Search results for: in vitro metabolites

Authors: Bello Gonzalez, Setten Van M., Brouwer M.

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The chicken small intestine represents a dynamic and complex organ in which the enzymatic digestion and absorption of nutrients take place. The development of an in vitro fermentation chicken small intestinal model could be used as an alternative to explore the interaction between the microbiota and nutrient metabolism and to enhance the efficacy of targeting interventions to improve animal health. In the present study we have developed an in vitro fermentation chicken ileum microbiota model for unrevealing the complex interaction of ileum microbial community under physiological conditions. A two-vessel continuous fermentation process simulating in real-time the physiological conditions of the ileum content (pH, temperature, microaerophilic/anoxic conditions, and peristaltic movements) has been standardized as a proof of concept. As inoculum, we use a pool of ileum microbial community obtained from chicken broilers at the age of day 14. The development and validation of the model provide insight into the initial characterization of the ileum microbial community and its dynamics over time-related to nutrient assimilation and fermentation. Samples can be collected at different time points and can be used to determine the microbial compositional structure, dynamics, and diversity over time. The results of studies using this in vitro model will serve as the foundation for the development of a whole small intestine in vitro fermentation chicken gastrointestinal model to complement our already established in vitro fermentation chicken caeca model. The insight gained from this model could provide us with some information about the nutritional strategies to restore and maintain chicken gut homeostasis. Moreover, the in vitro fermentation model will also allow us to study relationships between gut microbiota composition and its dynamics over time associated with nutrients, antimicrobial compounds, and disease modelling.

Keywords: broilers, in vitro model, ileum microbiota, fermentation

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Authors: A. N.Ukwuani, A. K. Abdulfatah

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G. crenata is used locally for the treatment of fractured bones, wound healing and inflammatory conditions. In vitro and in vivo antioxidant activity of hydromethanolic extracts of the leaves of G. crenata were assessed. The phytochemical analysis shows the presence of phenols, flavonoids, saponins, cardiac glycosides and tannins. An in vitro quantitative analysis of phenols, flavonoids and tannins respectively were (164±1.20, 199±0.88 and 88.67±0.88 mg/100g FW). In vivo studies of hydromethanolic extract demonstrated a dose dependent increase in hepatic superoxide dismutase (1.14±0.14, 2.13±0.11, 2.55±0.11 U/mg Protein) with improvement in hepatic glutathione (6.98±0.42, 8.91±0.37, 11.07±0.46 µM/mg Protein) and Catalase (4.47±0.05, 6.24±0.02, 7.17±0.04 U/mg Protein) and Total protein (6.18±0.08, 6.69±0.18, 7.27±0.16 mg/ml) respectively at 100-300mg/kg body weight Grewia crenata leaves when compared to the control and standard drug. It can be concluded from the present findings of that G. crenata leaves possess antioxidant potential.

Keywords: Grewia crenata, antioxidant, hydromethanolic extract, in vivo, in vitro

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Authors: Roya Razavizadeh, Razieh Soltaninejad, Hakimeh Oloumi

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Glycyrrhiza glabra L. (Licorice) is one of the oldest medicinal plants in Iran and secondary metabolites present in the plant root is used in food and pharmaceutical industries. With the use of heavy metals as elicitors, plant secondary metabolite production can be increased. In this study, the effects of the concentrations of 1 and 10 μM of zinc oxide and copper oxide on the contents of reducing sugars (as precursor of secondary metabolites), proline, glycyrrhizin, total phenolic compounds, flavonoids and anthocyanin in Glycyrrhiza glabra seedlings were investigated. Also, the correlation between the content of these metabolites in the treated seedlings was examined using Pearson's test. The amount of reducing sugars at concentration of 10 μM zinc oxide was decreased. Whereas, the amounts of proline and glycyrrhizin under treatment 1 and 10 μM copper oxide and 1 μM zinc oxide compared with the control plants was increased. The content of total phenolic compounds was increased with increasing concentrations of copper oxide. The highest amount of flavonoids was observed at concentrations of 1 and 10 μM copper oxide. Anthocyanin content was increased in concentration of 1 μM copper oxide. Also, the tannin content of the Glycyrrhiza glabra seedlings at concentrations of 10 μM zinc oxide was increased. Based on the result it seemed that at concentrations of 1 and 10 μM copper oxide the amount of glycyrrhizin, phenolic compounds, flavonoids, anthocyanins were significantly increased, whereas, zinc oxide had no significant impact on the levels of these metabolites.

Keywords: zinc oxide, copper oxide, phenolic compounds, licorice (glycyrrhiza glabra L.), glycyrrhizin

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Authors: Kehinde T. Kareem

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The need to increase the production of cucumber has led to the use of inorganic fertilizers. This chemical affects the ecological balance of nature by increasing the nitrogen and phosphorus contents of the soil. Surface runoffs into rivers and streams cause eutrophication which affects aquatic organisms as well as the consumers of aquatic animals. Therefore, this study was carried out in the screenhouse to investigate the use of a plant growth-promoting fungus; Trichoderma longibrachiatum for the growth promotion of conventional and in-vitro propagated Ashley and Marketmoor cucumber. Before planting of cucumber, spore suspension (108 cfu/ml) of Trichoderma longibrachiatum grown on Potato dextrose agar (PDA) was inoculated into the soil. Fruits were evaluated for the presence of Trichoderma longibrachiatum using a species-specific primer. Results revealed that the highest significant plant height produced by in-vitro propagated Ashley was 19 cm while the highest plant height of in-vitro propagated Marketmoor was 19.67 cm. The yield of the conventional propagated Ashley cucumber showed that the number of fruit/plant obtained from T. longibrachiatum-fertilized plants were significantly more than those of the control. The in-vitro Ashely had 7 fruits/plant while the control produced 4 fruits/plant. In-vitro Marketmoor had ten fruits/plant, and the control had a value of 4 fruits/plant. There were no traces of Trichoderma longibrachiatum genes in the harvested cucumber fruits. Therefore, the use of Trichoderma longibrachiatum as a plant growth-promoter is safe for human health as well as the environment.

