Search results for: in vitro assay

Authors: Abobaker Abrahem M. Saad, Asma Abudasalam

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The stem node segments were cultured on Murashige and Skoog (MS) medium. In the case of using MS+ Zeatin (1 mg/l), small shoot buds were formed directly in 70% of explants after 15 days, their length range between 0.1 to 0.3 cm after two weeks and reached 0.3 cm in length and three shoots in numbers after 4 weeks. When those small shoots were sub cultured on the same medium, they increased in length, number and reached 0.4 cm with 4 shoots, 0.4 cm with 5 shoots after six, eight and ten weeks respectively. In the case of using MS free hormones, MS+IAA (0.2mg/l) +BA (0.5mg/l), MS + kin(0.5mg/l), MS + kin (3mg/l) and MS +NAA (3mg/l) +BA (1mg/l), no sign of responses were noticed and only change in color in some cases. Different types of parenchyma cells and many layers of thick wall sclerenchyma cells were observed on MS+BA (1mg/l).

Keywords: Maerua, stem node, shoots, buds, In vitro

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Authors: Ipsita Pujari, Abitha Thomas, Vidhu S. Babu, K. Satyamoorthy

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Orchids are esteemed as celebrities in cut flower industry globally, due to their long-lasting fragrance and freshness. Apart from splendor, the unique metabolites endowed with pharmaceutical potency have made them one of the most hunted in plant kingdom. This had led to their trafficking, resulting in habitat loss, subsequently making them occupiers of IUCN red list as RET species. Many of the orchids especially wild varieties still remain undiscovered. In view to protect and conserve the wild germplasm, researchers have been inventing novel micropropagation protocols; thereby conserving Orchids. India is overflowing with exclusive wild cultivars of Orchids, whose pharmaceutical properties remain untapped and are not marketed owing to relatively small flowers. However, their germplasm is quite pertinent to be preserved for making unusual hybrids. Dendrobium genus is the second largest among Orchids exists in India and has highest demand attributable to enduring cut flowers and significant therapeutic uses in traditional medicinal system. Though the genus is quite endemic in Western Ghat regions of the country, many species are still anonymous with their unknown curative properties. A standard breeding cycle in Orchids usually takes five to seven years (Dendrobium hybrids taking a long juvenile phase of two to five years reaching maturity and flowering stage) and this extensive life cycle has always hindered the development of Dendrobium breeding. Dendrobium is reported with essential therapeutic plant bio-chemicals and ‘Moscatilin’ is one, found exclusive to this famous Dendrobium genus. Moscatilin is reported to have anti-mutagenic and anti-cancer properties, whose positive action has very recently been demonstrated against a range of cancers. Our preliminary study here established a simple and economic small-scale propagation protocol of Dendrobium ovatum describing in vitro production of Moscatilin. Subsequently for enhancing the content of Moscatilin, an efficient experimental related to the organization of transgenic (hairy) D. ovatum root cultures through infection of Agrobacterium rhizogenes 2364 strain on MS basal medium is being reported in the present study. Hairy roots generated on almost half of the explants used (spherules, in vitro plantlets and calli) maintained through suspension cultures, after 8 weeks of co-cultivation with Agrobacterium rhizogenes. GFP assay performed with isolated hairy roots has confirmed the integrative transformation which was further positively confirmed by PCR using rolB gene specific primers. Reverse phase-high performance liquid chromatography and mass spectrometry techniques were used for quantification and accurate identification of Moscatilin respectively from transgenic systems. A noticeable ~3 fold increase in contents were observed in transformed D. ovatum root cultures as compared to the simple in vitro culture, callus culture and callus regeneration plantlets. Role of elicitors e.g., Methyl jasmonate, Salicylic acid, Yeast extract and Chitosan were tested for elevating the Moscatilin content to obtain a comprehensive optimized protocol facilitating the in vitro production of valuable Moscatilin with larger yield. This study would provide evidence towards the in vitro assembly of Moscatilin within a short time-period through not a so-expensive technology for the first time. It also serves as an appropriate basis for bioreactor scale-up resulting in commercial bioproduction of Moscatilin.

Keywords: bioproduction, Dendrobium ovatum, hairy root culture, moscatilin

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Authors: R. Mehta, M. Ghogomu, B. Schoel

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Reports appeared in 2020 about China detecting SARS-Cov-2 (Covid-19) on frozen meat, shrimp, and food packaging material. In this study, we examined the use of swabs for the detection of Covid-19 on meat samples, and chicken breast (CB) was used as a model. Methods: Heat inactivated SARS-Cov-2 virus (IV) from Microbiologics was loaded onto the CB, swabbing was done, and the recovered inactivated virus was subjected to the Machery & Nagel NucleoSpin RNAVirus kit for RNA isolation according to manufacturer's instructions. For RT-PCR, the IDT 2019-nCoV RUO Covid-19 test kit was used with the Taqman Fast Virus 1-step master mix. The limit of detection (LOD) of viral load recovered from the CB was determined under various conditions: first on frozen CB where the IV was introduced on a defined area, then on frozen CB, with IV spread-out, and finally, on thawed CB. Results: The lowest amount of IV which can be reliably detected on frozen CB was a load of 1,000 - 2,000 IV copies where the IV was loaded on one spot of about 1 square inch. Next, the IV was spread out over a whole frozen CB about 16 square inches. The IV could be recovered at a lowest load of 4,000 to 8,000 copies. Furthermore, the effects of temperature change on viral load recovery was investigated i.e., if raw unfrozen meat became contaminated and remains for 1 hour at 4°C or gets refrozen. The amount of IV recovered successfully from CB kept at 4°C and the refrozen CB was similar to the recovery gotten from loading the IV directly on the frozen CB. In conclusion, an assay using swabs was successfully established for the detection of SARS-Cov-2 on frozen or raw (unfrozen) CB with a minimal load of up to 8,000 copies spread over 16 square inches.

