Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 193

Search results for: in vitro

193 In silico Simulations for DNA Shuffling Experiments

Authors: Luciana Montera

Abstract:

DNA shuffling is a powerful method used for in vitro evolute molecules with specific functions and has application in areas such as, for example, pharmaceutical, medical and agricultural research. The success of such experiments is dependent on a variety of parameters and conditions that, sometimes, can not be properly pre-established. Here, two computational models predicting DNA shuffling results is presented and their use and results are evaluated against an empirical experiment. The in silico and in vitro results show agreement indicating the importance of these two models and motivating the study and development of new models.

Keywords: Computer simulation, DNA shuffling, in silico andin vitro comparison.

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192 Cultivation of Thymus by In Vitro And Hydroponics Combined Method

Authors: E. Sargsyan, A. Vardanyan, L. Ghalachyan, S. Bulgadaryan

Abstract:

Our results showed that for the growth of qualitative seedling and vegetative raw material of ðó. marschallianus Willd. and T. serphyllum L. it is more profitable to use the in vitro and hydroponics combined method. In in vitro culture it is possible to do micro-propagation whole year with 98-99% rhizogenesis. 30000 micro-plants were obtained from one explant during 9 months. Hydroponic conditions provide the necessary microclimate for microplants where the survival rate without acclimatization was 93.3%. The essential oil content in hydroponic dry herb of both species in vegetative and blossom phase was 1.3% whereas in wild plants it was 1.2%, the content of extractive substances and vitamin C also exceeded wild plants. Our biochemical and radiochemical investigations indicated that the medicinal raw materials obtained from hydroponic and wild plants of Thymus species correspond to the demands of SPh XI, and the content of artificial radionuclides does not exceed the MACL.

Keywords: Hydroponics, In vitro, Micro-propagation, Thymus

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191 Endothelial Specificity of ICAM2, Flt-1, and Tie2 Promoters In Vitro and In Vivo

Authors: Jing Lei, Yoram Vodovotz, Timothy R. Billiar

Abstract:

To identify an endothelial cell-specific promoter suitable for vascular-specific targeting, we tested five promoters in vitro--Tie2SE, Tie2LE, ICAM2, Flt-1 and vWF--for promoter activity and specificity in endothelial cells, smooth muscle cells and non-vascular resident cells as well as tissues. These promoters, except for vWF, exhibited good endothelial activity and specificity in vitro. In a syngenic heart transplantation model, the ICAM2 promoter was variably functional in coronary endothelial cells of donor hearts. Thus, the ICAM2, Flt-1, Tie2SE and Tie2LE promoters hold promise for endothelial-specific targeting, but in vitro expression may not predict in vivo expression.

Keywords: vascular-specific targeting, endothelial cell-specificpromoter, endothelial specificity.

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190 In vitro Antioxidant Activity of Two Selected Herbal Medicines

Authors: S. Vinotha, I. Thabrew, S. Sri Ranjani

Abstract:

Hot aqueous and methanol extracts of the two selected herbal medicines such are Vellarugu chooranam (V.C) and Amukkirai Chooranam (A.C) were examined for total phenolic and flavonoid contents and in vitro antioxidant activity using four different methods. The total phenolic and flavonoid contents in methanol extract of V.C were found to be higher (44.41±1.26mg GAE/g; 174.44±9.32mg QE/g) than in the methanol extract of A.C (20.56±0.67mg GAE/g; 7.21±0.85mg QE/g). Hot methanol and aqueous extracts of both medicines showed low antioxidant activity in DPPH, ABTS, and FRAP methods and Iron chelating activity not found at highest possible concentration. V.C contains higher concentrations of total phenolic and flavonoid contents than A.C and can also exert greater antioxidant activity than A.C, although the activities demonstrated were lower than the positive control Trolox. The in vitro antioxidant activity was not related with the total phenolic and flavonoid contents of the methanol and aqueous extracts of both herbal medicines (A.C and V.C).

Keywords: Activity, Different extracts, Herbal medicine, in vitro antioxidant.

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189 Effect of Recombinant Human Follicle Stimulating Hormone on Meiotic Competence of In Vitro Grown Nili Ravi Buffalo Oocytes

Authors: Muhammad Ijaz Khan, Samina Jalali, Beenish Shahid, S. A. Shami, Muhammad Ikramullah

Abstract:

In the present study, the response of Nili Ravi buffalo oocytes to recombinant human follicle stimulating hormone (rhFSH) (Organon) on meiotic maturation in vitro was examined. Oocytes were matured in vitro in medium containing either 0 or 0.05 IU/ ml rhFSH and the stage of nuclear maturation recorded after 24 hours. The percentage of oocytes in the control group undergoing germinal vesicle breakdown (GVBD) observed after 24 hours of culture was 29 % whereas as in rhFSH group the percentage was 10 % were at this stage (P< 0.001).Thus in the presence of rhFSH, a significantly greater number of oocytes had progressed to the more advanced stages of nuclear maturation. Indeed, the maturation of GV (Germinal Vesicle) stage oocytes to the metaphase II (M II) stage after 24 hours was significantly (P< 0.0001) increased by the addition of rhFSH (82 % VS 47 %). The percentage of degenerated oocytes after 24 hours of culture was 24 % in control group, whereas in rhFSH group the percentage was 8 % after 24 hours. Degeneration of the oocytes after 24 hours was not significantly (P = 0. 9361) decreased.

