Search results for: metabolite analysis

Authors: Imam Taufik, Hilkatul Ilmi, Puryani, Mochammad Yuwono, Aty Widyawaruyanti

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Artocarpus champeden Spreng stembark (Moraceae) in Indonesia well known as ‘cempedak’ had been traditionally used for malarial remedies. The difference of growth locations could cause the difference of metabolite profiling. As a consequence, there were difference antimalarial activities in spite of the same plants. The aim of this research was to obtain the profile of metabolites that contained in A. champeden stembark from different locations in Indonesia for authentication and quality control purpose of this extract. The profiling had been performed by HPTLC-Densitometry technique and antimalarial activity had been also determined by HRP2-ELISA technique. The correlation between metabolite fingerprinting and antimalarial activity had been analyzed by Principle Component Analysis, Hierarchical Clustering Analysis and Partial Least Square. As a result, there is correlation between the difference metabolite fingerprinting and antimalarial activity from several different growth locations.

Keywords: antimalarial, artocarpus champeden spreng, metabolite fingerprinting, multivariate analysis

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Authors: Amanina Iymia Jeffree, Reena Thriumani, Mohammad Iqbal Omar, Ammar Zakaria, Yumi Zuhanis Has-Yun Hashim, Ali Yeon Md Shakaff

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Metabolite profiling is a strategy to be approached in the pattern recognition method focused on three types of cancer cell line that driving the most to death specifically lung, breast, and colon cancer. The purpose of this study was to discriminate the VOCs pattern among cancerous and control group based on metabolite profiling. The sampling was executed utilizing the cell culture technique. All culture flasks were incubated till 72 hours and data collection started after 24 hours. Every running sample took 24 minutes to be completed accordingly. The comparative metabolite patterns were identified by the implementation of headspace-solid phase micro-extraction (HS-SPME) sampling coupled with gas chromatography-mass spectrometry (GCMS). The optimizations of the main experimental variables such as oven temperature and time were evaluated by response surface methodology (RSM) to get the optimal condition. Volatiles were acknowledged through the National Institute of Standards and Technology (NIST) mass spectral database and retention time libraries. To improve the reliability of significance, it is of crucial importance to eliminate background noise which data from 3rd minutes to 17th minutes were selected for statistical analysis. Targeted metabolites, of which were annotated as known compounds with the peak area greater than 0.5 percent were highlighted and subsequently treated statistically. Volatiles produced contain hundreds to thousands of compounds; therefore, it will be optimized by chemometric analysis, such as principal component analysis (PCA) as a preliminary analysis before subjected to a pattern classifier for identification of VOC samples. The volatile organic compound profiling has shown to be significantly distinguished among cancerous and control group based on metabolite profiling.

Keywords: in vitro cancer cell line, metabolite profiling, pattern recognition, volatile organic compounds

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Authors: Manohar Salla, Mark S. Butler, Ruby Pelingon, Geraldine Kaeslin, Daniel E. Croker, Janet C. Reid, Jong Min Baek, Paul V. Bernhardt, Elizabeth M. J. Gillam, Matthew A. Cooper, Avril A. B. Robertson

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MCC950 is a potent and selective inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that shows early promise for treatment of inflammatory diseases. The identification of major metabolites of lead molecule is an important step during drug development process. It provides an information about the metabolically labile sites in the molecule and thereby helping medicinal chemists to design metabolically stable molecules. To identify major metabolites of MCC950, the compound was incubated with human liver microsomes and subsequent analysis by (+)- and (−)-QTOF-ESI-MS/MS revealed a major metabolite formed due to hydroxylation on 1,2,3,5,6,7-hexahydro-s-indacene moiety of MCC950. This major metabolite can lose two water molecules and three possible regioisomers were synthesized. Co-elution of major metabolite with each of the synthesized compounds using HPLC-ESI-SRM-MS/MS revealed the structure of the metabolite (±) N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. Subsequent synthesis of individual enantiomers and coelution in HPLC-ESI-SRM-MS/MS using a chiral column revealed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. To study the possible cytochrome P450 enzyme(s) responsible for the formation of major metabolite, MCC950 was incubated with a panel of cytochrome P450 enzymes. The result indicated that CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C18, CYP2C19, CYP2J2 and CYP3A4 are most likely responsible for the formation of the major metabolite. The biological activity of the major metabolite and the other synthesized regioisomers was also investigated by screening for for NLRP3 inflammasome inhibitory activity and cytotoxicity. The major metabolite had 170-fold less inhibitory activity (IC50-1238 nM) than MCC950 (IC50-7.5 nM). Interestingly, one regioisomer had shown nanomolar inhibitory activity (IC50-232 nM). However, no evidence of cytotoxicity was observed with any of these synthesized compounds when tested in human embryonic kidney 293 cells (HEK293) and human liver hepatocellular carcinoma G2 cells (HepG2). These key findings give an insight into the SAR of the hexahydroindacene moiety of MCC950 and reveal a metabolic soft spot which could be blocked by chemical modification.

