Search results for: spectrophotometric assay

Authors: A. Boveiri Monji, H. Yousefnia, S. Zolghadri, B. Salimi

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The main goal of this paper was to make use of green reagents as a substitute of perilous synthetic reagents and organic solvents for spectrophotometric determination of hafnium(IV). The extracts taken from six different kinds of tree leaves including Acer negundo, Ficus carica, Cerasus avium, Chimonanthus, Salix babylonica and Pinus brutia, were applied as green reagents for the experiments. In 6-M hydrochloric acid, hafnium reacted with the reagent to form a yellow product and showed maximum absorbance at 421 nm. Among tree leaves, Chimonanthus showed satisfactory results with a molar absorptivity value of 0.61 × 10⁴ l mol^-1 cm^-1 and the method was linear in the 0.3-9 µg mL ^-1 concentration range. The detection limit value was 0.064 µg mL^-1. The proposed method was simple, low cost, clean, and selective.

Keywords: hafnium, spectrophotometric determination, synthetic reagents, tree leaves

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Authors: A. Boveiri Monji, H. Yousefnia, M. Haji Hosseini, S. Zolghadri

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A clean spectrophotometric method for the determination of hafnium by using a green reagent, acidic extract of Ficus carica tree leaves is developed. In 6-M hydrochloric acid, hafnium reacts with this reagent to form a yellow product. The formed product shows maximum absorbance at 421 nm with a molar absorptivity value of 0.28 × 104 l mol⁻¹ cm⁻¹, and the method was linear in the 2-11 µg ml⁻¹ concentration range. The detection limit value was found to be 0.312 µg ml⁻¹. Except zirconium and iron, the selectivity was good, and most of the ions did not show any significant spectral interference at concentrations up to several hundred times. The proposed method was green, simple, low cost, and selective.

Keywords: spectrophotometric determination, Ficus caricatree leaves, synthetic reagents, hafnium

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Authors: Sulaiman Gafar Muhamad

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A rapid spectrophotometric method for the micro-determination of phenylephrine-HCl (PHE) has been developed. The proposed method involves the coupling of phenylephrine-HCl with diazotized 2,4-dinitroaniline in alkaline medium at λmax 455 nm. Under the present optimum condition, Beer’s law was obeyed in the range of 1.0-20 μg/ml of PHE with molar absorptivity of 1.915 ×104 l. mol-1.cm-1, with a relative error of 0.015 and a relative standard deviation of 0.024%. The current method has been applied successfully to estimate phenylephrine-HCl in pharmaceutical preparations (nose drop and syrup).

Keywords: diazo-coupling, 2, 4-dinitroaniline, phenylephrine-HCl, spectrophotometry

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Authors: Nesrine T. Lamie

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Four, accurate, precise, and sensitive spectrophotometric methods are developed for the simultaneous determination of a binary mixture containing amlodipine besylate (AM) and atenolol (AT) where AM is determined at its λmax 360 nm (0D), while atenolol can be determined by different methods. Method (A) is absorpotion factor (AFM). Method (B) is the new Ratio Difference method(RD) which measures the difference in amplitudes between 210 and 226 nm of ratio spectrum., Method (C) is novel constant center spectrophotometric method (CC) Method (D) is mean centering of the ratio spectra (MCR) at 284 nm. The calibration curve is linear over the concentration range of 10–80 and 4–40 μg/ml for AM and AT, respectively. These methods are tested by analyzing synthetic mixtures of the cited drugs and they are applied to their commercial pharmaceutical preparation. The validity of results was assessed by applying standard addition technique. The results obtained were found to agree statistically with those obtained by a reported method, showing no significant difference with respect to accuracy and precision.

Keywords: amlodipine, atenolol, absorption factor, constant center, mean centering, ratio difference

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Authors: Kristina Sakalauskiene, Renaldas Raisutis, Gintare Linkeviciute, Skaidra Valiukeviciene

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Cutaneous melanoma is a melanocytic skin tumour, which has a very poor prognosis while is highly resistant to treatment and tends to metastasize. Thickness of melanoma is one of the most important biomarker for stage of disease, prognosis and surgery planning. In this study, we hypothesized that the automatic analysis of spectrophotometric images and high-frequency ultrasonic 2D data can improve differential diagnosis of cutaneous melanoma and provide additional information about tumour penetration depth. This paper presents the novel complex automatic system for non-invasive melanocytic skin tumour differential diagnosis and penetration depth evaluation. The system is composed of region of interest segmentation in spectrophotometric images and high-frequency ultrasound data, quantitative parameter evaluation, informative feature extraction and classification with linear regression classifier. The segmentation of melanocytic skin tumour region in ultrasound image is based on parametric integrated backscattering coefficient calculation. The segmentation of optical image is based on Otsu thresholding. In total 29 quantitative tissue characterization parameters were evaluated by using ultrasound data (11 acoustical, 4 shape and 15 textural parameters) and 55 quantitative features of dermatoscopic and spectrophotometric images (using total melanin, dermal melanin, blood and collagen SIAgraphs acquired using spectrophotometric imaging device SIAscope). In total 102 melanocytic skin lesions (including 43 cutaneous melanomas) were examined by using SIAscope and ultrasound system with 22 MHz center frequency single element transducer. The diagnosis and Breslow thickness (pT) of each MST were evaluated during routine histological examination after excision and used as a reference. The results of this study have shown that automatic analysis of spectrophotometric and high frequency ultrasound data can improve non-invasive classification accuracy of early-stage cutaneous melanoma and provide supplementary information about tumour penetration depth.

