Search results for: enzyme assay

Authors: Drago Bratkovic, Curtis Gravance, David Ketteridge, Ravi Krishnan, Michael Imperiale

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The mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases that have a common defect in the catabolism of glycosaminoglycans (GAGs). MPS I is the most common of the MPS diseases. Manifestations of MPS I include coarsening of facial features, corneal clouding, developmental delay, short stature, skeletal manifestations, hearing loss, cardiac valve disease, hepatosplenomegaly, and umbilical and inguinal hernias. Treatments for MPS I restore or activate the missing or deficient enzyme in the case of enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT). Pentosan polysulfate sodium (PPS) is a potential treatment to improve bone and joint manifestations of MPS I. The mechanisms of action of PPS that are relevant to the treatment of MPS I are the ability to: (i) Reduce systemic and accumulated GAG, (ii) Reduce inflammatory effects via the inhibition of NF-kB, resulting in the reduction in pro-inflammatory mediators. (iii) Reduce the expression of the pain mediator nerve growth factor in osteocytes from degenerating joints. (iv) Inhibit the cartilage degrading enzymes related to joint dysfunction in MPS I. PPS is being evaluated as an adjunctive therapy to ERT and/or HSCT in an open-label, single-centre, phase 2 study. Patients are ≥ 5 years of age with a diagnosis of MPS I and previously received HSCT and/or ERT. Three white, female, patients with MPS I-Hurler, ages 14, 15, and 19 years, and one, white male patient aged 15 years are enrolled. All were diagnosed at ≤2 years of age. All patients received HSCT ≤ 6 months after diagnosis. Two of the patients were treated with ERT prior to HSCT, and 1 patient received ERT commencing 3 months prior to HSCT. Two patients received 0.75mg/kg and 2 patients received 1.5mg/kg of PPS. PPS was well tolerated at doses of 0.75 and 1.5 mg/kg to 47 weeks of continuous dosing. Of the 19 adverse events (AEs), 2 were related to PPS. One AE was moderate (pre-syncope) and 1 was mild (injection site bruising), experienced in the same patient. All AEs were reported as mild or moderate. There have been no SAEs. One subject experienced a COVID-19 infection and PPS was interrupted. The MPS I signature GAG fragments, sulfated disaccharide and UA-HNAc S, tended to decrease in 3 patients from baseline through Week 25. Week 25 GAG data are pending for the 4th patient. Overall, most biomarkers (inflammatory, cartilage degeneration, and bone turnover) evaluated in the 3 patients with 25-week assessments have indicated either no change or a reduction in levels compared to baseline. In 3 patients, there was a trend toward improvement in the 2MWT from baseline to Week 48 with > 100% increase in 1 patient (01-201). In the 3 patients that had Week 48 assessments, patients and proxies reported improvement in PGIC, including “worthwhile difference” (n=1), or “made all the difference” (n=2).

Keywords: MPS I, pentosan polysulfate sodium, clinical study, 2MWT, QoL

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Authors: Ali Akbar Kazemi Asl, Mansour Rahsepar

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Mesoporous carbon nanospheres (MCNs) with uniform particle size distribution having an average of 290 nm and large specific surface area (274.4 m²/g) were synthesized by a one-step hydrothermal method followed by the calcination process and then utilized as an enzyme-free glucose biosensor. Morphology, crystal structure, and porous nature of the synthesized nanospheres were characterized by scanning electron microscopy (SEM), X-Ray diffraction (XRD), and Brunauer–Emmett–Teller (BET) analysis, respectively. Also, the electrochemical performance of the MCNs@GCE electrode for the measurement of glucose concentration in alkaline media was investigated by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and chronoamperometry (CA). MCNs@GCE electrode shows good sensing performance, including a rapid glucose oxidation response within 3.1 s, a wide linear range of 0.026-12 mM, a sensitivity of 212.34 μA.mM⁻¹.cm⁻², and a detection limit of 25.7 μM with excellent selectivity.

Keywords: biosensor, electrochemical, glucose, mesoporous carbon, non-enzymatic

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Authors: Shashi Bala, Neha A. Chugh, Subhash C. Bansal, Mohal L. Garg, Ashwani Koul

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Purpose: The present study was designed to evaluate the radioprotective efficacy of Aloe vera from whole body X-ray exposure in rodents. Materials and Methods: For this purpose, after on week’s acclimatization, male balb/c mice procured from Central Animal House, Panjab University, Chandigarh (India), were divided into four groups: Group I mice served as control. Group II mice were orally administrated Aloe vera pulp extract (50 mg/ kg body weight) on alternate days for 30 days. Group III mice were subjected to whole body X-ray irradiation to cumulative dose of 2Gy (0.258Gy twice a day for four days in the last week). Group IV animals were pretreated with Aloe vera pulp extract on alternate days as in Group II and in the last week of the study, they were exposed to X-ray as in Group III. Results: Spleen of X-ray irradiated mice showed histopathological alterations accompanied with enhanced activity of lactate dehydrogenase (LDH) in serum. Elevated levels of reactive oxygen species (ROS), lipid peroxidation (LPO), enhanced activities in Glutathione based enzymes such as Glutathione peroxidase (GSH-Px), Glutathione reductase (GR), Catalase (CAT), Superoxide dismutase (SOD) associated with depletion in reduced Glutathione (GSH) concentration were observed after X-ray exposure in blood plasma and spleen.. Pro-inflammatory cytokines like tumor necrosis factors (TNF-α) and Inteleukin-6 (IL-6) levels were also found to be enhanced in serum of irradiated mice. Irradiation-induced significant elevation in Total leucocyte counts (TLC), neutrophil counts and decline in platelet counts, associated with unaltered levels of red blood cell counts (RBC’s) and haemoglobin (Hb) in various treatment groups. Clastogenic damage and apoptosis was also found to be increase in splenic tissue of X-ray exposed mice as assessed by micronucleus and TUNEL assay. However, X-ray irradiated animals administered with Aloe vera revealed significant improvement in levels of ROS/ LPO, LDH activity, and antioxidant mechanism. Aloe vera pretreated animals exhibited less severe damage, and early recovery in micronucleated cells, hematological parameters, apoptotic cells and inflammatory markers as compared to X-ray exposed mice. Conclusion: These results indicate that the radioprotective potential of Aloe vera against X-ray induced damage. This may be due to its free radical scavenging, antioxidant, anti-apoptotic and anti-inflammatory properties.