Keywords: Cytochrome P450, inflammasome, MCC950, metabolite, microsome, NLRP3

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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: Khaled S. Al Salhen

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

Keywords: aldehyde oxidase, fish, phenanthridine, specificity

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Authors: Madhusmita Dehingia, Supriyo Sen, Bhuwan Bhaskar, Tulsi Joishy, Mojibur R. Khan

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Human gut microbes and their metabolites are important for maintaining homeostasis in the gut and are responsible for many metabolic and immune mediated diseases. In the present study, we determined the profiles of the gut metabolites of five different ethnic groups (Bodo, Tai-Phake, Karbi, Tea tribe and Tai-Aiton) of Assam. Fecal metabolite profiling of the 39 individuals belonging to the ethnic groups was carried out using Gas chromatography – Mass spectrometry (GC-MS), and comparison was performed among the tribes for common and unique metabolites produced within their gut. Partial Least Squares Discriminant Analysis (PLS-DA) of the metabolites suggested that the individuals grouped according to their ethnicity. Among the 66 abundant metabolites, 12 metabolites were found to be common among the five ethnic groups. Additionally, ethnicity wise some unique metabolites were also detected. For example, the tea tribe of Assam contained the tea components, Aniline and Benzoate more in their gut in comparison to others. Metabolites of microbial origin were also correlated with the already published metagenomic data of the same ethnic group and functional analysis were carried out based on human metabolome database.

Keywords: ethnicity, gut microbiota, GC-MS, metabolites

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Authors: Zafar Iqbal, Sana Irshad Khan

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Organic extract from newly isolated plant growth promoting fungus (PGPF) Penicillium sp EU0013 was subjected to bioassays including anti fungal (disc diffusion) cytotoxicity (brine shrimp lethality), herbicidal (Lemna minor) and nematicidal activities. Metabolite profile of the extract was also assessed using HPLC analysis with the aim to identify bioactive natural products in the extract as new drug candidate(s). The extract showed anti fungal potential against tested fungal pathogens. Growth of the Wilt pathogen Fusarium oxyosproum was inhibited up to 63% when compared to negative reference. Activity against brine shrimps was weak and mortality up to 10% was observed at concentration of 200 µg. mL-1. The extract exhibited no toxicity against Lemna minor frond at 200 µg. mL-1. Nematicidal activity was observed very potent against root knot nematode and LC50 value was calculated as 52.5 ug. mL-1 using probit analysis. Methodically assessment of metabolites profile by HPLC showed the presence of kojic acid (Rt 1.4 min) and aflatoxin B1 (Rt 5.9 min) in the mycellial extract as compared with standards. The major unidentified metabolite was eluted at Rt 8.6 along with other minor peaks. The observed high toxicity against root knot nematode was attributed to the unidentified compounds that make fungal extract worthy of further exploration for isolation and structural characterization studies for development of future commercial nematicidal compound(s).