Keywords: cutaneous melanoma, differential diagnosis, high-frequency ultrasound, melanocytic skin tumours, spectrophotometric imaging

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Authors: Siobhan O’Shea, Sangeetha Vijaysri Nair, Hee Cheol Kim, Charles Thomas Nugent, Cheuk Yan William Tong, Sam Douthwaite, Andrew Worlock

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The Aptima® HIV-1 Quant Dx Assay is a fully automated assay on the Panther system. It is based on Transcription-Mediated Amplification and real time detection technologies. This assay is intended for monitoring HIV-1 viral load in plasma specimens and for the detection of HIV-1 in plasma and serum specimens. Nine-hundred and seventy nine specimens selected at random from routine testing at St Thomas’ Hospital, London were anonymised and used to compare the performance of the Aptima HIV-1 Quant Dx assay and Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0. Two-hundred and thirty four specimens gave quantitative HIV-1 viral load results in both assays. The quantitative results reported by the Aptima Assay were comparable those reported by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 with a linear regression slope of 1.04 and an intercept on -0.097. The Aptima assay detected HIV-1 in more samples than the Roche assay. This was not due to lack of specificity of the Aptima assay because this assay gave 99.83% specificity on testing plasma specimens from 600 HIV-1 negative individuals. To understand the reason for this higher detection rate a side-by-side comparison of low level panels made from the HIV-1 3rd international standard (NIBSC10/152) and clinical samples of various subtypes were tested in both assays. The Aptima assay was more sensitive than the Roche assay. The good sensitivity, specificity and agreement with other commercial assays make the HIV-1 Quant Dx Assay appropriate for both viral load monitoring and detection of HIV-1 infections.

Keywords: HIV viral load, Aptima, Roche, Panther system

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Authors: Hawraa Zbeeb, Hala Khalifeh, Mohamad Khalil, Francesca Storace, Francesca Baldini, Giulio Lupidi, Laura Vergani

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Sarcopoterium spinosum, a widely distributed spiny shrub belonging to the Rosaceae family, is rich in essential and beneficial constituents. In fact, S. spinosum fruits and roots are traditionally used as herbal medicine in the eastern Mediterranean landscape, and this shrub is mentioned as a medicinal plant in a large number of ethnobotanical surveys. Aqueous root extracts from S. spinosum are used by traditional medicinal practitioners for weight loss treatment of diabetes and pain. Moreover, the anti-diabetic activity of S. spinosum root extract has been reported in different studies, but the beneficial effects of aerial parts, especially fruits, have not been elucidated yet. The aim of the present study was to investigate the in vitro antioxidant and lipid-lowering properties of an ethanolic extract from S. spinosum fruits using both hepatic (FaO) and endothelial (HECV) cells in an attempt to evaluate its possible employment as a nutraceutical supplement. First of all, in vitro spectrophotometric assays were employed to characterize the extract. The total phenol content (TPC) was evaluated by Folin–Ciocalteu spectrophotometric method and the radical scavenging activity was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. After that, the beneficial effects of the extract were tested on cells. FaO cells treated for 3 hours with 0.75 mM oleate/palmitate mix (1:2 molar ratio) mimic in vitro a moderate hepato-steatosis. HECV cells exposed for 1 hour to 100 µM H₂O₂ mimic an oxidative insult leading to oxidative stress conditions. After the metabolic and oxidative insult, both cell lines were treated with increasing concentrations of the S. spinosum extract (1, 10, 25 µg/mL) for 24 hours. The results showed the S. spinosum ethanolic extract is rather rich in phenols (TPC of 18.6 mgGAE/g dry extracts). Moreover, the extract showed a good scavenging ability in vitro (IC₅₀ 15.9 µg/ml and 10.9 µg/ml measured by DPPH and ABTS assays, respectively). When the extract was tested on cells, the results showed that it could ameliorate some markers of cell dysfunction. The three concentrations of the extract led to a significant decrease in the intracellular triglyceride (TG) content in steatotic FaO cells measured by spectrophotometric assay. On the other hand, HECV cells treated with increasing concentrations of the extract did not result in a significant decrease in both lipid peroxidation measured by the Thiobarbituric Acid Reactive Substances (TBARS) assay, and in reactive oxygen species (ROS) production measured by fluorometric analysis after DCF staining. Interestingly, the ethanolic extract was able to accelerate the wound repair of confluent HECV cells with respect to H₂O₂-insulted cells as measured by T-scratch assay. Taken together, these results seem to indicate that the ethanol extract from S. spinosum fruits is rich in phenol compounds and plays considerable lipid-lowering activity in vitro on steatotic hepatocytes and accelerates wound healing repair on endothelial cells. In light of that, the ethanolic extract from S. spinosum fruits could be a potential candidate for nutraceutical applications.