Keywords: aloe vera, antioxidant defense system, lactate dehydrogenase (LDH), micronucleus assay, x-ray

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Authors: İsmail Çeli̇k, Gülgün Ayhan-Kılcıgi̇l, Arzu Onay-Beşi̇kçi̇

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In this study, some 2-(2-phenyl/substitutedphenyl)- lH-benzo[d]'imidazol-l-yl)-N'-(alkylthiosemicarbazide were designed and prepared. Firstly, 2-phenyl/ suhstitutedphenyl-lH-Benzo[d]imidazole was prepared via oxidative condensation of o-phenylenediamine, benzaldehyde and sodium metabisulfite. Treatment of the benzimidazole compound with ethyl chloroacetate in KOH/DMSO gave the ester compound ethyl 2-(2-substitutedphenyl)-1H-benzo[d]imidazol-l-yl)acetate. Hydrazine hydrate and the ester in ethanol were refluxed for 4 h to give 2-(2-phenyl/substitutedphenyl)-1H-benzo[d]imidazol-l-yl)acetohydrazide. Thiosemicarbazides were obtained by condensing acyl hydrazide with the alkylisothiocyanate in ethanol. Following the structure elucidation, benzimidazole compounds were tested for their EGFR kinase inhibitory activities by using ADP-GloTM Kinase Assay.

Keywords: benzimidazole, EGFR kinase inhibitor, synthesis, thiosemicarbazide

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Authors: Roihatul Mutiah, Sukardiman, Aty Widyawaruyanti

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Calotropis gigantiea (L.) R. Br (Apocynaceae) commonly called as “Biduri” or “giant milk weed” is a well-known weed to many cultures for treating various disorders. Several studies reported that C.gigantea roots has anticancer activity. The main aim of this research was to isolate and identify an active marker compound of C.gigantea roots for quality control purpose of its extract in the development as anticancer natural product. The isolation methods was bioactivity guided column chromatography, TLC, and HPLC. Evaluated anticancer activity of there substances using MTT assay methods. Identification structure active compound by UV, 1HNMR, 13CNMR, HMBC, HMQC spectral and other references. The result showed that the marker active compound was identical as Calotropin.

Keywords: calotropin, Calotropis gigantea, anticancer, marker active

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Authors: K. Verma, v. S. Moholkar

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In developed countries, water pollution caused by industrial discharge has emerged as a significant environmental concern over the past decades. However, despite ongoing efforts, a fully effective and sustainable remediation strategy has yet to be identified. This paper describes how enzymatic and sonochemical treatments have demonstrated great promise in degrading bio-refractory pollutants. Mainly, a compelling area of interest lies in the combined technique of sono-enzymatic treatment, which has exhibited a synergistic enhancement effect surpassing that of the individual techniques. This study employed the covalent attachment method to immobilize Laccase from Trametes versicolor onto amino-functionalized magnetic Fe₃O₄ nanoparticles. To comprehensively characterize the synthesized free nanoparticles and the laccase-immobilized nanoparticles, various techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), vibrating sample magnetometer (VSM), and surface area through Brunauer-Emmett-Teller (BET) were employed. The size of immobilized Fe₃O₄@Laccase was found to be 60 nm, and the maximum loading of laccase was found to be 24 mg/g of nanoparticle. An investigation was conducted to study the effect of various process parameters, such as immobilized Fe₃O₄ Laccase dose, temperature, and pH, on the % Chemical oxygen demand (COD) removal as a response. The statistical design pinpointed the optimum conditions (immobilized Fe₃O₄ Laccase dose = 1.46 g/L, pH = 4.5, and temperature = 66 oC), resulting in a remarkable 65.58% COD removal within 60 minutes. An even more significant improvement (90.31% COD removal) was achieved with ultrasound-assisted enzymatic reaction utilizing a 10% duty cycle. The investigation of various kinetic models for free and immobilized laccase, such as the Haldane, Yano, and Koga, and Michaelis-Menten, showed that ultrasound application impacted the kinetic parameters Vmax and Km. Specifically, Vmax values for free and immobilized laccase were found to be 0.021 mg/L min and 0.045 mg/L min, respectively, while Km values were 147.2 mg/L for free laccase and 136.46 mg/L for immobilized laccase. The lower Km and higher Vmax for immobilized laccase indicate its enhanced affinity towards the substrate, likely due to ultrasound-induced alterations in the enzyme's confirmation and increased exposure of active sites, leading to more efficient degradation. Furthermore, the toxicity and Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that after the treatment process, the wastewater exhibited 70% less toxicity than before treatment, with over 25 compounds degrading by more than 75%. At last, the prepared immobilized laccase had excellent recyclability retaining 70% activity up to 6 consecutive cycles. A straightforward manufacturing strategy and outstanding performance make the recyclable magnetic immobilized Laccase (Fe₃O₄ Laccase) an up-and-coming option for various environmental applications, particularly in water pollution control and treatment.

