Search results for: in-vitro antidiabetic assays

Authors: Rutanachai Thaipratum

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Free radicals are atoms or molecules with unpaired electrons. Many diseases are caused by free radicals. Normally, free radical formation is controlled naturally by various beneficial compounds known as antioxidants. Several analytical methods have been used for qualitative and quantitative determination of antioxidants, and each has its own specificity. This project aimed to evaluate antioxidant activity of ethanolic and aqueous extracts from the rice paddy herb (Limnophila aromatica (Lam.) Merr.) measured by DPPH and Hydroxyl radical scavenging method. The results showed that averaged antioxidant activity measured in ethanolic extract (µmol Ascorbic acid equivalent/g fresh mass) were 67.09± 4.99 and 15.55±4.82 as determined by DPPH and Hydroxyl radical scavenging activity assays, respectively. Averaged antioxidant activity measured in aqueous extract (µmol Ascorbic acid equivalent/g fresh mass) were 21.08±1.25 and 10.14±3.94 as determined by DPPH and Hydroxyl radical scavenging activity assays respectively.

Keywords: free radical, antioxidant, rice paddy herb, Limnophila aromatica (Lam.) Merr.

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Authors: Stela Papa, Klementina Puto, Migena Pllaha

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HCV is a hepatotropic RNA virus, transmitted primarily via the blood route, which causes progressive disease such as chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma. HCV nowadays is a global healthcare problem. A variety of immunoassays including old and new technologies are being applied to detect HCV in our country. These methods include Immunochromatography assays (ICA), Fluorescence immunoassay (FIA), Enzyme linked fluorescent assay (ELFA), and Enzyme linked immunosorbent assay (ELISA) to detect HCV antibodies in blood serum, which lately is being slowly replaced by more sensitive methods such as rapid automated analyzer chemiluminescence immunoassay (CLIA). The aim of this study is to estimate HCV infection in carriers and chronic acute patients and to evaluate the use of new diagnostic methods. This study was realized from September 2016 to May 2018. During this study period, 2913 patients were analyzed for the presence of HCV by taking samples from their blood serum. The immunoassays performed were ICA, FIA, ELFA, ELISA, and CLIA assays. Concluding, 82% of patients taken in this study, resulted infected with HCV. Diagnostic methods in clinical laboratories are crucial in the early stages of infection, in the management of chronic hepatitis and in the treatment of patients during their disease.

Keywords: CLIA, ELISA, Hepatitis C virus, immunoassay

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Authors: Ei Ei Hlaing, Narissara Lailerd, Sittiruk Roytrakul, Pichapat Piamrojanaphat

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Type 2 diabetes is one of the most common metabolic diseases all over the world. The pathogenesis of type 2 diabetes is not the only dysfunction of pancreatic beta cells but also insulin resistance in muscle, liver and adipose tissue. High levels of circulating free fatty acids, an increased lipid content of muscle cells, impaired insulin-mediated glucose uptake and diminished mitochondrial functioning are pathophysiological hallmarks of diabetic skeletal muscles. Purple rice (Oryza sativa L. indica) has been shown to have antidiabetic effects. However, the underlying mechanism(s) of antidiabetic activity of purple rice is still unraveled. In this research, to explore in-depth cellular mechanism(s), proteomic profile of purple rice supplemented type 2 diabetic rats’ skeletal muscle were analyzed contract with non-supplemented rats. Diabetic rats were induced high-fat diet combined with streptozotocin injection. By using one- dimensional gel electrophoresis (1-DE) and LC-MS/MS quantitative proteomic method, we analyzed proteomic profiles in skeletal muscle of normal rats, normal rats with purple rice supplementation, type 2 diabetic rats, and type 2 diabetic rats with purple rice supplementation. Total 2676 polypeptide expressions were identified. Among them, 24 peptides were only expressed in type 2 diabetic rats, and 24 peptides were unique peptides in type 2 diabetic rats with purple rice supplementation. Acetyl CoA carboxylase 1 (ACACA) found as unique protein in type 2 diabetic rats which is the major enzyme in lipid synthesis and metabolism. Interestingly, DNA damage response protein, heterogeneous nuclear ribonucleoprotein K [Mus musculus] (Hnrnpk), was upregulated in type 2 diabetic rats’ skeletal muscle. Meanwhile, unique proteins of type 2 diabetic rats with purple rice supplementation (bone morphogenetic 7 protein preproprotein, BMP7; and forkhead box protein NX4, Foxn4) involved with muscle cells growth through the regulation of TGF-β/Smad signaling network. Moreover, BMP7 may effect on insulin signaling through the downstream signaling of protein kinase B (Akt) which acts in protein synthesis, glucose uptake, and glycogen synthesis. In conclusion, our study supports that type 2 diabetes impairs muscular lipid metabolism. In addition, purple rice might recover the muscle cells growth and insulin signaling.

Keywords: proteomics, purple rice bran, skeletal muscle, type 2 diabetic rats

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Authors: M. Shemis, O. Maher, G. Casterou, F. Gauffre

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Hepatitis C virus (HCV) is a major cause of chronic liver disease with a global 170 million chronic carriers at risk of developing liver cirrhosis and/or liver cancer. Egypt reports the highest prevalence of HCV worldwide. Currently, two classes of assays are used in the diagnosis and management of HCV infection. Despite the high sensitivity and specificity of the available diagnostic assays, they are time-consuming, labor-intensive, expensive, and require specialized equipment and highly qualified personal. It is therefore important for clinical and economic terms to develop a low-tech assay for the direct detection of HCV RNA with acceptable sensitivity and specificity, short turnaround time, and cost-effectiveness. Such an assay would be critical to control HCV in developing countries with limited resources and high infection rates, such as Egypt. The unique optical and physical properties of gold nanoparticles (AuNPs) have allowed the use of these nanoparticles in developing simple and rapid colorimetric assays for clinical diagnosis offering higher sensitivity and specificity than current detection techniques. The current research aims to develop a detection assay for HCV RNA using gold nanoparticles (AuNPs). Methods: 200 anti-HCV positive samples and 50 anti-HCV negative plasma samples were collected from Egyptian patients. HCV viral load was quantified using m2000rt (Abbott Molecular Inc., Des Plaines, IL). HCV genotypes were determined using multiplex nested RT- PCR. The assay is based on the aggregation of AuNPs in presence of the target RNA. Aggregation of AuNPs causes a color shift from red to blue. AuNPs were synthesized using citrate reduction method. Different sets of probes within the 5’ UTR conserved region of the HCV genome were designed, grafted on AuNPs and optimized for the efficient detection of HCV RNA. Results: The nano-gold assay could colorimetrically detect HCV RNA down to 125 IU/ml with sensitivity and specificity of 91.1% and 93.8% respectively. The turnaround time of the assay is < 30 min. Conclusions: The assay allows sensitive and rapid detection of HCV RNA and represents an inexpensive and simple point-of-care assay for resource-limited settings.