Keywords: antioxidant activity, ethanolic extract, lipid-lowering activity, phenolic compounds, Sarcopoterium spinosum fruits

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Authors: Chand Pasha

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A Simple, accurate and precise spectrophotometric method for the quantitative analysis of determination of 5-aminosalicylic acid is described. This method is based on the reaction of 5-aminosalicylic acid with nitrite in acid medium to form diazonium ion, which is coupled with acetylacetone in basic medium to form azo dyes, which shows absorption maxima at 470 nm. The method obeys Beer’s law in the concentration range of 0.5-11.2 gml-1 of 5-aminosalicylic acid with acetylacetone. The molar absorptivity and Sandell’s sensitivity of 5-aminosalicylic acid -acetylacetone azo dye is 2.672 ×104 lmol-1cm-1, 5.731 × 10-3 gcm-2 respectively. The dye formed is stable for 10 hrs. The optimum reaction conditions and other analytical parameters are evaluated. Interference due to foreign organic compounds have been investigated. The method has been successfully applied to the determination of 5-aminosalicylic acid in pharmaceutical samples.

Keywords: spectrophotometry, diazotization, mesalazine, nitrite, acetylacetone

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Authors: Balgabay Sadepovich Maikanov, Laura Tyulegenovna Auteleyeva, Seidenova Simbat Polatbekovna

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In this article reduced nutritional value of the rabbits’ meat at 1, 1 dimethylhydrazine experimental toxicosis is shown. The assay was performed on liquid chromatograph SHIMADZU LC-20 Prominence (Japan) with fluorometric and spectrophotometric detector. This research has revealed that samples of rabbit meat of the experimental group had significant differences from the control group:in amino acids concentration from 1.2% to 9.1%; vitamin concentration from 11.2% to 60.5%, macro – minerals concentration from 17.4% to 78.1% and saturated fatty acids concentration from 17,1% to 34.5%, respectively. The decrease in the chemical composition of rabbits’ meat at 1,1 dimethylhydrazine toxicosis may be due to changes in the internal processes associated with impaired metabolic homeostasis of animals.

Keywords: 1, 1-dimethylhydrazine, metabolic homeostasis, nutritional value, rabbit meat

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Authors: Chand Pasha, Yasser Turki Alharbi, Krasamira Stancheva

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A simple spectrophotometric method for the determination of folic acid in pharmaceutical dosage samples was developed. The method is based on the diazotization reaction of thiourea with sodium nitrite in acidic medium yields diazonium compounds, which is then coupled with folic acid in basic medium yields yellow coloured azo dyes. Beer’s Lamberts law is observed in the range 0.5 – 16.2 μgmL-1 at a maximum wavelength of 416nm. The molar absorbtivity, sandells sensitivity, linear regression equation and detection limit and quantitation limit were found to be 5.695×104 L mol-1cm-1, 7.752×10-3 g cm-2, y= 0.092x - 0.018, 0.687 g mL-1 and 2.083 g mL-1. This method successfully determined Folate in Pharmaceutical formulations.

Keywords: folic acid determination, spectrophotometry, diazotization, thiourea, pharmaceutical dosage samples

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Authors: P. Singh, R. M. Banik

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Artificial neural network (ANN) was employed to optimize assay parameters viz., time, temperature, pH of reaction mixture, enzyme volume and substrate concentration of L-glutaminase from Bacillus cereus MTCC 1305. ANN model showed high value of coefficient of determination (0.9999), low value of root mean square error (0.6697) and low value of absolute average deviation. A multilayer perceptron neural network trained with an error back-propagation algorithm was incorporated for developing a predictive model and its topology was obtained as 5-3-1 after applying Levenberg Marquardt (LM) training algorithm. The predicted activity of L-glutaminase was obtained as 633.7349 U/l by considering optimum assay parameters, viz., pH of reaction mixture (7.5), reaction time (20 minutes), incubation temperature (35˚C), substrate concentration (40mM), and enzyme volume (0.5ml). The predicted data was verified by running experiment at simulated optimum assay condition and activity was obtained as 634.00 U/l. The application of ANN model for optimization of assay conditions improved the activity of L-glutaminase by 1.499 fold.

Keywords: Bacillus cereus, L-glutaminase, assay parameters, artificial neural network

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Authors: V. Nigam, V. Nambiar

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Aegle Marmelos leaf has been used as a remedy for various gastrointestinal infections and lowering blood sugar level in traditional system of medicine in India due to the presence of various constituents such as flavonoids, tannins and alkaloids (eg. Aegelin, Marmelosin, Luvangetin).The objective of the present study was to evaluate the total antioxidant activity, total and individual phenol content of the wild and cultivated variety of Aegle marmelos leaves to assess the role of this plant in ethanomedicine in India. The methanolic extracts of the leaves were screened for total antioxidant capacity through Ferric Reducing Antioxidant Potential (FRAP) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay; Total Phenol content (TPC) through spectrophotometric technique based on Folin Ciocalteau assay and for qualitative estimation of phenols, High performance Liquid Chromatography was used. The TPC of wild and cultivated variety was 7.6% and 6.5% respectively whereas HPLC analysis for quantification of individual polyphenol revealed the presence of gallic acid, chlorogenic acid and Ferullic acid in wild variety whereas gallic acid, Ferullic acid and pyrocatechol in cultivated variety. FRAP values and IC 50 value (DPPH) for wild and cultivated variety was 14.65 μmol/l and 11.80μmol/l; 437 μg/ml and 620μg/ml respectively and thus it can be used as potential inhibitor of free radicals. The wild variety was having more antioxidant capacity than the cultivated one it can be exploited further for its therapeutic application. As Aegle marmelos is rich in antioxidant, it can be used as food additives to delay the oxidative deterioration of foods and as nutraceutical in medicinal formulation against degenerative diseases like diabetes.