Keywords: Buffalo, in vitro, oocytes, recombinant FSH.

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188 Changes of in vitro Cytokine Production induced by δ-Lactams

Authors: Y. Baba hamed, A. Medjdoub, H. Merzouk, M. Narce

Abstract:

The aim of this work was to study the in vitro effects of δ-lactam 1 and its 4-chlorophenyl derivative 2, on the proliferative responses of human lymphocytes and Th1 and Th2 cytokine secretion. The possible protective role of vitamin E on intracellular stress oxidative induced by these compounds was also investigated. Peripheral blood lymphocytes were isolated using differential centrifugation on a density gradient of Histopaque. They were cultured with mitogen concanavalin A, vitamin E (10 μM) and with different concentrations of the compounds 1 and 2 (0.1 to 10 μM). Proliferation (MTT assay), IL-2, INFγ and IL-4 (Elisa kits), intracellular superoxide anion were determined. 1 and 2 were immunostimulant and increased cytokine secretion with a shift away from Th1 response to Th2. These properties were however accompanied by an increase in intracellular oxidative stress. The presence of vitamin E exhibited protective effects by reducing δ- lactam-induced superoxide anion generation in lymphocytes.

Keywords: Cytokines, δ-Lactams, In vitro Lymphocyte Proliferation, Superoxide Anion

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187 Novel Solid Lipid Nanoparticles for Oral Delivery of Oxyresveratrol: Effect of the Formulation Parameters on the Physicochemical Properties and in vitro Release

Authors: Y. Sangsen, K. Likhitwitayawuid, B. Sritularak, K. Wiwattanawongsa, R. Wiwattanapatapee

Abstract:

Novel solid lipid nanoparticles (SLNs) were developed to improve oral bioavailability of oxyresveratrol (OXY). The SLNs were prepared by a high speed homogenization technique, at an effective speed and time, using Compritol® 888 ATO (5% w/w) as the solid lipid. The appropriate weight proportions (0.3% w/w) of OXY affected the physicochemical properties of blank SLNs. The effects of surfactant types on the properties of the formulations such as particle size and entrapment efficacy were also investigated. Conclusively, Tween 80 combined with soy lecithin was the most appropriate surfactant to stabilize OXY-loaded SLNs. The mean particle size of the optimized formulation was 134.40 ± 0.57 nm. In vitro drug release study, the selected S2 formulation showed a retarded release profile for OXY with no initial burst release compared to OXY suspension in the simulated gastrointestinal fluids. Therefore, these SLNs could provide a suitable system to develop for the oral OXY delivery.

Keywords: Solid lipid nanoparticles, Physicochemical properties, in vitro drug release, Oxyresveratrol.

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186 In vitro Environmental Factors Controlling Root Morphological Traits of Pineapple (Ananas comosus L. Merr)

Authors: S. Mohajer, R. M. Taha, M. Adel

Abstract:

Developing our knowledge of when pineapple roots grow can lead to improved water, fertilizer applications, and more precise culture management. This paper presents current understanding of morphological traits in pineapple roots, highlighting studies using incubation periods and various solid MS media treated with different sucrose concentrations and pH, which directly assess in vitro environmental factors. Rooting parameters had different optimal sucrose concentrations and incubation periods. All shoots failed to root in medium supplemented with sucrose at 5 g/L and no roots formed within the first 45 days in medium enriched with sucrose at 10 g/L. After 75 days, all shoots rooted in medium enriched with 10 and 20 g/L sucrose. Moreover, MS medium supplied with 20 g/L sucrose resulted in the longest and the highest number of roots with 27.3 mm and 4.7, respectively. Root function, such as capacity for P and N uptake, declined rapidly with root length. As a result, the longer the incubation period, the better the rooting responses would be.

Keywords: Environmental factors, in vitro rooting, pineapple, tissue culture.

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185 Effect of Different Media and Mannitol Concentrations on Growth and Development of Vandopsis lissochiloides (Gaudich.) Pfitz. under Slow Growth Conditions

Authors: J. Linjikao, P. Inthima, A. Kongbangkerd

Abstract:

In vitro conservation of orchid germplasm provides an effective technique for ex situ conservation of orchid diversity. In this study, an efficient protocol for in vitro conservation of Vandopsis lissochiloides (Gaudich.) Pfitz. plantlet under slow growth conditions was investigated. Plantlets were cultured on different strength of Vacin and Went medium (½VW and ¼VW) supplemented with different concentrations of mannitol (0, 2, 4, 6 and 8%), sucrose (0 and 3%) and 50 g/L potato extract, 150 mL/L coconut water. The cultures were incubated at 25±2 °C and maintained under 20 µmol/m2s light intensity for 24 weeks without subculture. At the end of preservation period, the plantlets were subcultured to fresh medium for growth recovery. The results found that the highest leaf number per plantlet could be observed on ¼VW medium without adding sucrose and mannitol while the highest root number per plantlet was found on ½VW added with 3% sucrose without adding mannitol after 24 weeks of in vitro storage. The results showed that the maximum number of leaves (5.8 leaves) and roots (5.0 roots) of preserved plantlets were produced on ¼VW medium without adding sucrose and mannitol. Therefore, ¼VW medium without adding sucrose and mannitol was the best minimum growth conditions for medium-term storage of V. lissochiloides plantlets.

