Search results for: opiates metabolites

Authors: Sudik S. D., Raymon J. B., Maidala A., Lawan A., Bagudu I. A.

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An experiment was conducted with 240 Abor Acre broilers to determine the effect of replacing maize with acha offal (Digitaria exilis) on performance, haematology, and serum biochemical. Chicks were allotted to six diets (T1, T2, T3, T4, T5, and T6) with acha offal (AO) at 0.0%, 5.0%, 7.5%, 10.0%, 12.5% and 15.0% respectively as replacement of maize with 4 replicates consisting of 10 birds per replicate in a completely randomized design. They were allowed ad libitum accessed to feed and water throughout a 42 days experiment. The results showed that at the starter phase, only feed conversion ratio (FCR) was significantly affected (p < 0.05). Chicks fed T5 had best FCR more than those fed T1 while those fed T2, T3, T4, and T6 had similar FCR comparable with T1. At the finisher stage, final weight (FW), total weight change (TWC), average daily gain (ADG), and FCR were significantly affected (p < 0.05). Chickens fed T3, T4, T5, and T6 had similar FW, TWC, and ADG and higher than those fed T1; those fed T2 had similar FW, TWG, and DWG with T1. Chickens fed T6 had best FCR, followed by those fed T3, T4, and T5, while those T2 had worse FCR similar with those fed T1. Eviscerated weight was significantly affected (p < 0.05) by treatment. Birds fed T4, T5, and T6 had higher eviscerated weight followed by T3 while those fed T2 had least eviscerated weight comparable with those fed T1. The entire organs (Gizzard, heart, kidneys, liver, lungs, pancreas, and proventriculus) were not significantly affected (p > 0.05) by treatments. Packed cell volume (PCV) and red blood cell (RBC) were significantly (p < 0.05) affected by treatment. Birds fed T4, T5, and T6 had higher and similar PCV and RBC with those fed T1 while those fed T2 and T3 had lower PCV and RBC. The entire serum metabolites were not significantly affected (p > 0.05) by treatments. In conclusion, acha offal can replace maize in starter and finisher broilers’ diets at 12.5% and 15.0%, respectively, without an adverse effect.

Keywords: broiler, acha offal, maize, performance, eviscerated, haematology, serum

Procedia PDF Downloads 118

Authors: Nafiseh Noormohammadi, Ahmad Sobhani Najafabadi

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Hypericins (hypericin and pseudohypericin) are natural napthodianthrone derivatives produced by Hypericum perforatum (St. John’s Wort), which have many medicinal properties such as antitumor, antineoplastic, antiviral, and antidepressant activities. Production and accumulation of hypericin in the plant are influenced by both genetic and environmental conditions. Despite the existence of different high-throughput data on the plant, genetic dimensions of hypericin biosynthesis have not yet been completely understood. In this research, 21 high-quality RNA-seq data on different parts of the plant were integrated into metabolic data to reconstruct a coexpression network. Results showed that a cluster of 30 transcripts was correlated with total hypericin. The identified transcripts were divided into three main groups based on their functions, including hypericin biosynthesis genes, transporters, detoxification genes, and transcription factors (TFs). In the biosynthetic group, different isoforms of polyketide synthase (PKSs) and phenolic oxidative coupling proteins (POCPs) were identified. Phylogenetic analysis of protein sequences integrated into gene expression analysis showed that some of the POCPs seem to be very important in the biosynthetic pathway of hypericin. In the TFs group, six TFs were correlated with total hypericin. qPCR analysis of these six TFs confirmed that three of them were highly correlated. The identified genes in this research are a rich resource for further studies on the molecular breeding of H. perforatum in order to obtain varieties with high hypericin production.

Keywords: hypericin, St. John’s Wort, data mining, transcription factors, secondary metabolites

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Authors: T. C. Machaba, S. M. Mahlo

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The current study investigates the antifungal properties of crude plant extracts from selected medicinal plant species. Eight plant species used by the traditional healers and local people to treat fungal infections were selected for further phytochemical analysis and biological assay. The selected plant species were extracted with solvent of various polarities such as acetone, methanol, ethanol, hexane, dichloromethane, ethyl acetate and water. Leaf, roots and bark extracts of Maerua juncea Pax, Albuca seineri (Engl & K. Krause) J.C Manning & Goldblatt, Senna italica Mill., Elephantorrhiza elephantina (Burch.) Skeels, Indigofera circinata Benth., Schinus molle L., Asparagus buchananii Bak., were screened for antifungal activity against three animal fungal pathogens (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans). All plant extracts were active against the tested microorganisms. Acetone, dichloromethane, hexane and ethanol extracts of Senna italica and Elephantorrhiza elephantine had excellent activity against Candida albicans and A. fumigatus with the lowest MIC value of 0.02 mg/ml. Bioautography assay was used to determine the number of antifungal compounds presence in the plant extracts. No active compounds were observed in plant extracts of Indigofera circinnata, Schinus molle and Pentarrhinum insipidum with good antifungal activity against C. albicans and A. fumigatus indicating possible synergism between separated metabolites.

Keywords: antifungal activity, bioautography, ethnobotanical survey, minimum inhibitory concentration

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Authors: Asma Lamin, Anna H. Kaksonen, Ivan Cole, Paul White, Xiao-Bo Chen

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Microbiologically influenced corrosion (MIC) is a process in which microorganisms initiate, facilitate, or accelerate the electrochemical corrosion reactions of metallic components. Several reports documented that MIC accounts for about 20 to 40 % of the total cost of corrosion. Biofilm formation due to the presence of microorganisms on the surface of metal components is known to play a vital role in MIC, which can lead to severe consequences in various environmental and industrial settings. Quorum sensing (QS) system plays a major role in regulating biofilm formation and control the expression of some microbial enzymes. QS is a communication mechanism between microorganisms that involves the regulation of gene expression as a response to the microbial cell density within an environment. This process is employed by both Gram-positive and Gram-negative bacteria to regulate different physiological functions. QS involves production, detection, and responses to signalling chemicals, known as auto-inducers. QS controls specific processes important for the microbial community, such as biofilm formation, virulence factor expression, production of secondary metabolites and stress adaptation mechanisms. The use of QS inhibitors (QSIs) has been proposed as a possible solution to biofilm related challenges in many different applications. Although QSIs have demonstrated some strength in tackling biofouling, QSI-based strategies to control microbially influenced corrosion have not been thoroughly investigated. As such, our research aims to target the QS mechanisms as a strategy for mitigating MIC on metal surfaces in engineered systems.