Keywords: antioxidant activity, aegle marmelos, antidiabetic, nutraceutical

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Authors: Shashikant R. Kuchekar, Somnath D. Bhumkar, Haribhau R. Aher, Bhaskar H. Zaware, Ponnadurai Ramasami

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A precise, sensitive, rapid and selective method for the solvent extraction, spectrophotometric determination of palladium(II) using para-methylphenyl thiourea (PMPT) as an extractant is developed. Palladium(II) forms yellow colored complex with PMPT which shows an absorption maximum at 300 nm. The colored complex obeys Beer’s law up to 7.0 µg ml^-1 of palladium. The molar absorptivity and Sandell’s sensitivity were found to be 8.486 x 10³ l mol^-1cm^-1 and 0.0125 μg cm^-2 respectively. The optimum conditions for the extraction and determination of palladium have been established by monitoring the various experimental parameters. The precision of the method has been evaluated and the relative standard deviation has been found to be less than 0.53%. The proposed method is free from interference from large number of foreign ions. The method has been successfully applied for the determination of palladium from alloy, synthetic mixtures corresponding to alloy samples.

Keywords: solvent extraction, PMPT, Palladium (II), spectrophotometry

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Authors: Shimayali Kaushal, Nitesh Priyadarshi, Nitin Kumar Singhal

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Food borne bacterial species have been identified as major pathogens in most of the severe pathogen-related diseases among humans which result in great loss to human health and food industry. Conventional methods like plating and enzyme-linked immune sorbent assay (ELISA) are time-consuming, laborious and require specialized instruments. Nanotechnology has emerged as a great field in case of rapid detection of pathogens in recent years. The AuNRs material has good electro-optical properties due to its larger light absorption band and scattering in surface plasmon resonance wavelength regions. By exploiting the sugar-based adhesion properties of microorganism, we can use the glycoconjugates capped gold nanorods as a potential nanobiosensor to detect the foodborne pathogen. In the present study, polyethylene glycol (PEG) coated gold nanorods (AuNRs) were prepared and functionalized with different types of carbohydrates and further characterized by UV-Visible spectrophotometry, dynamic light scattering (DLS), transmission electron microscopy (TEM). The reactivity of above said nano-biosensor was probed by lectin binding assay and also by different strains of foodborne bacteria by using spectrophotometric and microscopic techniques. Due to the specific interaction of probe with foodborne bacteria (Escherichia coli, Pseudomonas aeruginosa), our nanoprobe has shown significant and selective ablation of targeted bacteria. Our findings suggest that our nanoprobe can be an ideal candidate for selective optical detection of food pathogens and can reduce loss to the food industry.

Keywords: glyco-conjugates, gold nanorods, nanobiosensor, nanoprobe

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Authors: Mohamed Korany, Azza Gazy, Essam Khamis, Marwa Adel, Miranda Fawzy

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Background: Two new, simple and specific methods; First, a Zero-crossing first-derivative technique and second, a chemometric-assisted spectrophotometric artificial neural network (ANN) were developed and validated in accordance with ICH guidelines. Both methods were used for the simultaneous estimation of the two closely related antioxidant nutraceuticals ; Coenzyme Q10 (Q) ; also known as Ubidecarenone or Ubiquinone-10, and Vitamin E (E); alpha-tocopherol acetate, in their pharmaceutical binary mixture. Results: For first method: By applying the first derivative, both Q and E were alternatively determined; each at the zero-crossing of the other. The D1 amplitudes of Q and E, at 285 nm and 235 nm respectively, were recorded and correlated to their concentrations. The calibration curve is linear over the concentration range of 10-60 and 5.6-70 μg mL-1 for Q and E, respectively. For second method: ANN (as a multivariate calibration method) was developed and applied for the simultaneous determination of both analytes. A training set (or a concentration set) of 90 different synthetic mixtures containing Q and E, in wide concentration ranges between 0-100 µg/mL and 0-556 µg/mL respectively, were prepared in ethanol. The absorption spectra of the training sets were recorded in the spectral region of 230–300 nm. A Gradient Descend Back Propagation ANN chemometric calibration was computed by relating the concentration sets (x-block) to their corresponding absorption data (y-block). Another set of 45 synthetic mixtures of the two drugs, in defined range, was used to validate the proposed network. Neither chemical separation, preparation stage nor mathematical graphical treatment were required. Conclusions: The proposed methods were successfully applied for the assay of Q and E in laboratory prepared mixtures and combined pharmaceutical tablet with excellent recoveries. The ANN method was superior over the derivative technique as the former determined both drugs in the non-linear experimental conditions. It also offers rapidity, high accuracy, effort and money saving. Moreover, no need for an analyst for its application. Although the ANN technique needed a large training set, it is the method of choice in the routine analysis of Q and E tablet. No interference was observed from common pharmaceutical additives. The results of the two methods were compared together

Keywords: coenzyme Q10, vitamin E, chemometry, quantitative analysis, first derivative spectrophotometry, artificial neural network

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Authors: Puchita Chokcharoenying