Keywords: Preservation, Vandopsis, germplasm, in vitro.

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184 Metoprolol Tartrate-Ethylcellulose Tabletted Microparticles: Development of a Validated Invitro In-vivo Correlation

Authors: Fatima Rasool, Mahmood Ahmad, Ghulam Murtaza, Haji M. S. Khan, Shujaat A. Khan, Sonia Khiljee, Muhammad Qamar-Uz-Zaman

Abstract:

This study describes the methodology for the development of a validated in-vitro in-vivo correlation (IVIVC) for metoprolol tartrate modified release dosage forms with distinctive release rate characteristics. Modified release dosage forms were formulated by microencapsulation of metoprolol tartrate into different amounts of ethylcellulose by non-solvent addition technique. Then in-vitro and in-vivo studies were conducted to develop and validate level A IVIVC for metoprolol tartrate. The values of regression co-efficient (R2-values) for IVIVC of T2 and T3 formulations were not significantly (p<0.05) different from 1 while the values of R2 for IVIVC of T1 and Mepressor® were significantly (p<0.05) different from 1. Internal prediction errors of IVIVC, calculated from observed Area under Curve (AUC) and predicted AUC, were less than 10%. This study successfully presents a valid level A IVIVC for metoprolol tartrate modified dosage forms.

Keywords: Metoprolol tartrate, Dissolution, Bioavailability, Validated in-vitro in-vivo correlation.

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183 Screening and Evaluation of in vivo and in vitro Generated Insulin Plant (Vernonia divergens) for Antimicrobial and Anticancer Activities

Authors: Santosh Kumar, Anand Prakash, Kanak Sinha, Anita K Verma

Abstract:

Vernonia divergens Benth., commonly known as “Insulin Plant” (Fam: Asteraceae) is a potent sugar killer. Locally the leaves of the plant, boiled in water are successfully administered to a large number of diabetic patients. The present study evaluates the putative anti-diabetic ingredients, isolated from the in vivo and in vitro grown plantlets of V. divergens for their antimicrobial and anticancer activities. Sterilized explants of nodal segments were cultured on MS (Musashige and Skoog, 1962) medium in presence of different combinations of hormones. Multiple shoots along with bunch of roots were regenerated at 1mg l-1 BAP and 0.5 mg l-1 NAA. Micro-plantlets were separated and sub-cultured on the double strength (2X) of the above combination of hormones leading to increased length of roots and shoots. These plantlets were successfully transferred to soil and survived well in nature. The ethanol extract of plantlets from both in vivo & in vitro sources were prepared in soxhlet extractor and then concentrated to dryness under reduced pressure in rotary evaporator. Thus obtainedconcentrated extracts showed significant inhibitory activity against gram negative bacteria like Escherichia coli and Pseudomonas aeruginosa but no inhibition was found against gram positive bacteria. Further, these ethanol extracts were screened for in vitro percentage cytotoxicity at different time periods (24 h, 48 h and 72 h) of different dilutions. The in vivo plant extract inhibited the growth of EAC mouse cell lines in the range of 65, 66, 78, and 88% at 100, 50, 25 & 12.5μg mL-1 but at 72 h of treatment. In case of the extract of in vitro origin, the inhibition was found against EAC cell lines even at 48h. During spectrophotometric scanning, the extracts exhibited different maxima (ʎ) - four peaks in in vitro extracts as against single in in vivo preparation suggesting the possible change in the nature of ingredients during micropropagation through tissue culture techniques.

Keywords: Anti-cancer, Anti-microbial, EAC mouse cell, Tissue culture, Vernonia divergens.

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182 The Effect of Carboxymethyl Cellulose on the Stability of Emulsions Stabilized by Whey Proteins under Digestion in vitro and in vivo

Authors: D. Leskauskaite, I. Jasutiene, M. Kersiene, E. Malinauskyte, P. Matusevicius

Abstract:

In vitro gastro-duodenal digestion model was used to investigate the changes of emulsions under digestion conditions. Oil in water emulsions stabilized by whey proteins (2%) and stabilized by whey proteins (2%) with addition of carboxymethyl cellulose (0.75%) as gelling agent of continuous phase were prepared at pH7. Both emulsions were destabilized under gastric conditions; however the protective role of carboxymethyl cellulose was indicated by recording delay of fat digestibility of this emulsion. In the presence of carboxymethyl cellulose whey proteins on the interfacial surface of droplets were more resistant to gastric degradation causing limited hydrolysis of fat due to the poor acceptability of lipids for the enzymes. Studies of emulsions using in vivo model supported results from in vitro studies. Lower content of triglycerides in blood serum and higher amount of fecal fat of rats were determined when rats were fed by diet containing emulsion made with whey proteins and carboxymethyl cellulose. 

Keywords: Digestibility, emulsions, lipids, rats.

