuhan, China. COVID-19 lacked an established conventional pharmaceutical therapy, and as a result, the outbreak quickly became an epidemic affecting the entire World. Only a qPCR assay is reliable for diagnosing COVID-19. Clustered, regularly interspaced short palindromic repeats (CRISPR) technology is being researched for speedy and specific identification of COVID-19, among other therapeutic techniques. Apart from its therapeutic capabilities, the CRISPR technique is being evaluated to develop antiviral therapies; nevertheless, no CRISPR-based medication has been approved for human use to date. Prophylactic antiviral CRISPR in living being cells, a Cas 13-based approach against coronavirus, has been developed. While this method can be evolved into a treatment approach, it may face substantial obstacles in human clinical trials for licensure. This study discussed the potential applications of CRISPR-based techniques for developing a speedy and accurate feasible treatment alternative for the COVID-19 virus.

Keywords: COVID-19, CRISPR technique, Cas13, SARS-CoV-2, prophylactic antiviral

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Authors: S. N. T. I. Sampath, J. M. S. Jayasinghe, A. P. Attanayake, V. Karunaratne

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Diabetes mellitus is one of the major public health posers throughout the world today that incidence and associated with increasing mortality. Insufficient regulation of the blood glucose level might be serious effects for health and its necessity to identify new therapeutics that have ability to reduce hyperglycaemic condition in the human body. Even though synthetic antidiabetic drugs are more effective to control diabetes mellitus, there are considerable side effects have been reported. Thus, there is an increasing demand for searching new natural products having high antidiabetic activity with lesser side effects. The purposes of the present study were to evaluate different physical parameters and in-vitro and in-vivo antidiabetic potential of the selected combined medicinal plant extracts mixture composed of leaves of Murraya koenigii, cloves of Allium sativum, fruits of Garcinia queasita and seeds of Piper nigrum. The selected plants parts were mixed and ground together and extracted sequentially into the hexane, ethyl acetate and methanol. Solvents were evaporated and they were further dried by freeze-drying to obtain a fine powder of each extract. Various physical parameters such as moisture, total ash, acid insoluble ash and water soluble ash were evaluated using standard test procedures. In-vitro antidiabetic activity of combined plant extracts mixture was screened using enzyme assays such as α-amylase inhibition assay and α-glucosidase inhibition assay. The acute anti-hyperglycaemic activity was performed using oral glucose tolerance test for the streptozotocin induced diabetic Wistar rats to find out in-vivo antidiabetic activity of combined plant extracts mixture and it was assessed through total oral glucose tolerance curve (TAUC) values. The percentage of moisture content, total ash content, acid insoluble ash content and water soluble ash content were ranged of 7.6-17.8, 8.1-11.78, 0.019-0.134 and 6.2-9.2 respectively for the plant extracts and those values were less than standard values except the methanol extract. The hexane and ethyl acetate extracts exhibited highest α-amylase (IC50 = 25.7 ±0.6; 27.1 ±1.2 ppm) and α-glucosidase (IC50 = 22.4 ±0.1; 33.7 ±0.2 ppm) inhibitory activities than methanol extract (IC50 = 360.2 ±0.6; 179.6 ±0.9 ppm) when compared with the acarbose positive control (IC50 = 5.7 ±0.4; 17.1 ±0.6 ppm). The TAUC values for hexane, ethyl acetate, and methanol extracts and glibenclamide (positive control) treated rats were 8.01 ±0.66; 8.05 ±1.07; 8.40±0.50; 5.87 ±0.93 mmol/L.h respectively, whereas in diabetic control rats the TAUC value was 13.22 ±1.07 mmol/L.h. Administration of plant extracts treated rats significantly suppressed (p<0.05) the rise in plasma blood glucose levels compared to control rats but less significant than glibenclamide. The obtained results from in-vivo and in-vitro antidiabetic study showed that the hexane and ethyl acetate extracts of selected combined plant mixture might be considered as a potential source to isolate natural antidiabetic agents and physical parameters of hexane and ethyl acetate extracts will helpful to develop antidiabetic drug with further standardize properties.

Keywords: diabetes mellitus, in-vitro antidiabetic assays, medicinal plants, standardization

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Authors: Reza Sadeghi

Abstract:

In this study, microwave heating was employed for controlling Oryzaephilus mercator. adults infesting stored Iranian dried fig. For this purpose, the dried fig samples were artificially infested with O. mercator and then heated in a microwave oven (2450 MHz) at the power outputs of 450, 720, and 900 W for 10, 20, 30, and 40 s, respectively. Subsequently, changes in the colors of the product samples under the effects of the varied microwave applications were investigated in terms of lightness (ΔL*), redness (Δa*), and yellowness (Δb*) using an image processing technique. The results revealed that both parameters of microwave power and exposure time had significant impacts on the pest mortality rates (p<0.01). In fact, a direct positive relationship was obtained between the mortality rate and microwave irradiation power. Complete mortality was achieved for the pest at the power of 900 W and exposure time of 40 s. The dried fig samples experienced fewer changes in their color parameters. Considering the successful pest control and acceptable changes in the product quality, microwave irradiation can be introduced as an appropriate alternative to chemical fumigants.

Keywords: colorimetric assay, microwave heating, Oryzaephilus mercator, mortality

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