Keywords: quorum sensing, quorum quenching, biofilm, biocorrosion

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Authors: Ammani K., Glory M.

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Demands of traditional herbal medicines are increasing day by day over the world. Considering the growing demand of medicinal plants in curative treatments and the role of VAM fungi in augmentation of the production of active secondary metabolites by the medicinal plants, the present work has been undertaken to survey the mycorrhizal status in 30 different medicinal plants belonging to various families from Krishna district, Andhra Pradesh. The roots were collected carefully and stained by the Phillips & Hayman technique. Basing on the occurrence of vesicles and arbuscules, categorized into four grades; Excellent: mycelia, vesicles or arbuscules present more than 75% of root bits, Good: mycelia, vesicles or arbuscules present 50-75% in surface of root bits, moderate: mycelia, vesicles or arbuscules present 25-50% in surface of root bits, and poor: mycelia, vesicles or arbuscules present 1-25% in surface of root bits. The study reveals that the roots of all plants were colonized by AM fungi. Percentage of root colonization by AM fungi was more in Aloe vera, Phylanthus emblica, Azadiracta indica and least in plants such as Aerva lanata, Vinca rosea, Crotalaria verrucosa among the 30 medicinal plants in present study. The enhancement of growth and vigour and increased production of bioactive compounds of the medicinal plants is desirable which may be achieved by inoculation of the roots with Arbuscular mycorrhizal fungi. There is a steady increase in the cultivation of medicinal plants to maintain a steady supply to support the increasing demand but corresponding researches of VAM fungi and their association in medicinal plants have received very little attention as compared to the studies on forest species and field crops. So a vast research on this field is necessary for a better tomorrow.

Keywords: Arbuscular mycorrhizae, colonization, categories, medicinal plants

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Authors: Ali Bahri Lubis

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Tryptophan oxidation by Heme-dioxygenase enzyme is the initial rate-limiting step in the kynurenine pathway, which leads to the formation of NADH and dangerous metabolites, implicating several severe diseases such as Parkinson’s Disease, Huntington's Disease, poliomyelitis and cataract. This oxidation, generally, allows tryptophan to convert to N-Formylkynurenine (NFK). Observing the catalytic mechanism of Heme dioxygenase in tryptophan oxidation has been a debatably scientific interest since no one has yet proven the mechanism obviously. In this research we have attempted to prove mechanistic steps of tryptophan oxidation via human indoleamine dioxygenase (h-IDO) utilising various substrates: L-tryptophan, L-tryptophan (indole-ring-2-¹³C), L-fully-labelled¹³C-tryptophan, L-N-methyl-tryptophan, L-tryptophanol and 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid. All enzyme assay experiments were measured using a UV-Vis spectrophotometer, LC-MS, 1H-NMR and HSQC. We also successfully synthesised enzyme products as our control in NMR measurements. The result exhibited that all substrates produced N-formyl kynurenine (NFK), and a side, the minor product of hydroxypyrrolloindoleamine carboxylic acid (HPIC) in cis and trans isomer, except 1-methyl tryptophan only generating cis HPIC. Interestingly, L- tryptophanol was oxidised to form HPIC derivative as a major product and 5-hydroxy tryptophan was converted to NFK derivative instead without any HPIC derivative. The bizarre result of oxidation underwent in 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid, which produced epoxide cyclic. Those phenomena have been explainable in our research based on the proposed mechanism of how tryptophan is oxidised by human indoleamine dioxygenase.

Keywords: tryptophan oxidation, heme-dioxygenases, human indoleamine dioxygenases, N-formylkynurenine, hydroxypyrroloindoleamine carboxylic acid

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Authors: J. Pazourek, K. Šmejkal, P. Kollár, J. Rajchard, J. Šinko, Z. Balounová, E. Vlková, H. Salmonová

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Pectinatella magnifica (Leidy, 1851) is an invasive freshwater animal that lives in colonies. A colony of Pectinatella magnifica (a gelatinous blob) can be up to several feet in diameter large and under favorable conditions it exhibits an extreme growth rate. Recently European countries around rivers of Elbe, Oder, Danube, Rhine and Vltava have confirmed invasion of Pectinatella magnifica, including freshwater reservoirs in South Bohemia (Czech Republic). Our project (Czech Science Foundation, GAČR P503/12/0337) is focused onto biology and chemistry of Pectinatella magnifica. We monitor the organism occurrence in selected South Bohemia ponds and sandpits during the last years, collecting information about physical properties of surrounding water, and sampling the colonies for various analyses (classification, maps of secondary metabolites, toxicity tests). Because the gelatinous matrix is during the colony lifetime also a host for algae, bacteria and cyanobacteria (co-habitants), in this contribution, we also applied a high performance liquid chromatography (HPLC) method for determination of potentially present cyanobacterial toxins (microcystin-LR, microcystin-RR, nodularin). Results from the last 3-year monitoring show that these toxins are under limit of detection (LOD), so that they do not represent a danger yet. The final goal of our study is to assess toxicity risks related to fresh water resources invaded by Pectinatella magnifica, and to understand the process of invasion, which can enable to control it.