Keywords: assay, COVID-19, meat, SARS-Cov-2

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Authors: Benlaifa Meriem, Berredjem Hajira, Bouasla Radia, Berredjem Malika, Djebar Med Reda

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Toxoplasmosis is a cosmopolitan infection due to Toxoplasma gondii (T.gondii). It is a significant cause of congenital disease and an important opportunistic pathogen which has become a worldwide increasing problem due to the AIDS epidemic. Current available drugs do not give satisfactory results and often have only a static and several adverse side effects as it is the case of pyrimethamine. So, the need to develop and evaluate new drugs is critical. The purpose of this study is to investigate the in vitro and in vivo effects of the new chiral N-acylsulfonamide bis-oxazolidin-2-ones on T.gondii. In this study, anti-T.gondii RH strain activities, of two new chiral N-acylsulfonamide bis-oxazolidin-2-ones were evaluated in vitro, using a MRC-5 fibroblast tissue cultures to determine the concentration that inhibit parasite multiplication by 50% (IC50) of each drug and in vivo, by PCR detection of the tachyzoites in mice ascites after new molecules treatment, using the 35-fold repetitive B1 gene of T.gondii. The in vitro results demonstrated that the treatment with the tested molecules decreased the amount of tachyzoites in cell culture in a dose-dependent manner. The inhibition was complete for concentrations over 4 mg/ml. The IC50 of Mol 1 and Mol 2 were 1.5 and 3 mg/ml, respectively, and were quite similar to the control one (2 mg/ml). The Mol 1 was highly active against T.gondii in cell cultures than Mol 2; these results were similar to those of sulfadiazine-treated group (p < 0.05). Toxoplasma-specific DNA was demonstrated in all ascites samples from infected mice of the different tested groups. Mol 1 showed better effect than Mol 2, but it did not completely inhibit the parasite proliferation. The intensity of amplification products increased when the treatment started late after infection. These findings suggest continuous parasite replication despite the treatment. In conclusion, our results showed a promising treatment effect of the tested molecules and suggest that in vitro, the Mol 1, and Mol 2 have a dose-dependent effect and a high cytotoxicity on the studied cells. The present study revealed that concentration and duration of tested molecules treatment are major factors that influence the course of Toxoplasma infection in infected mice.

Keywords: cytotoxicity, PCR, sulfonamide, Toxoplasma gondii

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Authors: Azza T. Tahera, Eman M. Ahmeda, Nadia A. Khalila, Yassin M. Nissanb

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New 3-(pyridin-4-yl)-[1,2,4] triazolo [4,3-b] pyridazine derivatives 2a-i, 4a,b and 6a,b were designed, synthesized and evaluated as cytotoxic agents. All compounds were investigated for their in vitro cytotoxicity at a single dose 10-5M concentration towards 60 cancer cell lines according to USA NCI protocol. The preliminary screening results showed that the majority of tested compounds exhibited remarkable activity against SR (leukemia) cell panel. Molecular docking for all synthesized compounds was performed on the active site of c-Met kinase. The most active compounds, 2f and 4a were further evaluated at a seven dose level screening and their IC50 as a c-Met kinase inhibitors were determined in vitro.

Keywords: triazolopyridazines, pyridazines, cytotoxic activity, cell panel

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Authors: Alsulami H., Alghamdi N., Alasker A., Almohen N., Shome D.

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Most conventional chemotherapeutic agents which are used for the treatment of cancers not only eradicate cancer cells but also affect normal hematopoietic Stem cells (HSCs) that leads to severe pancytopenia during treatment. Therefore, a need exists for novel approaches to treat cancer without or with minimum effect on normal HSCs. Parthenolide (PTL), a herbal product occurring naturally in the plant Feverfew, is a potential new chemotherapeutic agent for the treatment of many cancers such as acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). In this study we investigated the effect of different PTL concentrations on the viability of normal HSCs and also on the ability of these cells to form colonies after they have been treated with PTL in vitro. Methods: In this study, 24 samples of bone marrow and cord blood were collected with consent, and mononuclear cells were separated using density gradient separation. These cells were then exposed to various concentrations of PTL for 24 hours. Cell viability after culture was determined using 7ADD in a flow cytometry test. Additionally, the impact of PTL on hematopoietic stem cells (HSCs) was evaluated using a colony forming unit assay (CFU). Furthermore, the levels of NFҝB expression were assessed by using a PE-labelled anti-pNFκBP65 antibody. Results: this study showed that there was no statistically significant difference in the percentage of cell death between untreated and PTL treated cells with 5 μM PTL (p = 0.7), 10 μM PTL (p = 0.4) and 25 μM (p = 0.09) respectively. However, at higher doses, PTL caused significant increase in the percentage of cell death. These results were significant when compared to untreated control (p < 0.001). The response of cord blood cells (n=4) on the other hand was slightly different from that for bone marrow cells in that the percentage of cell death was significant at 100 μM PTL. Therefore, cord blood cells seemed more resistant than bone marrow cells. Discussion &Conclusion: At concentrations ≤25 μM PTL has a minimum or no effect on HSCs in vitro. Cord blood HSCs are more resistant to PTL compared to bone marrow HSCs. This could be due to the higher percentage of T-lymphocytes, which are resistant to PTL, in CB samples (85% in CB vs. 56% in BM. Additionally, CB samples contained a higher proportion of CD34+ cells, with 14.5% of brightly CD34+ cells compared to only 1% in normal BM. These bright CD34+ cells in CB were mostly negative for early-stage stem cell maturation antigens, making them young and resilient to oxidative stress and high concentrations of PTL.

Keywords: stem cell, parthenolide, NFKB, CLL

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Authors: Mehieddine Bouatrous

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Recently bioactive ceramic materials have been applied in the biomedical field as bulk, granular, or coating materials for more than half a century. More recently, bone tissue engineering scaffolds made of highly porous bioactive ceramic, glass-ceramic, and composite materials have also been created. As a result, recent bioactive ceramic structures have a high bioactivity rate, an open pores network, and good mechanical characteristics simulating cortical bone. Cyclowollastonite frameworks are also suggested for use as a graft material. As a porogenous agent, various amounts of the polymethyl methacrylate (PMMA) powders were used in this study successfully to synthesize a highly interrelated, nanostructured porous cyclowollastonite with a large specific surface area where the morphology and porosity were investigated. Porous cyclowollastonite bioactive ceramics were synthesized with a cost-effective and eco-friendly wet chemical method. The synthesized biomaterial is bioactive according to in vitro tests and can be used for bone tissue engineering scaffolds where cyclowollastonite sintered dense discs were submerged in simulated body fluid (S.B.F.) for various periods of time (1-4 weeks), resulting in the formation of a dense and consistent layer of hydroxyapatite on the surface of the ceramics, indicating its good in vitro bioactivity. Therefore, the cyclowollastonite framework exhibits good in vitro bioactivity due to its highly interconnecting porous structure and open macropores. The results demonstrate that even after soaking for several days, the surface of cyclowollastonite ceramic can generate a dense and consistent layer of hydroxyapatite. The results showed that cyclowollastonite framework exhibits good in vitro bioactivity due to highly interconnecting porous structure and open macropores.