Keywords: biofertilizer, cucumber, genes, growth-promoter, in-vitro, propagation

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Authors: Vadim V. Yanshole, Lyudmila V. Yanshole, Alexey S. Kiryutin, Timofey D. Verkhovod, Yuri P. Tsentalovich

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Cataract, or clouding of the eye lens, is the leading cause of vision impairment in the world. The lens tissue have very specific structure: It does not have vascular system, the lens proteins – crystallins – do not turnover throughout lifespan. The protection of lens proteins is provided by the metabolites which diffuse inside the lens from the aqueous humor or synthesized in the lens epithelial layer. Therefore, the study of changes in the metabolite composition of a cataractous lens as compared to a normal lens may elucidate the possible mechanisms of the cataract formation. Quantitative metabolomic profiles of normal and cataractous human lenses were obtained with the combined use of high-frequency nuclear magnetic resonance (NMR) and ion-pairing high-performance liquid chromatography with high-resolution mass-spectrometric detection (LC-MS) methods. The quantitative content of more than fifty metabolites has been determined in this work for normal aged and cataractous human lenses. The most abundant metabolites in the normal lens are myo-inositol, lactate, creatine, glutathione, glutamate, and glucose. For the majority of metabolites, their levels in the lens cortex and nucleus are similar, with the few exceptions including antioxidants and UV filters: The concentrations of glutathione, ascorbate and NAD in the lens nucleus decrease as compared to the cortex, while the levels of the secondary UV filters formed from primary UV filters in redox processes increase. That confirms that the lens core is metabolically inert, and the metabolic activity in the lens nucleus is mostly restricted by protection from the oxidative stress caused by UV irradiation, UV filter spontaneous decomposition, or other factors. It was found that the metabolomic composition of normal and age-matched cataractous human lenses differ significantly. The content of the most important metabolites – antioxidants, UV filters, and osmolytes – in the cataractous nucleus is at least ten fold lower than in the normal nucleus. One may suppose that the majority of these metabolites are synthesized in the lens epithelial layer, and that age-related cataractogenesis might originate from the dysfunction of the lens epithelial cells. Comprehensive quantitative metabolic profiles of the human eye lens have been acquired for the first time. The obtained data can be used for the analysis of changes in the lens chemical composition occurring with age and with the cataract development.

Keywords: cataract, lens, NMR, LC-MS, metabolome

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Authors: Neelma Ashraf

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Actinomycetes, particularly genus Streptomyces is of great importance due to their role in the discovery of new natural products, particularly antimicrobial secondary metabolites in the medicinal science and biotechnology industry. Different Streptomyces strains were isolated from Helianthus annuus plants and tested for antibacterial and antifungal activities. The most promising five strains were chosen for further investigation, and growth conditions for antibiotic synthesis were optimised. The supernatants were extracted in different solvents, and the extracted products were analyzed using liquid chromatography-mass spectrometry (LC-MS) and biological testing. From one of the potent strains Streptomyces globusus sp. BR123, a compound lavendamycin was identified using these analytical techniques. In addition, this potent strain also produces a strong antifungal polyene compound with a quasimolecular ion of 2072. Streptomyces sp. BR123 was genome sequenced because of its promising antimicrobial potential in order to identify the gene cluster responsible for analyzed compound “lavendamycin”. The genome analysis yielded candidate genes responsible for the production of this potent compound. The genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 with a GC content of 72.63% and 8103 protein coding genes was attained. Many antimicrobial, antiparasitic, and anticancerous compounds were detected through multiple biosynthetic gene clusters predicted by in-Silico analysis. Though, the novelty of metabolites was determined through the insignificant resemblance with known biosynthetic gene clusters. The current study gives insight into the bioactive potential of Streptomyces sp. isolate BR123 with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study revealed the connection of isolate BR123 with other Streptomyces strains, which could expand the knowledge of this genus and the mechanism involved in the discovery of new antimicrobial metabolites.

Keywords: streptomyces, secondary metabolites, genome, biosynthetic gene clusters, high performance liquid chromatography, mass spectrometry

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Authors: Alexis Grandcoin, Stephanie Piel, Estelle Baures

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Brittany (France) is an agricultural region, where emerging pollutants are highly at risk to reach water bodies. Among them, pesticides metabolites are frequently detected in surface waters. The Vilaine watershed (11 000 km²) is of great interest, as a large drinking water treatment plant (100 000 m³/day) is located at the extreme downstream of it. This study aims to provide an evaluation of the pesticides metabolites pollution in the Vilaine watershed, and an understanding of their availability, in order to protect the water resource. Hydroclimatic conditions, land use, and water quality parameters controlling metabolites availability are emphasized. Later this knowledge will be used to understand the favoring conditions resulting in metabolites export towards surface water. 19 sampling points have been strategically chosen along the 220 km of the Vilaine river and its 3 main influents. Furthermore, the intakes of two drinking water plants have been sampled, one is located at the extreme downstream of the Vilaine river and the other is the riparian groundwater under the Vilaine river. 5 sampling campaigns with various hydroclimatic conditions have been carried out. Water quality parameters and hydroclimatic conditions have been measured. 15 environmentally relevant pesticides and metabolites have been analyzed. Also, these compounds are recalcitrant to classic water treatment that is why they have been selected. An evaluation of the watershed contamination has been done in 2016-2017. First observations showed that aminomethylphosphonic acid (AMPA) and metolachlor ethanesulfonic acid (MESA) are the most detected compounds in surface waters samples with 100 % and 98 % frequency of detection respectively. They are the main pollutants of the watershed regardless of the hydroclimatic conditions. AMPA concentration in the river strongly increases downstream of Rennes agglomeration (220k inhabitants) and reaches a maximum of 2.3 µg/l in low waters conditions. Groundwater contains mainly MESA, Diuron and metazachlor ESA at concentrations close to limits of quantification (LOQ) (0.02 µg/L). Metolachlor, metazachlor and alachlor due to their fast degradation in soils were found in small amounts (LOQ – 0.2 µg/L). Conversely glyphosate was regularly found during warm and sunny periods up to 0.6 µg/L. Soil uses (agricultural cultures types, urban areas, forests, wastewater treatment plants implementation), water quality parameters, and hydroclimatic conditions have been correlated to pesticides and metabolites concentration in waters. Statistical treatments showed that chloroacetamides metabolites and AMPA behave differently regardless of the hydroclimatic conditions. Chloroacetamides are correlated to each other, to agricultural areas and to typical agricultural tracers as nitrates. They are present in waters the whole year, especially during rainy periods, suggesting important stocks in soils. Also Chloroacetamides are negatively correlated with AMPA, the different forms of phosphorus, and organic matter. AMPA is ubiquitous but strongly correlated with urban areas despite the recent French regulation, restricting glyphosate to agricultural and private uses. This work helps to predict and understand metabolites present in the water resource used to craft drinking water. As the studied metabolites are difficult to remove, this project will be completed by a water treatment part.