Keywords: HCV, gold nanoparticles, point of care, viral load

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Authors: Dana A. Thal, Heike Kahlert, Fritz Scholz

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Thiol molecules are known to easily form self-assembled monolayers (SAM) on Au surfaces. Depending on the thiol’s structure, surface modifications via SAM can be used for electrode sensor development. In the presented work, 1-decanethiol coated polycrystalline Au electrodes were applied to indirectly assess the radical scavenging potential of plant compounds and extracts. Different plant compounds with reported antioxidant properties as well as an extract from the plant Gynostemma pentaphyllum were tested for their effectiveness to prevent SAM degradation on the sensor electrodes via photolytically generated radicals in aqueous media. The SAM degradation was monitored over time by differential pulse voltammetry (DPV) measurements. The results were compared to established antioxidant assays. The obtained data showed an exposure time and concentration dependent degradation process of the SAM at the electrode’s surfaces. The tested substances differed in their capacity to prevent SAM degradation. Calculated radical scavenging activities of the tested plant compounds were different for different assays. The presented method poses a simple system for radical scavenging evaluation and, considering the importance of the test system in antioxidant activity evaluation, might be taken as a bridging tool between in-vivo and in-vitro antioxidant assay in order to obtain more biologically relevant results in antioxidant research.

Keywords: alkanethiol SAM, plant antioxidant, polycrystalline Au, radical scavenger

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Authors: Ana M. Guzman, Claudia M. Rodriguez, Pedro F. B. Brandao, Elianna Castillo

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Cadmium (Cd) is a carcinogenic metal to which humans are exposed mainly due to its presence in the food chain. Lactic acid bacteria have the capability to bind cadmium and thus the potential to be used as probiotics to treat this metal toxicity in the human body. The main objective of this study is to evaluate the potential of native lactic acid bacteria, isolated from Colombian fermented cocoa, to remove cadmium from aqueous solutions. An initial screening was made with the Lactobacillus plantarum JCM 1055 type strain, and Cd was quantified by atomic absorption spectroscopy (AAS). Lb. plantarum JCM 1055 was grown in ½ MRS medium to follow growth kinetics during 32 h at 37 °C, by measuring optical density at 600 nm. Washed cells, grown for 18 h, were adjusted to obtain dry biomass concentrations of 1.5 g/L and 0.5 g/L for removal assays in 10 mL of Cd(NO₃)₂ solution with final concentrations of 10 mg/Kg or 1.0 mg/Kg. The assays were performed at two different pH values (2.0 and 5.0), and results showed better adsorption abilities at higher pH. After incubation for 1 h at 37 °C and 150 rpm, the removal percentages for 10 mg/Kg Cd with 1.5 g/L and 0.5 g/L biomass concentration at pH 5.0 were, respectively, 71% and 50%, while the efficiency was 9.15 and 4.52 mg Cd/g dry biomass, respectively. For the assay with 1.0 mg/Kg Cd at pH 5.0, the removal was 100% and 98%, respectively for the same biomass concentrations, and the efficiency was 1.63 and 0.56 mg Cd/g dry biomass, respectively. These results suggest the efficiency of Lactobacillus strains to remove cadmium and their potential to be used as probiotics to treat cadmium toxicity and reduce its accumulation in the human body.

Keywords: cadmium removal, fermented cocoa, lactic acid bacteria, probiotics

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Authors: Ghebremedhin Tsegay, Weldu Tesfagaber, Yuanmao Zhu, Xijun He, Wan Wang, Zhenjiang Zhang, Encheng Sun, Jinya Zhang, Yuntao Guan, Fang Li, Renqiang Liu, Zhigao Bu, Dongming Zhao*

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African swine fever (ASF) is a highly infectious viral disease of pigs, resulting in significant economic loss worldwide. As there is no approved vaccines and treatments, the control of ASF entirely depends on early diagnosis and culling of infected pigs. Thus, highly specific and sensitive diagnostic assays are required for accurate and early diagnosis of ASF virus (ASFV). Currently, only a few recombinant proteins have been tested and validated for use as reagents in ASF diagnostic assays. The most promising ones for ASFV antibody detection were p72, p30, p54, and pp62. So far, three ELISA kits based on these recombinant proteins have been commercialized. Due to the complex nature of the virus and variety forms of the disease, robust serodiagnostic assays are still required. ASFV p22 protein, encoded by KP177R gene, is located in the inner membrane of viral particle and appeared transiently in the plasma membrane early after virus infection. The p22 protein interacts with numerous cellular proteins, involved in processes of phagocytosis and endocytosis through different cellular pathways. However, p22 does not seem to be involved in virus replication or swine pathogenicity. In this study, E.coli expressed recombinant p22 protein was used to generate a monoclonal antibody (mAb), and its potential use for the development of blocking ELISA (bELISA) was evaluated. A total of 806 pig serum samples were tested to evaluate the bELISA. Acording the ROC (Reciever operating chracteristic) analysis, 100% sensitivity and 98.10% of specificity was recorded when the PI cut-off value was set at 47%. The novel assay was able to detect the antibodies as early as 9 days post infection. Finaly, a highly sensitive, specific and rapid novel p22-mAb based bELISA assay was developed, and optimized for detection of antibodies against genotype I and II ASFVs. It is a promising candidate for an early and acurate detection of the antibodies and is highly expected to have a valuable role in the containment and prevention of ASF.

Keywords: ASFV, blocking ELISA, diagnosis, monoclonal antibodies, sensitivity, specificity

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Authors: Paam Bidaya

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The red seaweed Gracilaria fisheri, widely distributed along Thailand's southern coastlines, has been discovered to be edible. Sulfated polysaccharides from G. fisheri were extracted in low-temperature (25 °C) water. Seaweed polysaccharides (SPs) have been shown to have various advantageous biological effects. This study aims to investigate total phenolic content and antioxidant capacity of G. fisheri extract. The total phenolic content of G. fisheri extract was determined using Folin-Cioucalteu method and calculated as gallic acid equivalents (GAE). The antioxidant activity of G. fisheri extract was performed via 2, 2-diphenyl-1- picrylhydrazyl (DPPH) free radical scavenging assay and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity assays. The findings exhibited a strong correlation between antioxidant activity and the total phenol contents. In addition, DPPH and ABTS assays showed that G. fisheri extract showed antioxidant activities as a concentration-dependent manner. The IC50 values of G. fisheri extract were 902.19 μg/mL ± 0.785 and 727.98 μg/mL ± 0.822 for DPPH and ABTS, respectively. Vitamin C was used as a positive control in DPPH assay, while Trolox was used as a positive control in ABTS assay. To conclude, G. fisheri extract consists of a high amount of total phenolic content, which exhibit a significant antioxidant activity. However, further investigation regarding antioxidant activity should be performed in order to identify the mechanism of Gracilaria fisheri action.