Keywords: porous, bioactive, biomaterials, S.B.F, cyclowollastonite, biodegradability

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Authors: Tayyaba Bari, Muhammad Hamza Anjum, Samra Kanwal, Fakhera Ikram

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Osteoarthritis, a degenerative condition, affects more than 213 million individuals globally. Since articular cartilage has no or limited vessels, therefore, after deteriorating, it is unable to rejuvenate. Traditional approaches for cartilage repair, like autologous chondrocyte implantation, microfracture and cartilage transplantation are often associated with postoperative complications and lead to further degradation. Decellularized human umbilical cord has gained interest as a viable treatment for cartilage repair. Decellularization removes all cellular contents as well as debris, leaving a biologically active 3D network known as extracellular matrix (ECM). This matrix is biodegradable, non-immunogenic and provides a microenvironment for homeostasis, growth and repair. UC derived bioink function as 3D scaffolding material, not only mediates cell-matrix interactions but also adherence, proliferation and propagation of cells for 3D organoids. This study comprises different physical, chemical and biological approaches to optimize the decellularization of human umbilical cord (UC) tissues followed by the solubilization of these tissues to bioink formation. The decellularization process consisted of two cycles of freeze thaw where the umbilical cord at -20˚C was thawed at room temperature followed by dissection in small sections from 0.5 to 1cm. Similarly decellularization with ionic and non-ionic detergents Sodium dodecyl sulfate (SDS) and Triton-X 100 revealed that both concentrations of SDS i.e 0.1% and 1% were effective in complete removal of cells from the small UC tissues. The results of decellularization was further confirmed by running them on 1% agarose gel. Histological analysis revealed the efficacy of decellularization, which involves paraffin embedded samples of 4μm processed for Hematoxylin-eosin-safran and 4,6-diamidino-2-phenylindole (DAPI). ECM preservation was confirmed by Alcian Blue, and Masson’s trichrome staining on consecutive sections and images were obtained. Sulfated GAG’s content were determined by 1,9-dimethyl-methylene blue (DMMB) assay, similarly collagen quantification was done by hydroxy proline assay. This 3D bioengineered scaffold will provide a typical atmosphere as in the extracellular matrix of the tissue, which would be seeded with the mesenchymal cells to generate the desired 3D ink for in vitro and in vivo cartilage regeneration applications.

Keywords: umbilical cord, 3d printing, bioink, tissue engineering, cartilage regeneration

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Authors: Wanbao Chen, Qianqian Yao, Zhenming Zhou

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Whole grains with intact pericarp are largely resistant to digestion by ruminants because entire kernels are not conducive to bacterial attachment. But processing methods makes the starch more accessible to microbes, and increases the rate and extent of starch degradation in the rumen. To estimate the feasibility of applying a steam-flaking as the processing technique of grains for ruminants, cereal grains (maize, wheat, barley and sorghum) were processed by steam-flaking (steam temperature 105°C, heating time, 45 min). And chemical analysis, in vitro gas production, volatile fatty acid concentrations, and energetic values were adopted to evaluate the effects of steam-flaking. In vitro cultivation was conducted for 48h with the rumen fluid collected from steers fed a total mixed ration consisted of 40% hay and 60% concentrates. The results showed that steam-flaking processing had a significant effect on the contents of neutral detergent fiber and acid detergent fiber (P < 0.01). The concentration of starch gelatinization degree in all grains was also great improved in steam-flaking grains, as steam-flaking processing disintegrates the crystal structure of cereal starch, which may subsequently facilitate absorption of moisture and swelling. Theoretical maximum gas production after steam-flaking processing showed no great difference. However, compared with intact grains, total gas production at 48 h and the rate of gas production were significantly (P < 0.01) increased in all types of grain. Furthermore, there was no effect of steam-flaking processing on total volatile fatty acid, but a decrease in the ratio between acetate and propionate was observed in the current in vitro fermentation. The present study also found that steam-flaking processing increased (P < 0.05) organic matter digestibility and energy concentration of the grains. The collective findings of the present study suggest that steam-flaking processing of grains could improve their rumen fermentation and energy utilization by ruminants. In conclusion, the utilization of steam-flaking would be practical to improve the quality of common cereal grains.

Keywords: cereal grains, gas production, in vitro rumen fermentation, steam-flaking processing

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Authors: Er-Yuan Chuang

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Bio-mimetic matters have biological functionalities. This might be valuable in the development of versatile biomaterials. At biological fields, protein-based materials might be components to form a 3D network of extracellular biomolecules, containing growth factors. Also, the protein-based biomaterial provides biochemical and structural assistance of adjacent cells. In this study, we try to prepare protein based biomaterial, which was harvested from living animal. We analyzed it’s chemical, physical and biological property in vitro. Besides, in vivo bio-interaction of the prepared biomimetic matrix was tested in an animal model. The protein-based biomaterial has degradability and biocompatibility. This development could be used for tissue regenerations and be served as platform technologies.

Keywords: protein based, in vitro study, in vivo study, biomaterials

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Authors: Harshani Uggallage, Kapila D. Dissanayaka

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A Sulforhodamine-B assay-guided fractionation on Nigella sativa seeds was conducted to determine the presence of cytotoxic compounds against human hepatoma (HepG2) cells. Initially, a freeze-dried sample of Nigella sativa seeds was sequentially extracted into solvents of increasing polarities. Crude extracts from the sequential extraction of Nigella sativa seeds in chloroform and ethyl acetate showed the highest cytotoxicity. The combined mixture of these two extracts was subjected to bioassay guided fractionation using a modified Kupchan method of partitioning, followed by Sephadex® LH-20 chromatography. This chromatographic separation process resulted in a column fraction with a convincing IC50 (half-maximal inhibitory concentration) value of 13.07µg/ml, which is considerable for developing therapeutic drug leads against human hepatoma. Reversed phase High-Performance Liquid Chromatography (HPLC) was finally conducted for the same column fraction, and the result indicates the presence of one or several main cytotoxic compounds against human HepG2 cells.