Keywords: agricultural watershed, AMPA, metolachlor-ESA, water resource

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Authors: Van Huong Do, Simon Walton, German Amaya, Madeline Batsiokis, Sally Catt, Andrew Taylor-Robinson

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Identification of the most suitable stages of bovine in vitro-derived blastocysts (early, expanded and hatching) prior to vitrification is a straightforward process that facilitates the decision as to which blastocyst stage to use for transfer of fresh and vitrified embryos. Research on in vitro evaluation of suitable stages has shown that the more advanced developmental stage of blastocysts is recommended for fresh embryo transfer while the earlier stage is proposed for embryo transfer following vitrification. There is, however, limited information on blastocyst stages using in vivo assessment. Hence, the aim of the present study was to determine the optimal stage of a blastocyst for vitrification and embryo transfer through a two-step procedure of embryo transfer followed by pregnancy testing at 35, 60 and 90 days of pregnancy. 410 good quality oocytes aspirated by the ovum pick-up technique from 8 donor cows were subjected to in vitro embryo production, vitrification and embryo transfer. Good quality embryos were selected, subjected to vitrification and embryo transfer. Subsequently, 77 vitrified embryos at different blastocyst stages were transferred to synchronised recipient cows. The overall cleavage and blastocyst rates of oocytes were 68.8% and 41.7%, respectively. In addition, the fertility and blastocyst production of 6 bulls used for in vitro fertilization was examined and shown to be statistically different (P<0.05). Results of ongoing pregnancy trials conducted at 35 days, 60 days and 90 days will be discussed. However, preliminary data indicate that individual bulls demonstrate distinctly different fertility performance in vitro. Findings from conception rates would provide a useful tool to aid selection of bovine in vitro-derived embryos for vitrification and embryo transfer in commercial settings.

Keywords: blastocyst, embryo transfer, in vitro-derived embryos, ovum pick-up, vitrification

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Authors: Hadriana Bansi, Elizabeth Wina, Toto Toharmat

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Acacia villosa is thornless shrub legume which contents high crude protein. However, the utilization of A. villosa as ruminant feed is limited by its secondary compounds. The aim of this article is to find out the maximum of substitution A. villosa in sheep ration. The nutritional evaluation consisted of in vitro two stages, in vivo, and in vitro gas production trials. The secondary compounds of A. villosa also were analyzed. Evaluating digestibility of increasing level of substitution A. villosa replacing Pennisetum purpureum was using in vitro two stages. The substitution of 30% A. villosa was compared to 100% P. purpureum by in vitro gas production technique and in vivo digestibility. The results of two stages in vitro showed that total phenol, condensed tannin, and non-protein amino acid (NPAA) were high. Substitution 15% A. villosa reached the highest digestibility for both dry matter (DM) and crude protein (CP) which were 67% and 86% respectively, but it was shown that DM and CP digestibility of substitution 30% of A. villosa was still high which were 61.82% and 75-67% respectively. The pattern of gas production showed that first 8 hours total gas production substitution of 30% A. villosa was higher than 100% P. purpureum and declined after 10 hours incubation. In vivo trials showed that substitution of 30% A. villosa significantly increased CP intake, CP digestibility, and nitrogen retention. It can be concluded that substitution A. villosa until 30% still gave the good impact even though it has high secondary compounds.

Keywords: Acacia villosa, digestibility, gas production, secondary compounds

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

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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±1.26 mg GAE⁄g; 174.44±9.32 mg QE⁄g) than in the methanol extract of A.C (20.56±0.67 mg GAE⁄g;7.21±0.85 mg 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 medicines, in-vitro antioxidant

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Authors: Dmitriy Yu. Korulkin, Raissa A. Muzychkina

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The article represents the results of research of antitumor activity of different structural types of plant flavonoids extracted by authors from Polygonum L. plants in commercial reserves at the territory of the Republic of Kazakhstan. For the first time ever the results comparative research of antitumor activity of plant flavonoids of different structural groups and their synthetic derivatives have been represented. The results of determination of toxicity of flavonoids in single parenteral infusion conditions have been represented. Experimental substantiation of possible mechanisms of antiproliferative and cytotoxic action of flavonoids has been suggested. The perspectives of usage of plant flavonoids as medications and creation of effective dosage forms of antitumor medicines on their basis have been substantiated.

Keywords: antitumor activity, cytotoxicity, flavonoids, Polygonum L., secondary metabolites

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Authors: Reine Suci Wulandari, Rosa Suryantini

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Albizia (Paraserianthes falcataria) is a woody plant species that has a high economic value and multifunctional. Albizia is important timber, medicinal plants and can also be used as a plant to rehabilitate critical lands. The demand value of Albizia is increased so that the large quantities and high quality of seeds are required. In vitro propagation techniques are seed propagation that can produce more seeds and quality in a short time. In vitro cultures require growth regulators that can be obtained from biological agents such as endophytic fungi. Endophytic fungi are micro fungi that colonize live plant tissue without producing symptoms or other negative effects on host plants and increase plant growth. The purposes of this research were to isolate and identify endophytic fungi isolated from the root of Albizia and to study the effect of endophytic fungus on the growth of Albizia in vitro. The methods were root isolation, endophytic fungal identification, and inoculation of endophytic fungi to Albizia plants in vitro. Endophytic fungus isolates were grown on PDA media before being inoculated with Albizia sprouts. Incubation is done for 4 (four) weeks. The observed growth parameters were live explant percentage, percentage of explant shoot, and percentage of explant rooted. The results of the research showed that 6 (six) endophytic fungal isolates obtained from the root of Albizia, namely Aspergillus sp., Verticillium sp, Penicillium sp., Trichoderma sp., Fusarium sp., and Acremonium sp. Statistical analysis found that Trichoderma sp. and Fusarium sp. affect in vitro growth of Albizia. Endophytic fungi from the results of this research were potential as plant growth promoting. It can be applied to increase productivity either through increased plant growth and increased endurance of Albizia seedlings to pests and diseases.