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Polyphenols are the most abundant antioxidants found in plants, and they are highly effective at scavenging oxidative free radicals. Antioxidants are substances found in medicinal plants to help prevent heart disease, stroke, and some cancers. This study focused on evaluating the flavonoids content of Chrysanthemum Indicum and determine their antioxidant capacity by using DPPH and ABTS radical scavenging capacity assay. The total flavonoid content of C. indicumextract was determined and expressed as quercetin equivalents (QE)/g measured by an aluminiumchloride colorimetric method. The results showed that the IC50 of C. indicum extract were 83.57μg/mL ± 0.875 and52.57μg/mL ± 0.632for DPPH and ABTS, respectively. C. indicumextract exhibited antioxidant activities as a concentration dependent manner. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. In summary, C. indicum extract is rich in flavonoids, which have potent antioxidant properties. Thus, C. indicum extract is a good source of antioxidants and can be developed for medicinal purposes. Nevertheless, more research on the antioxidant activity of C. indicum extract and in vivo antioxidant studies are still needed.

Keywords: ABTS assay, antioxidant, chrysanthemum indicum, DPPH assay, total flavonoid content

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Authors: Ajay Pratap Singh Lodhi, Deepak Kumar

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Vegetable oil-based environmentally friendly metalworking fluids (MWFs) are formulated. The tribological performance, cytotoxicity, and corrosion resistance of the formulated fluids (FFs) are evaluated and benchmarked with commercial mineral oil-based MWFs (CF). Results show that FFs exhibited better machining characteristics (roughness, cutting forces, and surface morphology) during machining than CF. MTT assay and Live dead cell assay confirm the cytocompatibility nature of the FFs relative to the toxic CF. Electrochemical analysis shows that FFs and CF exhibited comparable corrosion current density.

Keywords: corrosion inhibitors, cytotoxicity, machining, MTT assay, Taguchi method, vegetable oil

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Authors: Dawit Gebreegzabher Hagos, Yazezew Kebede Kiro, Mahmud Abdulkader, Henk H. D. F. Schallig, Dawit Wolday

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Rapid and accurate visceral leishmaniasis (VL) diagnosis is needed to initiate prompt treatment to reduce morbidity and mortality. Here, we evaluated the performance of loop-mediated isothermal amplification (LAMP) assay for the diagnosis of VL from blood in an endemic area in Ethiopia. LAMP was positive in 117/122 confirmed VL cases and negative in 149/152 controls, resulting in a sensitivity of 95.9% (95% CI: 90.69–98.66) and a specificity of 98.0% (95% CI: 94.34–99.59), respectively. The sensitivity of the LAMP assay was 95.0% (95% CI: 88.61–98.34) in HIV-negatives and 100% (95% CI: 85.18–100.0) in HIV-positives. Compared with microscopy, LAMP detected 82/87 (94.3%, 95% CI: 87.10–98.11) of the microscopy1 cases and was negative in 11/27 (40.7%, 95% CI: 22.39–61.20) of the microscopy2 cases. Compared with the rK39 serology, LAMP detected 113/120 (94.2%, 95% CI: 88.35–97.62) of the rK391 cases and was negative in 149/154 (96.8%, 95% CI: 92.59–98.94) of the rK392 cases. However, when compared with microscopy only, rK39 detected 83/87 (95.4%, 95% CI: 88.64–98.73) of the microscopy1 cases and negative in only 12/27 (44.4%, 95% CI: 25.48–64.67) of the microscopy– cases. There was an excellent agreement between rK39 and LAMP (Kappa 5 0.91, 95% CI: 0.86–0.96). Furthermore, an algorithm using rK39 followed by LAMP would yield a sensitivity of 99.2% (95%CI: 95.52–99.89) and a specificity of 98.0% (95% CI: 94.34–99.59). The findings demonstrate that the LAMP assay is an accurate and rapid molecular assay for VL diagnosis, including in HIV-1 co-infected patients, in an endemic setting.

Keywords: visceral leishmaniasis, HIV, diagnosis, LAMP, Ethiopia

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Authors: Jirathan Pongchababnapa

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Polyphenols are the most common antioxidant found in plants and are efficient in capturing oxidative free radicals. Antioxidants are substances found in medicinal plants which may have a protective role to play in certain conditions such as heart disease, stroke and some cancers. By relying on these benefits, we have traced out the presence of antioxidant in Camellia sinensis leaves extract. This study aims to evaluate flavonoids content in C. sinensisextract and investigate antioxidant activities by using DPPH and ABTS radical scavenging capacity assay. The total flavonoid content of C. Sinensis extract was determined and expressed as quercetin equivalents (QE)/g measured by the aluminum chloride colorimetric method. The results showed that the IC₅₀ of C. Sinensis leaves extract were 40.90 μg/mL ± 0.755 and32.96 μg/mL ± 0.679 for DPPH and ABTS, respectively. C. Sinensis extract at increasing concentration showed antioxidant activities as a concentration dependent manner. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. In conclusion, C. Sinensis extract consisted of a high amount of flavonoids content which possesses potent antioxidant activity. However, further investigation on the identification of pure compound of this plant and molecular antioxidant assays are still required.