Keywords: cytotoxic compounds, half-maximal inhibitory concentration, high-performance liquid chromatography, human HepG2 cells, nigella sativa seeds, Sulforhodamine-B assay

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Authors: Abdelkrim Cheriti, Mebarka Belboukhari, Nasser Belboukhari

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Launaea Cass. is a small genus of the family Asteraceae (tribe Lactuceae, subtribe Sonchinae), consisting of 54 species, of which 9 are presented in the flora of Algeria and is mainly distributed in the South Mediterranean, Africa and SW Asia. Plants in the Launaea genus have been used ethnobotanically as bitter stomachic, for treating diarrhea, gastrointestinal tracts, as anti-inflammatory, for skin diseases, treatment of infected wounds, hepatic pains, children fever, as soporific, lactagogue, diuretic and as insecticidal. Antioxidants are vital substances, which possess the ability to protect the body from damages caused by free radical induced oxidative stress. A variety of free radical scavenging antioxidants is found in a number of dietary sources. The main objective of this study focused on the screening of antioxidant activity of Launaea nudicaulis (Asteraceae) extracts. The in vitro antioxidant activity was investigated with DPPH radical scavenging assay. The quantitative evaluation of DPPH scavenging activity showed that n-BuOH and EtOAc extracts are the most active extracts with a percentage of antiradical activity of 89,62% and 71,57% respectively.

Keywords: Launaea, phytochemical, South Algeria, Sahara, endemic specie

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Authors: Mathias Twizeyimana, Urmila Adhikari, Julius P. Sserumaga, David Ingham

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The management of aflatoxins (naturally occurring toxins produced by certain fungi, most importantly Aspergillus flavus and A. parasiticus) relies mostly on the use of best cultural practices and, in some cases, the use of the biological control consisting of atoxigenic strains inhibiting the toxigenic strains through competition resulting in considerable toxin reduction. At AgBiome, we have built a core collection of over 100,000 fully sequenced microbes from diverse environments and employ both the microbes and their sequences in the discovery of new biological products for disease and pest control. The most common approach to finding beneficial microbes consists of isolating microorganisms from samples collected from diverse environments, selecting antagonistic strains through empirical screening, studying modes of action, and stabilization through the formulation of selected microbial isolates. A total of 608 diverse bacterial strains were screened using a high-throughput assay (48-well assay) to identify strains that inhibit toxigenic A. flavus growth on maize kernels. Active strains in 48-well assay had their pathogen inhibiting activity confirmed using the Flask Assay and were concurrently tested for their ability to reduce the aflatoxin content in maize grains. Strains with best growth inhibition and reduction of aflatoxin were tested in the greenhouse and field trials. From the field trials, three bacterial strains, AFS000009 (Pseudomonas chlororaphis), AFS032321 (Bacillus subtilis), AFS024683 (Bacillus velezensis), had aflatoxin concentrations (ppb) values that were significantly lower than those of inoculated control. The identification of biological products with high efficacy in inhibiting pathogen growth and eventually reducing the aflatoxin content will provide a valuable alternative to control strategies used in aflatoxin contamination management.

Keywords: aflatoxin, microorganism bacteria, biocontrol, beneficial microbes

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Authors: D. R. Masvodza, G. Coetzer, E. van der Watt

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Microorganisms usually have a prolific growth nature and may cause major problems on in-vitro cultures. For in vitro propagation to be successful explants need to be sterile. In order to determine the best sterilization method for potato explants cv. Amerthyst, five sterilization methods were applied separately to 24 shoots. The first sterilization method was the use of 20% sodium hypochlorite with 1 ml Tween 20 for 15 minutes. The second, third and fourth sterilization methods were the immersion of explants in 70% ethanol in a beaker for either 30 seconds, 1 minute or 2 minutes, followed by 1% sodium hypochlorite with 1 ml Tween 20 for 5 minutes. For the control treatment, no chemicals were used. Finally, all the explants were rinsed three times with autoclaved distilled water and trimmed to 1-2 cm. Explants were then cultured on MS medium with 0.01 mg L-1 NAA and 0.1 mg L-1 GA3 and supplemented with 2 mg L-1 D-calcium pentothenate. The trial was laid out as a complete randomized design, and each treatment combination was replicated 24 times. At 7, 14 and 21 days after culture, data on explant color, survival, and presence or absence of contamination was recorded. Best results were obtained when 20% sodium hypochlorite was used with 1 ml Tween 20 for 15 minutes which is sterilization method 1. Method 2 was comparable to method 1 when explants were cultured in glass vessels. Explants in glass vessels were significantly less contaminated than explants in polypropylene vessel. Therefore at times, ideal methods for sterilization should be coupled with ideal culture conditions such as good quality culture vessel, rather than the addition of more stringent sterilants.

Keywords: culture containers, explants, sodium hypochlororite, sterilization

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Authors: Mardiati Zain, W. S. N. Rusmana, Erpomen, Malik Makmur, Ezi Masdia Putri

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Background and Aim: The leaves of the Leucaenaleucocephala tree have potential as a nitrogen source for ruminants. Leucaena leaf meal as protein supplement has been shown to improve the feed quality of ruminants. The effects of different levels of Leucaena leucocephala supplementation as substitute of concentrate on fiber digestibility and in vitro rumen fermentation characteristics were investigated. This research was conducted in vitro. The study used a randomized block design consisting of 3 treatments and 5 replications. The treatments were A. 40% rice straw ammoniated + 60% concentrate, B. 40% rice straw ammoniated + 50% concentrate + 10% Leucaena leuchephala, C. 40% rice straw ammoniated + 40% concentrate + 20% Leucaena leuchephala, Result: The results showed that the addition of Leucaena leucocephala increased the digestibility of Neutral detergent Fiber NDF and Acid Detergent Fiber (ADF) (p < 0.05). In this study, rumen NH3, propionate, amount of escape protein and total Volatyl Fatty Acid (VFA) were found increased significantly at treatment B. No significant difference was observed in acetate and butyrate production. The populations of total protozoa and methane production had significantly decreased (P < .05) in supplemented group. Conclusion: Supplementation of leuchaena leucochepala on completed feed based on ammoniated rice straw in vitro can increase fiber digestibility, VFA production and decreased protozoa pupulataion and methane production. Supplementation of 10% and 20% L. leucochepala were suitable to be used for further studies, therefore in vivo experiment is required to study the effects on animal production.