Keywords: cyanobacteria, fresh water resources, Pectinatella magnifica invasion, toxicity monitoring

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Authors: Marzieh Fotovvat, Farzaneh Najafi, Ramazan Ali Khavari-Nejad, Daryush Talei, Farhad Rejali

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Salvia leriifolia Benth. is one of the valuable and perennial medicinal plants of the Lamiaceae family, geographically growing in the south and tropical regions of Khorassan and Semnan provinces in Iran. In recent years, several medicinal properties such as antimicrobial, antifungal, anti-diabetic, analgesic, and anti-inflammatory effects have been reported from this plant. The use of elicitors such as chitosan and Arbuscular mycorrhizal fungi (AMF) symbiosis are the main methods for increasing the production of secondary metabolites, growth, and physiological factors in plants. The main aim of this study was to investigate the effects of foliar spraying applications by chitosan and/or the contribution of AMF (Glomus interaradices) on some growth factors and chlorophyll content of S. leriifolia under glasshouse conditions. The sterilized seeds were germinated by placing them into a cocopeat. After one month, seedlings that were in the 2-4 leaf stage were transferred to plastic pots (garden soil and pumice at 2:1) with or without mycorrhizal fungi. Chitosan (0, 50, 100, 200, and 400 mg L-1) was sprayed four times in the fourth month of the vegetative period. The results showed that fresh leaf weight, fresh root weight, root height, and chlorophyll content could change in the plant treated with chitosan and AMF symbiosis. So that the highest chlorophyll content and fresh weight of roots and leaves were observed in the interaction of chitosan and G. interaradices. In general, by optimizing the chitosan concentration and the use of appropriate AMF symbiosis, it is possible to improve the growth and quality of the medicinal plant S. leriifolia.

Keywords: chitosan, chlorophyll, growth factors, mycorrhiza

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Authors: Anja Gräff, Stefan Holzer, Manfred Höld, Jörn Stumpenhausen, Heinz Bernhardt

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In view of the increasing quantity of 'green energy' from renewable raw materials and photovoltaic facilities, it is quite conceivable that power supply variations may occur, so that constantly working machines like automatic milking systems (AMS) may break down temporarily. The usage of farm-made energy is steadily increasing in order to keep energy costs as low as possible. As a result, power cuts are likely to happen more frequently. Current work in the framework of the project 'stable 4.0' focuses on possible stress reactions by simulating power cuts up to four hours in dairy farms. Based on heart activity it should be found out whether stress on dairy cows increases under these circumstances. In order to simulate a power cut, 12 random cows out of 2 herds were not admitted to the AMS for at least two hours on three consecutive days. The heart rates of the cows were measured and the collected data evaluated with HRV Program Kubios Version 2.1 on the basis of eight parameters (HR, RMSSD, pNN50, SD1, SD2, LF, HF and LF/HF). Furthermore, stress reactions were examined closely via video analysis, milk yield, ruminant activity, pedometer and measurements of cortisol metabolites. Concluding it turned out, that during the test only some animals were suffering from minor stress symptoms, when they tried to get into the AMS at their regular milking time, but couldn´t be milked because the system was manipulated. However, the stress level during a regular “time-dependent milking rejection” was just as high. So the study comes to the conclusion, that the low psychological stress level in the case of a 2-4 hours failure of an AMS does not have any impact on animal welfare and health.

Keywords: dairy cow, heart activity, power cut, stable 4.0

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Authors: Amir Saber Gharamaleki, Beitollah Alipour, Zeinab Faghfoori, Ahmad YariKhosroushahi

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Colorectal cancer (CRC) is one of the most prevalent cancers and intestinal microbial community plays an important role in colorectal tumorigenesis. Probiotics have recently been assessed as effective anti-proliferative agents and thus this study was performed to examine whether CRC undergo apoptosis by treating with isolated Iranian native dairy yeast, Kluyveromyces marxianus YAS, secretion metabolites. The cytotoxicity assessments on cells (HT-29, Caco-2) were accomplished through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as well as qualitative DAPI (4',6-diamidino-2-phenylindole staining) and quantitative (flow cytometry assessments) evaluations of apoptosis. To evaluate the main mechanism of apoptosis, Real time PCR method was applied. Kluyveromyces marxianus YAS secretions (IC50) showed significant cytotoxicity against HT-29 and Caco-2 cancer cell lines (66.57 % and 66.34 % apoptosis) similar to 5-Fluorouracil (5-FU) while apoptosis only was developed in 27.57 % of KDR normal cells. The prophylactic effects of Kluyveromyces marxianus (PTCC 5195), as a reference yeast, was not similar to Kluyveromyces marxianus YAS indicating strain dependency of bioactivities on CRC disease prevention. Based on real time PCR results, the main cytotoxicity is related to apoptosis phenomenon and the core related mechanism is depended on the overexpression of BAX, CASP 9, CASP 8 and CASP 3 inducing apoptosis genes. However, several investigations should be conducted to precisely determine the effective compounds to be used as anticancer therapeutics in the future.

Keywords: anticancer, anti-proliferative, apoptosis, cytotoxicity, yeast

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Authors: Himanshi Rathore, Shalinee Prasad, Satyawati Sharma, Ajay Singh Yadav

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Medicinal mushrooms are acceptable all over the world not only because they have a unique flavour and texture but also due to the presence of great nutritional, nutraceutical and functional properties. High content of physiologically active substances like ergosterol, vitamin D, phenolic compounds, triterpenoids and steroids make these medicinal mushrooms a key source of nutraceuticals. Calocybe indica is a popular medicinal mushroom of India which is known to possess high amount of secondary metabolites including ergosterol (vitamin D2). The ergosterol gets converted to vitamin D in the presence of UV rays by a photochemical reaction. In lieu of the above facts the present study was undertaken to investigate the effect of UV-B light treatment on the vitamin D2 concentration, phenolic content and non volatile compounds in Calocybe indica. For this study, UV-B light source of intensity 5.3w/m2 was used to expose mushrooms for the time period of 0min, 30min, 60min and 90 min. It was found that the vitamin D2 concentration increased with the time duration i.e. 85±0.15 (0 min), 182±1.6 (30 min), 187±0.4 (60 min) and 182 ±0.8 (90 min) μg/g (dry weight). Highest concentration of vitamin D2 was found at 60 min duration. No discoloration in sliced mushrooms was observed during the exposure time. The results revealed that the exposure of mushrooms for a minimum of 30 min duration under UVB source can be a novel, convenient and cheapest way to increase the vitamin D content in mushrooms. This can be one of richest source to fulfil the recommended dietary allowances of vitamin D in our daily diets. The paper provides information on the enhancement of vitamin D content by UV lights and its effects on the non volatile (soluble sugars, free amino acids, 5′-nucleotides and phenolics) compounds will also be presented.