Keywords: Albizia, endophytic fungi, propagation, in vitro

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Authors: Rasha Saad, Jean Frederic Weber, Fatimah Bebe, Sadia Sultan

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The goal of study was to screen the effects of candesartan and four endophytic fungi for their potential in microbial biotransformation. In this experiment, four types of unidentified fungi with the codes of TH2L1, TH2R10, TH1P35 and TH1S46 were used in screening process by MECFUS (Microtiter plate, Elicitors, Combination, Freeze-drying, UHPLC, Statistical analysis) protocol. The experiment was carried out by using 96-well microtiter plate (MTP) with different media and elicitors. Various media with two concentrations of Potato Dextrose Broth (PDB) and elicitors used were to induce the production of secondary metabolites from the fungi as well as the biotransformation of the drug compound. After incubation, cultures were extracted by freeze drying method and finally analyzed by ultra-High performance Liquid Chromatography (uHPLC). The extracts analyzed by uHPLC followed by LC/Ms, demonstrated the presence of biotransformation products from the drug compound and elicitation of the secondary metabolism from the fungi by the occurrence of the additional peaks. From the four fungi, TH1S46 showed highly potential produced secondary metabolites as well as the biotransformation of candesartan. For other fungi, they responded when candesartan was introduced. Moreover, the additional peaks produced in uHPLC need to be further investigation by using LC-MS or NMR.

Keywords: biotransformation, candesartan, endophytes, secondary metabolites

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Authors: Wai Prathumpai, Pranee Rachtawee, Sutamat Khajeeram, Pariya Na Nakorn

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The  β-glucan produced by Ophiocordyceps dipterigena BCC 2073 is a (1, 3)-β-D-glucan with highly branching O-6-linkedside chains that is resistant to acid hydrolysis (by hydrochloric acid and porcine pancreatic alpha-amylase). This β-glucan can be utilized as a prebiotic due to its advantageous structural and biological properties. The effects of using this β-glucan as the sole carbon source for the in vitro growth of two probiotic bacteria (L. acidophilus BCC 13938 and B. animalis ATCC 25527) were investigated. Compared with the effect of using 1% glucose or fructo-oligosaccharide (FOS) as the sole carbon source, using 1% β-glucan for this purpose showed that this prebiotic supported and stimulated the growth of both types of probiotic bacteria and induced them to produce the highest levels of metabolites during their growth. The highest levels of lactic and acetic acid, 10.04 g·L^-1 and 2.82 g·L^-1, respectively, were observed at 2 h of cultivation using glucose as the sole carbon source. Furthermore, the fermentation broth obtained using 1% β-glucan as the sole carbon source had greater antibacterial activity against selected pathogenic bacteria (B. subtilis TISTR 008, E. coli TISTR 780, and S. typhimurium TISTR 292) than did the broths prepared using glucose or FOS as the sole carbon source. The fermentation broth obtained by growing L. acidophilus BCC 13938 in the presence of β-glucan inhibited the growth of B. subtilis TISTR 008 by more than 70% and inhibited the growth of both S. typhimurium TISTR 292 and E. coli TISTR 780 by more than 90%. In conclusion, O. dipterigena BCC 2073 is a potential source of a β-glucan prebiotic that could be used for commercial production in the near future.

Keywords: beta-glucan, Ophiocordyceps dipterigena, prebiotic, probiotic, antimicrobial

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Authors: L. Correia-Sá, S. Norberto, Conceição Calhau, C. Delerue-Matos, V. F. Domingues

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Endocrine disruptor chemicals (EDCs) are synthetic compounds that even though being initially designed for a specific function are now being linked with a wide range of side effects. The list of possible EDCs is growing and includes phthalates and bisphenol A (BPA). Phthalates are one of the most widely used plasticizers to improve the extensibility, elasticity and workability of polyvinyl chloride (PVC), polyvinyl acetates, etc. Considered non-toxic and harmless additives for polymers, they were used unrestrainedly all over the world for several decades. However, recent studies have indicated that some phthalates and their metabolic products are reproductive and developmental toxicants in animals and suspected endocrine disruptors in humans. BPA (2,2-bis(4-hydroxyphenyl)propane) is a high production volume chemical mainly used in the production of polycarbonate plastics and epoxy resins. Although BPA was initially considered to be a weak environmental estrogen, nowadays it is known that this compound can stimulate several cellular responses at very low levels of concentrations. The aim of this study was to develop a method based on tandem SPE to evaluate the presence of phthalates, metabolites and BPA in human urine samples. The analyzed compounds included: dibutyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP), BPA, mono-isobutyl phthalate (MiBP), monobutyl phthalate (MBP) and. mono-(2-ethyl-5-oxohexyl) (MEOHP). Two SPE cartridges were applied both from Phenomenex, the strata X polymeric reversed phase and the strata X A (Strong anion). Chromatographic analyses were carried out in a Thermo GC ULTRA GC-MS/MS. Good recoveries and linear calibration curves were obtained. After validation, the methodology was applied to human urine samples for phthalates, metabolites and BPA evaluation.

Keywords: Bisphenol A (BPA), gas chromatography, metabolites, phtalates, SPE, tandem mode

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Authors: Prabir Kumar Paul, Joyeeta Mitra

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Ergosterol, is an important fungal metabolite on phylloplane which is not synthesised by plants. However, the functional requirement of ergosterol to the plants is still an enigma. Being ubiquitously present in all plants except algae needs an insight into its physiological implication. The present study aimed at understanding if and how ergosterol influences the physiology of chloroplast particularly the activity of RuBisCo and carbonic anhydrase. The concept of the study was based on one of our earlier observation of enhanced Hills reaction in plants treated with fungal metabolites which contained ergosterol. The fungal metabolite treated plants had a significantly high concentration of photosynthetic pigments. Eight-week-old tomato plants raised under aseptic conditions at 25 + 10 C, 75 % relative humidity and 12 hour L/D photoperiod. Metabolites of Aspergillus niger and Fusarium oxysporum were sprayed on plants either singly or in a 1: 1 combination. A separate group of plants was also treated with 0.5, 1.0, 3.0, 5.0. 7.0 mg ergosterol / ml of n- heptane. Control plants were treated with sterile distilled water only. Plants were sampled at 24, 48, 72 and 96 hours of treatment. RuBisCo and carbonic anhydrase was estimated from sampled leaves. RuBisCo was separated on 1D SDS-PAGE and subjected to MALDI – TOF- TOF – MS analysis. The presence of ergosterol in fungal metabolites was confirmed. Fungal metabolites significantly enhanced the concentration and activity of RuBisCo and carbonic anhydrase. The Vmax activity of the enzymes was significantly high in metabolite treated plants. 1:1 mix of metabolite was more effective than when applied individually. Insilico analysis revealed, RuBisCo subunits had a binding site for ergosterol and in its presence affinity of Co2 to the enzyme increased by several folds. Invivo activity of RuBisCo was significantly elicited by ergosterol. Results of the present study indicate that ergosterol from phylloplane microfungi probably regulates the binding of Co2 to RuBisCo along with activity of carbonic anhydrase thereby modulating the physiology of choloroplast.