Keywords: kinetic, laccase enzyme, sonoenzymatic, ultrasound irradiation

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Authors: Jana Chakrabarti, Madhushrita Das, Ankhi Haldar, Roshni Chatterjee, Tanmoy Dey, Pubali Dhar

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High incidences of Protein Energy Malnutrition as a consequence of low protein intake are quite prevalent among the children in developing countries. Thus prevention of under-nutrition has emerged as a critical challenge to India’s developmental Planners in recent times. Increase in population over the last decade has led to greater pressure on the existing animal protein sources. But these resources are currently declining due to persistent drought, diseases, natural disasters, high-cost of feed, and low productivity of local breeds and this decline in productivity is most evident in some developing countries. So the need of the hour is to search for efficient utilization of unconventional low-cost animal protein resources. Molluscs, as a group is regarded as under-exploited source of health-benefit molecules. Bivalve is the second largest class of phylum Mollusca. Annual harvests of bivalves for human consumption represent about 5% by weight of the total world harvest of aquatic resources. The freshwater mussel Lamellidens marginalis is widely distributed in ponds and large bodies of perennial waters in the Indian sub-continent and well accepted as food all over India. Moreover, ethno-medicinal uses of the flesh of Lamellidens among the rural people to treat hypertension have been documented. Present investigation thus attempts to evaluate the potential of Lamellidens marginalis as functional food. Mussels were collected from freshwater ponds and brought to the laboratory two days before experimentation for acclimatization in laboratory conditions. Shells were removed and fleshes were preserved at- 20oC until analysis. Tissue homogenate was prepared for proximate studies. Fatty acids and amino acids composition were analyzed. Vitamins, Minerals and Heavy metal contents were also studied. Mussel Protein hydrolysate was prepared using Alcalase 2.4 L and degree of hydrolysis was evaluated to analyze its Functional properties. Ferric Reducing Antioxidant Power (FRAP) and DPPH Antioxidant assays were performed. Anti-hypertensive property was evaluated by measuring Angiotensin Converting Enzyme (ACE) inhibition assay. Proximate analysis indicates that mussel meat contains moderate amount of protein (8.30±0.67%), carbohydrate (8.01±0.38%) and reducing sugar (4.75±0.07%), but less amount of fat (1.02±0.20%). Moisture content is quite high but ash content is very low. Phospholipid content is significantly high (19.43 %). Lipid constitutes, substantial amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) which have proven prophylactic values. Trace elements are found present in substantial amount. Comparative study of proximate nutrients between Labeo rohita, Lamellidens and cow’s milk indicates that mussel meat can be used as complementary food source. Functionality analyses of protein hydrolysate show increase in Fat absorption, Emulsification, Foaming capacity and Protein solubility. Progressive anti-oxidant and anti-hypertensive properties have also been documented. Lamellidens marginalis can thus be regarded as a functional food source as this may combine effectively with other food components for providing essential elements to the body. Moreover, mussel protein hydrolysate provides opportunities for utilizing it in various food formulations and pharmaceuticals. The observations presented herein should be viewed as a prelude to what future holds.

Keywords: functional food, functional properties, Lamellidens marginalis, protein hydrolysate

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Authors: M. S. Kilic, S. Korkut, B. Hazer

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Newly synthesized Polypropylene-g-Polyethylene glycol polymer was first time used for a compartment-less enzymatic fuel cell. Working electrodes based on Polypropylene-g-Polyethylene glycol were operated as unmediated and mediated system (with ferrocene and gold/cobalt oxide nanoparticles). Glucose oxidase and bilirubin oxidase was selected as anodic and cathodic enzyme, respectively. Glucose was used as fuel in a single-compartment and membrane-less cell. Maximum power density was obtained as 0.65 nW cm-2, 65 nW cm-2, and 23500 nW cm-2 from the unmediated, ferrocene and gold/cobalt oxide modified polymeric film, respectively. Power density was calculated to be ~16000 nW cm-2 for undiluted wastewater sample with gold/cobalt oxide nanoparticles including system.

Keywords: bilirubin oxidase, enzymatic fuel cell, glucose oxidase, nanoparticles

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Authors: F. Bouchouka, S. Benamara

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The present work is an attempt to prepare a fresh cheese using lemon juice and lemon juice / orange juice mixture as acidifying / clotting agents. A reference cheese is obtained by acidification with commercial vinegar. The analysis performed on the final product (fat, cheese yield, sensory analysis, rheological and bacteriological properties) confirmed the technical feasibility of a natural cheese, using a lemon juice and / or lemon juice / orange juice mixture as acidifying / clotting agents. In addition, a general acceptance test allowed to select the cheese sample acidified with lemon juice as the best, compared to the two other samples (lemon juice/orange juice acidification and commercial vinegar acidification).

Keywords: clotting agent, fresh cheese, juice, lemon, orange

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Authors: A. Nusrath Unissa, Sameer Hassan, Luke Elizabeth Hanna

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Altered drug binding may be an important factor in isoniazid (INH) resistance, rather than major changes in the enzyme’s activity as a catalase or peroxidase (KatG). The identification of structural or functional defects in the mutant KatGs responsible for INH resistance remains as an area to be explored. In this connection, the differences in the binding affinity between wild-type (WT) and mutants of KatG were investigated, through the generation of three mutants of KatG, Ser315Thr [S315T], Ser315Asn [S315N], Ser315Arg [S315R] and a WT [S315]) with the help of software-MODELLER. The mutants were docked with INH using the software-GOLD. The affinity is lower for WT than mutant, suggesting the tight binding of INH with the mutant protein compared to WT type. These models provide the in silico evidence for the binding interaction of KatG with INH and implicate the basis for rationalization of INH resistance in naturally occurring KatG mutant strains of Mycobacterium tuberculosis.