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181 In vitro Effects of Salvia officinalis on Bovine Spermatozoa

Authors: Eva Tvrdá, Boris Botman, Marek Halenár, Tomáš Slanina, Norbert Lukáč

Abstract:

In vitro storage and processing of animal semen represents a risk factor to spermatozoa vitality, potentially leading to reduced fertility. A variety of substances isolated from natural sources may exhibit protective or antioxidant properties on the spermatozoon, thus extending the lifespan of stored ejaculates. This study compared the ability of different concentrations of the Salvia officinalis extract on the motility, mitochondrial activity, viability and reactive oxygen species (ROS) production by bovine spermatozoa during different time periods (0, 2, 6 and 24 h) of in vitro culture. Spermatozoa motility was assessed using the Computer-assisted sperm analysis (CASA) system. Cell viability was examined using the metabolic activity MTT assay, the eosin-nigrosin staining technique was used to evaluate the sperm viability and ROS generation was quantified using luminometry. The CASA analysis revealed that the motility in the experimental groups supplemented with 0.5-2 µg/mL Salvia extract was significantly lower in comparison with the control (P<0.05; Time 24 h). At the same time, a long-term exposure of spermatozoa to concentrations ranging between 0.05 µg/mL and 2 µg/mL had a negative impact on the mitochondrial metabolism (P<0.05; Time 24 h). The viability staining revealed that 0.001-1 µg/mL Salvia extract had no effects on bovine male gametes, however 2 µg/mL Salvia had a persisting negative effect on spermatozoa (P<0.05). Furthermore 0.05-2 µg/mL Salvia exhibited an immediate ROS-promoting effect on the sperm culture (P>0.05; Time 0 h and 2 h), which remained significant throughout the entire in vitro culture (P<0.05; Time 24 h). Our results point out to the necessity to examine specific effects the biomolecules present in Salvia officinalis may have individually or collectively on the in vitro sperm vitality and oxidative profile.

Keywords: Bulls, CASA, MTT test, reactive oxygen species, sage, Salvia officinalis, spermatozoa.

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180 Micropropagation and in vitro Conservation via Slow Growth Techniques of Prunus webbii (Spach) Vierh: An Endangered Plant Species in Albania

Authors: Valbona Sota, Efigjeni Kongjika

Abstract:

Wild almond is a woody species, which is difficult to propagate either generatively by seed or by vegetative methods (grafting or cuttings) and also considered as Endangered (EN) in Albania based on IUCN criteria. As a wild relative of cultivated fruit trees, this species represents a source of genetic variability and can be very important in breeding programs and cultivation. For this reason, it would be of interest to use an effective method of in vitro mid-term conservation, which involves strategies to slow plant growth through physicochemical alterations of in vitro growth conditions. Multiplication of wild almond was carried out using zygotic embryos, as primary explants, with the purpose to develop a successful propagation protocol. Results showed that zygotic embryos can proliferate through direct or indirect organogenesis. During subculture, stage was obtained a great number of new plantlets identical to mother plants derived from the zygotic embryos. All in vitro plantlets obtained from subcultures underwent in vitro conservation by minimal growth in low temperature (4ºC) and darkness. The efficiency of this technique was evaluated for 3, 6, and 10 months of conservation period. Maintenance in these conditions reduced micro cuttings growth. Survival and regeneration rates for each period were evaluated and resulted that the maximal time of conservation without subculture on 4ºC was 10 months, but survival and regeneration rates were significantly reduced, specifically 15.6% and 7.6%. An optimal period of conservation in these conditions can be considered the 5-6 months storage, which can lead to 60-50% of survival and regeneration rates. This protocol may be beneficial for mass propagation, mid-term conservation, and for genetic manipulation of wild almond.

Keywords: Micropropagation, minimal growth, storage, wild almond.

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179 Manipulation of Probiotics Fermentation of Yogurt by Cinnamon and Licorice: Effects on Yogurt Formation and Inhibition of Helicobacter Pylori Growth in vitro

Authors: S. Behrad, M.Y. Yusof, K. L. Goh, A.S. Baba

Abstract:

Probiotic bacteria especially Lactobacillus spp. and Bifidobacterium exert suppressive effect on Helicobacter pylori. Cinnamon and licorice have been traditionally used for the treatment of gastric ulcer. The objectives of this study were to determine the effects of herbs on yogurt fermentation, the level of probiotic bacteria in yogurt during 28 days storage and the effect of herbal yogurt on the growth of H. pylori in vitro. Cinnamon or licorice was mixed with milk and the mixture was fermented with probiotic bacteria to form herbal-yogurt. Changes of pH and total titratable acids were monitored and the viability of probiotic bacteria was evaluated during and after refrigerated storage. The in vitro inhibition of H. pylori growth was determined using agar diffusion and minimum inhibitory concentration (MIC) method. The presence of herbs did not affect the probiotic population during storage. There were no significant differences in pH and TTA between herbal-yogurts and plain-yogurt during fermentation and storage. Water extract of cinnamon-yogurt showed the highest inhibition effect (13.5mm) on H. pylori growth in comparison with licorice-yogurt (11.2mm). The present findings indicate cinnamon and licorice has bioactive components to decrease the growth of H. pylori.

Keywords: Cinnamon, Helicobacter pylori, Herbal-Yogurt, Licorice, Probiotics

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178 Sterilisation of in vitro Culture Medium of Chrysanthemum by Plant Essential Oils without Autoclaving

Authors: Chockpisit Thepsithar, Aree Thongpukdee, Apichya Daorat

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The alternative technique for sterilization of culture medium to replace autoclaving was carried out. For sterilization of culture medium without autoclaving, some commercial pure essential oils, bergamot oil, betel oil, cinnamon oil, lavender oil and turmeric oil, were tested alone or in combinations with some disinfectants, 10% povidone-iodine and 2% iodine + 2.4% potassium iodide. Each essential oil or combination was added to 25-mL Murashige and Skoog (MS) medium before medium was solidified in a 120-mL container, kept for 2 weeks before evaluating sterile conditions. Treated media, supplemented with essential oils, were compared to control medium, autoclaved at 121 degree Celsius for 15 min. In vitro sterile conditions were found 20 – 100% from these treated media compared to 100% sterile condition from autoclaved medium. Treated media obtained 100% sterile conditions were chosen for culturing chrysanthemum shoots. It was found that 10% povidoneiodine in combination with cinnamon oil (3:1) and 2% iodine + 2.4% potassium iodide in combination with lavender oil (1:3) at the concentration of 36 3L/25 mL medium provided the promising growth of shoot explants.