Keywords: carbonic anhydrase, ergosterol, phylloplane, RuBisCo

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Authors: Sri Wahjuningsih, Nur Ihsan, Hadiah

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In the embryo transfer program, to address the limited production of embryos in vivo, in vitro embryo production has become an alternative approach that is relatively inexpensive. One potential source of embryos that can be developed is to use immature oocytes then conducted in vitro maturation and in vitro fertilization. However, obstacles encountered were oocyte viability mammals have very limited that it cannot be stored for a long time, so we need oocyte cryopreservation. The research was conducted to know the optimal concentration use of ethylene glycol as a cryoprotectant on oocytes freezing.Material use in this research was immature oocytes; taken from abbatoir which was aspirated from follicle with diameter 2-6 mm. Concentration ethylen glycol used were 0,5 M, I M, 1,5 M and 2M. The freezing method used was conventional method combined with a five-step protocol washing oocytes from cryoprotectant after thawing. The result showed that concentration ethylen glycol have the significant effect (P<0.05) on oocytes quality after thawing and in vitro maturation. It was concluded that concentration 1,5 M was the best concentration for freezing oocytes using conventional method.

Keywords: bovine, conventional freezing, ethylen glycol, oocytes

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Authors: Sakinah Abdullah, Rosna Mat Taha

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Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen species such as singlet oxygen, superoxide, peroxyl radicals, and peroxynitrite which result in oxidative stress leading to cellular damage. Natural antioxidant are in high demand because of their potential in health promotion and disease prevention and their improved safety and consumer acceptability. Plants are rich sources of natural antioxidant. Dioscorea alata L. known as 'ubi badak' in Malaysia were well known for their antioxidant content, but this plant was seasonal. Thus, tissue culture technique was used to mass propagate this plant. In the present work, a comparative study between in vitro (from tissue culture) and in vivo (from intact plant) samples of Dioscorea alata L. for their antioxidant potential by 2,2-diphenil -1- picrylhydrazyl (DPPH) radical scavenging activity method and their total phenolic and flavonoid contents were carried out. All samples had better radical scavenging activity but in vivo samples had the strongest radical scavenging activity compared to in vitro samples. Furthermore, tubers from in vivo samples showed the greatest free radical scavenging effect and comparatively greater phenolic content than in vitro samples.

Keywords: Dioscorea alata, tissue culture, antioxidant, in vivo, in vitro, DPPH

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Authors: Yogesh Dalvi, Ruby Varghese, Nibu Varghese, C. K. Krishnan Nair

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Introduction: The Genus Phellinus, locally known as Phansomba is a well-known traditional folk medicine. Especially, in Western Ghats of India, many tribes use several species of Phellinus for various ailments related to teeth, throat, tongue, stomach and even wound healing. It is one of the few mushrooms which play a pivotal role in Ayurvedic Dravyaguna. Aim: The present study focuses on to investigate phytochemical analysis, antioxidant, and antitumor (in vitro and in vivo) potential of Phellinus robinae from South India, Kerala Material and Methods: The present study explores the following: 1. Phellinus samples were collected from Ranni, Pathanamthitta district of Kerala state, India from Artocarpus heterophyllus Lam. and species were identified using rDNA region. 2. The fruiting body was shadow dried, powdered and extracted with 50% alcohol using water bath at 60°C which was further condensed by rotary evaporator and lyophilized at minus 40°C temperature. 3. Secondary metabolites were analyzed by using various phytochemical screening assay (Hager’s Test, Wagner’s Test, Sodium hydroxide Test, Lead acetate Test, Ferric chloride Test, Folin-ciocalteu Test, Foaming Test, Benedict’s test, Fehling’s Test and Lowry’s Test). 4. Antioxidant and free radical scavenging activity were analyzed by DPPH, FRAP and Iron chelating assay. 5. The antitumor potential of Water alcohol extract of Phellinus (PAWE) is evaluated through In vitro condition by Trypan blue dye exclusion method in DLA cell line and In vivo by murine model. Result and Discussion: Preliminary phytochemical screening by various biochemical tests revealed presence of a variety of active secondary molecules like alkaloids, flavanoids, saponins, carbohydrate, protein and phenol. In DPPH and FRAP assay PAWE showed significantly higher antioxidant activity as compared to standard Ascorbic acid. While, in Iron chelating assay, PAWE exhibits similar antioxidant activity that of Butylated Hydroxytoluene (BHT) as standard. Further, in the in vitro study, PAWE showed significant inhibition on DLA cell proliferation in dose dependent manner and showed no toxicity on mice splenocytes, when compared to standard chemotherapy drug doxorubicin. In vivo study, oral administration of PAWE showed dose dependent tumor regression in mice and also raised the immunogenicity by restoring levels of antioxidant enzymes in liver and kidney tissue. In both in vitro and in vivo gene expression studies PAWE up-regulates pro-apoptotic genes (Bax, Caspases 3, 8 and 9) and down- regulates anti-apoptotic genes (Bcl2). PAWE also down regulates inflammatory gene (Cox-2) and angiogenic gene (VEGF). Conclusion: Preliminary phytochemical screening revealed that PAWE contains various secondary metabolites which contribute to its antioxidant and free radical scavenging property as evaluated by DPPH, FRAP and Iron chelating assay. PAWE exhibits anti-proliferative activity by the induction of apoptosis through a signaling cascade of death receptor-mediated extrinsic (Caspase8 and Tnf-α), as well as mitochondria-mediated intrinsic (caspase9) and caspase pathways (Caspase3, 8 and 9) and also by regressing angiogenic factor (VEGF) without any inflammation or adverse side effects. Hence, PAWE serve as a potential antioxidant and antitumor agent.

Keywords: antioxidant, antitumor, Dalton lymphoma ascites (DLA), fungi, Phellinus robinae

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Authors: Christian A. Romero, Tanja Grkovic, John. R. J. French, D. İpek Kurtböke, Ronald J. Quinn

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A high-throughput and automated 1H NMR metabolic fingerprinting dereplication approach was used to accelerate the discovery of unknown bioactive secondary metabolites. The applied dereplication strategy accelerated the discovery of natural products, provided rapid and competent identification and quantification of the known secondary metabolites and avoided time-consuming isolation procedures. The effectiveness of the technique was demonstrated by the isolation and elucidation of arglecins B (1), C (2) and actinofuranosin A (3) from a termite-gut associated Streptomyces sp. (USC 597) grown under solid state fermentation. The structures of these compounds were elucidated by extensive interpretation of 1H, 13C and 2D NMR spectroscopic data. These represent the first report of arglecin analogs isolated from a termite gut-associated Streptomyces species.