Keywords: digestibility, Leucaena leucocephala, complete feed, rice straw ammoniated

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Authors: Melanie Ostermann, Eivind Birkeland, Ying Xue, Alexander Sauter, Mihaela R. Cimpan

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Background: New reliable and robust techniques to assess biological effects of nanomaterials (NMs) in vitro are needed to speed up safety analysis and to identify key physicochemical parameters of NMs, which are responsible for their acute cytotoxicity. The central aim of this study was to validate and evaluate the applicability and reliability of an impedance-based flow cytometry (IFC) technique for the high-throughput screening of NMs. Methods: Eight inorganic NMs from the European Commission Joint Research Centre Repository were used: NM-302 and NM-300k (Ag: 200 nm rods and 16.7 nm spheres, respectively), NM-200 and NM- 203 (SiO₂: 18.3 nm and 24.7 nm amorphous, respectively), NM-100 and NM-101 (TiO₂: 100 nm and 6 nm anatase, respectively), and NM-110 and NM-111 (ZnO: 147 nm and 141 nm, respectively). The aim was to assess the biological effects of these materials on human monoblastoid (U937) cells. Dispersions of NMs were prepared as described in the NANOGENOTOX dispersion protocol and cells were exposed to NMs at relevant concentrations (2, 10, 20, 50, and 100 µg/mL) for 24 hrs. The change in electrical impedance was measured at 0.5, 2, 6, and 12 MHz using the IFC AmphaZ30 (Amphasys AG, Switzerland). A traditional toxicity assay, Trypan Blue Dye Exclusion assay, and dark-field microscopy were used to validate the IFC method. Results: Spherical Ag particles (NM-300K) showed the highest toxic effect on U937 cells followed by ZnO (NM-111 ≥ NM-110) particles. Silica particles were moderate to non-toxic at all used concentrations under these conditions. A higher toxic effect was seen with smaller sized TiO2 particles (NM-101) compared to their larger analogues (NM-100). No interferences between the IFC and the used NMs were seen. Uptake and internalization of NMs were observed after 24 hours exposure, confirming actual NM-cell interactions. Conclusion: Results collected with the IFC demonstrate the applicability of this method for rapid nanotoxicity assessment, which proved to be less prone to nano-related interference issues compared to some traditional toxicity assays. Furthermore, this label-free and novel technique shows good potential for up-scaling in directions of an automated high-throughput screening and for future NM toxicity assessment. This work was supported by the EC FP7 NANoREG (Grant Agreement NMP4-LA-2013-310584), the Research Council of Norway, project NorNANoREG (239199/O70), the EuroNanoMed II 'GEMN' project (246672), and the UH-Nett Vest project.

Keywords: cytotoxicity, high-throughput, impedance, nanomaterials

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

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After cold pressing of pumpkin oil, the defatted oil cake (PUOC) was utilized 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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Authors: Benjaporn Buranrat, Nootchanat Mairuae

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Cratoxylum formosum (Jack) Dyer (CF) has been used for the traditional medicines in South East Asian and Thailand. Normally, northeast Thai vegetables have proven cytotoxic to many cancer cells. Therefore, the present study aims to explore the molecular mechanisms underlying CF-induced cancer cell death and apoptosis on breast and liver cancer cells. The cytotoxicity and antiproliferative effects of CF on the human breast MCF-7 and liver HepG2 cancer cell lines were evaluated using sulforhodamine B assay and colony formation assay. Cell migration assay was measured using wound healing assay. The apoptosis induction mechanisms were investigated through reactive oxygen species formation, caspase 3 activity, and JC-1 activity. Gene expression by real-time PCR and apoptosis related protein levels by Western blot analysis. CF induced MCF-7 and HepG2 cell death by time- and dose-dependent manner. Furthermore, CF had the greater cytotoxic potency on MCF-7 more than HepG2 cells with IC50 values of 85.70+4.52 μM and 219.03±9.96 μM respectively, at 24 h. Treatment with CF also caused a dose-dependent decrease in colony forming ability and cell migration, especially on MCF-7 cells. CF induced ROS formation, increased caspase 3 activities, and decreased the mitochondrial membrane potential, and causing apoptotic body production and DNA fragmentation. CF significantly decreased expression of the cell cycle regulatory protein RAC1 and downstream proteins, cdk6. Additionally, CF enhanced p21 and reduced cyclin D1 protein levels. CF leaf extract induced cell death, apoptosis, antimigration in both of MCF-7 and HepG2 cells. CF could be useful for developing to anticancer drug candidate for breast and liver cancer therapy.

Keywords: cratoxylum formosum (jack) dyer, breast cancer, liver cancer, cell death

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Authors: Samir Dekali, David Crouzier

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Due to their new physico-chemical properties engineered nanoparticles (ENPs) are increasingly employed in numerous industrial sectors (such as electronics, textile, aerospace, cosmetics, pharmaceuticals, food industry, etc). These new physico-chemical properties can also represent a threat for the human health. Consumers can notably be exposed involuntarily by different routes such as inhalation, ingestion or through the skin. Several studies recently reported a possible biodistribution of these ENPs on the blood-brain barrier (BBB). Consequently, there is a great need for developing BBB in vitro models representative of the in vivo situation and capable of rapidly and accurately assessing ENPs toxic effects and their potential translocation through this barrier. In this study, several in vitro models established with micro-endothelial brain cell lines of different origins (bEnd.3 mouse cell line or a new human cell line) co-cultivated or not with astrocytic cells (C6 rat or C8-B4 mouse cell lines) on Transwells® were compared using different endpoints: trans-endothelial resistance, permeability of the Lucifer yellow and protein junction labeling. Impact of NIST diesel exhaust particles on BBB cell viability is also discussed.