Keywords: ABTS assay, DPPH assay, sulfated polysaccharides, total phenolic content

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Authors: Ewa Flaczyk, Monika Przeor, Joanna Kobus-Cisowska, Józef Korczak

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The aim of this paper was to collect the information concerning the most popular raw plant materials of antidiabetic activity, in a context of functional food developing production. The elaboration discusses morphological elements possible for an application in functional food production of the plants such as: common bean, ginger, Ceylon cinnamon, white mulberry, fenugreek, French lilac, ginseng, jambolão, and bitter melon. An activity of bioactive substances contained in these raw plant materials was presented, pointing their antiglycemic and also hypocholesterolemic, antiarthritic, antirheumatic, antibacterial, and antiviral activity in the studies on humans and animals. Also the genesis of functional food definition was presented.

Keywords: antiglycemic activity, raw plant materials, functional food, food, nutritional sciences

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Authors: Aruma Baduge Kithma Hansanee De Silva

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Haskap (Lonicera caerulea L.), also known as blue honeysuckle, is a recently commercialized berry crop in Canada. Haskap berries are rich in polyphenols, including anthocyanins, which are known for potential health-promoting effects. Cyanidin-3-O-glucoside (C3G) is the most prominent anthocyanin of haskap berries. Recent literature reveals the efficacy of C3G in reducing the risk of type 2 diabetes (T2D), which has become an increasingly common health issue around the world. The T2D is characterized as a metabolic disorder of hyperglycemia and insulin resistance. It has been demonstrated that C3G has anti-diabetic effects in various ways, including improvement in insulin sensitivity, and inhibition of activities of carbohydrate-hydrolyzing enzymes, including alpha-amylase and alpha-glucosidase. The goal of this study was to investigate the influence of variety and harvesting date on haskap composition, biological properties, and antidiabetic properties. The polyphenolic compounds present in four commercially grown haskap cultivars, Aurora, Rebecca, Larissa and Evie among five harvesting stages (H1-H5), were extracted separately in 80% ethanol and analyzed to characterize their phenolic profiles. The haskap berries contain different types of polyphenols including flavonoids and phenolic acids. Anthocyanin is the major type of flavonoid. C3G is the most prominent type of anthocyanin, which accounts for 79% of total anthocyanin in all extracts. The variety Larissa at H5 contained the highest average C3G content, and its ethanol extract had the highest (1212.3±63.9 mg/100g FW) while, Evie at H1 contained the lowest C3G content (96.9±40.4 mg/100g FW). The average C3G content of Larissa from H1 – H5 varies from 208 – 1212 mg/100g FW. Quarcetin-3-Rutinoside (Q3Rut) is the major type of flavonol and highest is observed in Rebecca at H4 (47.81 mg/100g FW). The haskap berries also contained phenolic acids, but approximately 95% of the phenolic acids consisted of chlorogenic acid. The cultivar Larissa has a higher level of anthocyanin than the other four cultivars. The highest total phenolic content is observed in Evie at H5 (2.97±1.03 mg/g DW) while the lowest in Rebecca at H1 (1.47±0.96 mg/g DW). The antioxidant capacity of Evie at H5 was higher (14.40±2.21 µmol TE/ g DW) among other cultivars and the lowest observed in Aurora at H3 (5.69±0.34 µmol TE/ g DW). Furthermore, Larissa H5 shows the greatest inhibition of carbohydrate-hydrolyzing enzymes including alpha-glucosidase and alpha-amylase. In conclusion Larissa, at H5 demonstrated highest polyphenol composition and antidiabetic properties.

Keywords: anthocyanin, cyanidin-3-O-glucoside, haskap, type 2 diabetes

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Authors: J. O. Ezekwesili-Ofili, L. I. Okorafor, S. C. Nsofor

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Diabetes mellitus is a carbohydrate metabolism disorder due to an absolute or relative deficiency of insulin secretion, action or both. Many known hypoglycaemic drugs are known to produce serious side effects. However, the search for safer and more effective agents has shifted to plant products, including foods and spices. One of such is the rhizome of Curcuma longa or turmeric, which is a spice with high medicinal value. A drawback in the use of C. longa is the poor bioavailability of curcumin, the active ingredient. It has been reported that piperine, an alkaloid present in peppers increases the bioavailability of curcumin. This work therefore investigated the hypoglycaemic and antioxidant effects of ethanolic extract of C. longa rhizomes, alone and with two pepper adjuvants in alloxan-induced diabetic rats. A total of 48 rats were divided into 6 groups of 8 rats each. Groups A–E were induced with diabetes using 150mg/kg body weight of alloxan monohydrate, while group F was normoglycaemic: Group A: Diabetic; fed with 400 mg/g body weight of turmeric extract; group B: Diabetic, fed with 400 mg/kg b. w. and 200mg/kg b. w of ethanolic extract of seeds of Piper guinensee; group C: Diabetic, fed with 400 mg/kg b. w. and 200 mg /kg b. w. of ethanolic extract of seeds of Capsicum annum var cameroun, group D: Diabetic, treated with standard drug, glibenclamide (0.3mg/kg body weight), group E: Diabetic; no treatment i.e. Positive control and group F: non diabetic, no treatment i.e. Negative control. Blood glucose levels were monitored for 14 days using a glucometer. The levels of the antioxidant enzymes; glutathione peroxidase, catalase and superoxide dismutase were also assayed in serum. The ethanolic extracts of C. longa rhizomes at the dose given (400 mg/kg b. w) significantly reduced the blood glucose levels of the diabetic rats (p<0.05) comparable to the standard drug. Co administration of extract of the peppers did not significantly increase the efficiency of the extract, although C. annum var cameroun showed greater effect, though not significantly. The antioxidant effect of the extract was significant in diabetic rats. The use of piperine-containing peppers enhanced the antioxidant effect. Phytochemical analyses of the ethanolic extract of C. longa showed the presence of alkaloids, flavonoids, steroids, saponins, tannins, glycosides, and terpenoids. These results suggest that the ethanolic extract of C. longa had antidiabetic with antioxidant effects and could thus be of benefit in the treatment and management of diabetes as well as ameliorate pro-oxidant effects that may lead to diabetic complications. However, while the addition of piperine did not affect the antidiabetic effect of C. longa, the antioxidant effect was greatly enhanced.