Keywords: Sterilizing agents, essential oils, disinfectants, MS medium, in vitro culture, chrysanthemum, sterilization of medium without autoclaving

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177 Role of Fish Hepatic Aldehyde Oxidase in Oxidative in vitro Metabolism of Phenanthridine Heterocyclic Aromatic Compound

Authors: Khaled S. Al Salhen

Abstract:

Aldehyde oxidase is molybdo-flavoenzyme involved in the oxidation of hundreds of endogenous and exogenous and N-heterocyclic compounds and environmental pollutants. Uncharged N-heterocyclic aromatic compounds such phenanthridine are commonly distributed pollutants in soil, air, sediments, surface water and groundwater, and in animal and plant tissues. Phenanthridine as uncharged N-heterocyclic aromatic compound was incubated with partially purified aldehyde oxidase from rainbow trout fish liver. Reversed-phase HLPC method was used to separate the oxidation products from phenanthridine and the metabolite was identified. The 6(5H)-phenanthridinone was identified the major metabolite by partially purified aldehyde oxidase from fish liver. Kinetic constant for the oxidation reactions were determined spectrophotometrically and showed that this substrate has a good affinity (Km = 78 ± 7.6µM) for hepatic aldehyde oxidase, will be a significant pathway. This study confirms that partially purified aldehyde oxidase from fish liver is indeed the enzyme responsible for the in vitro production 6(5H)-phenanthridinone metabolite as it is a major metabolite by mammalian aldehyde oxidase, coupled with a relatively high oxidation rate (0.77± 0.03 nmol/min/mg protein). In addition, the kinetic parameters of hepatic fish aldehyde oxidase towards the phenanthridine substrate indicate that in vitro biotransformation by hepatic fish aldehyde oxidase will be a significant pathway. This study confirms that partially purified aldehyde oxidase from fish liver is indeed the enzyme responsible for the in vitro production 6(5H)-phenanthridinone metabolite as it is a major metabolite by mammalian aldehyde oxidase.

Keywords: Aldehyde oxidase, Fish, Phenanthridine, Specificity.

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176 In vitro Effects of Viscum album on the Functionality of Rabbit Spermatozoa

Authors: Marek Halenár, Eva Tvrdá, Simona Baldovská, Ľubomír Ondruška, Peter Massányi, Adriana Kolesárová

Abstract:

This study aimed to assess the in vitro effects of different concentrations of the Viscum album extract on the motility, viability, and reactive oxygen species (ROS) production by rabbit spermatozoa during different time periods (0, 2, and 8h). Spermatozoa motility was assessed by using the CASA (Computer aided sperm analysis) system. Cell viability was evaluated by using the metabolic activity MTT assay, and the luminol-based luminometry was applied to quantify the ROS formation. The CASA analysis revealed that low Viscum concentrations were able to prevent a rapid decline of spermatozoa motility, especially in the case of concentrations ranging between 1 and 5 µg/mL (P<0.05 with respect to time 8h). At the same time, concentrations ranging between 1 and 100 µg/mL of the extract led to a significant preservation of the cell viability (P<0.05 in case of 5, 50 and 100 µg/mL; P<0.01 with respect to 1 and 10 µg/mL, time 8h). 1 and 5 µg/mL of the extract exhibited antioxidant characteristics, translated into a significant reduction of the ROS production, particularly notable at time 8h (P<0.01). The results indicate that the Viscum extract is capable of delaying the damage inflicted to the spermatozoon by the in vitro environment.

Keywords: CASA, mistletoe, mitochondrial activity, motility, reactive oxygen species, rabbits, spermatozoa, Viscum album.

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175 In vitro Susceptibility of Madurella mycetomatis to the Extracts of Anogeissus leiocarpus Leaves

Authors: Ikram Mohamed Eltayeb Elsiddig, Abdel Khalig Muddather, Hiba Abdel Rahman Ali, Saad Mohamed Hussein Ayoub

Abstract:

Anogeissus leiocarpus (Combretaceae) is well known for its medicinal uses in African traditional medicine, for treating many human diseases mainly skin diseases and infections. Mycetoma disease is a fungal and/ or bacterial skininfection, mainly cause by Madurella mycetomatis fungus. This study was carried out in vitro to investigate the antifungal activity of Anogeissus leiocarpus leaf extracts against the isolated pathogenic Madurella mycetomatis, by using the NCCLS modified method compared to Ketoconazole standard drug, and MTT assay. The bioactive fraction was subjected to chemical analysis implementing different chromatographic analytical methods (TLC, HPLC, and LC-MS/MS). The results showed significance antifungal activity of A. leiocarpus leaf extracts against the isolated pathogenic M. mycetomatis, compared to negative and positive controls. The chloroform fraction showed the highest antifungal activity. The chromatographic analysis of the chloroform fraction with the highest activity showed the presence of important bioactive compounds such as ellagic and flavellagic acids derivatives, flavonoids and stilbenoid, which are well known for their antifungal activity.