Keywords: Calocybe indica, ergosterol, nutraceutical, phenolics

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Authors: Gajanan V. Mane, Rashmi More, Mahesh S. Sonawane, Tushar Lodha, Rohit Sharma

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The diversity of fungi isolated from two different termite species viz., Odontoterms assmuthi and O. abesus was investigated by dilution- plate method, combined with morphological characteristics and sequencing of internal transcribed spacer region. In total, ninety-six fungi were isolated and purified, out of which 69 isolates were obtained from O. assmuthi belonging to 18 genera and 31 species, whereas 27 isolates were obtained from O. abesus belonging to 15 genera and 17 species. The fungal strains were screened for laccase, amylase, cellulase and pectinase enzymes production. Twenty-seven strains were positive for laccase, 59 strains were positive for amylase, 71 strains were positive for cellulase and 72 strains were positive for pectinase enzymes. The antimicrobial activities of the isolated fungi were tested by the dual plate culture method against standard pathogens. Bioactive secondary metabolites were identified by HPLC and LCMS. Four isolates viz., Penicillium goetzii MG 57, Epicoccum sp. MG 39, Penicillium tanzanicum MG 30, Aspergillus polyporicola MG 54, showed positive antimicrobial activity against standard pathogens, Streptococcus pneumonia MCC 2425, Staphylococcus aureus MCC 2408, Pseudomonas aeruginosa MCC 2080, Escherichia coli MCC 2412, Enterococcus faecalis MCC 2409, Klebsiella pneumonia MCC 2451, Micrococcus luteus MCC 2155 and Candida albicans MCC 1151. In conclusion, the study showed that the insect gut harbor fungal diversity, which is futuristic with biotechnological potential and could be a good source of enzymes and antibiotics.

Keywords: termites, fungi, its, enzyme, antimicrobial activity

Procedia PDF Downloads 75

Authors: Ghulam Mustafa Kamal, Xiaohua Wang, Bin Yuan, Abdullah Ijaz Hussain, Jie Wang, Shahzad Ali Shahid Chatha, Xu Zhang, Maili Liu

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Organic food products are becoming increasingly popular in recent years, as consumers have turned more health conscious and environmentally aware. A lot of consumers have understood that the organic foods are healthier than conventionally produced food stuffs. Price difference between conventional and organic foods is very high. So, it is very common to cheat the consumers by mislabeling and adulteration. Our study describes the 1H NMR based approach to characterize and differentiate soy sauce prepared from organically and conventionally grown raw materials (wheat and soybean). Commercial soy sauce samples fermented from organic and conventional raw materials were purchased from local markets. Principal component analysis showed clear separation among organic and conventional soy sauce samples. Orthogonal partial least squares discriminant analysis showed a significant (p < 0.01) separation among two types of soy sauce yielding leucine, isoleucine, ethanol, glutamate, lactate, acetate, β-glucose, sucrose, choline, valine, phenylalanine and tyrosine as important metabolites contributing towards this separation. Abundance ratio of 13C to 12C was also evaluated by 1H NMR spectroscopy which showed an increased ratio of 13C isotope in organic soy sauce samples indicating the organically grown wheat and soybean used for the preparation of organic soy sauce. Results of the study can be helpful to the end users to select the soy sauce of their choice. This information could also pave the way to further trace and authenticate the raw materials used in production of soy sauce.

Keywords: 1H NMR, multivariate analysis, organic, conventional, 13C isotopic ratio, soy sauce

Procedia PDF Downloads 237

Authors: Vanessa G. P. Severino, JOÃO Gabriel M. Junqueira, Michelle N. G. do Nascimento, Francisco W. B. Aquino, João B. Fernandes, Ana P. Terezan

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The scientific interest in analyzing VOCs represents a significant modern research field as a result of importance in most branches of the present life and industry. Therefore it is extremely important to investigate, identify and isolate volatile substances, since they can be used in different areas, such as food, medicine, cosmetics, perfumery, aromatherapy, pesticides, repellents and other household products through methods for extracting volatile constituents, such as solid phase microextraction (SPME), hydrodistillation (HD), solvent extraction (SE), Soxhlet extraction, supercritical fluid extraction (SFE), stream distillation (SD) and vacuum distillation (VD). The Chemometrics is an area of chemistry that uses statistical and mathematical tools for the planning and optimization of the experimental conditions, and to extract relevant chemical information multivariate chemical data. In this context, the focus of this work was the study of the chemical VOCs by SPME of the specie X. aromatica, in search of constituents that can be used in the industrial sector as well as in food, cosmetics and perfumery, since these areas industrial has a considerable role. In addition, by chemometric analysis, we sought to maximize the answers of this research, in order to search for the largest number of compounds. The investigation of flowers from X. aromatica in vitro and in alive mode proved consistent, but certain factors supposed influence the composition of metabolites, and the chemometric analysis strengthened the analysis. Thus, the study of the chemical composition of X. aromatica contributed to the VOCs knowledge of the species and a possible application.