Keywords: antioxidant, Curcuma longa rhizome, hypoglycaemic, pepper adjuvants, piperine

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Authors: Akanksha Bhatnagar, Sandhya Kortegare, Felice Elefant

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Context: Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by progressive cognitive decline and memory loss. The cause of AD is not fully understood, but it is thought to be caused by a combination of genetic, environmental, and lifestyle factors. One of the hallmarks of AD is the loss of neurons in the hippocampus, a brain region that is important for memory and learning. This loss of neurons is thought to be caused by a decrease in histone acetylation, which is a process that regulates gene expression. Research Aim: The research aim of the study was to develop mall molecule compounds that can enhance the activity of Tip60, a histone acetyltransferase that is important for memory and learning. Methodology/Analysis: The researchers used in silico structural modeling and a pharmacophore-based virtual screening approach to design and synthesize small molecule compounds strongly predicted to target and enhance Tip60’s HAT activity. The compounds were then tested in vitro and in vivo to assess their ability to enhance Tip60 activity and rescue cognitive deficits in AD models. Findings: The researchers found that several of the compounds were able to enhance Tip60 activity and rescue cognitive deficits in AD models. The compounds were also developed to cross the blood-brain barrier, which is an important factor for the development of potential AD therapeutics. Theoretical Importance: The findings of this study suggest that Tip60 HAT activators have the potential to be developed as therapeutic agents for AD. The compounds are specific to Tip60, which suggests that they may have fewer side effects than other HDAC inhibitors. Additionally, the compounds are able to cross the blood-brain barrier, which is a major hurdle for the development of AD therapeutics. Data Collection: The study collected data from a variety of sources, including in vitro assays and animal models. The in vitro assays assessed the ability of compounds to enhance Tip60 activity using histone acetyltransferase (HAT) enzyme assays and chromatin immunoprecipitation assays. Animal models were used to assess the ability of the compounds to rescue cognitive deficits in AD models using a variety of behavioral tests, including locomotor ability, sensory learning, and recognition tasks. The human clinical trials will be used to assess the safety and efficacy of the compounds in humans. Questions: The question addressed by this study was whether Tip60 HAT activators could be developed as therapeutic agents for AD. Conclusions: The findings of this study suggest that Tip60 HAT activators have the potential to be developed as therapeutic agents for AD. The compounds are specific to Tip60, which suggests that they may have fewer side effects than other HDAC inhibitors. Additionally, the compounds are able to cross the blood-brain barrier, which is a major hurdle for the development of AD therapeutics. Further research is needed to confirm the safety and efficacy of these compounds in humans.

Keywords: Alzheimer's disease, cognition, neuroepigenetics, drug discovery

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Authors: Jillian M. Hagel, Joseph E. Tucker, Peter J. Facchini

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With the aim of establishing a holistic approach for the treatment of central nervous system (CNS) disorders, we are pursuing a drug development program rapidly progressing through discovery and characterization phases. The drug candidates identified in this program are referred to as neuroplastogens owing to their ability to mediate neuroplasticity, which can be beneficial to patients suffering from anxiety, depression, or posttraumatic stress disorder. These and other related neuropsychiatric conditions are associated with the onset of neuronal atrophy, which is defined as a reduction in the number and/or productivity of neurons. The stimulation of neuroplasticity results in an increase in the connectivity between neurons and promotes the restoration of healthy brain function. We have synthesized a substantial catalogue of proprietary indolethylamine derivatives based on the general structures of serotonin (5-hydroxytryptamine) and psychedelic molecules such as N,N-dimethyltryptamine (DMT) and psilocin (4-hydroxy-DMT) that function as neuroplastogens. A primary objective in our screening protocol is the identification of derivatives associated with a significant reduction in hallucination, which will allow administration of the drug at a dose that induces neuroplasticity and triggers other efficacious outcomes in the treatment of targeted CNS disorders but which does not cause a psychedelic response in the patient. Both neuroplasticity and hallucination are associated with engagement of the 5HT2A receptor, requiring drug candidates differentially coupled to these two outcomes at a molecular level. We use novel and proprietary artificial intelligence algorithms to predict the mode of binding to the 5HT2A receptor, which has been shown to correlate with the hallucinogenic response. Hallucination is tested using the mouse head-twitch response model, whereas mouse marble-burying and sucrose preference assays are used to evaluate anxiolytic and anti-depressive potential. Neuroplasticity is assays using dendritic outgrowth assays and cell-based ELISA analysis. Pharmacokinetics and additional receptor-binding analyses also contribute the selection of lead candidates. A summary of the program is presented.

Keywords: neuroplastogen, non-hallucinogenic, drug development, anxiety, depression, PTSD, indolethylamine derivatives, psychedelic-inspired, 5-HT2A receptor, computational chemistry, head-twitch response behavioural model, neurite outgrowth assay

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Authors: Ginpreet Kaur, Mohammad Yasir Usmani, Mohammed Kamil Khan

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Diabetes mellitus is a disease with a high global burden and results in significant morbidity and mortality. In India, the number of people suffering with diabetes is expected to rise from 19 to 57 million in 2025. At present, interest in herbal remedies is growing to reduce the side effects associated with conventional dosage form like oral hypoglycemic agents and insulin for the treatment of diabetes mellitus. Our aim was to investigate the antidiabetic activities of combinatorial extract of N. sativa & C. cassia in Streptozotocin induced type-I Diabetic Rats. Thus, the present study was undertaken to screen postprandial glucose excursion potential through α- glucosidase inhibitory activity (In Vitro) and effect of combinatorial extract of N. sativa & C. cassia in Streptozotocin induced type-I Diabetic Rats (In Vivo). In addition changes in body weight, plasma glucose, lipid profile and kidney profile were also determined. The IC50 values for both extract and Acarbose was calculated by extrapolation method. Combinatorial extract of N. sativa & C. cassia at different dosages (100 and 200 mg/kg orally) and Metformin (50 mg/kg orally) as the standard drug was administered for 28 days and then biochemical estimation, body weights and OGTT (Oral glucose tolerance test) were determined. Histopathological studies were also performed on kidney and pancreatic tissue. In In-Vitro the combinatorial extract shows much more inhibiting effect than the individual extracts. The results reveals that combinatorial extract of N. sativa & C. cassia has shown significant decrease in plasma glucose (p<0.0001), total cholesterol and LDL levels when compared with the STZ group The decreasing level of BUN and creatinine revealed the protection of N. sativa & C. cassia extracts against nephropathy associated with diabetes. Combination of N. sativa & C. cassia significantly improved glucose tolerance to exogenously administered glucose (2 g/kg) after 60, 90 and 120 min interval on OGTT in high dose streptozotocin induced diabetic rats compared with the untreated control group. Histopathological studies shown that treatment with N. sativa & C. cassia extract alone and in combination restored pancreatic tissue integrity and was able to regenerate the STZ damaged pancreatic β cells. Thus, the present study reveals that combination of N. sativa & C. cassia extract has significant α- glucosidase inhibitory activity and thus has great potential as a new source for diabetes treatment.