Keywords: Anogeissus leiocarpus, crude extracts and fractions of Anogeissus leiocarpus, in vitro susceptibility of Madurella mycetomatis, Madurella mycetomatis.

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174 Bioactivity Evaluation of Cucurbitin Derived Enzymatic Hydrolysates

Authors: Ž. Vaštag, Lj. Popović, S. Popović

Abstract:

After cold pressing of pumpkin oil, the defatted oil cake (PUOC) was utilised as raw material for processing of bio-functional hydrolysates. In this study, the in vitro bioactivity of an alcalase (AH) and a pepsin hydrolysate (PH) prepared from the major pumpkin 12S globulin (cucurbitin) are compared. The hydrolysates were produced at optimum reaction conditions (temperature, pH) for the enzymes, during 60min. The bioactivity testing included antioxidant and angiotensin I converting enzyme inhibitory activity assays. The hydrolysates showed high potential as natural antioxidants and possibly antihypertensive agents in functional food or nutraceuticals. Additionally, preliminary studies have shown that both hydrolysates could exhibit modest α-amylase inhibitory activity, which indicates on their hypoglycemic potential.

Keywords: Cucurbitin, alcalase, pepsin, protein hydrolysates, in vitro bioactivity.

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173 In vitro Effects of Amygdalin on the Functional Competence of Rabbit Spermatozoa

Authors: Marek Halenár, Eva Tvrdá, Tomáš Slanina, Ľubomír Ondruška, Eduard Kolesár, Peter Massányi, Adriana Kolesárová

Abstract:

The present in vitro study was designed to reveal whether amygdalin (AMG) is able to cause changes to the motility, viability and mitochondrial activity of rabbit spermatozoa. New Zealand White rabbits (n = 10) aged four months were used in the study. Semen samples were collected from each animal and used for the in vitro incubation. The samples were divided into five equal parts and diluted with saline supplemented with 0, 0.5, 1, 2.5 and 5 mg/mL AMG. At times 0h, 3h and 5h spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system, cell viability was examined with the metabolic activity (MTT) assay, and the eosin-nigrosin staining technique was used to evaluate the viability of rabbit spermatozoa. All AMG concentrations exhibited stimulating effects on the spermatozoa activity, as shown by a significant preservation of the motility (P<0.05 with respect to 0.5 mg/mL and 1 mg/mL AMG; Time 5 h) and mitochondrial activity (P< 0.05 in case of 0.5 mg/mL AMG; P< 0.01 in case of 1 mg/mL AMG; P < 0.001 with respect to 2.5 mg/mL and 5 mg/mL AMG; Time 5 h). None of the AMG doses supplemented had any significant impact of the spermatozoa viability. In conclusion, the data revealed that short-term co-incubation of spermatozoa with AMG may result in a higher preservation of the sperm structural integrity and functional activity.

Keywords: Amygdalin, CASA, mitochondrial activity, motility, rabbits, spermatozoa, viability.

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172 Comparative Study of Calcium Content on in vitro Biological and Antibacterial Properties of Silicon-Based Bioglass

Authors: Morteza Elsa, Amirhossein Moghanian

Abstract:

The major aim of this study was to evaluate the effect of CaO content on in vitro hydroxyapatite formation, MC3T3 cells cytotoxicity and proliferation as well as antibacterial efficiency of sol-gel derived SiO2–CaO–P2O5 ternary system. For this purpose, first two grades of bioactive glass (BG); BG-58s (mol%: 60%SiO2–36%CaO–4%P2O5) and BG-68s (mol%: 70%SiO2–26%CaO–4%P2O5)) were synthesized by sol-gel method. Second, the effect of CaO content in their composition on in vitro bioactivity was investigated by soaking the BG-58s and BG-68s powders in simulated body fluid (SBF) for time periods up to 14 days and followed by characterization inductively coupled plasma atomic emission spectrometry (ICP-AES), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM) techniques. Additionally, live/dead staining, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity assays were conducted respectively, as qualitatively and quantitatively assess for cell viability, proliferation and differentiations of MC3T3 cells in presence of 58s and 68s BGs. Results showed that BG-58s with higher CaO content showed higher in vitro bioactivity with respect to BG-68s. Moreover, the dissolution rate was inversely proportional to oxygen density of the BG. Live/dead assay revealed that both 58s and 68s increased the mean number live cells which were in good accordance with MTT assay. Furthermore, BG-58s showed more potential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) bacteria. Taken together, BG-58s with enhanced MC3T3 cells proliferation and ALP activity, acceptable bioactivity and significant high antibacterial effect against MRSA bacteria is suggested as a suitable candidate in order to further functionalizing for delivery of therapeutic ions and growth factors in bone tissue engineering.

Keywords: Antibacterial, bioactive glass, hydroxyapatite, proliferation, sol-gel processes.