Keywords: chemometrics, flowers, HS-SPME, Xylopia aromatica

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Authors: V. A. Doss, R. Sowndarya, K. Juila Rose Mary

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Objective: Amino acid neurotransmitter system dysfunction plays a major role in the pathophysiology of depression. The objective of the present study was to identify the amino acids as possible metabolite biomarkers for depression using GCMS (Gas Chromatography Mass Spectrometry) before and after exercise regimen in brain samples of depression induced animal models. Methods: Depression-like behaviour was induced by Chronic Unpredictable mild stress (CUMS). Severity of depression was measured by forced swim test (FST) and sucrose consumption test (SCT). Swimming protocol was followed for 4 weeks of exercise treatment. Brain obtained from depressed and exercise treated rats were used for the metabolite analysis by GCMS. Subsequent statistical analysis obtained by ANOVA followed by post hoc test revealed significant metabolic changes. Results: Amino acids such as alanine, glycine, serine, glutamate, homocysteine, proline and branched chain aminoacids (BCAs) Leucine, Isoleucine, Valine were determined in brain samples of control, depressed and exercised groups. Among these amino acids, the levels of D-Serine and branched chain amino acids were found to be decreased in depression induced rats. After four weeks of swimming exercise regimen, there were improvements in the levels of serine and Branched chain amino acids. Conclusion: We suggest that Serine and BCAs may be investigated as potential metabolite markers using GCMS and their beneficial metabolic changes in Exercise.

Keywords: metabolomics, depression, forced swim test, exercise, amino acid metabolites, GCMS, biomarker

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Authors: Guillaume Caulier, Lola Brasseur, Patrick Flammang, Pascal Gerbaux, Igor Eeckhaut

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Saponins and quinones are two major groups of secondary metabolites widely distributed in the biosphere. More specifically, triterpenoid saponins and anthraquinones are mainly found in a wide variety of plants, bacteria and fungi. In the animal kingdom, these natural organic compounds are rare and only found in small quantities in arthropods, marine sponges and echinoderms. In this last group, triterpenoid saponins are specific to holothuroids (sea cucumbers) while anthraquinones are the chemical signature of crinoids (feather stars). Depending on the species, they present different molecular cocktails. Despite presenting different chemical properties, these molecules share numerous similarities. This study compares the biological distribution, the pharmacological effects and the ecological roles of holothuroid saponins and crinoid anthraquinones. Both of them have been defined as allomones repelling predators and parasites (i.e. chemical defense) and have interesting pharmacological properties (e.g. anti-bacterial, anti-fungal, anti-cancer). Our study investigates the chemical ecology of two symbiotic associations models; between the snapping shrimp Synalpheus stimpsonii associated with crinoids and the Harlequin crab Lissocarcinus orbicularis associated with holothuroids. Using behavioral experiments in olfactometers, chemical extractions and mass spectrometry analyses, we discovered that saponins and anthraquinones present a second ecological role: the attraction of obligatory symbionts towards their hosts. They can, therefore, be defined as kairomones. This highlights a new paradigm in marine chemical ecology: Chemical repellents are attractants to obligatory symbionts because they constitute host specific chemical signatures.

Keywords: anthraquinones, kairomones, marine symbiosis, saponins, attractant

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Authors: Madhuri Kanala, Geoffrey Savage

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Worldwide CO₂ levels are slowly rising, and this may have effects on the growth and nutritional composition of many food plants. The production of secondary metabolites such as oxalates has not been investigated in depth. The oxalate content of many food plants are known to have adverse nutritional effects on humans and reduction in the oxalate contents of food plants is a very positive move. Recent studies had shown that the oxalate content of the leaves of spinach and silver beet reduced when the plants were grown in an environment where CO₂ was increased. The response of amaranth and strawberry spinach leaves to changes in the high CO₂ environment have not been understood though it is known that the plants do contain appreciable oxalate contents. A study was conducted where amaranth and strawberry spinach plants were grown in identical plant growth chambers with the same environmental conditions except that one chamber was supplied with ambient air (CO₂ 405 ppm) while the other chamber had the CO₂ level increased to 650 ppm. The total and soluble oxalate content of the leaves of raw and cooked amaranth and strawberry spinach were determined by HPLC and calcium levels were determined using ICP following 6 weeks of growth. The total oxalate content of the fresh leaves of amaranth and strawberry spinach were reduced by 29.5 % and 24.6% respectively in the leaves of the plants grown in increased CO₂ conditions compared to ambient levels. The soluble oxalate content of amaranth leaves grown under ambient and increased CO₂ conditions were future reduced by 42% and 26.8% respectively following cooking as the soluble oxalate was leached into the cooking water and discarded. The reduction of the oxalate and calcium levels of raw and cooked amaranth and strawberry spinach leaves following an increase in CO₂ content in the air is an interesting positive response to an otherwise significant environmental problem.

Keywords: amaranth, calcium oxalate, enriched CO₂, oxalates, strawberry spinach

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Authors: Arieh Moussaieff, Bénédicte Elena-Herrmann, Yaakov Nahmias, Daniel Aberdam

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Differentiation of pluripotent stem cells is a slow process, marked by the gradual loss of pluripotency factors over days in culture. While the first few days of differentiation show minor changes in the cellular transcriptome, intracellular signaling pathways remain largely unknown. Recently, several groups demonstrated that the metabolism of pluripotent mouse and human cells is different from that of somatic cells, showing a marked increase in glycolysis previously identified in cancer as the Warburg effect. Here, we sought to identify the earliest metabolic changes induced at the first hours of differentiation. High-resolution NMR analysis identified 35 metabolites and a distinct, gradual transition in metabolism during early differentiation. Metabolic and transcriptional analyses showed the induction of glycolysis toward acetate and acetyl-coA in pluripotent cells, and an increase in cholesterol biosynthesis during early differentiation. Importantly, this metabolic pathway regulated differentiation of human and mouse embryonic stem cells. Acetate delayed differentiation preventing differentiation-induced histone de-acetylation in a dose-dependent manner. Glycolytic inhibitors upstream of acetate caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our data suggests that a rapid loss of glycolysis in early differentiation down-regulates acetate and acetyl-coA production, causing a loss of histone acetylation and concomitant loss of pluripotency. It demonstrate that pluripotent stem cells utilize a novel metabolism pathway to maintain pluripotency through acetate/acetyl-coA and highlights the important role metabolism plays in pluripotency and early differentiation of stem cells.