Keywords: ABTS assay, antioxidant, camellia sinensis, DPPH assay, total flavonoid content

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Authors: Tawqeer Ali Syed, Prakash Chandra

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This study aims to evaluate the antileishmanial activity of Nigella sativa black seed extract. This well-known plant extract was taken from the botanical garden of Kashmir. Materials and Methods: The methanolic extracts of these plants were screened for their antileishmanial activity against Leishmania major using 3‑(4.5‑dimethylthiazol‑2yl)‑2.5‑diphenyltetrazolium bromide assay or MTT assay. Results: The methanolic extract of Nigella sativa showed potential antileishmanial activity at an inhibition% value of 80.29% ± 0.65%. IC 50 was calculated after 48 hours to be 964.3 µg/ml. Conclusion: Considering these results, these medicinal plants from Kashmir could serve as potential drug sources for antileishmanial compounds.

Keywords: MTT assay, antileishmanial, cell viability, Nigella sativa

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Authors: Hadiatullah, T. S. D. Desak Ketut, A. A. Ayu, A. N. Isna, D. P. Ririn

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Spirogyra sp. is genus of microalgae which has a high carbohydrate content that used as a best medium for bacterial fermentation to produce cellulose. This study objective to determine the effect of pulp banana in the fermented paper production process using Spirogyra sp. and characterizing of the paper product. The method includes the production of bacterial cellulose, assay of the effect fermented paper interpolating with banana pulp using Spirogyra sp., and the assay of paper characteristics include gram-mage paper, water assay absorption, thickness, power assay of tensile resistance, assay of tear resistance, density, and organoleptic assay. Experiments were carried out with completely randomized design with a variation of the concentration of sewage treatment in the fermented paper production interpolating banana pulp using Spirogyra sp. Each parameter data to be analyzed by Anova variance that continued by real difference test with an error rate of 5% using the SPSS. Nata production results indicate that different carbon sources (glucose and sugar) did not show any significant differences from cellulose parameters assay. Significantly different results only indicated for the control treatment. Although not significantly different from the addition of a carbon source, sugar showed higher potency to produce high cellulose. Based on characteristic assay of the fermented paper showed that the paper gram-mage indicated that the control treatment without interpolation of a carbon source and a banana pulp have better result than banana pulp interpolation. Results of control gram-mage is 260 gsm that show optimized by cardboard. While on paper gram-mage produced with the banana pulp interpolation is about 120-200 gsm that show optimized by magazine paper and art paper. Based on the density, weight, water absorption assays, and organoleptic assay of paper showing the highest results in the treatment of pulp banana interpolation with sugar source as carbon is 14.28 g/m2, 0.02 g and 0.041 g/cm2.minutes. The conclusion found that paper with nata material interpolating with sugar and banana pulp has the potential formulation to produce super-quality paper.

Keywords: cellulose, fermentation, grammage, paper, Spirogyra sp.

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Authors: Monthon Tangjitmungman

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Numerous diseases have been linked to oxidative stress, in which a disproportion of free radicals in the body leads to tissue or cell damage. Polyphenols are the most abundant antioxidants found in plants, and they are highly effective at scavenging oxidative free radicals. Due to the presence of phenolic compounds in Caesalpinia sappan has been discovered to have antioxidant activity. It has several health benefits, the most important of which is preventing cardiovascular and cancer diseases. This study aimed to determine the phenolic content and antioxidant activity of C. sappan extract using a variety of antioxidant assays. The extract of C. sappan was made using a mixture of solvents (ethyl alcohol: water in ratio 8:2). The total phenolic content of C. sappan extract was determined and expressed as gallic acid equivalents using the Folin-Cioucalteu method (GAE). The antioxidant activity of C. sappan extract was assessed using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and the ABTS radical scavenging capacity assay. An association was found between antioxidant activity and total phenol content. The antioxidant activity of C. sappan extract was also determined by DPPH and ABTS assays. The IC50 values for C. sappan extract from DPPH and ABTS assays were 54.48 μg/mL ± 0.545 and 25.46 μg/mL ± 0.790, respectively, in the DPPH assay. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. In conclusion, C. sappan extract contains a high level of total phenolics and exhibits significant antioxidant activity. Nevertheless, more research should be done on the antioxidant activity, such as SOD and ROS scavenging assays and in vivo experiments, to determine whether the compound has antioxidant activity.

Keywords: ABTS assay, antioxidant activity, Caesalpinia sappan, DPPH assays, total phenolic content

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Authors: Eun Ko, Moonsung Choi, Sooim Shin

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4,4’-Dichlorodiphenyltrichloroethane (4,4’-DDT) is colorless, odorless organochlorine and known as persistent toxic organic pollutant accumulated in organs. In this study, effects of 4,4’-DDT on activities of mitochondrial electron transport chain system was analyzed. 4,4’-DDT is directly treated to isolated mitochondria from eyes of zebrafish and then activities of mitochondrial complex I, II, III, IV were measured spectrophotometrically. The reaction was proceeded immediately after adding 4,4’-DDT to examine the short-term exposing effects of persistent organic pollutant. As a result, high concentration of 4,4’-DDT treated mitochondria exhibited slightly enhanced activity in all complexes than non-treated one except complex III in male. Particularly, 4,4’-DDT was more effective on enzymatic activity in mitochondria isolated from eyes of male zebrafish. These results represented that 4,4’-DDT might temporarily induce to open up ion channel on isolated mitochondria resulting in increasing the functional activity of mitochondrial electron transport chain system.