Keywords: nanoparticles, blood-brain barrier, diesel exhaust particles, toxicology

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Authors: Archana Sharma, Vijayashree Nayak, Ashok Kumar

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Three-dimensional matrices were fabricated from blend of natural-natural polymers like carrageenan-gelatin and synthetic -natural polymers such as PEG- gelatin (PEG of different molecular weights (2,000 and 6,000) using two different crosslinkers; glutaraldehyde and EDC-NHS by cryogelation technique. Blends represented a feasible approach to design 3-D scaffolds with controllable mechanical, physical and biochemical properties without compromising biocompatibility and biodegradability. These matrices possessed interconnected porous structure, good mechanical strength, biodegradable nature, constant swelling kinetics, ability to withstand high temperature and visco-elastic behavior. Hemocompatibility of cryogel matrices was determined by coagulation assays and hemolytic activity assay which demonstrated that these cryogels have negligible effects on coagulation time and have excellent blood compatibility. In vitro biocompatibility (cell-matrix interaction) inferred good cell adhesion, proliferation, and secretion of ECM on matrices. These matrices provide a microenvironment for the growth, proliferation, differentiation and secretion of ECM of different cell types such as IMR-32, C2C12, Cos-7, rat bone marrow derived MSCs and human bone marrow MSCs. Hoechst 33342 and PI staining also confirmed that the cells were uniformly distributed, adhered and proliferated properly on the cryogel matrix. An ideal scaffold used for tissue engineering application should allow the cells to adhere, proliferate and maintain their functionality. Neurotransmitter analysis has been done which indicated that IMR-32 cells adhered, proliferated and secreted neurotransmitters when they interacted with these matrices which showed restoration of their functionality. The cell-matrix interaction up to molecular level was also evaluated so to check genotoxicity and protein expression profile which indicated that these cryogel matrices are non-genotoxic and maintained biofunctionality of cells growing on these matrices. All these cryogels, when implanted subcutaneously in balb/c mice, showed no adverse systemic or local toxicity effects at implantation site. There was no significant increase in inflammatory cell count has otherwise been observed after scaffold implantation. These cryogels are supermacroporous and this porous structure allows cell infiltration and proliferation of host cells. This showed the integration and presence of infiltrated cells into the cryogel implants. Histological analysis confirmed that the implanted cryogels do not have any adverse effect in spite of host immune system recognition at the site of implantation, on its surrounding tissues and other vital host organs. In vivo biocompatibility study after in vitro biocompatibility analysis has also concluded that these synthesized cryogels act as important biological substitutes, more adaptable and appropriate for transplantation. Thus, these cryogels showed their potential for soft tissue engineering applications.

Keywords: cryogelation, hemocompatibility, in vitro biocompatibility, in vivo biocompatibility, soft tissue engineering applications

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Authors: Qiao Yushuang, Shao Longyi

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This paper presents the result of plasmid assay of inhalable particulates PM10 and PM2.5 that were collected during the period of the 11th Hanyuan temple fair of ancestor worship in Kaifeng City. By use of a high-resolution Field Emission Scanning Electron Microscopy (FESEM) and image analysis (IA) technology, the morphological characteristics and Particle Size Distribution (PSD) of each were analyzed and the Bioreactivity of PM10 was evaluated by using plasmid DNA assay. The result shows that, as the dominant component of the samples taken in the urban area of Kaifeng City, the mineral particles, compared with the other components including the soot aggregates, coal ash, and unidentified particles, have a much greater amount and volume. The mineral particles exhibited a decentralized quantity - size distribution, whose presence could be available among the particles sizing 2.5μm or smaller. In contrast, the volume-size distribution of mineral particles is scattered in a relatively narrow range of between1μm and 2.5μm. According to the plasmid assay the TD50 (toxic dose of PM causing 50% of plasmid damage, expressed in μg/ml) of water-soluble PM10 and whole fraction of Kaifeng airborne PM10 was measured respectively at 220-208μg/ml and 300-400μg/ml versus 160μg/ml and 190μg/ml for PM2.5. It can be seen that the whole fraction of airborne particles caused more oxidative damage than the water-soluble fractions, and the PM2.5 has a greater oxidative capacity than the PM10.

Keywords: inhalable particulates (PM10 and PM2.5), morphological features, bioreactivity, Kaifeng

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Authors: Athanasios Triantafyllou, Georgios Papagiannis, Vassilios Nikolaou, Panayiotis J. Papagelopoulos, George C. Babis

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In vitro measurements are widely used in order to predict THAs wear rate implementing gait kinematic and kinetic parameters. Clinical tests of materials and designs are crucial to prove the accuracy and validate such measurements. The purpose of this study is to examine the affection of THA gait kinematics and kinetics on wear during gait, the essential functional activity of humans, by comparing in vivo gait data to in vitro results. Our study hypothesis is that both implants will present the same hip joint kinematics and kinetics during gait. 127 unilateral primary cementless total hip arthroplasties were included in the research. Independent t-tests were used to identify a statistically significant difference in kinetic and kinematic data extracted from 3D gait analysis. No statistically significant differences observed at mean peak abduction, flexion and extension moments between the two groups (P.abduction= 0,125, P.flexion= 0,218, P.extension= 0,082). The kinematic measurements show no statistically significant differences too (Prom flexion-extension= 0,687, Prom abduction-adduction= 0,679). THA kinematics and kinetics during gait are important biomechanical parameters directly associated with implants wear. In vitro studies report less wear in CoC than CoXLPE when tested with the same gait cycle kinematic protocol. Our findings confirm that both implants behave identically in terms of kinematics in the clinical environment, thus strengthening in vitro results of CoC advantage. Correlated to all other significant factors that affect THA wear could address in a complete prism the wear on CoC and CoXLPE.

Keywords: total hip arthroplasty biomechanics, THA gait analysis, ceramic on ceramic kinematics, ceramic on XLPE kinetics, total hip replacement wear

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Authors: Ishmael Ntlhamu

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In Limpopo Province, the use of herbal concoctions is becoming very popular. These concoctions are claimed to be capable of treating ulcers, diabetes, certain STDs, blood cleansing, and many more types of diseases. The aim of this study was to evaluate the phytochemical composition, evaluate the pharmacological effects and consumption safety in herbal concoctions to treat various kinds of ailments in Limpopo. The concoctions were extracted with 80% acetone. Microorganisms in the concoctions were identified using the Vitek 2 compact system. Qualitative phytochemical analysis was determined using standard chemical tests and thin layer chromatography (TLC). Total polyphenol content was quantified. Antioxidant activity was quantified using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay and ferric reducing power. Antimicrobial activities were determined using a broth micro-dilution assay and bioautography. Cell viability assay was used to determine the cytotoxicity. Results showed that concoctions had antioxidant activity. Presence of different phytoconstituents was observed. Isolated microorganisms were identified as Burkholderia pseudomallei, Staphylococcus vitulimus, Enterococcus columbae, Kocuria kristanae, Staphylococcus intermedius, Cryptococcus laurenti. and Burkholderia pseudomallei (highly pathogenic). Therefore, phytochemicals prove that the concoctions can heal as the antimicrobial tests also displayed activity. Moreover, the concoctions did not exhibit cytotoxic effects. However, contaminants raise concerns, not only for consumer safety but also the quality of herbal concoctions available as part of the traditional medicinal practice in Limpopo.