Keywords: actinomycetes, actinofuranosin, antibiotics, arglecins, NMR spectroscopy

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Authors: A. Jamsheera, F. Arjmand, D. K. Mohapatra

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Small molecules binding to specific sites along DNA molecule are considered as potential chemotherapeutic agents. Their role as mediators of key biological functions and their unique intrinsic properties make them particularly attractive therapeutic agents. Keeping in view, novel dipeptide complexes Cu(II)-Val-Pro (1), Zn(II)-Val-Pro (2), Cu(II)-Ala-Pro (3) and Zn(II)-Ala-Pro (4) were synthesized and thoroughly characterized using different spectroscopic techniques including elemental analyses, IR, NMR, ESI–MS and molar conductance measurements. The solution stability study carried out by UV–vis absorption titration over a broad range of pH proved the stability of the complexes in solution. In vitro DNA binding studies of complexes 1–4 carried out employing absorption, fluorescence, circular dichroism and viscometric studies revealed the binding of complexes to DNA via groove binding. UV–vis titrations of 1–4 with mononucleotides of interest viz., 5´-GMP and 5´-TMP were also carried out. The DNA cleavage activity of the complexes 1 and 2 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents and the cleavage mechanism involved a hydrolytic pathway. Furthermore, in vitro antitumor activity of complex 1 was screened against human cancer cell lines of different histological origin.

Keywords: dipeptide Cu(II) and Zn(II) complexes, DNA binding profile, pBR322 DNA cleavage, in vitro anticancer activity

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Authors: Susan Hall, Gary D. Grant, Catherine McDermott, Devinder Arora

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Introduction /Aim: The kynurenine pathway is thought to play an important role in the pathophysiology of numerous neurodegenerative diseases including depression, Alzheimer’s disease, and Parkinson’s disease. Numerous neuroactive compounds, including the neurotoxic 3-hydroxyanthranilic acid, 3-hydroxykynurenine and quinolinic acid and the neuroprotective kynurenic acid and picolinic acid, are produced through the metabolism of kynurenine and are thought to be the causative agents responsible for neurodegeneration. The toxicity of 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid has been widely evaluated and demonstrated in primary cell cultures but to date only 3-hydroxykynurenine and 3-hydroxyanthranilic acid have been shown to cause toxicity in immortal tumour cells. The aim of this study was to evaluate the toxicity of kynurenine metabolites, both individually and in combination, on SH-SY5Y neuroblastoma cells after 24 and 72 h exposure in order to explore a cost-effective model to study their neurotoxic effects and potential protective agents. Methods: SH-SY5Y neuroblastoma cells were exposed to various concentrations of the neuroactive kynurenine metabolites, both individually and in combination, for 24 and 72 h, and viability was subsequently evaluated using the Resazurin (Alamar blue) proliferation assay. Furthermore, the effects of these compounds, alone and in combination, on specific death pathways including apoptosis, necrosis and free radical production was evaluated using various assays. Results: Consistent with literature, toxicity was shown with short-term 24-hour treatments at 1000 μM concentrations for both 3-hydroxykynurenine and 3-hydroxyanthranilic acid. Combinations of kynurenine metabolites showed modest toxicity towards SH-SY5Y neuroblastoma cells in a concentration-dependent manner. Specific cell death pathways, including apoptosis, necrosis and free radical production were shown to be increased after both 24 and 72 h exposure of SH-SY5Y neuroblastoma cells to 3-hydroxykynurenine and 3-hydroxyanthranilic acid and various combinations of neurotoxic kynurenine metabolites. Conclusion: It is well documented that neurotoxic kynurenine metabolites show toxicity towards primary human neurons in the nanomolar to low micromolar concentration range. Results show that the concentrations required to show significant cell death are in the range of 1000 µM for 3-hydroxykynurenine and 3-hydroxyanthranilic acid and toxicity of quinolinic acid towards SH-SY5Y was unable to be shown. This differs significantly from toxicities observed in primary human neurons. Combinations of the neurotoxic metabolites were shown to have modest toxicity towards these cells with increased toxicity and activation of cell death pathways observed after 72 h exposure. This study suggests that the 24 h model is unsuitable for use in neurotoxicity studies, however, the 72 h model better represents the observations of the studies using primary human neurons and may provide some benefit in providing a cost-effective model to assess possible protective agents against kynurenine metabolite toxicities.

Keywords: kynurenine metabolites, neurotoxicity, quinolinic acid, SH-SY5Y neuroblastoma

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Authors: Nishtha, Richa, Anju Rao

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Plants are the source of herbal medicine and medicinal value of the plants lies in the bioactive phytochemical constituents that produce definite physiological effects on human body. Angiospermic families are known to produce such phytochemical constituents which are termed as secondary plant metabolites. These metabolites include alkaloids, saponins, phenolic compounds, flavonoids, tannins, terpenoids and so on. Solanaceae is one of the important families of Angiosperms known for medicinally important alkaloids such as hyoscyamine, scopolamine, solanine, nicotine, capsaicin etc. Medicinally important species of this family mostly belong to the genera of Datura,Atropa,Solanum,Withania and Nicotiana.Six species such as Datura metel, Solanum torvum, Physalis minima, Cestrum nocturnum, Cestrum diurnum and Nicotiana plumbaginifolia have been collected from different localities of Chandigarh and adjoining areas.Field and anatomical studies helped to identify the plants and their parts used for the study of secondary plant metabolites. Preliminary phytochemcial studies have been done on various parts of plants such as roots, stem and leaves by making aqueous and alcoholic extracts from their powdered forms which showed the presence of alkaloids in almost all the species followed by steroids, flavonoids, terpenoids, tannins etc. HPLC profiles of leaves of Datura metel showed the presence of active compounds such as scopalamine and hyoscyamine and Solanum torvum showed the presence of solanine and solasodine. These alkaloids are important source of drug based medicine used in pharmacognosy. The respective compounds help in treating vomiting, nausea, respiratory disorders, dizziness, asthma and many heart problems.