Keywords: Mycobacterium tuberculosis, KatG, INH resistance, mutants, modelling, docking

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Authors: Chiun-C.R. Wang, Po-Wen Yen, Chien-Chun Huang

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Banana is produced in large quantities in tropical and subtropical areas. Banana is one of the important fruits which constitute a valuable source of energy, vitamins and minerals. The ripening and maturity standards of banana vary from country to country depending on the expected shelf life of market. The compositions of bananas change dramatically during ethylene-induced ripening that are categorized as nutritive values and commercial utilization. Nevertheless, there is few study reporting the changes of physicochemical properties of banana starch during ethylene-induced ripening of green banana. The objectives of this study were to investigate the changes of chemical composition and enzyme activity of banana and physicochemical properties of banana starch during ethylene-induced ripening. Green bananas were harvested and ripened by ethylene gas at low temperature (15℃) for seven stages. At each stage, banana was sliced and freeze-dried for banana flour preparation. The changes of total starch, resistant starch, chemical compositions, physicochemical properties, activity of amylase, polyphenolic oxidase (PPO) and phenylalanine ammonia lyase (PAL) of banana were analyzed each stage during ripening. The banana starch was isolated and analyzed for gelatinization properties, pasting properties and microscopic appearance each stage of ripening. The results indicated that the highest total starch and resistant starch content of green banana were 76.2% and 34.6%, respectively at the harvest stage. Both total starch and resistant starch content were significantly declined to 25.3% and 8.8%, respectively at the seventh stage. Soluble sugars content of banana increased from 1.21% at harvest stage to 37.72% at seventh stage during ethylene-induced ripening. Swelling power of banana flour decreased with the progress of ripening stage, but solubility increased. These results strongly related with the decreases of starch content of banana flour during ethylene-induced ripening. Both water insoluble and alcohol insoluble solids of banana flour decreased with the progress of ripening stage. Both activity of PPO and PAL increased, but the total free phenolics content decreased, with the increases of ripening stages. As ripening stage extended, the gelatinization enthalpy of banana starch significantly decreased from 15.31 J/g at the harvest stage to 10.55 J/g at the seventh stage. The peak viscosity and setback increased with the progress of ripening stages in the pasting properties of banana starch. The highest final viscosity, 5701 RVU, of banana starch slurry was found at the seventh stage. The scanning electron micrograph of banana starch showed the shapes of banana starch appeared to be round and elongated forms, ranging in 10-50 μm at the harvest stage. As the banana closed to ripe status, some parallel striations were observed on the surface of banana starch granular which could be caused by enzyme reaction during ripening. These results inferred that the highest resistant starch was found in the green banana at the harvest stage could be considered as a potential application of healthy foods. The changes of chemical composition and physicochemical properties of banana could be caused by the hydrolysis of enzymes during the ethylene-induced ripening treatment.

Keywords: ethylene-induced ripening, banana starch, resistant starch, soluble sugars, physicochemical properties, gelatinization enthalpy, pasting characteristics, microscopic appearance

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Authors: O. R. Raimi, A. Lateef

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Inoculum developments of A. niger were used for inoculation of medium for submerged fermentation and solid state fermentation. The filtrate obtained were used as sources of the extra-cellular enzymes. The FTase activities and the course of pH in submerged fermentation ranged from 7.53-24.42µ/ml and 4.4-4.8 respectively. The maximum FTase activity was obtained at 48 hours fermentation. In solid state fermentation, FTase activities ranged from 2.41-27.77µ/ml. Using ripe plantain peel and kola nut pod respectively. Both substrates supported the growth of the fungus, producing profuse growth during fermentation. In the control experiment (using kolanut pod) that lack supplementation, appreciable FTase activity of 16.92µ/ml was obtained. The optimum temperature range was 600C. it was also active at broad pH range of 1-9 with optimum obtain at pH of 5.0. FTase was stable within the range of investigated pH showing more than 60% activities. FTase can be used in the production of fructooligosaccharide, a functional food.

Keywords: Aspergillus niger, solid state fermentation, kola nut pods, Fructosyltransferase (FTase)

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Authors: Shahram Nanekarani, Majid Goodarzi, Majid Khosravi

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The present study aimed at analyzing of ovine major histocompatibility complex class II (Ovar II) DRB1 gene second exon in Lori Sheep breed. The MHC plays a central role in the control of disease resistance and immunological response. Genomic DNA from blood samples of 124 sheep was extracted and a 296 bp MHC exon 2 fragment was amplified using polymerase chain reaction. PCR products were characterized by the restriction fragment length polymorphism technique using Hin1I restriction enzyme. The PCRRFLP patterns showed three genotypes, AA, AB and BB with frequency of 0.282, 0.573 and 0.145, respectively. There was no significant (P > 0.05) deviation from Hardy–Weinberg equilibrium for this locus in this population. The results of the present study indicate that exon 2 of the Ovar-DRB1 gene is highly polymorphic in Lori sheep and could be considered as an important marker assisted selection, for improvement of immunity in sheep.