Keywords: antimicrobials, concoctions, cytotoxicity, phytochemicals

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Authors: Sihem Benmimoune, Abdelbaki Lemgharbi, Ahmed Ait Yahia, Abdelkrim Kameli

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Our study is based on chemical and phytochemical characterization of Artemisia absinthium L and in vitro tests to demonstrate the biological activities of essential oil and natural extract. A qualitative and quantitative comparison of the essential oil extracted by two extraction procedures was performed by analysis of CG/SM and the yield calculation. The method of hydrodistillation has a chemical composition and provides oil content than the best training water vapor. These oils are composed mainly of thujone followed chamazulene and ρ-cymene. The antimicrobial activity of wormwood oil was tested in vitro by two methods (agar diffusion and microdilution) on four plant pathogenic fungi (Aspergillus sp, Botrytis cinerea, Fusarium culmorum and Helminthosporium sp). The study of the antifungal effect showed that this oil has an inhibitory effect counterpart the microorganisms tested in particular the strain Botrytis cinerea. Otherwise, this activity depends on the nature of the oil and the germ itself. The antioxidant activity in vitro was studied with the DPPH method. The activity test shows that the oil and extract of Artemisia absinthium have a very low antioxidant capacity compared to the antioxidants used as a reference. The extract has a potentially high antiradical power not from its oil. The quantitative determinations of phenolic compounds by the Folin-Ciocalteu revealed that absinthe is low in total polyphenols and tannins.

Keywords: artemisia absinthium, biological activities, essential oil, extraction processes

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Authors: Yohannes Kelifa, Gomathi Periasamy, Aman Karim

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Introduction: Diabetes mellitus has become a major public health and economical problem across the globe. Modern antidiabetic drugs have a number of limitations, and scientific investigation of traditional herbal remedies used for diabetes may provide novel leads for the development of new antidiabetic drugs that can be used as alternative or complementary to available antidiabetic allopathic medications. Though Myrica salicifolia Hochst. ex A. Rich. is used for the management of diabetes in Ethiopian traditional medicine, there was no previous scientific evidence about its antidiabetic effect to the authors’ knowledge. This study was undertaken to evaluate the antidiabetic activity the root extracts of Myrica salicifolia in streptozotocin (STZ)-induced diabetic mice. Methods: Experimental diabetes was induced by intraperitoneal administration of STZ (150 mg/kg) in male mice. Diabetic mice were treated with oral doses of M. salicifolia root extracts at 200, 400 and 600 mg/kg, and its fractions (chloroform, ethyl acetate, n-butanol and aqueous) at a dose of 400 mg/kg daily for 15 days. Fasting blood glucose level (BGL) was measured at 0, 5th,10th, and 15th day. The free radical scavenging activity of the crude extract was determined using in vitro by DPPH assay. The statistical significance was assessed by one-way ANOVA, followed by Tukey’s multiple comparison tests. Results were considered significant when p < 0.05. Results: Daily administration of the M. salicifolia 80% methanol root extracts (at three different doses (200, 400 and 600 mg/kg) significantly (p < 0.05, p < 0.01 and p < 0.001) reduced fasting BGL compared with diabetic control. The aqueous and butanol fractions at a dose of 400 mg/kg resulted in maximum reduction of fasting BGL by 42.39%, and 52.13%, respectively at the 15th day in STZ-induced diabetic mice. Free radical scavenging activity of the 80% methanol extract of M. salicifolia was comparable to ascorbic acid. The IC50 values of the crude extract and ascorbic acid (a reference compound) were found to be 4.54 μg/ml and 4.39 μg/ml, respectively. Conclusion: These findings demonstrated that the methanolic extracts of M. salicifolia root and its fractions (n-butanol and aqueous) exhibit a significant antihyperglycemic activity in STZ-induced diabetic mice. Furthermore, the result of the present study indicates that M. salicifolia root extract is a potential source of natural antioxidants.

Keywords: antidiabetic, diabetes mellitus, DPPH, mice, Myrica salicifolia, streptozotocin

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Authors: Borokini Funmilayo Boede

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B. alba L., commonly called ‘Amunututu’ and Solanecio biafrae called ‘Worowo’ among the Yoruba tribe in the southwest part of Nigeria are reported to be of great ethnomedicinal importance but are among many underutilized green leafy vegetables in the country. Many studies have established the nutritional values of these vegetables, utilization are very poor and indepth information on their chemical profiles is scarce. The aqueous, methanolic and ethanolic extracts of these vegetables were subjected to phytochemical screening and phenolic profiles of the alcoholic extracts were characterized by using high-performance liquid chromatography coupled with diode array detector (HPLC-DAD). Total phenol and flavonoid contents were determined, antioxidant activities were evaluated using five in vitro assays to assess DPPH, nitric oxide and hydroxyl radical-scavenging abilities, as well as reducing power with ferric reducing antioxidant assay and phosphomolybdate method. The antibacterial activities of the extracts against Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhi were evaluated by using agar well diffusion method and the antifungal activity evaluated against food-associated filamentous fungi by using poisoned food technique with the aim of assessing their nutraceutical potentials to encourage their production and utilization. The results revealed the presence of saponnin, steroids, tannin, terpenoid and flavonoid as well as phenolic compounds: gallic acid, chlorogenic acid, caffeic acid, coumarin, rutin, quercitrin, quercetin and kaemferol. The vegetables showed varying concentration dependent reducing and radical scavenging abilities from weak to strong compared with gallic acid, rutin, trolox and ascorbic acid used as positive controls; the aqueous extracts which gave higher concentrations of total phenol displayed higher ability to reduce Fe (lll) to Fe (ll) and stronger inhibiting power against hydroxyl radical than the alcoholic extracts and in most cases exhibited more potency than the ascorbic acids used as positive controls, at the same concentrations, whereas, methanol and / or ethanol extracts were found to be more effective in scavenging 2, 2-diphenyl-1-picryl hydrazyl radical and showed higher ability to reduce Mo (VI) to Mo (V) in total antioxidant assay than the aqueous extracts. However, the inhibition abilities of all the extracts against nitric oxide were comparable with the ascorbic acid control at the same concentrations. There were strong positive correlations with total phenol (mg GAE/g) and total flavonoid (mg RE/g) contents in the range TFC (r=0.857- 0999 and r= 0.904-1.000) and TPC (r= 0.844- 0.992 and r= 0.900 -0.999) for Basella alba and Senecio biafrae respectively. Inhibition concentration at 50 % (IC50) for each extract to scavenge DPPH, OH and NO radicals ranged from 32.73 to 1.52 compared with control (0.846 - -6.42) mg/ml. At 0.05g/ml, the vegetables were found to exhibit mild antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi compared with streptomycin sulphate used as control but appreciable antifungi activities against (Trichoderma rubrum and Aspergillus fumigates) compared with bonlate antibiotic positive control. The vegetables possess appreciable antioxidant and antimicrobial properties for promoting good health, their cultivation and utilization should be encouraged especially in the face of increasing health and economic challenges and food insecurity in many parts of the world.