Keywords: alkaloids, flavanoids, phytochemical constituents, pharmacognosy, secondary metabolites

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Authors: S. Beekrum, B. Odhav, R. Lalloo, E. A. Amonsou

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Marine microalgae are rich sources of novel and biologically active metabolites; therefore they may be used in the food industry as natural food ingredients and functional foods. They have several biological applications related to health benefits, among others. The aim of the study focused on the screening of phytochemicals from Amphora sp. biomass extracts, and to examine the in vitro antioxidant and antimicrobial potential. Amphora sp. biomass was obtained from CSIR (South Africa) and methanol, hexane and water extracts were prepared. The in vitro antimicrobial effect of extracts were tested against some pathogens (Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis, Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa and Candida albicans), using the disc diffusion assay. Qualitative analyses of phytochemicals were conducted by chemical tests. The present investigation revealed that all extracts showed relatively strong antibacterial activity against most of the tested bacteria. The highest phenolic content was found in the methanolic extract. Results of the DPPH assay showed that the biomass contained strong antioxidant capacity, 79% in the methanolic extract and 85% in the hexane extract. Extracts have displayed effectively reducing power and superoxide anion radical scavenging activity. Results of this study have highlighted potential antioxidant activity in the methanol and hexane extracts. The results of the phytochemical screening showed the presence of terpenoids and sterols with potential applications as food flavorants and functional foods, respectively. The use of Amphora sp. as a natural antioxidant source and a potential source of antibacterial compounds and phytochemicals in the food industry appears promising and should be investigated further.

Keywords: antioxidants, antimicrobial, microalgae, phytochemicals, cymbella

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Authors: Jeisson Alejandro Triana, Maria Fernanda Quiceno Vallejo, Patricia del Portillo, Juan Manuel Anzola

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Most of the known antimicrobials so far discovered are secondary metabolites. The potential for new natural products of this category increases as new microbial genomes and metagenomes are being sequenced. Despite the advances, there is no systematic way to interrogate metagenomic clones for their potential to contain clusters of genes related to these pathways. Here we analyzed 52 biosynthetic pathways from the AntiSMASH database at the protein domain level in order to identify domains of high specificity and sensitivity with respect to specific biosynthetic pathways. These domains turned out to have various degrees of divergence at the DNA level. We propose PCR assays targetting such domains in-silico and corroborated one by Sanger sequencing.

Keywords: bioinformatic, anti smash, antibiotics, secondary metabolites, natural products, protein domains

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Authors: Afsheen Aman, Shah Ali Ul Qader

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Dextransucrase [EC 2.4.1.5] is a glucosyltransferase that catalysis the biosynthesis of a natural biopolymer called dextran. It can catalyze the transfer of D-glucopyranosyl residues from sucrose to the main chain of dextran. This unique biopolymer has multiple applications in several industries and the key utilization of dextran lies on its molecular weight and the type of branching. Extracellular dextransucrase from Leuconostoc mesenteroides is most extensively studied and characterized. Limited data is available regarding cell-bound or intracellular dextransucrase and on the characterization of dextran produced by in-vitro reaction of intracellular dextransucrase. L. mesenteroides AA1 is reported to produce extracellular dextransucrase that catalyzes biosynthesis of a high molecular weight dextran with only α-(1→6) linkage. Current study deals with the characterization of an intracellular dextransucrase and in vitro biosynthesis of low molecular weight dextran from L. mesenteroides AA1. Intracellular dextransucrase was extracted from cytoplasm and purified to homogeneity for characterization. Kinetic constants, molecular weight and N-terminal sequence analysis of intracellular dextransucrase reveal unique variation with previously reported extracellular dextransucrase from the same strain. In vitro synthesized biopolymer was characterized using NMR spectroscopic techniques. Intracellular dextransucrase exhibited Vmax and Km values of 130.8 DSU ml-1 hr-1 and 221.3 mM, respectively. Optimum catalytic activity was detected at 35°C in 0.15 M citrate phosphate buffer (pH-5.5) in 05 minutes. Molecular mass of purified intracellular dextransucrase is approximately 220.0 kDa on SDS-PAGE. N-terminal sequence of the intracellular enzyme is: GLPGYFGVN that showed no homology with previously reported sequence for the extracellular dextransucrase. This intracellular dextransucrase is capable of in vitro synthesis of dextran under specific conditions. This intracellular dextransucrase is capable of in vitro synthesis of dextran under specific conditions and this biopolymer can be hydrolyzed into different molecular weight fractions for various applications.

Keywords: characterization, dextran, dextransucrase, leuconostoc mesenteroides

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Authors: Elene Zhuravliova, Tamar Barbakadze, Irina Kalandadze, Elnari Zaalishvili, Lali Shanshiashvili, David Mikeladze

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Background: Multiple sclerosis (MS) is an inflammatory, neurodegenerative disease, which is accompanied by demyelination and autoimmune response to myelin proteins. Among post-translational modifications, which mediate the modulation of inflammatory pathways during MS, methylation is the main one. The methylation of DNA, also amino acids lysine and arginine, occurs in the cell. It was found that decreased trans-methylation is associated with neuroinflammatory diseases. Therefore, abnormal regulation of the methyl cycle could induce demyelination through the action on PAD (peptidyl-arginine-deiminase) gene promoter. PAD takes part in protein citrullination and targets myelin basic protein (MBP), which is affected during demyelination. To determine whether MBP charge isomers are changing the methyl cycle, we have estimated the concentrations of methyl cycle metabolites in MBP-activated primary astrocytes and oligodendrocytes. For this purpose, the action of the citrullinated MBP- C8 and the most cationic MBP-C1 isomers on the primary cells were investigated. Methods: Primary oligodendrocyte and astrocyte cell cultures were prepared from whole brains of 2-day-old Wistar rats. The methyl cycle metabolites, including homocysteine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH), were estimated by HPLC analysis using fluorescence detection and prior derivatization. Results: We found that the action of MBP-C8 and MBP-C1 induces a decrease in the concentration of both methyl cycle metabolites, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), in astrocytes compared to the control cells. As for oligodendrocytes, the concentration of SAM was increased by the addition of MBP-C1, while MBP-C8 has no significant effect. As for SAH, its concentration was increased compared to the control cells by the action of both MBP-C1 and MBP-C8. A significant increase in homocysteine concentration was observed by the action of the MBP-C8 isomer in both oligodendrocytes and astrocytes. Conclusion: These data suggest that MBP charge isomers change the concentration of methyl cycle metabolites. MBP-C8 citrullinated isomer causes elevation of homocysteine in astrocytes and oligodendrocytes, which may be the reason for decreased astrocyte proliferation and increased oligodendrocyte cell death which takes place in neurodegenerative processes. Elevated homocysteine levels and subsequent abnormal regulation of methyl cycles in oligodendrocytes possibly change the methylation of DNA that activates PAD gene promoter and induces the synthesis of PAD, which in turn provokes the process of citrullination, which is the accompanying process of demyelination. Acknowledgment: This research was supported by the SRNSF Georgia RF17_534 grant.