Keywords: MHC-DRB1 gene, polymorphism, PCR-RFLP, lori sheep

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Authors: Sarwar Beg, Gajanand Sharma, O. P. Katare, Bhupinder Singh

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The current studies entail development of self-nano emulsifying drug delivery systems (SNEDDS) of rosuvastatin, employing rational QbD-based approach for enhancing its oral bioavailability. SNEDDS were prepared using the blend of lipidic and emulsifying excipients, i.e., Peceol, Tween 80, and Transcutol HP. The prepared formulations evaluated for in vitro drug release, ex vivo permeation, in situ perfusion studies and in vivo pharmacokinetic studies in rats, which demonstrated 3-4 fold improvement in biopharmaceutical performance of the developed formulations. Cytotoxicity studies using MTT assay and histopathological studies in intestinal cells revealed the lack of cytotoxicity and thereby safety and efficacy of the developed formulations.

Keywords: SNEDDS, bioavailability, solubility, Quality by Design (QbD)

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Authors: B. T. Naveen Kumar, Anuj Tyagi, Niraj Kumar Singh, Visanu Boonyawiwat, A. H. Shanthanagouda, Orawan Boodde, K. M. Shankar, Prakash Patil, Shubhkaramjeet Kaur

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Early mortality syndrome (EMS)/Acute Hepatopancreatic Necrosis Disease (AHPND) has emerged as a major obstacle for the shrimp farming around the world. It is caused by a strain of Vibrio parahaemolyticus. The possible preventive and control measure is, early and rapid detection of the pathogen in the broodstock, post-larvae and monitoring the shrimp during the culture period. Polymerase chain reaction (PCR) based early detection methods are good, but they are costly, time taking and requires a sophisticated laboratory. The present study was conducted to develop a simple, sensitive and rapid diagnostic farmer level kit for the reliable detection of AHPND in shrimp. A panel of monoclonal antibodies (MAbs) were raised against the recombinant Pir B protein (rPirB). First, an immunodot was developed by using MAbs G3B8 and Mab G3H2 which showed specific reactivity to purified r-PirB protein with no cross-reactivity to other shrimp bacterial pathogens (AHPND free Vibrio parahaemolyticus (Indian strains), V. anguillarum, WSSV, Aeromonas hydrophila, and Aphanomyces invadans). Immunodot developed using Mab G3B8 is more sensitive than that with the Mab G3H2. However, immunodot takes almost 2.5 hours to complete with several hands-on steps. Therefore, the flow-through assay (FTA) was developed by using a plastic cassette containing the nitrocellulose membrane with absorbing pads below. The sample was dotted in the test zone on the nitrocellulose membrane followed by continuos addition of five solutions in the order of i) blocking buffer (BSA) ii) primary antibody (MAb) iii) washing Solution iv) secondary antibody and v) chromogen substrate (TMB) clear purple dots against a white background were considered as positive reactions. The FTA developed using MAbG3B8 is more sensitive than that with MAb G3H2. In FTA the two MAbs showed specific reactivity to purified r-PirB protein and not to other shrimp bacterial pathogens. The FTA is simple to farmer/field level, sensitive and rapid requiring only 8-10 min for completion. Tests can be developed to kits, which will be ideal for use in biosecurity, for the first line of screening (at the port or pond site) and during monitoring and surveillance programmes overall for the good management practices to reduce the risk of the disease.

Keywords: acute hepatopancreatic necrosis disease, AHPND, flow-through assay, FTA, farmer level, immunodot, pond site, shrimp

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Authors: Özlem Arpacı, Zeliha Soysal, Nuran Diril

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Benzoxazoles are heterocyclic compounds that have a fused benzene and an oxazole ring. These heterocyclic compounds are reported to have antibacterial, antitubercular, antifungal, antiviral, antioxidant and anticancer activities. In this study, some benzoxazole derivatives that have broad antimicrobial and potent antitumoral activities, are investigated their mutagenic activities with using the Ames Test. The Ames test was conducted using Salmonella typhimurium TA98, TA100 and TA102 tester strains in the standard plate incorporation assay in the absence of liver S9 fraction. The results are evaluated using SPSS and none of the benzoxazole derivatives showed mutagenic activity using the Ames test in the absence of S9 liver fraction.

Keywords: benzoxazoles, ames test, mutagenic activity, antimutagenic activity, antitumoral activity

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Authors: Görkem Cemali̇, Avram Aruh, Gamze Torun Köse, Erde Can ŞAfak

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In order to heal bone disorders, the conventional methods which involve the use of autologous and allogenous bone grafts or permanent implants have certain disadvantages such as limited supply, disease transmission, or adverse immune response. A biodegradable material that acts as structural support to the damaged bone area and serves as a scaffold that enhances bone regeneration and guides bone formation is one desirable solution. Poly(propylene fumarate) (PPF) which is an unsaturated polyester that can be copolymerized with appropriate vinyl monomers to give biodegradable network structures, is a promising candidate polymer to prepare bone tissue engineering scaffolds. In this study, hydroxyl-terminated PPF was synthesized and thermally cured with vinyl phosphonic acid (VPA) and diethyl vinyl phosphonate (VPES) in the presence of radical initiator benzoyl peroxide (BP), with changing co-monomer weight ratios (10-40wt%). In addition, the synthesized PPF was cured with VPES comonomer at body temperature (37oC) in the presence of BP initiator, N, N-Dimethyl para-toluidine catalyst and varying amounts of Beta-tricalcium phosphate (0-20 wt% ß-TCP) as filler via radical polymerization to prepare composite materials that can be used in injectable forms. Thermomechanical properties, compressive properties, hydrophilicity and biodegradability of the PPF/VPA and PPF/VPES copolymers were determined and analyzed with respect to the copolymer composition. Biocompatibility of the resulting polymers and their composites was determined by the MTS assay and osteoblast activity was explored with von kossa, alkaline phosphatase and osteocalcin activity analysis and the effects of VPA and VPES comonomer composition on these properties were investigated. Thermally cured PPF/VPA and PPF/VPES copolymers with different compositions exhibited compressive modulus and strength values in the wide range of 10–836 MPa and 14–119 MPa, respectively. MTS assay studies showed that the majority of the tested compositions were biocompatible and the overall results indicated that PPF/VPA and PPF/VPES network polymers show significant potential for applications as bone tissue engineering scaffolds where varying PPF and co-monomer ratio provides adjustable and controllable properties of the end product. The body temperature cured PPF/VPES/ß-TCP composites exhibited significantly lower compressive modulus and strength values than the thermal cured PPF/VPES copolymers and were therefore found to be useful as scaffolds for cartilage tissue engineering applications.