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171 In vitro Plant Regeneration of Java Vetiver (Vetiveria zizanioides)

Authors: Iriawati, R. R. Esyanti, W. Natalia, N. Zahya

Abstract:

In vitro plant regeneration has been successfully obtained from basal shoot explant of Vetiveria zizanioides through indirect organogenesis. The explant was cultured in Murashige & Skoog’s (MS) media supplemented with 2,4-D, IAA, and kinetin in various concentrations. Callus was well induced in media supplemented with 2 ppm 2,4-D, 1 ppm IAA, and 1 ppm kinetin. This callus was then transferred to MS media supplemented with 1 - 5 ppm of BAP for shoot regeneration. The media supplemented with 3 ppm BAP was a suitable medium for shoot induction, as well as for shoot multiplication. Rooting was well developed in shoot following transferred to half MS media containing 0.2 ppm IBA. Plantlet was then transferred to husk charcoal for acclimatization, and almost all (90%) of plantlets were survived during acclimatization.

Keywords: Callus, plantlet regeneration, shoot induction, Vetiveria zizanioides.

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170 Morphological Interaction of Porcine Oocyte and Cumulus Cells Study on in vitro Oocyte Maturation Using Electron Microscopy

Authors: M. Areekijseree, W. Pongsawat, M. Pumipaiboon, C. Thepsithar, S. Sengsai, T. Chuen-Im

Abstract:

Morphological interaction of porcine cumulus-oocyte complexes (pCOCs) was investigated on in vitro condition using electron microscope (SEM and TEM). The totals of 1,923 oocytes were round in shape, surrounded by Zona pellucida with layer of cumulus cells ranging between 59.29-202.14 μm in size. They were classified into intact-, multi-, partial cumulus cell layer oocyte, and completely denuded oocyte, at the percentage composition of 22.80% 32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as intact- and multi cumulus cell layer oocytes were further culturing at 37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2 mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 μg/mL porcine follicle-stimulating hormone, 1 μg/mL LH, 1μg/mL estradiol with ethanol, and 50 μg/mL gentamycin sulfate. On electron microscope study, cumulus cells were found to stick their processes to secrete substance from the sac-shape end into Zona pellucida of the oocyte and also communicated with the neighboring cells through their microvilli on the beginning of incubation period. It is believed that the cumulus cells communicate with the oocyte by inserting the microvilli through this gap and embedded in the oocyte cytoplasm before secreting substance, through the sac-shape end of the microvilli, to inhibit primary oocyte development at the prophase I. Morphological changes of the complexes were observed after culturing for 24-44 h. One hundred percentages of the cumulus layers were expanded and cumulus cells were peeling off from the oocyte surface. In addition, the round-shape cumulus cells transformed themselves into either an elongate shape or a columnar shape, and no communication between cumulus neighboring cells. After 44 h of incubation time, diameter of oocytes surrounded by cumulus cells was larger than 0 h incubation. The effect of hormones in culture medium is exerted by their receptors present in porcine oocyte. It is likely that all morphological changes of the complexes after hormone treatment were to allow maturation of the oocyte. This study demonstrated that the association of hormones in M199 could promote porcine follicle activation in 44 h in vitro condition. This culture system should be useful for studying the regulation of early follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell technology.

Keywords: Cumulus cells, electron microscopy (SEM and TEM), in vitro, porcine oocyte.

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169 Leaf Pigments Help Almond Explants Tolerating Osmotic Stress

Authors: Soheil Karimi, Abbas Yadollahi, Kazem Arzani, Ali Imani

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This study was conducted to evaluate the response of almond genotypes to osmotic stress in vitro in order to screen drought tolerance. Explants subjected to polyethyleneglycol osmotic stress (0, 3.5, and 7.0% WV) on the MS medium. Concentrations of photosynthesis pigments, anthocyanins, and carothenoids were significantly reduced under osmotic stress. Under osmotic stress, leaf water content, cellular membrane stability and pigments concentrations were significantly higher in the leaves of drought tolerant genotypes. The results revealed that carotenoids and anthocyanins may act as photoprotectant compounds in almond leaves and involved in drought tolerance system of the plant.

Keywords: Almond, Anthocianins, Carotenoids, in vitro; Leaf Osmotic Stress, Leaf Pigments, Polyethylene Glycol.

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168 Effects of a Nectandra Membranacea Extract on Labeling of Blood Constituents with Technetium-99m and on the Morphology of Red Blood Cells

Authors: Silvana R.F. Moreno, Jorge J. Carvalho, Ana L. Nascimento, Mario Pereira, Luiz Q. A. Caldas, Mário Bernardo-Filho

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The aim of this in vitro study was to evaluate the possible interference of a Nectandra membranacea extract (i) on the labeling of blood cells (BC), (ii) on the labeling process of BC and plasma (P) proteins with technetium-99m (Tc-99m) and (iii) on the morphology of red blood cells (RBC). Blood samples were incubated with a Nectandra membranacea crude extract, stannous chloride, Tc- 99m (sodium pertechnetate) was added, and soluble (SF) and insoluble (IF) fractions were isolated. Morphometry studies were performed with blood samples incubated with Nectandra membranacea extract. The results show that the Nectandra membranacea extract does not promote significant alteration of the labeling of BC, IF-P and IF-BC. The Nectandra membranacea extract was able to alter the erythrocyte membrane morphology, but these morphological changes were not capable to interfere on the labeling of blood constituents with Tc-99m.