Keywords: myelin basic protein, astrocytes, methyl cycle metabolites, homocysteine, oligodendrocytes

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Authors: Guem-Jae Chung, Jin-Hui Lee, Myung-Min Oh

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Environmental control of in vitro-propagated apple plantlets is required for successful acclimation to ex vitro due to its low survival rate. This study aimed to determine the proper lighting condition for ex vitro acclimation of the apple plantlets in plant factories. In vitro-propagated M9 apple plantlets treated with pre-acclimatization for 1 week were exposed to following light treatments for additional 6 weeks; 60 μmol·m⁻²·s⁻¹ (A), 100 μmol·m⁻²·s⁻¹ (B), 140 μmol·m⁻²·s⁻¹ (C), 180 μmol·m⁻²·s⁻¹ (D), 60 μmol·m⁻²·s⁻¹ → 100 μmol·m⁻²·s⁻¹ at 2 weeks (E) or 4 weeks (F), 60 μmol·m⁻²·s⁻¹ → 100 μmol·m⁻²·s⁻¹ at 2 weeks → 140 μmol·m⁻²·s⁻¹ at 4 weeks (G) and 60 μmol·m⁻²·s⁻¹ → 140 μmol·m⁻²·s⁻¹ at 4 weeks (H). Shoot height, total leaf area, soil-plant analysis development (SPAD) value, root length, fresh and dry weights of shoots and roots were measured every 2 weeks after transplanting. In addition, the photosynthetic rate was measured at 5 weeks after transplanting. At 6 weeks after transplanting, shoot height of B was significantly higher than the other treatments. SPAD value, total leaf area and root length of B and F were relatively higher than the other treatments. Root fresh weights of B, D, F, and G were relatively higher than those in the other treatments. D induced the highest value in shoot fresh weight probably due to stem hardening, but it also resulted in shoot damage in the early stage of acclimation. Photosynthetic rate at 5 weeks after the transplanting was significantly increased as the light intensity increased. These results suggest that 100 μmol·m⁻²·s⁻¹ for 6 weeks (B) or gradually increased treatment from 60 μmol·m⁻²·s⁻¹ to 140 μmol·m⁻²·s⁻¹ at 2 weeks interval (F) were the proper lighting conditions for successful acclimation of in vitro-propagated apple plantlets. Acknowledgment: This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (315003051SB020).

Keywords: acclimation, in vitro-propagated apple plantlets, light intensity, plant factory

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Authors: Neha Sahu, Awantika Singh, Brijesh Kumar, K. R. Arya

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Taraxacum officinale Weber or dandelion (Asteraceae) is an important Indian traditional herb used to treat liver detoxification, digestive problems, spleen, hepatic and kidney disorders, etc. The plant is well known to possess important phenolic and flavonoids to serve as a potential source of antioxidative and chemoprotective agents. Biosynthesis of bioactive compounds through in vitro cultures is a requisite for natural resource conservation and to provide an alternative source for pharmaceutical applications. Thus an efficient and reproducible protocol was developed for in vitro biosynthesis of bioactive antioxidative compounds from leaf derived callus and in vitro regenerated cultures of Taraxacum officinale using MS media fortified with various combinations of auxins and cytokinins. MS media containing 0.25 mg/l 2, 4-D (2, 4-Dichloro phenoxyacetic acid) with 0.05 mg/l 2-iP [N6-(2-Isopentenyl adenine)] was found as an effective combination for the establishment of callus with 92 % callus induction frequency. Moreover, 2.5 mg/l NAA (α-Naphthalene acetic acid) with 0.5 mg/l BAP (6-Benzyl aminopurine) and 1.5 mg/l NAA showed the optimal response for in vitro plant regeneration with 80 % regeneration frequency and rooting respectively. In vitro regenerated plantlets were further transferred to soil and acclimatized. Quantitative variability of accumulated bioactive compounds in cultures (in vitro callus, plantlets and acclimatized) were determined through UPLC-MS/MS (ultra-performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry) and compared with wild plants. The phytochemical determination of in vitro and wild grown samples showed the accumulation of 6 compounds. In in vitro callus cultures and regenerated plantlets, two major antioxidative compounds i.e. chlorogenic acid (14950.0 µg/g and 4086.67 µg/g) and umbelliferone (10400.00 µg/g and 2541.67 µg/g) were found respectively. Scopoletin was found to be highest in vitro regenerated plants (83.11 µg/g) as compared to wild plants (52.75 µg/g). Notably, scopoletin is not detected in callus and acclimatized plants, but quinic acid (6433.33 µg/g) and protocatechuic acid (92.33 µg/g) were accumulated at the highest level in acclimatized plants as compared to other samples. Wild grown plants contained highest content (948.33 µg/g) of flavonoid glycoside i.e. luteolin-7-O-glucoside. Our data suggests that in vitro callus and regenerated plants biosynthesized higher content of antioxidative compounds in controlled conditions when compared to wild grown plants. These standardized cultural conditions may be explored as a sustainable source of plant materials for enhanced production and adequate supply of oxidative polyphenols.

Keywords: anti-oxidative compounds, in vitro cultures, Taraxacum officinale, UPLC-MS/MS

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Authors: James Sinclair, Katy Soapi, Brad Carte

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The marine environment has continuously demonstrated to be a rich source of secondary metabolites and bioactive compounds that can address the many pharmaceutical problems facing mankind. The emergence of multidrug resistant pathogens has caused scientists to explore contemporary ways of combating these super bugs. A red-pigmented bacterial strain isolated from a marine alga collected in Fiji was identified to be Pseudoalteromonas rubra from 16s rRNA sequencing. This bacterial strain was cultured using a yeast-peptone media and incubated for five days. The ethyl acetate extract of this bacterium was subjected to chromatographic separation techniques such as vacuum liquid chromatography, flash chromatography, size exclusion chromatography and high-pressure liquid chromatography to yield the pure compound and a number of semi-pure fractions. The crude extract and subsequent purified fractions were analyzed by ultraviolet/visible spectroscopy and mass spectroscopy and was found to contain the compounds ivermectin, stenothricin, cyclo-L-pro-L-val, prodigiosin, mycophenolic acid, phenazine-1-carboxylic acid, eplerenone, staurosporine and pseudoalteromone A. The structure of the pure compound, pseudoalteromone A, was elucidated using NMR 1H, 13C, 1H-1H COSY, HSQC and HMBC spectroscopic data.

Keywords: Pseudoalteromonas rubra, Pseudoalteromone A, secondary metabolites, structure elucidation

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