Keywords: antimicrobial, antioxidants, extracts, phytochemicals

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Authors: D. H. Tejavathi

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Medicinal plants are globally valuable sources of herbal products. Wild populations of many medicinal plants are facing threat of extinction because of their narrow distribution, endemicity, and degradation of specific habitats. Micropropagation is an established in vitro technique by which large number of clones can be obtained from a small bit of explants in a short span of time within a limited space. Mycorrhization can minimize the transient transplantation shock, experienced by the micropropagated plants when they are transferred from lab to land. AM fungal association improves the physiological status of the host plants through better uptake of water and nutrients, particularly phosphorus. Consequently, the growth performance and biosynthesis of active principles are significantly enhanced in AM fungal treated plants. Bacopa monnieri, Andrographis paniculata, Agave vera-curz, Drymaria cordata and Majorana hortensis, important medicinal plants used in various indigenous systems of medicines, are selected for the present study. They form the main constituents of many herbal formulations. Standard in vitro techniques were followed to obtain the micropropagated plants. Shoot tips and nodal segments were used as explants. Explants were cultured on Murashige and Skoog, and Phillips and Collins media supplemented with various combinations of growth regulators. Multiple shoots were obtained on a media containing both auxins and cytokinins at various concentrations and combinations. Multiple shoots were then transferred to rooting media containing auxins for root induction. Thus, obtained in vitro regenerated plants were subjected to brief acclimatization before transferring them to land. One-month-old in vitro plants were treated with AM fungi, and the symbiotic effect on the overall growth parameters was analyzed. It was found that micropropagation coupled with mycorrhization has significant effect on the enhancement of biomass and biosynthesis of active principles in these selected medicinal plants. In vitro techniques coupled with mycorrhization have opened a possibility of obtaining better clones in respect of enhancement of biomass and biosynthesis of active principles. Beneficial effects of AM fungal association with medicinal plants are discussed.

Keywords: cultivation, medicinal plants, micropropagation, mycorrhization

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Authors: Khaled S. Al Salhen

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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 ± 7.6 µM) for hepatic 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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Authors: Qiuyue Peng, Vladimir Zachar

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The application of adipose-derived stromal/stem cells (ASCs) in regenerative medicine is gaining more awareness due to their advanced translational potential and abundant source preparations. However, ASC-based translation has been confounded by high subpopulation heterogeneity, causing ambiguity about its precise therapeutic value. Some phenotypes defined by a unique combination of positive and negative surface markers have been found beneficial to the required roles. Therefore, the immunophenotypic repertoires of cultured ASCs and temporal changes of distinct subsets were investigated in this study. ASCs from three donors undergoing cosmetic liposuction were cultured in standard culturing methods, and the co-expression patterns based on the combination of selected markers at passages 1, 4, and 8 were analyzed by multi-chromatic flow cytometry. The results showed that the level of heterogeneity of subpopulations of ASCs became lower by in vitro expansion. After a few passages, most of the CD166⁺/CD274⁺/CD271⁺ based subpopulations converged to CD166 single positive cells. Meanwhile, these CD29⁺CD201⁺ double-positive cells, in combination with CD36/Stro-1 expression or without, feathered only the major epitopes and maintained prevailing throughout the whole process. This study suggested that, upon in vitro expansion, the phenotype repertoire of ASCs redistributed and stabilized in a way that cells co-expressing exclusively the strong markers remained dominant. These preliminary findings provide a general overview of the distribution of heterogeneous subsets residents within human ASCs during expansion in vitro. It is a critical step to fully characterize ASCs before clinical application, although the biological effects of heterogeneous subpopulations still need to be clarified.

Keywords: adipose-derived stromal/stem cells, heterogeneity, immunophenotype, subpopulations

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Authors: Asad Ali, Asif Jamal

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The agriculture productivity losses due to microbial pathogens have been a serious issue in Pakistan and rest of the world. Present work was designed to isolate soil borne microorganisms having the antagonistic ability against notorious phytopathogens. From the initial collection of 23 bacterial isolates, two potent strains of Bacillus were screened on the basis of their comparative efficacy against devastating fungal pathogens. The strains AK-1 and AK-5 showed excellent inhibitory indexes against the majority of tested fungal strains. It was noted that both strains of Bacillus showed significant biocontrolling activity against Aspergillus flavus, Fusarium moniliforme, Colletotricum falcatum, Botrytis cinerea, Aspergillus niger, Fusarium oxysporum, Phythopthora capsici and Rhizopus oryzae. The strain AK-1 was efficient to suppress Aspergillus species and Rhizopus oryzae while AK-5 expressed significant antagonistic activity against Fusarium, Botrytis and Colletotricum species. On the basis of in vitro assay, it can be postulated that the Bacillus strains AK-1 and AK-5 can be used as bio-protective agent against various plant diseases. In addition, their applications as natural pesticides could be very helpful to prevent the adverse effects of chemical pesticides.

Keywords: bacillus species, biocontrol agent, biopesticides, phytopathogens

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