Keywords: lipid levels, OGTT, diabetes, herbs, glucosidase

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Authors: K. Rasool

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Sulfate-reducing bacteria (SRBs) induced biofilm formation is a global industrial concern due to its role in the development of microbial-induced corrosion (MIC). Herein, we have developed a biodegradable chitosan/lignosulfonate nanocomposite (CS@LS) as an efficient green biocide for the inhibition of SRBs biofilms. We investigated in detail the inhibition mechanism of SRBs by CS@LS in seawater media. Stable CS@LS-1:1 with 150–200 nm average size and zeta potential of + 34.25 mV was synthesized. The biocidal performance of CS@LS was evaluated by sulfate reduction profiles coupled with analysis of extracted extracellular polymeric substances (EPS) and lactate dehydrogenase (LDH) release assays. As the nanocomposite concentration was increased from 50 to 500 µg/mL, the specific sulfate reduction rate (SSRR) decreased from 0.278 to 0.036 g-sulfate/g-VSS*day showing a relative sulfate reduction inhibition of 86.64% as compared to that of control. Similarly, the specific organic uptake rate (SOUR) decreased from 0.082 to 0.039 0.036 g-TOC/g-VSS*day giving a relative co-substrate oxidation inhibition of 52.19% as compared to that of control. The SRBs spiked with 500 µg/mL CS@LS showed a reduction in cell viability to 1.5 × 106 MPN/mL. To assess the biosafety of the nanocomposite on the marine biota, the 72-hours acute toxicity assays using the zebrafish embryo model revealed that the LC50 for the CS@LS was 103.3 µg/mL. Thus, CS@LS can be classified as environmentally friendly. The nanocomposite showed long-term stability and excellent antibacterial properties against SRBs growth and is thus potentially useful for combating the problems of biofilm growth in harsh marine and aquatic environments.

Keywords: green biocides, chitosan/lignosulfonate nanocomposite, SRBs, toxicity

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Authors: Nicholas Mavrogiannis, Francesca Crivellari, Zachary Gagnon

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Development of low-cost, rapid, sensitive and portable biosensing systems are important for the detection and prevention of disease in developing countries, biowarfare/antiterrorism applications, environmental monitoring, point-of-care diagnostic testing and for basic biological research. Currently, the most established commercially available and widespread assays for portable point of care detection and disease testing are paper-based dipstick and lateral flow test strips. These paper-based devices are often small, cheap and simple to operate. The last three decades in particular have seen an emergence in these assays in diagnostic settings for detection of pregnancy, HIV/AIDS, blood glucose, Influenza, urinary protein, cardiovascular disease, respiratory infections and blood chemistries. Such assays are widely available largely because they are inexpensive, lightweight, and portable, are simple to operate, and a few platforms are capable of multiplexed detection for a small number of sample targets. However, there is a critical need for sensitive, quantitative and multiplexed detection capabilities for point-of-care diagnostics and for the detection and prevention of disease in the developing world that cannot be satisfied by current state-of-the-art paper-based assays. For example, applications including the detection of cardiac and cancer biomarkers and biothreat applications require sensitive multiplexed detection of analytes in the nM and pM range, and cannot currently be satisfied with current inexpensive portable platforms due to their lack of sensitivity, quantitative capabilities and often unreliable performance. In this talk, inexpensive label-free biomolecular detection at liquid interfaces using a newly discovered electrokinetic phenomenon known as fluidic dielectrophoresis (fDEP) is demonstrated. The electrokinetic approach involves exploiting the electrical mismatches between two aqueous liquid streams forced to flow side-by-side in a microfluidic T-channel. In this system, one fluid stream is engineered to have a higher conductivity relative to its neighbor which has a higher permittivity. When a “low” frequency (< 1 MHz) alternating current (AC) electrical field is applied normal to this fluidic electrical interface the fluid stream with high conductivity displaces into the low conductive stream. Conversely, when a “high” frequency (20MHz) AC electric field is applied, the high permittivity stream deflects across the microfluidic channel. There is, however, a critical frequency sensitive to the electrical differences between each fluid phase – the fDEP crossover frequency – between these two events where no fluid deflection is observed, and the interface remains fixed when exposed to an external field. To perform biomolecular detection, two streams flow side-by-side in a microfluidic T-channel: one fluid stream with an analyte of choice and an adjacent stream with a specific receptor to the chosen target. The two fluid streams merge and the fDEP crossover frequency is measured at different axial positions down the resulting liquid

Keywords: biodetection, fluidic dielectrophoresis, interfacial polarization, liquid interface

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Authors: Simona Pileviciene

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On 24th January 2014, Lithuania notified two primary cases of African swine fever (ASF) in wild boars. The animals were tested positive for ASF virus (ASFV) genome by real-time PCR at the National Reference Laboratory for ASF in Lithuania (NRL), results were confirmed by the European Union Reference Laboratory for African swine fever (CISA-INIA). Intensive wild and domestic animal monitoring program was started. During the period of 2014-2017 ASF was confirmed in two large commercial pig holding with the highest biosecurity. Pigs were killed and destroyed. Since 2014 ASF outbreak territory from east and south has expanded to the middle of Lithuania. Diagnosis by PCR is one of the highly recommended diagnostic methods by World Organization for Animal Health (OIE) for diagnosis of ASF. The aim of the present study was to compare singleplex real-time PCR assays to a duplex assay allowing the identification of ASF and internal control in a single PCR tube and to compare primers, that target the p72 gene (ASF 250 bp and ASF 75 bp) effectivity. Multiplex real-time PCR assays prove to be less time consuming and cost-efficient and therefore have a high potential to be applied in the routine analysis. It is important to have effective and fast method that allows virus detection at the beginning of disease for wild boar population and in outbreaks for domestic pigs. For experiments, we used reference samples (INIA, Spain), and positive samples from infected animals in Lithuania. Results show 100% sensitivity and specificity.

Keywords: African swine fewer, real-time PCR, wild boar, domestic pig

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Authors: H. Aldewachi, M. Hines, M. McCulloch, N. Woodroofe, P. Gardiner

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DPP-IV is an enzyme whose expression is affected in a variety of diseases, therefore, has been identified as possible diagnostic or prognostic marker for various tumours, immunological, inflammatory, neuroendocrine, and viral diseases. Recently, DPP-IV enzyme has been identified as a novel target for type II diabetes treatment where the enzyme is involved. There is, therefore, a need to develop sensitive and specific methods that can be easily deployed for the screening of the enzyme either as a tool for drug screening or disease marker in biological samples. A variety of assays have been introduced for the determination of DPP-IV enzyme activity using chromogenic and fluorogenic substrates, nevertheless these assays either lack the required sensitivity especially in inhibited enzyme samples or displays low water solubility implying difficulty for use in vivo samples in addition to labour and time-consuming sample preparation. In this study, novel strategies based on exploiting the high extinction coefficient of gold nanoparticles (GNPs) are investigated in order to develop fast, specific and reliable enzymatic assay by investigating synthetic peptide sequences containing a DPP IV cleavage site and coupling them to GNPs. The DPP IV could be detected by colorimetric response of peptide capped GNPs (P-GNPS) that could be monitored by a UV-visible spectrophotometer or even naked eyes, and the detection limit could reach 0.01 unit/ml. The P-GNPs, when subjected to DPP IV, showed excellent selectivity compared to other proteins (thrombin and human serum albumin) , which led to prominent colour change. This provided a simple and effective colorimetric sensor for on-site and real-time detection of DPP IV.