Keywords: pluripotency, metabolomics, epigenetics, acetyl-coA

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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

Procedia PDF Downloads 114

Authors: Ana Vulic, Tina Lesic, Nina Kudumija, Maja Kis, Manuela Zadravec, Nada Vahcic, Tomaz Polak, Jelka Pleadin

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Mycotoxins are toxic secondary metabolites produced by numerous types of molds. They can contaminate both food and feed so that they represent a serious public health concern. Production of dry-fermented meat products involves ripening, during which molds can overgrow the product surface, produce mycotoxins, and consequently contaminate the final product. Citrinin is a mycotoxin produced mainly by the Penicillium citrinum. Data on citrinin occurrence in both food and feed are limited. Therefore, there is a need for research on citrinin occurrence in these types of meat products. The LC-MS/MS method for citrinin determination was developed and validated. Sample preparation was performed using immunoaffinity columns, which resulted in clean sample extracts. Method validation included the determination of the limit of detection (LOD), the limit of quantification (LOQ), recovery, linearity, and matrix effect in accordance to the latest validation guidance. The determined LOD and LOQ were 0.60 µg/kg and 1.98 µg/kg, respectively, showing a good method sensitivity. The method was tested for its linearity in the calibration range of 1 µg/L to 10 µg/L. The recovery was 100.9 %, while the matrix effect was 0.7 %. This method was employed in the analysis of 47 samples of dry-fermented sausages collected from local households. Citrinin wasn’t detected in any of these samples, probably because of the short ripening period of the tested sausages that takes three months tops. The developed method shall be used to test other types of traditional dry-cured products, such as prosciuttos, whose surface is usually more heavily overgrown by surface molds due to the longer ripening period.

Keywords: citrinin, dry-fermented meat products, LC-MS/MS, mycotoxins

Procedia PDF Downloads 92

Authors: Leona Ondrejikova, Martin Hill, Antonin Parizek

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The incidence of gestational diabetes mellitus (GDM) is higher in women predisposed to developing intrahepatic cholestasis of pregnancy (ICP). Both diseases are associated with altered steroidogenesis when compared with none-ICP controls. However, the effect of GDM on circulating steroids in ICP patients remains unclear. The question remains, whether the levels of circulating steroids differ between ICP patients with and without GDM. In total 10 ICP patients without GDM (ICP+GDM-), 7 ICP patients with GDM (ICP+GDM+), and 15 controls (ICP-GDM-) were monitored during late gestation, at labor, and during three periods postpartum (day 5, week 3, and week 6 postpartum) (Šimják et al., 2018). The relationships between steroid profiles and patients’ status were evaluated using the ANOVA model consisting of subject factor, between-subject factors Group (ICP+GDM+, ICP+GDM-, ICP-GDM-), gestational age at the diagnosis of ICP and gestational age at labor, and within-subject factor Stage and ICP × Stage interaction. The levels of the C21 and C19 Δ5 steroids and 5α/β-reduced C19 steroids were highest in ICP+GDM+, while those for the ICP-GDM-, and ICP+GDM- groups were lower. In the C21 Δ4 steroids and their 5α/β-reduced metabolites, the steroid levels were highest in the ICP+GDM-, intermediate in the ICP-GDM- and lowest in the ICP+GDM+ group. This higher concentration in ICP+GDM- group may be of importance as the 5α-pregnane-3α,20α-diol disulfate, is considered as the substance inducing ICP. In general, these data show that the comorbidity with GDM substantially changes the steroidome in ICP patients towards the higher activity of steroid CYP17A1 lyase step in adrenal zona reticularis reduced CYP17A1 hydroxylase step in zona fasciculata. This is consistent with our previously published hypothesis about the critical role of maternal zona reticularis in the pathophysiology of ICP. Our present data also indicate that the comorbidity with GDM might moderate the gravity of the ICP in this way.

Keywords: CYP17A1, GC-MS, gestational diabetes mellitus, intrahepatic cholestasis of pregnancy

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Authors: Omar Ali, Adesh Ramsubhag, Jayaraj Jayaraman

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Seaweed extracts have been reported as plant biostimulants which could be a safer, organic alternative to harsh pesticides. The incentive to use seaweed-based biostimulants is becoming paramount in sustainable agriculture. The current study, therefore, screened a commercial extract of A. nodosum in tomatoes, cultivated in Trinidad to showcase the multiple beneficial effects. Foliar treatment with an A. nodosum commercial extract led to significant increases in fruit yield and a significant reduction of incidence of bacterial spots and early blight diseases under both greenhouse and field conditions. Investigations were carried out to reveal the possible mechanisms of action of this biostimulant through defense enzyme assays and transcriptome profiling via RNA sequencing of tomato. Studies into disease control mechanisms by A. nodosum showed that the extract stimulated the activity of enzymes such as peroxidase, phenylalanine ammonia-lyase, chitinase, polyphenol oxidase, and β-1,3-glucanase. Additionally, the transcriptome survey revealed the upregulation and enrichment of genes responsible for the biosynthesis of growth hormones, defense enzymes, PR proteins and defense-related secondary metabolites, as well as genes involved in the nutrient mobilization, photosynthesis and primary and secondary metabolic pathways. The results of the transcriptome study also demonstrated the cross-talks between growth and defense responses, confirming the bioelicitor and biostimulant value of seaweed extracts in plants. These effects could potentially implicate the benefits of seaweed extract and validate its usage in sustainable crop production.