Keywords: biodegradable, bone tissue, copolymer, poly(propylene fumarate), scaffold

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Authors: Rachid Kacem, Sara Talbi, Yasmina Hemissi, Sofia Bouguattoucha

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The aim of the present work is the evaluation of the antioxidant activity of the Peganum harmala (P. harmala) seeds extracts. The antioxidant activity was evaluated by applying two methods, the method of ß-carotene bleaching and DPPH (2, 2-Diphenyl-1-Picryl-Hydrazyl). Using Folin-Ciocalteu assay, these results revealed that the concentration of polyphenols in EthOH E. (122.28 ± 2.24 µg GAE/mg extract) is the highest. The antiradical activity of the P. harmala seeds extracts on DPPH was found to be dose dependent with polyphenols concentration. The E. EthOH extract showed the highest antioxidant activity (IC = 252.10 ± 11.18 μg /ml). The test of β-carotene bleaching indicates that the E. EthOH of P. harmala showed the highest percentage of the antioxidant activity (49.88 %).

Keywords: antioxidant activity, Peganum harmala, polyphenols, flavonoids

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Authors: Hairin Taha, P. Narrima, M. A. Hapipah, A. M. Mustafa

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The chemical constituents and antioxidant activity of the leaf essential oils of Pseuduvaria monticola and Pseuduvaria macrophylla from the Annonaceae family were investigated. GC-TOFMS analyses identified 46 compounds from Pseuduvaria monticola and 11 compounds from Pseuduvaria macrophylla. The major constituents in the leaf essential oil of Pseuduvaria monticola were a-cadinol (13.0%), calamenene-cis (6.9%), alfa copaene (4%), and epizonarene (3.8%), while in the leaf essential oil of Pseuduvaria macrophylla were caryophyllene oxide (29.7%) and elimicin (28%). The antioxidant activity of both the essential oils were determined using the 2,2'-diphenyl-1-picrylhydrazyl assay (DPPH). The present study suggests that both essential oils demonstrated good antioxidant activity.

Keywords: Pseuduvaria monticola, Pseuduvaria macrophylla, leaf essential oils, GC-MSTOF, antioxidant activity

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Authors: Mustafa Ümi̇t Ünal, Nafi̇z Çeli̇ktaş, Aysun Şener, Sara Betül Dolgun, Duygu Keser

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The aim of this study was to compare acid and base pretreatment of switchgrass for bioethanol production. Switchgrass was pretreated with sulfuric acid and sodium hydroxide at 0.5, 1.0 and 1.5% (v/v) at 120, 140, 180 °C for 10, 60 and 90. Optimization of enzymatic hydrolysis of the pretreated switchgrass samples were carried out using three different enzyme mixtures (22.5 mg cellulase and 75 mg cellobiase /g biomass; 45 mg cellulase and 150 mg cellobiase /g biomass; 90 mg cellulase and 300 mg cellobiase /g biomass). Samples were removed at 24-h interval for fermentable sugar analyses with HPLC. The results showed that use of 90 mg cellulase and 300 mg cellobiase/g biomass resulted in the highest fermentable sugar formation. Furthermore, the highest fermentable sugar yield was obtained by pretreatment at 120 °C for 10 min using 1.0 % sodium hydroxide.

Keywords: switchgrass, acid pretreatment, enzymatic hydrolysis, base pretreatment, ethanol production

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Authors: Patrícia Severino, Luciana Nalone, Daniele Martins, Marco Chaud, Classius Ferreira, Cristiane Bani, Ricardo Albuquerque

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Hydrogels produced with polymers have been used in the development of dressings for wound treatment and tissue revitalization. Our study on polymer nanocomposites containing silver nanoparticles shows antimicrobial activity and applications in wound healing. The effects are linked with the slow oxidation and Ag⁺ liberation to the biological environment. Furthermore, bacterial cell membrane penetration and metabolic disruption through cell cycle disarrangement also contribute to microbial cell death. The silver antimicrobial activity has been known for many years, and previous reports show that low silver concentrations are safe for human use. This work aims to develop a hydrogel using natural polymers (sodium alginate and gelatin) combined with silver nanoparticles for wound healing and with antimicrobial properties in cutaneous lesions. The hydrogel development utilized different sodium alginate and gelatin proportions (20:80, 50:50 and 80:20). The silver nanoparticles incorporation was evaluated at the concentrations of 1.0, 2.0 and 4.0 mM. The physico-chemical properties of the formulation were evaluated using ultraviolet-visible (UV-Vis) absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and thermogravimetric (TG) analysis. The morphological characterization was made using transmission electron microscopy (TEM). Human fibroblast (L2929) viability assay was performed with a minimum inhibitory concentration (MIC) assessment as well as an in vivo cicatrizant test. The results suggested that sodium alginate and gelatin in the (80:20) proportion with 4 mM of AgNO₃ in the (UV-Vis) exhibited a better hydrogel formulation. The nanoparticle absorption spectra of this analysis showed a maximum band around 430 - 450 nm, which suggests a spheroidal form. The TG curve exhibited two weight loss events. DSC indicated one endothermic peak at 230-250 °C, due to sample fusion. The polymers acted as stabilizers of a nanoparticle, defining their size and shape. Human fibroblast viability assay L929 gave 105 % cell viability with a negative control, while gelatin presented 96% viability, alginate: gelatin (80:20) 96.66 %, and alginate 100.33 % viability. The sodium alginate:gelatin (80:20) exhibited significant antimicrobial activity, with minimal bacterial growth at a ratio of 1.06 mg.mL⁻¹ in Pseudomonas aeruginosa and 0.53 mg.mL⁻¹ in Staphylococcus aureus. The in vivo results showed a significant reduction in wound surface area. On the seventh day, the hydrogel-nanoparticle formulation reduced the total area of injury by 81.14 %, while control reached a 45.66 % reduction. The results suggest that silver-hydrogel nanoformulation exhibits potential for wound dressing therapeutics.