Keywords: gold nanoparticles, synthetic peptides, colorimetric detection, DPP-IV enzyme

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Authors: Hsiang-Ru Lin

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Cholesterol plays an essential role in the regulation of the progression of numerous important diseases including atherosclerosis and Alzheimer disease so the generation of suitable cholesterol-lowering reagents is urgent to develop. Liver X receptor (LXR) is a ligand-activated transcription factor whose natural ligands are cholesterols, oxysterols and glucose. Once being activated, LXR can transactivate the transcription action of various genes including CYP7A1, ABCA1, and SREBP1c, involved in the lipid metabolism, glucose metabolism and inflammatory pathway. Essentially, the upregulation of ABCA1 facilitates cholesterol efflux from the cells and attenuates the production of beta-amyloid (ABeta) 42 in brain so LXR is a promising target to develop the cholesterol-lowering reagents and preventative treatment of Alzheimer disease. Engelhardia roxburghiana is a deciduous tree growing in India, China, and Taiwan. However, its chemical composition is only reported to exhibit antitubercular and anti-inflammatory effects. In this study, four compounds, engelheptanoxides A, C, engelhardiol A, and B isolated from the root of Engelhardia roxburghiana were evaluated for their agonistic activity against LXR by the transient transfection reporter assays in the HepG2 cells. Furthermore, their interactive modes with LXR ligand binding pocket were generated by molecular modeling programs. By using the cell-based biological assays, engelheptanoxides A, C, engelhardiol A, and B showing no cytotoxic effect against the proliferation of HepG2 cells, exerted obvious LXR agonistic effects with similar activity as T0901317, a novel synthetic LXR agonist. Further modeling studies including docking and SAR (structure-activity relationship) showed that these compounds can locate in LXR ligand binding pocket in the similar manner as T0901317. Thus, LXR is one of nuclear receptors targeted by pharmaceutical industry for developing treatments of Alzheimer and atherosclerosis diseases. Importantly, the cell-based assays, together with molecular modeling studies suggesting a plausible binding mode, demonstrate that engelheptanoxides A, C, engelhardiol A, and B function as LXR agonists. This is the first report to demonstrate that the extract of Engelhardia roxburghiana contains LXR agonists. As such, these active components of Engelhardia roxburghiana or subsequent analogs may show important therapeutic effects through selective modulation of the LXR pathway.

Keywords: Liver X receptor (LXR), Engelhardia roxburghiana, CYP7A1, ABCA1, SREBP1c, HepG2 cells

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Authors: Abigail Tan, Heng Liang Tan, Vanessa Ding, James Hui, Eng Hin Lee, Andre Choo

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Introduction: Comparative oncology investigates the similarities in spontaneous carcinogenesis between humans and animals, in order to identify treatments that can benefit these patients. Companion animals (CA), like canines and felines, are of special interest when it comes to studying human cancers due to their exposure to the same environmental factors and develop tumours with similar features. The purpose of this study is to explore the cross-reactivity of monoclonal antibodies (mAbs) across cancers in humans and CA. Material and Methods: A panel of CA mAbs generated in the lab was screened on multiple human cancer cell lines through flow cytometry to identify for positive binders. Shortlisted candidates were then characterised by biochemical and functional assays e.g., antibody-drug conjugate (ADC) and western blot assays, including glycan studies. Results: Candidate mAb KP52 was generated from whole-cell immunisation using feline mammary carcinoma. KP52 showed strong positive binding to human cancer cells, such as breast cancer and ovarian cancer. Furthermore, KP52 demonstrated strong killing ( > 50%) as an ADC with Saporin as the payload. Western blot results revealed the molecular weight of the antigen targets to be approximately 45kD and 50kD under reduced conditions. Glycan studies suggest that the epitope is glycan in nature, specifically an O-linked glycan. Conclusion: Candidate mAb KP52 has a therapeutic potential as an ADC against feline mammary cancer, human ovarian cancer, human mammary cancer, human pancreatic cancer, and human gastric cancer.

Keywords: ADC, comparative oncology, mAb, therapeutic

Procedia PDF Downloads 158

Authors: Daniel Mueller, Melanie Breitbach, Stefan Cornelius, Sarah Pakulla-Dickel, Margaretha Koenig, Anke Prochnow, Mario Scherer

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The European STR standard set (ESS) of loci as well as the new expanded CODIS core loci set as recommended by the CODIS Core Loci Working Group, has led to a higher standardization and harmonization in STR analysis across borders. Various multiplex PCRs assays have since been developed for the analysis of these 17 ESS or 23 CODIS expansion STR markers that all meet high technical demands. However, forensic analysts are often faced with difficult STR results and the questions thereupon. What is the reason that no peaks are visible in the electropherogram? Did the PCR fail? Was the DNA concentration too low? QIAGEN’s newest Investigator STR kits contain a novel Quality Sensor (QS) that acts as internal performance control and gives useful information for evaluating the amplification efficiency of the PCR. QS indicates if the reaction has worked in general and furthermore allows discriminating between the presence of inhibitors or DNA degradation as a cause for the typical ski slope effect observed in STR profiles of such challenging samples. This information can be used to choose the most appropriate rework strategy.Based on the latest PCR chemistry called FRM 2.0, QIAGEN now provides the next technological generation for STR analysis, the Investigator ESSplex SE QS and Investigator 24plex QS Kits. The new PCR chemistry ensures robust and fast PCR amplification with improved inhibitor resistance and easy handling for a manual or automated setup. The short cycling time of 60 min reduces the duration of the total PCR analysis to make a whole workflow analysis in one day more likely. To facilitate the interpretation of STR results a smart primer design was applied for best possible marker distribution, highest concordance rates and a robust gender typing.

Keywords: PCR, QIAGEN, quality sensor, STR

Procedia PDF Downloads 477

Authors: Francisco Dos Santos, Diogo Fonseca-Pereira, Sílvia Arroz-Madeira, Henrique Veiga-Fernandes

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Haematopoiesis is a developmental process that generates all blood cell lineages in health and disease. This relies on quiescent haematopoietic stem cells (HSCs) that are able to differentiate, self renew and expand upon physiological demand. HSCs have great interest in regenerative medicine, including haematological malignancies, immunodeficiencies and metabolic disorders. However, the limited yield from existing HSC sources drives the global need for reliable techniques to expand harvested HSCs at high quality and sufficient quantities. With the extensive use of cord blood progenitors for clinical applications, there is a demand for a safe and efficient expansion protocol that is able to overcome the limitations of the cord blood as a source of HSC. StemCell2MAXTM developed a technology that enhances the survival, proliferation and transplantation efficiency of HSC, leading the way to a more widespread use of HSC for research and clinical purposes. StemCell2MAXTM MIX is a solution that improves HSC expansion up to 20x, while preserving stemness, when compared to state-of-the-art. In a recent study by a leading cord blood bank, StemCell2MAX MIX was shown to support a selective 100-fold expansion of CD34+ Hematopoietic Stem and Progenitor Cells (when compared to a 10-fold expansion of Total Nucleated Cells), while maintaining their multipotent differentiative potential as assessed by CFU assays. The technology developed by StemCell2MAXTM opens new horizons for the usage of expanded hematopoietic progenitors for both research purposes (including quality and functional assays in Cord Blood Banks) and clinical applications.