Keywords: electron transport chain, mitochondrial function, persistent organic pollutant, spectrophotometric assay, zebrafish

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Authors: Ibrahim Aly, Rabab Zalat, Bahaa EL Deen W. El Aswad, Ismail M. Moharm, Basam M. Masoud, Tarek Diab

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The objective of the present study was to evaluate the novel nanomagnetic beads based–latex agglutination assay (NMB-LAT) as a simple test for diagnosis of S. haematobium as well as standardize the novel nanomagnetic beads based –ELISA (NMB-ELISA). According to urine examination this study included 85 S. haematobium infected patients, 30 other parasites infected patients and 25 negative control samples. The sensitivity of novel NMB-LAT was 82.4% versus 96.5% and 88.2% for NMB-ELISA and currently used sandwich ELISA respectively. The specificity of NMB-LAT was 83.6% versus 96.3% and 87.3% for NMB-ELISA and currently used sandwich ELISA respectively. In conclusion, the novel NMB-ELISA is a valuable applicable diagnostic technique for diagnosis of human schistosomiasis haematobium. The novel NMB-ELISA assay is a suitable applicable diagnostic method in field survey especially when followed by ELISA as a confirmatory test in query false negative results. Trials are required to increase the sensitivity and specificity of NMB-ELISA assay.

Keywords: diagnosis, iatex agglutination, nanomagnetic beads, sandwich ELISA

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Authors: Shayma Munqith Al-Baker, Fadhl Ahmed Saeed Al-Gasha’a, Samira Hamid Hanash, Ahmed Ali Al-Hazmi

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A total of 200 samples (180 fecal materials and 20 organ samples) were collected from (5 different poultry farms, 10 local poultry shops, 5 houses poultry, 5 Eggs stores shops and 5 hand slaughters centers) in Ibb city, Yemen, 2014. According to morphological, cultural, as well as biochemical characterization and serological tests, 59 29.5% isolates were identified as Salmonella spp. and all Salmonella isolates were categorized by serotype, which comprised of, 37 62.71% Salmonella Typhimurium serovar, 21 35.59%. Salmonella Enteritidis serovar and 11.69% Salmonella Heidelberg serovar. Antibiotic sensitivity test was done for bacterial isolates and the results showed there were clear differences in antibiotic resistant. Antimicrobial susceptibility of the isolates varies as follows: Ofloxacin 79.66%, Ciprofloxacin 67.80%, Colistin 59.32% and Gentamycin 52.54%. All of isolates were resistant to Erythromycin, Penicillin and Lincomycin. Antibacterial activity was done for both aqueous and ethanol extracts of Dodonaea viscosa plant by using well and disc diffusion assay. The results indicated that well diffusion assay had best results than disc diffusion assay, the highest inhibition zone was 22 mm for well diffusion and 15 mm for disc diffusion assay, the results observed that ethanol extract had best antibacterial effect than aqueous extract which the percentage of bacterial isolates affected with ethanol extract was 71.19% comparing with aqueous extract 28.81% by using disc diffusion assay, while the percentage of bacterial isolates affected with ethanol extract was 88.13% comparing with aqueous extract 52.54% by using will diffusion assay.

Keywords: Salmonella spp, Dodonaea viscosa, antimicrobial and salmonellosis

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Authors: Ayman K. El Essawy, Amal M. Hosny, Hala M. Abu Shady

Abstract:

The rapid detection of TB and drug resistance, both optimizes treatment and improves outcomes. In the current study, respiratory specimens were collected from 155 patients. Conventional susceptibility testing and MIC determination were performed for rifampicin (RIF) and isoniazid (INH). Genotype MTBDRplus assay, which is a molecular genetic assay based on the DNA-STRIP technology and specific gene sequencing with primers for rpoB, KatG, and mab-inhA genes were used to detect mutations associated with resistance to rifampicin and isoniazid. In comparison to other categories, most of rifampicin resistant (61.5%) and isoniazid resistant isolates (47.1%) were from patients relapsed in treatment. The genotypic profile (using Genotype MTBDRplus assay) of multi-drug resistant (MDR) isolates showed missing of katG wild type 1 (WT1) band and appearance of mutation band katG MUT2. For isoniazid mono-resistant isolates, 80% showed katG MUT1, 20% showed katG MUT1, and inhA MUT1, 20% showed only inhA MUT1. Accordingly, 100% of isoniazid resistant strains were detected by this assay. Out of 17 resistant strains, 16 had mutation bands for katG distinguished high resistance to isoniazid. The assay could clearly detect rifampicin resistance among 66.7% of MDR isolates that showed mutation band rpoB MUT3 while 33.3% of them were considered as unknown. One mono-resistant rifampicin isolate did not show rifampicin mutation bands by Genotype MTBDRplus assay, but it showed an unexpected mutation in Codon 531 of rpoB by DNA sequence analysis. Rifampicin resistance in this strain could be associated with a mutation in codon 531 of rpoB (based on molecular sequencing), and Genotype MTBDRplus assay could not detect the associated mutation. If the results of Genotype MTBDRplus assay and sequencing were combined, this strain shows hetero-resistance pattern. Gene sequencing of eight selected isolates, previously tested by Genotype MTBDRplus assay, could detect resistance mutations mainly in codon 315 (katG gene), position -15 in inhA promotes gene for isoniazid resistance and codon 531 (rpoB gene) for rifampicin resistance. Genotyping techniques allow distinguishing between recurrent cases of reinfection or reactivation and supports epidemiological studies.