Keywords: nanocomposite, wound healing, hydrogel, silver nanoparticle

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Authors: Barbra Bhebhe, Melvyn Quan

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A serological survey was done to detect antibodies against Shuni virus (SHUV) from cattle in Western Kenya. In Kenya the disease status of SHUV in cattle has never been established. It is a zoonotic virus and even though studies have been carried out as early as the 1960s, little research has been published and SHUV is still not a well-recognised Orthobunyavirus. One hundred serum samples were collected from healthy cattle in Kenya and tested for antibodies against SHUV by a serum neutralization assay. All antibody titre values were greater than 1:160, with most of the samples greater than 1:320. Of the samples tested, 87 % had titres greater than 1:320, 12% had a titre of 1:320 and 2% had a titre of 1:160. Samples were classified as positive if the antibody titre was ≥ 1:10 and negative if < 1:10. This study suggests that cattle are exposed commonly to SHUV, which may be endemic in Kenya.

Keywords: Shuni virus, Orthobunyavuruses, serum neutralization test, cell-culture

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Authors: Sonu Gandhi, Neena Capalash, Prince Sharma, C. Raman Suri

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A sensitive chemiluminescence immunoassay (CIA) for heroin and its major metabolites is reported. The method is based on the competitive reaction of horseradish peroxidase (HRP)-labeled anti-MAM antibody and free drug in spiked urine samples. A hapten-protein conjugate was synthesized by using acidic derivative of monoacetyl morphine (MAM) coupled to carrier protein BSA and was used as an immunogen for the generation of anti-MAM (monoacetyl morphine) antibody. A high titer of antibody (1:64,0000) was obtained and the relative affinity constant (Kaff) of antibody was 3.1×107 l/mol. Under the optimal conditions, linear range and reactivity for heroin, mono acetyl morphine (MAM), morphine and codeine were 0.08, 0.09, 0.095 and 0.092 ng/mL respectively. The developed chemiluminescence inhibition assay could detect heroin and its metabolites in standard and urine samples up to 0.01 ng/ml.

Keywords: heroin, metabolites, chemiluminescence immunoassay, horse radish peroxidase

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Authors: W. Soufi, F. Boukli Hacene, S. Ghalem

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Alzheimer's disease (AD) is a neurodegenerative disease that leads to a progressive and permanent deterioration of nerve cells. This disease is progressively accompanied by an intellectual deterioration leading to psychological manifestations and behavioral disorders that lead to a loss of autonomy. It is the most frequent of degenerative dementia. Alzheimer's disease (AD), which affects a growing number of people, has become a major public health problem in a few years. In the context of the study of the mechanisms governing the evolution of AD disease, we have found that natural flavonoids are good acetylcholinesterase inhibitors that reduce the rate of ßA secretion in neurons. This work is to study the inhibition of acetylcholinesterase (AChE) which is an enzyme involved in Alzheimer's disease, by methods of molecular modeling. These results will probably help in the development of an effective therapeutic tool in the fight against the development of Alzheimer's disease. Our goal of the research is to study the inhibition of acetylcholinesterase (AChE) by molecular modeling methods.

Keywords: Alzheimer's disease, acetylcholinesterase, flavonoids, molecular modeling

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Authors: Seok Woo Chang

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Aim: Capseal I and Capseal II are calcium silicate and calcium phosphate based experimental root canal sealer. The aim of this study was to evaluate the biocompatibility and mineralization potential of Capseal I and Capseal II. Materials and Methods: The biocompatibility and mineralization-related gene expression (alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN)) of Capseal I and Capseal II were compared using methylthiazol tetrazolium assay and reverse transcription-polymerization chain reaction analysis, respectively. The results were analyzed by Kruskal-Wallis test. P-value of < 0.05 was considered significant. Result: Both Capseal I and Capseal II were favorable in biocompatibility and influenced the messenger RNA expression of ALP and BSP. Conclusion: Within the limitation of this study, Capseal is biocompatible and have mineralization promoting potential, and thus could be a promising root canal sealer.

Keywords: biocompatibility, mineralization-related gene expression, Capseal I, Capseal II

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Authors: Ibrahim Djelloul Berkane

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A survey was conducted to assess the presence of the virus in Citrus tristeza one of the main citrus regions of Algeria, namely the Chlef region, using the technique of Direct Tissue Print Immunoprinting Assay (DTBIA) and the Double Sandwich ELISA antibodies. A nursery citrus, lumber yards, and commercial orchards, which are the main varieties cultivated citrus were subjected to samples collected samples for laboratory analysis. 0.91% of the plants tested orchards were infected with CTV, while no positive case was detected at the nursery the yard, however, it is reported that an alarming rate of 10,5% of orchards tested at the common Chettia were infected with tristeza virus. The investigation was launched to identify the vector species tristeza revealed the presence of a vector is important Aphis gossypii.