Keywords: cord blood, expansion, hematopoietic stem cell, transplantation

Procedia PDF Downloads 246

Authors: Sharon N. Nuñal, May Flor S. Muegue, Nizzy Hope N. Cartago, Raymund B. Parcon, Sheina B. Logronio

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The antioxidant and anti-inflammatory potentials of Perna Viridis, Modiolus philippinarum, and Mytella charruana found in the Philippines were assessed. Mussel protein samples were hydrolyzed using trypsin, maturase, alcalase and pepsin at 1% and 2% concentrations and then fractionated through membrane filtration (<10 kDa and <30 kDa). Antioxidant assays showed that pepsin hydrolysate at 2% enzyme concentration exhibited the maximum activities for both 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity (155-176 µM TE/mg protein) and 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging (67-68 µM TE/mg protein) assays while trypsin hydrolysate dominated the Ferric Reducing Antioxidant Power (FRAP) for the three mussel species. Lower molecular weight peptide fractions at <10 kDa exhibited better antioxidant activities than the higher molecular weight fractions. The anti-inflammatory activities of M. philippinarum and M. charruana showed comparable protein denaturation inhibition potentials with the highest in P. Viridis samples (98.93%). The 5-Lipoxygenase (5-LOX) inhibitory activities of mussel samples showed no significant difference with inhibition exceeding 70%. P. Viridis demonstrated the highest inhibition against Cyclooxygenase-2 (COX-2) at 56.19%, while the rest showed comparable activities. This study showed that the three mussel species are potential sources of bioactive peptides and lipids with antioxidant and anti-inflammatory properties.

Keywords: anti-inflammatory, antioxidant, bioactive properties, mussel

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Authors: Jaco Oosthuizen, Nerina C. Van Der Merwe

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The National Health Laboratory Services in Bloemfontien has been the diagnostic testing facility for 1830 patients for familial breast cancer since 1997. From the cohort, 540 were comprehensively screened using High-Resolution Melting Analysis or Next Generation Sequencing for the presence of point mutations and/or indels. Approximately 90% of these patients stil remain undiagnosed as they are BRCA1/2 negative. Multiplex ligation-dependent probe amplification was initially added to screen for copy number variation detection, but with the introduction of next generation sequencing in 2017, was substituted and is currently used as a confirmation assay. The aim was to investigate the viability of utilizing internationally designed copy number variation detection assays based on mostly European/Caucasian genomic data for use within a South African context. The multiplex ligation-dependent probe amplification technique is based on the hybridization and subsequent ligation of multiple probes to a targeted exon. The ligated probes are amplified using conventional polymerase chain reaction, followed by fragment analysis by means of capillary electrophoresis. The experimental design of the assay was performed according to the guidelines of MRC-Holland. For BRCA1 (P002-D1) and BRCA2 (P045-B3), both multiplex assays were validated, and results were confirmed using a secondary probe set for each gene. The next generation sequencing technique is based on target amplification via multiplex polymerase chain reaction, where after the amplicons are sequenced parallel on a semiconductor chip. Amplified read counts are visualized as relative copy numbers to determine the median of the absolute values of all pairwise differences. Various experimental parameters such as DNA quality, quantity, and signal intensity or read depth were verified using positive and negative patients previously tested internationally. DNA quality and quantity proved to be the critical factors during the verification of both assays. The quantity influenced the relative copy number frequency directly whereas the quality of the DNA and its salt concentration influenced denaturation consistency in both assays. Multiplex ligation-dependent probe amplification produced false positives due to ligation failure when ligation was inhibited due to a variant present within the ligation site. Next generation sequencing produced false positives due to read dropout when primer sequences did not meet optimal multiplex binding kinetics due to population variants in the primer binding site. The analytical sensitivity and specificity for the South African population have been proven. Verification resulted in repeatable reactions with regards to the detection of relative copy number differences. Both multiplex ligation-dependent probe amplification and next generation sequencing multiplex panels need to be optimized to accommodate South African polymorphisms present within the genetically diverse ethnic groups to reduce the false copy number variation positive rate and increase performance efficiency.

Keywords: familial breast cancer, multiplex ligation-dependent probe amplification, next generation sequencing, South Africa

Procedia PDF Downloads 216

Authors: Kambiz Hassanzadeh, Seyedeh Shohreh Ebrahimi, Shahrbanoo Oryan, Arman Rahimmi, Esmael Izadpanah

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Parkinson’s disease is one of the most prevalent neurodegenerative diseases which occurs in elderly. There are convincing evidences that oxidative stress has an important role in both the initiation and progression of Parkinson’s disease. Thymoquinone (TQ) is shown to have antioxidant and anti-inflammatory properties in invitro and invivo studies. It is well documented that TQ acts as a free radical scavenger and prevents the cell damage. Therefore this study aimed to evaluate the effect of TQ on motor and non-motor symptoms in animal model of Parkinson’s disease. Male Wistar rats (10-12 months) received rotenone (1mg/kg/day, sc) to induce Parkinson’s disease model. Pretreatment with TQ (7.5 and 15 mg/kg/day, po) was administered one hour before the rotenone injection. Three motor tests (rotarod, rearing and bar tests) and two non-motor tests (forced swimming and elevated plus maze) were performed for behavioral assessment. Our results indicated that TQ significantly ameliorated the rotenone-induced motor dysfunction in rotarod and rearing tests also it could prevent the non-motor dysfunctions in forced swimming and elevated plus maze tests. In conclusion we found that TQ delayed the Parkinson's disease induction by rotenone and this effect might be related to its proved antioxidant effect.

Keywords: Parkinson's disease, thymoquinone, motor and non-motor symptoms, neurodegenerative disease

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Authors: Khalil Abdullah Ahmed Khalil, Elsadig Mohamed Ahmed

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Extracts of various plants material capable of decreasing blood sugar have been tested in experimental animal models, and their effects confirmed. Neem or Margose (AzadirachtaIndica) is an indigenous plant believed to have antiviral, antifungal, antidiabetic, and many other properties. In this paper deals with a comparative study of effect of aqueous Neem leaves extract alone or in combination with glibenclamide on alloxan diabetic rabbits. Administration of crude aqueous Neem extract (CANE) alone (1.5 ml/kg/day) as well as the combination of CANE (1.5 ml/kg/day) with glibenclamide (0.25 mg/kg/day) significantly decreased (P<0.05) the concentrations of serum lipids, blood glucose and lipoprotein VLDL and LDL but significantly increased (P<0.05) the concentration of HDL. The change was observed significantly greater when the treatment was given in combination of CANE and glibenclamid than with CANE alone.