Keywords: in vitro study, Nectandra membranacea, red bloodcell, technetium-99m

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167 Origanum vulgare as a Possible Modulator of Testicular Endocrine Function in Mice

Authors: Eva Tvrdá, Barbora Babečková, Michal Ďuračka, Róbert Kirchner, Július Árvay

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This study was designed to assess the in vitro effects of Origanum vulgare L. (oregano) extract on the testicular steroidogenesis. We focused on identifying major biomolecules present in the oregano extract, as well as to investigate its in vitro impact on the secretion of cholesterol, testosterone, dehydroepiandrosterone and androstenedione by murine testicular fragments. The extract was subjected to high performance liquid chromatography (HPLC) which identified cyranosid, daidzein, thymol, rosmarinic and trans-caffeic acid among the predominant biochemical components of oregano. For the in vitro experiments, testicular fragments from 20 sexually mature Institute of Cancer Research (ICR) mice were incubated in the absence (control group) or presence of the oregano extract at selected concentrations (10, 100 and 1000 μg/mL) for 24 h. Cholesterol levels were quantified using photometry and the hormones were assessed by ELISA (Enzyme-Linked Immunosorbent Assay). Our data revealed that the release of cholesterol and androstenedione (but not dehydroepiandrosterone and testosterone) by the testicular fragments was significantly impacted by the oregano extract in a dose-dependent fashion. Supplementation of the extract resulted in a significant decline of cholesterol (P < 0.05 in case of 100 μg/mL; P < 0.01 with respect 100 μg/mL extract), as well as androstenedione (P < 0.01 with respect to 100 and 1000 μg/mL extract). Our results suggest that the biomolecules present in Origanum vulgare L. could exhibit a dose-dependent impact on the secretion of male steroids, playing a role in the regulation of testicular steroidogenesis.

Keywords: Mice, Origanum vulgare L., steroidogenesis, testes.

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166 The Effect of Granule Size on the Digestibility of Wheat Starch Using an in vitro Model

Authors: Mee-Lin Lim Chai Teo, Darryl M. Small

Abstract:

Wheat has a bimodal starch granule population and the dependency of the rate of enzymatic hydrolysis on particle size has been investigated. Ungelatinised wheaten starch granules were separated into two populations by sedimentation and decantation. Particle size was analysed by laser diffraction and morphological characteristics were viewed using SEM. The sedimentation technique though lengthy, gave satisfactory separation of the granules. Samples (<10μm, >10μm and original) were digested with a-amylase using a dialysis model. Granules of <10μm showed significantly higher rate of reducing sugar release than those >10μm (p<0.05). In contrast, the rate was not significantly different between the original sample and granules >10μm. Moreover, the digestion rate was dependent on particle size whereby smaller granules produced higher rate of release. The methodology and results reported here can be used as a basis for further evaluations designed to delay the release of glucose during the digestion of native starches.

Keywords: in vitro Digestion, a-amylase, wheat starch, granule size.

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165 Inhibition Effect of Brazilin to Human Bladder Cancer Cell Line T24

Authors: Liansheng Ren, Xihua Yang, Guoping Wang, Hong Zhang, Lili Zhao, Zhenguo Mi

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The inhibition effect of brazilin to human bladder tumor cell line T24 in vitro and in vivo was studied. The results of the in vitro experiments showed that brazilin has strong inhibition activity on the target cells. The inhibition ratio of 100 μg/mL brazilin and 100 μg/mL mitomycin to the target cells was 90.90 % and 63.24 % respectively, which showed that brazilin has higher inhibition activity than mitomycin under the same concentration. Brazilin could induce cell apoptosis in T24 cells. Significant antitumor activity of brazilin was also showed in the animals experiments. The life extention rate of 200 mg/mL, 300 mg/kg, and 400 mg/kg brazilin intraperitoneally injected into Balb/c-nu-nu nude mice that with human bladder cancer were 51.50 %, 56.90 %, and 58.42 %(P<0.05). Our study showed that brazilin has significant inhibitory effect on human bladder tumor cell.

Keywords: bladder cancer, brazilin, inhibition, T24 cell line

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164 Metal-Based Anticancer Agents: In vitro DNA Binding, Cleavage and Cytotoxicity

Authors: Mala Nath, Nagamani Kompelli, Partha Roy, Snehasish Das

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Two new metal-based anticancer chemotherapeutic agents, [(Ph2Sn)2(HGuO)2(phen)Cl2] 1 and [(Ph3Sn)(HGuO)(phen)]- Cl.CH3OH.H2O 2, were designed, prepared and characterized by analytical and spectral (IR, ESI-Mass, 1H, 13C and 119Sn NMR) techniques. The proposed geometry of Sn(IV) in 1 and 2 is distorted octahedral and distorted trigonal-bipyramidal, respectively. Both 1 and 2 exhibit potential cytotoxicity in vitro against MCF-7, HepG-2 and DU-145 cell lines. The intrinsic binding constant (Kb) values of 1 (2.33 × 105 M-1) and 2 (2.46 × 105 M-1) evaluated from UV-Visible absorption studies suggest non-classical electrostatic mode of interaction via phosphate backbone of DNA double helix. The Stern- Volmer quenching constant (Ksv) of 1 (9.74 × 105 M-1) and 2 (2.9 × 106 M-1) determined by fluorescence studies suggests the groove binding and intercalation mode for 1 and 2, respectively. Effective cleavage of pBR322 DNA is induced by 1.Their interaction with DNA of cancer cells may account for potency.

Keywords: Anticancer agents, DNA binding studies, NMR spectroscopy, organotin.

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