Keywords: aphis, chlef, citrus, DAS-ELISA, DTBIA, tristeza

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Authors: Rajesh A. Rane, Shital S. Naphade, Rajshekhar Karpoormath

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Thiozolidin-4-one, a mimic of thiazolobenzimidazole (TBZ) has drawn many attentions due to its potent and selective inhibition against the HIV-1 and low toxicity by binding to the allosteric site of the reverse transcriptase (RT) as a non-nucleoside RT inhibitor (NNRTI). Similarly, marine bromopyrrole alkaloids are well known for their diverse array of anti-infective properties. Hence, we have reported synthesis and in vitro HIV-1 RT inhibitory activity of a series of 4-thiazolidinone-bromopyrrole alkaloid hybrids tethered with amide linker. The results of in vitro HIV-1 RT kit assay showed that some of the compounds, such as 4c, 4d, and 4i could effectively inhibit RT activity. Among them, compounds 4c having 4-chlorophenyl substituted 4-thiazolidione ring was the best one with the IC50 value of 0.26 µM. The sturdy emerges with key structure-activity relationship that pyrrole-NH-free core benefited inhibition against HIV-1 RT inhibition. This study identified conjugate 4c with potent activity and selectivity as promising compound for further drug development to HIV.

Keywords: antiviral drugs, bromopyrrole alkaloids, HIV-1 RT inhibition, 4-thiazolidinone

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Authors: Abdol-Hassan Doulah

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In this research, some of the inorganic complexes of uranyl with N- donor ligands were synthesized. Complexes were characteriezed by FT-IR and UV spectra, ¹HNMR, ¹³CNMR and some physical properties. The uranyl unit (UO2) is composed of a center of uranium atom with the charge (+6) and two oxygen atom by forming two U=O double bonds. The structure is linear (O=U=O, 180) and usually stable. So other ligands often coordinate to the U atom in the plane perpendicularly to the O=U=O axis. The antitumor activity of some of ligand and their complexes against a panel of human tumor cell lines (HT29: Haman colon adenocarcinoma cell line T47D: human breast adenocarcinoma cell line) were determined by MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. These data suggest that some of these compounds provide good models for the further design of potent antitumor compounds.

Keywords: inorganic, uranyl complex-donor ligands, Schiff bases, anticancer activity

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Authors: Nima Samie, Sekaran Muniandy, Zahurin Mohamed, M. S. Kanthimathi

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Although number of indole containing compounds have been reported to have anticancer properties in vitro but only a few of them show potential as anticancer compounds in vivo. The current study was to evaluate the mechanism of cytotoxicity of selected indolic compound in vivo and in vitro. In this context, we determined the potency of the compound in the induction of apoptosis, cell cycle arrest, and cytoskeleton rearrangement. HT-29, WiDr, CCD-18Co, human monocyte/macrophage CRL-9855, and B lymphocyte CCL-156 cell lines were used to determine the IC50 of the compound using the MTT assay. Analysis of apoptosis was carried out using immunofluorescence, acridine orange/ propidium iodide double staining, Annexin-V-FITC assay, evaluation of the translocation of NF-kB, oxygen radical antioxidant capacity, quenching of reactive oxygen species content, measurement of LDH release, caspase-3/-7, -8 and -9 assays and western blotting. The cell cycle arrest was examined using flowcytometry and gene expression was assessed using qPCR array. Results displayed a potent suppressive effect on HT-29 and WiDr after 24 h of treatment with IC50 value of 2.52±0.34 µg/ml and 2.13±0.65 µg/ml respectively. This cytotoxic effect on normal, monocyte/macrophage and B-cells was insignificant. Dipping in the mitochondrial membrane potential and increased release of cytochrome c from the mitochondria indicated induction of the intrinsic apoptosis pathway by the compound. Activation of this pathway was further evidenced by significant activation of caspase-9 and 3/7. The compound was also shown to activate the extrinsic pathways of apoptosis via activation of caspase-8 which is linked to the suppression of NF-kB translocation to the nucleus. Cell cycle arrest in the G1 phase and up-regulation of glutathione reductase, based on excessive ROS production were also observed. These findings were further investigated for inhibitory efficiency of the compound on colonic aberrant crypt foci in male rats. Rats were divided in to 5 groups: vehicle, cancer control, positive control groups and the groups treated with 25 and 50 mg/kg of compounds for 10 weeks. Administration of compound suppressed total colonic ACF formation up to 73.4%. The results also showed that treatment with the compound significantly reduced the level of malondialdehyde while increasing superoxide dismutase and catalase activities. Furthermore, the down-regulation of PCNA and Bcl2 and the up-regulation of Bax was confirmed by immunohistochemical staining. The outcome of this study suggest sthat the indolic compound is a potent anti-cancer agent against colon cancer and can be further evaluated by animal trial.

Keywords: indolic compound, chemoprevention, crypt, azoxymethane, colon cancer

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Authors: Kakali Bhadra

Abstract:

Harmalol administration caused remarkable reduction in proliferation of HepG₂ cells with GI₅₀ of 14.2 mM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism and differential scanning calorimetric analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 ^oC. Binding constant and stoichiometry was also calculated using the above biophysical techniques. Further, dose dependent apoptotic induction ability of harmalol was studied in HepG₂ cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub G_o/G₁ population made the cancer cell, HepG₂, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

Keywords: apoptosis, beta carboline alkaloid, comet assay, cytotoxicity, ROS

Procedia PDF Downloads 209