Keywords: aqueos neem leaves extract, hypoglycemic, hypolipidemic, cholesterol

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Authors: Miguel Pereira, Lígia Pimentel, Manuela Pintado

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Animal blood is a major by-product of slaughterhouses and still represents a cost and environmental problem in some countries. To be eliminated, blood should be stabilised by cooking and afterwards the slaughterhouses must have to pay for its incineration. In order to reduce the elimination costs and valorise the high protein content the aim of this study was the optimization of hydrolysis conditions, in terms of enzyme ratio and time, in order to obtain hydrolysates with biological activity. Two enzymes were tested in this assay: pepsin and proteases from Cynara cardunculus (cardosins). The latter has the advantage to be largely used in the Portuguese Dairy Industry and has a low price. The screening assays were carried out in a range of time between 0 and 10 h and using a ratio of enzyme/reaction volume between 0 and 5%. The assays were performed at the optimal conditions of pH and temperature for each enzyme: 55 °C at pH 5.2 for cardosins and 37 °C at pH 2.0 for pepsin. After reaction, the hydrolysates were evaluated by FPLC (Fast Protein Liquid Chromatography) and tested for their antioxidant activity by ABTS method. FPLC chromatograms showed different profiles when comparing the enzymatic reactions with the control (no enzyme added). The chromatogram exhibited new peaks with lower MW that were not present in control samples, demonstrating the hydrolysis by both enzymes. Regarding to the antioxidant activity, the best results for both enzymes were obtained using a ratio enzyme/reactional volume of 5% during 5 h of hydrolysis. However, the extension of reaction did not affect significantly the antioxidant activity. This has an industrial relevant aspect in what concerns to the process cost. In conclusion, the enzymatic blood hydrolysis can be a better alternative to the current elimination process allowing to the industry the reuse of an ingredient with biological properties and economic value.

Keywords: antioxidant activity, blood, by-products, enzymatic hydrolysis

Procedia PDF Downloads 492

Authors: Mohamed H. Khalifa, Fikry I. El-Shahawi, Nabil A. Mansour

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The cotton leafworm, Spodoptera littoralis (Boisduval) is a major insect pest of vegetables and cotton crops in Egypt, and exhibits different levels of tolerance to certain insecticides. Chlorantraniliprole has been registered recently in Egypt for control this insect. The susceptibilities of three S. littoralis populations collected from El Behaira governorate, north Egypt to chlorantraniliprole were determined by leaf-dipping technique on 4^th instar larvae. Obvious variation of toxicity was observed among the laboratory susceptible, and three field populations with LC₅₀ values ranged between 1.53 µg/ml and 6.22 µg/ml. However, all the three field populations were less susceptible to chlorantraniliprole than a laboratory susceptible population. The most tolerant populations were sampled from El Delengat (ED) Province where S. littoralis had been frequently challenged by insecticides. Certain enzyme activity assays were carried out to be correlated with the mechanism of the observed field population tolerance. All field populations showed significantly enhanced activities of detoxification enzymes compared with the susceptible strain. The regression analysis between chlorantraniliprole toxicities and enzyme activities revealed that the highest correlation is between α-esterase or β-esterase (α-β-EST) activity and collected field strains susceptibility, otherwise this correlation is not significant (P > 0.05). Synergism assays showed the ED and susceptible strains could be synergized by known detoxification inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.01-8.76-fold and 1.09-2.94 fold, respectively), TPP showed the maximum synergism in both strains. The results show that there is a correlation between the enzyme activity and tolerance, and carboxylic-esterase (Car-EST) is likely the main detoxification mechanism responsible for tolerance of S. littoralis to chlorantraniliprole.

Keywords: chlorantraniliprole, detoxification enzymes, Egypt, Spodoptera littoralis

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Authors: Kandi Sridhar, Charles Albert Linton

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Grapes are excellent sources of bioactive compounds, which have been suggested to be responsible for lowering the risk of chronic diseases. Fresh and freeze-dried extracts of Kyoho and Jubilee, commercial grape varieties available in Taiwan and attractive for their quality berries, were investigated for their total phenolics and total flavonoids contents and related dose-dependent antioxidants properties using various in vitro assays. The efficiency of the extraction yield ranged from 7.10 % to 25.53 % (w/w), depending on solvent used. Fresh samples of Kyoho and Jubilee exhibited total polyphenolic contents (351.56 ± 23.08 and 328.67 ± 16.54 µg GAE/mL, respectively), whereas Kyoho freeze-dried methanol: water extracts contains the good levels of total flavonoids (4767.82 ± 22.20 µg QE/mL). Kyoho and Jubilee freeze-dried extracts exhibited the highest total flavonoid contents. There was a weak correlation between total phenolic and flavonoid assays (r= -0.05, R2 = 0.02, p > 0.05). Kyoho fresh and freeze-dried samples showed the DPPH (11.51 – 77.82 %), superoxide scavenging activity (33.61 – 81.95 %), and total antioxidant inhibition (92.01 – 99.28 %), respectively. Total flavonoids were statistically correlated with EC50 DPPH scavenging radicals (r =0.91, p < 0.01), EC50 nitric oxide (r = 0.25, p > 0.05), and EC50 lipid peroxidation radicals (r = 0.38, p > 0.05). These results suggested that the two commercial grape cultivars in Taiwan could be used as a good source of natural antioxidants. Thus, consumption of grapes as a source antioxidant might lower the risk of chronic diseases. Moreover, future studies will investigate and develop phenolic acid profile for the cultivars in Taiwan.

Keywords: antioxidants, EC50 radical scavenging activity, grape cultivars, total phenolics

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Authors: H. R. Kelidari, M. Saeedi, J. Akbari, K. Morteza-Semnani, H. Valizadeh

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Spironolactoe (SP) a synthetic steroid diuretic is a poorly water-soluble drug with a low and variable oral bioavailability. Regarding to the good solubility of SP in lipid materials, SP loaded Solid lipid nanoparticles (SP-SLNs) and nanostructured lipid carrier (SP-SLNs) were thus prepared in this work for accelerating dissolution of this drug. The SP loaded NLC with stearic acid (SA) as solid lipid and different Oleic Acid (OA) as liquid lipid content and SLN without OA were prepared by probe ultrasonication method. With increasing the percentage of OA from 0 to 30 wt% in SLN/NLC, the average size and zeta potential of nanoparticles felled down and entrapment efficiency (EE %) rose dramatically. The obtained micrograph particles showed pronounced spherical shape. Differential Scanning Calorimeter (DSC) measurements indicated that the presence of OA reduced the melting temperature and melting enthalpy of solid lipid in NLC structure. The results reflected good long-term stability of the nanoparticles and the measurements show that the particle size remains lower in NLC compare to SLN formulations, 6 months after production. Dissolution of SP-SLN and SP-NLC was about 5.1 and 7.2 times faster than raw drugs in 120 min respectively. These results indicated that the SP loaded NLC containing 70:30 solid lipid to liquid lipid ratio is a suitable carrier of SP with improved drug EE and steady drug release properties.

Keywords: drug release, lipid nanoparticles, spironolactone, stability

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