Keywords: A. nodosum, biostimulants, elicitor, enzymes, growth responses, seaweeds, tomato, transcriptome analysis

Procedia PDF Downloads 137

Authors: Naoki Yamamoto, Hidenobu Ozaki, Taiichiro Ookawa, Youming Liu, Kazunori Okada, Aiping Zheng

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Rice sheath blight is one of the destructive fungal diseases in rice. We have thought that rice sheath blight resistance is a polygenic trait. Host-pathogen interactions and secondary metabolites such as lignin and phytoalexins are likely to be involved in defense against R. solani. However, to our knowledge, it is still unknown how sheath blight resistance can be enhanced in rice breeding. To seek for an alternative genetic factor that contribute to sheath blight resistance, we mined relevant allelic variations from rice core collections created in Japan. Based on disease lesion length on detached leaf sheath, we selected 30 varieties of the top tail-end and the bottom tail-end, respectively, from the core collections to perform genome-wide association mapping. Re-sequencing reads for these varieties were used for calling single nucleotide polymorphisms among the 60 varieties to create a SNP panel, which contained 1,137,131 homozygous variant sites after filitering. Association mapping highlighted a locus on the long arm of chromosome 11, which is co-localized with three sheath blight QTLs, qShB11-2-TX, qShB11, and qSBR-11-2. Based on the localization of the trait-associated alleles, we identified an ankyryn repeat-containing protein gene (ANK-M) as an uncharacterized candidate factor for rice sheath blight resistance. Allelic distributions for ANK-M in the whole rice population supported the reliability of trait-allele associations. Gene expression characteristics were checked to evaluiate the functionality of ANK-M. Since an ANK-M homolog (OsPIANK1) in rice seems a basal defense regulator against rice blast and bacterial leaf blight, ANK-M may also play a role in the rice immune system.

Keywords: allele mining, GWAS, QTL, rice sheath blight

Procedia PDF Downloads 47

Authors: Nasim Rasuli, Akram Ahmadi, Hossein Riahi, Zeinab Shariatmadari, Majid Ghorbani Nohooji, Pooyan Mehraban Joubani

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Cyanobacteria are one of the most promising sources of new biostimulants and have received much attention due to their diverse applications in biotechnology. These microorganisms enhance the growth and productivity of plants by producing plant growth stimulants and fixing atmospheric nitrogen. Thymus vulgaris L., a valuable medicinal plant from the Lamiaceae family, is widely distributed across the globe. essential oil of T. vulgaris is best characterized by the prominence of phenols, making them the key compounds in its composition. Lignin biosynthesis as a natural plant polyphenol plays a crucial role in promoting plant growth, strengthening cell walls, and increasing resistance to pathogens. In this study, the bioelicitor activity of five cyanobacterial suspensions including Anabaena torulosa ISB213, Nostoc calcicola ISB215, Nostoc ellipsosporum ISB217, Trichormus doliolum ISB214, and Oscillatoria sp. ISB2116 on the lignin content of the T. vulgaris L. was investigated. Pot experiments were performed by inoculation of a %2 algal extract to the soil of treated plants one week before planting and then every 20 days. After four months, the lignin content in the leaves of both treated and control plants was evaluated. The results demonstrated that the application of cyanobacteria significantly increased the lignin content in the leaves of treated plants compared to the control. The treatment with Oscillatoria sp. ISB216 and N. ellipsosporum ISB217 resulted in the highest lignin content, with an increase of 93.33% and 86.67%, respectively. These findings highlight the potential of cyanobacteria as bioelicitors, offering a viable alternative for enhancing the production of secondary metabolites in T. vulgaris. Consequently, this could contribute to the economic value of this medicinal plant.

Keywords: cyanobacteria, bioelicitor, thymus vulgaris, lignin

Procedia PDF Downloads 42

Authors: Shivankar Agrawal, Sunil Kumar Deshmukh, Colin Barrow, Alok Adholeya

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A variety of genetic and environmental factors cause various cosmetics and dermatological problems. There are already claimed drugs available in market for treating these problems. However, the challenge remains in finding more potent, environmental friendly, causing minimal side effects and economical cosmeceuticals. This leads to an increased demand for natural cosmeceutical products in the last few decades. Plant derived ingredients are limited because plants either contain toxic metabolites, grow too slow or seasonal harvesting is a problem. The research work carried out in this project aims at isolation, characterization of marine fungal secondary metabolite and evaluating their potential use in future cosmetic skin care products. We have isolated and purified 35 morphologically different fungal isolates from various marine habitats of the India. These isolates have been functionally characterized for anti-tyrosinase, antioxidant and anti-acne activities. For molecular characterization, the Internal Transcribed spacer (ITS) region of 15 functionally active marine fungal isolates was amplified using universal primers, ITS1 and ITS4 and sequenced. Out of 15 marine fungal isolates crude extract of strains D4 (Aspergillus terreus) and P2 (Talaromyces stipitatus) showed 70% and 57% tyrosinase inhibition at 1mg/mL respectively. Strain D5 (Simplicillium lamellicola) has showed significant inhibition against Propionibacterium acnes and Staphylococcus epidermidis. In addition, all these strains also displayed DPPH- radical scavenging activity and may be utilized as skin cosmeceutical applications. Purification and characterization of crude extracts for identification of active lead molecule is under process.

Keywords: anti-acne, anti-tyrosinase, cosmeceutical, marine fungi

Procedia PDF Downloads 251

Authors: Soufiane Fal, Abderahim Aasfar, Ali Ouhssain, Hasnae Choukri, Abelaziz Smouni, Hicham El Arroussi

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Heavy metals contamination is a major environmental concern around the world. It has a harmful impact on plant productivity and poses a serious risk to humans and animals health. In the present study, the effect of Microalgae Crude Extract (MCE) on tomato growth and nutrients uptake exposed to 2 mM Pb2+ and Cd2+ was investigated. In results, 2 mM Pb2+ and Cd2+ showed a significant reduction of tomatobiomass and perturbation in nutrients absorption. Moreover, MCE application in tomato plant exposed to Pb2+ and Cd2+ showed a significant enhancement of biomass compared to tomato plants under Pb2+ and Cd2+. On the other hand, MCE application favoured heavy metals accumulation in root and inhibited their translocation to shoot as phytostabilisation mechanism. Tomato plants showed biochemical responses to Pb2+ and Cd2+ stress with elevation of scavenging enzymes and molecules such as POD, CAT, SOD, Proline, and polyphenols, etc. In addition, the treatment by MCE showed a significant reduction level of the majority of these parameters. Furthermore, the metabolomic analysis revealed a significant change in important metabolites. Pb2+ and Cd2+ showed decrease in SFA and increase of UFA, VLFA, alkanes, alkenes, sterols, which known accumulated as tolerance and resistance mechanism to heavy metal (H.M) stress. However, MCE treatment showed the inverse of these response to return tomato plants to normal state and enhanced tolerance and resistance to heavy metal stress. In the present study, we emphasized that MCE can alleviate H.M stress, enhance tomato plant growth nutrients absorption and improve biochemical responses.