Keywords: M. tuberculosis, rpoB, KatG, inhA, genotype MTBDRplus

Procedia PDF Downloads 115

Authors: Aqeela Ashraf, Muhammad Imran, Tahir Yaqub, Muhammad Tayyab, Yung Fu Chang

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For the identification of bovine mastitic pathogen, an economical, rapid and sensitive molecular diagnostic assay is developed by PCR multiplexing of gene and pathogenic species specific DNA sequences. The multiplex PCR assay is developed for detecting nine important bacterial pathogens causing mastitis Worldwide. The bacterial species selected for this study are Streptococcus agalactiae, Streptococcus dysagalactiae, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, Staphylococcus haemolyticus, Staphylococcus chromogenes Mycoplasma bovis and Staphylococcus epidermidis. A single reaction assay was developed and validated by 27 reference strains and further tested on 276 bacterial strains obtained from culturing mastitic milk. The multiplex PCR assay developed here is further evaluated by applying directly on genomic DNA isolated from 200 mastitic milk samples. It is compared with bacterial culturing method and proved to be more sensitive, rapid, economical and can specifically identify 9 bacterial pathogens in a single reaction. It has detected the pathogens in few culture negative mastitic samples. Recognition of disease is the foundation of disease control and prevention. This assay can be very helpful for maintaining the udder health and milk monitoring.

Keywords: multiplex PCR, bacteria, mastitis, milk

Procedia PDF Downloads 293

Authors: J. Hidalgo de Quintana, I. Stoner, M. Tackett, G. Doran, C. Rafferty, A. Windemuth, J. Tytell, D. Pregibon

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We have developed the Multiplexed Circulating microRNA assay that allows the detection of up to 68 microRNA targets per sample. The assay combines particle­based multiplexing, using patented Firefly hydrogel particles, with single­ step RT-PCR signal. Thus, the Circulating microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target­-specific qPCR. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the Circulating microRNA assay are interpreted using Firefly Analysis Workbench, which allows visualization, normalization, and export of experimental data. To aid discovery and validation of biomarkers, we have generated fixed panels for Oncology, Cardiology, Neurology, Immunology, and Liver Toxicology. Here we present the data from several studies investigating circulating and tumor microRNA, showcasing the ability of the technology to sensitively and specifically detect microRNA biomarker signatures from fluid specimens.

Keywords: biomarkers, biofluids, miRNA, photolithography, flowcytometry

Procedia PDF Downloads 321

Authors: Eman Abdullah Morsi, Hend Okasha, Heba Abdel Hady, Mortada El-Sayed, Mohamed Abbas Shemis

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Context: Linum usitatissimum (Linn), known as Flaxseed, is one of the most important medicinal plants traditionally used for various health as nutritional purposes. Objective: Estimation of total phenolic and flavonoid contents as well as evaluate the antioxidant using α, α-diphenyl-β-picrylhydrazyl (DPPH), 2-2'azinobis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and total antioxidant capacity (TAC) assay and investigation of anti-inflammatory by Bovine serum albumin (BSA) and anticancer activities of hepatocellular carcinoma cell line (HepG2) and breast cancer cell line (MCF7) have been applied on hexane, ethyl acetate, n-butanol and methanol extracts and also, fractions of methonal extract (hexane, ethyl acetate and n-butanol). Materials and Methods: Phenolic and flavonoid contents were detected using spectrophotometric and colorimetric assays. Antioxidant and anti-inflammatory activities were estimated in-vitro. Anticancer activity of extracts and fractions of methanolic extract were tested on (HepG2) and (MCF7). Results: Methanolic extract and its ethyl acetate fraction contain higher contents of total phenols and flavonoids. In addition, methanolic extract had higher antioxidant activity. Butanolic and ethyl acetate fractions yielded higher percent of inhibition of protein denaturation. Meanwhile, ethyl acetate fraction and methanolic extract had anticancer activity against HepG2 and MCF7 (IC50=60 ± 0.24 and 29.4 ± 0.12µg.mL⁻¹) and (IC50=94.7 ± 0.21 and 227 ± 0.48µg.mL⁻¹), respectively. In Gas chromatography-mass spectrometry (GC-MS) analysis, methanolic extract has 32 compounds, whereas; ethyl acetate and butanol fractions contain 40 and 36 compounds, respectively. Conclusion: Flaxseed contains totally different biologically active compounds that have been found to possess good variable activities, which can protect human body against several diseases.

Keywords: phenolic content, flavonoid content, HepG2, MCF7, hemolysis-assay, flaxseed

Procedia PDF Downloads 93

Authors: Yeimy L. Quintana, Juan G. Zuluaga, Sandra S. Arango

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The comet assay is a method based on electrophoresis that is used to measure DNA damage in cells and has shown important results in the identification of substances with a potential risk to the human population as innumerable physical, chemical and biological agents. With this technique is possible to obtain images like a comet, in which the tail of these refers to damaged fragments of the DNA. One of the main problems is that the image has unequal luminosity caused by the fluorescence microscope and requires different processing to condition it as well as to know how many optimal comets there are per sample and finally to perform the measurements and determine the percentage of DNA damage. In this paper, we propose the design and implementation of software using Image Processing Toolbox-MATLAB that allows the automation of image processing. The software chooses the optimum comets and measuring the necessary parameters to detect the damage.

Keywords: artificial vision, comet assay, DNA damage, image processing

Procedia PDF Downloads 265