Keywords: microalgae crude extract, heavy metal stress, nutrient uptake, metabolomic analysis, solanum lycopersicum (Tomato), phytostabilisation

Procedia PDF Downloads 85

Authors: Hafiz Arbab Sakandar

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Time-restricted intermittent fasting (TRIF) impacts host’s physiology and health. Plenty of health benefits have been reported for TRIF in animal models. However, limited studies have been conducted on humans especially in underdeveloped economies. Here, we designed a study to investigate the impact of TRIF/Ramadan fasting (16:8) on the modulation of gut-microbiome structure, metabolic pathways, and predicted metabolites and explored the correlation among them at different time points (during and after the month of Ramadan) in Pakistani Expats living in China. We observed different trends of Shannon-Wiener index in different subjects; however, all subjects showed substantial change in bacterial diversity with the progression of TRIF. Moreover, the changes in gut microbial structure by the end of TRIF were higher vis-a-vis in the beginning, significant difference was observed among individuals. Additionally, metabolic pathways analysis revealed that amino acid, carbohydrate and energy metabolism, glycan biosynthesis metabolism of cofactors and vitamins were significantly affected by TRIF. Pyridoxamine, glutamate, citrulline, arachidonic acid, and short chain fatty acid showed substantial difference at different time points based on the predicted metabolism. In conclusion, these results contribute to further our understanding about the key relationship among, dietary intervention (TRIF), gut microbiome structure and function. The preliminary results from study demonstrate significant potential for elucidating the mechanisms underlying gut microbiome stability and enhancing the effectiveness of microbiome-tailored interventions among the Pakistani populace. Nonetheless, extensive, and rigorous large-scale research on the Pakistani population is necessary to expound on the association between diet, gut microbiome, and overall health.

Keywords: gut microbiome, health, fasting, functionality

Procedia PDF Downloads 39

Authors: Lilia Belkacem, Zahra Rouabah, Assia Allaoui, Karina Bachtarzi, Souhila Belkadi, Boubakeur Safsaf, Madjid Tlidjane

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The Damascus breed, known for its prolificacy and milking ability, is recently imported in Algeria. Farmers tend to improve the local native herds by crossbreeding with Damascus bucks. The aim of the current investigation was to study the effects of physiological status on blood progesterone and some biochemical parameters in Shami goats and their crosses with local breed in arid conditions of Algeria. Ten does with an age range of 1.5- 3 years and BSC between 2.5 and 3.5 were used. Female goats were divided into two groups of five animals each: Damascus, and crossbred (Damascus x Arbia). All females were estrus synchronized and naturally mated. Blood samples were collected before intravaginal sponge insertion (non- pregnant), in early (30 days after sponge removal), mid (90 days), late pregnancy (130 days) and after kidding (30 days post-partum). Results demonstrate a significant effect of the reproductive stage on progesterone (P4) levels in both groups, on glycemia and cholesterolemia in crossbred does (p<0.05) and on albuminemia and uremia in Damascus ones. Concentrations of triglycerides, total proteins, globulin and creatinine revealed no significant difference between physiological phases in both groups (p>0.05). Breed effect was detected in early and mid-pregnancy for P4, in early pregnancy and lactation for total proteins and in lactation for globulin with lower concentrations in Damascus compared to crossbred does. Changes in P4 and biochemical profiles of both groups reflect the female goat’s adaptation to increased requirement of gestation and lactation in arid conditions of Algeria.

Keywords: damascus goat, crossbred, reproductive status, progesterone, biochemical metabolites

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Authors: Ronald Villamar-Torres, Michael Staudt, Christopher Viot

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Cotton Gossypium hirsutum L. is one of the most important industrial crops, producing the world leading natural textile fiber, but is very prone to arthropod attacks that reduce crop yield and quality. Cotton cultivation, therefore, makes an outstanding use of chemical pesticides. In reaction to herbivorous arthropods, cotton plants nevertheless show natural defense reactions, in particular through volatile organic compounds (VOCs) emissions. These natural defense mechanisms are nowadays underutilized but have a very high potential for cotton cultivation, and elucidating their genetic bases will help to improve their use. Simulating herbivory attacks by mechanical wounding of cotton plants in greenhouse, we studied by qPCR the changes in gene expression for genes of the terpenoids biosynthesis pathway. Differentially expressed genes corresponded to higher levels of the terpenoids biosynthesis pathway and not to enzymes synthesizing particular terpenoids. The genes were mapped on the G. hirsutum L. reference genome; their global relationships inside the general metabolic pathways and the biosynthesis of secondary metabolites were visualized with iPath2. The chromatographic profiles of VOCs emissions indicated first monoterpenes and sesquiterpenes emissions, dominantly four molecules known to be involved in plant reactions to arthropod attacks. As a result, the study permitted to identify potential key genes for the emission of volatile terpenoids by cotton plants in reaction to an arthropod attack, opening possibilities for molecular-assisted cotton breeding in benefit of smallholder cotton growers.

Keywords: biosynthesis pathways, cotton, mechanisms of plant defense, terpenoids, volatile organic compounds

Procedia PDF Downloads 333

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

Procedia PDF Downloads 232