Keywords: penicillium, nematicidal activity, metabolites, HPLC

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Authors: Rani Abro, Ali Ata Moazzami, Jan Erik Lindberg, Torbjörn Lundh

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The effect of dietary starch level on liver metabolism in Arctic charr (Salvelinus alpinus) and tilapia (Oreochromis mossambicus) was studied using 1H-NMR based metabolomics. Fingerlings were fed iso-nitrogenous diets containing 0, 10 and 20 % starch for two months before liver samples were collected for metabolite analysis. Metabolite profiling was performed using 600 MHz NMR Chenomx software. In total, 48 metabolites were profiled in liver extracts from both fish species. Following the profiling, principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLC-DA) were performed. These revealed that differences in the concentration of significant metabolites were correlated to the dietary starch level in both species. The most prominent difference in metabolic response to starch feeding between the omnivorous tilapia and the carnivorous Arctic charr was an indication of higher anaerobic metabolism in Arctic charr. The data also indicated that amino acid and pyrimidine metabolism was higher in Artic charr than in tilapia.

Keywords: arctic charr, metabolomics, starch, tilapia

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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: Fereshteh Falah, Alireza Vasiee, Farideh Tabatabaei-Yazdi

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In this research, we investigated the usage of dairy sludge in the fermentation process and cyclosporin production. This bioactive compound is a metabolite produced by Tolypocladium inflatum. Results showed that about 200 ppm of cyclosporin can be produced in this fermentation. In order to have a proper and specific function, CyA must be free of any impurities, so we need purification. In this downstream processing, we used chromatographic extraction and evaluation of pharmacological activities of cyA. Results showed that the obtained metabolite has very high activity against Aspergilus niger (25mm clear zone). This cyclosporin was isolated for use as an antibiotic. The current research shows that this drug is very vital and commercially very important.

Keywords: fermentation, cyclosporin A, Tolypocladium inflatum, TLC

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Authors: Hossain A, Hossain S.

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Introduction: In a small cohort, we sought to assess the magnetic resonance spectroscopy's (MRS) ability to predict the presence of metastatic lesions. Method: A Popular Diagnostic Centre Limited enrolled patients with neuroepithelial tumors. The 1H CSI MRS of the brain allows us to detect changes in the concentration of specific metabolites caused by metastatic lesions. Among these metabolites are N-acetyl-aspartate (NNA), creatine (Cr), and choline (Cho). For Cho, NAA, Cr, and Cr₂, the metabolic ratio was calculated using the division method. Results: The NAA values were 0.63 and 5.65 for tumor cells, 1.86 and 5.66 for normal cells, and 1.86 and 5.66 for normal cells 2. NAA values for normal cells 1 were 1.84, 10.6, and 1.86 for normal cells 2, respectively. Cho levels were as low as 0.8 and 10.53 in the tumor cell, compared to 1.12 and 2.7 in the normal cell 1 and 1.24 and 6.36 in the normal cell 2. Cho/Cr₂ barely distinguished itself from the other ratios in terms of significance. For tumor cells, the ratios of Cho/NAA, Cho/Cr₂, NAA/Cho, and NAA/Cr₂ were significant. Normal cell 1 had significant Cho/NAA, Cho/Cr, NAA/Cho, and NAA/Cr ratios. Conclusion: The clinical result can be improved by using 1H-MRSI to guide the size of resection for metastatic lesions. Even though it is non-invasive and doesn't present any difficulties during the procedure, MRS has been shown to predict the detection of metastatic lesions.

Keywords: metabolite ratio, MRI images, metastatic lesion, MR spectroscopy, N-acetyl-aspartate

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Authors: Ankita Pathak, Ashok Munjal, Ravi Parkash

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Plastic responses to multiple environmental stressors in wet or dry seasonal populations of tropical Drosophila species have received less attention. We tested plastic effects of heat hardening, acclimation to drought or starvation; and changes in trehalose, proline and body lipids in D. ananassae flies reared under wet or dry season specific conditions. Wet season flies revealed significant increase in heat knockdown, starvation resistance and body lipids after heat hardening. However, accumulation of proline was observed only after desiccation acclimation of dry season flies while wet season flies elicited no proline but trehalose only. Therefore, drought-induced proline can be a marker metabolite for dry season flies. Further, partial utilization of proline and trehalose under heat hardening reflects their possible thermoprotective effects. Heat hardening elicited cross-protection to starvation stress. Stressor-specific accumulation or utilization, as well as rates of metabolic change for each energy metabolite, were significantly higher in wet season flies than dry season flies. Energy metabolite changes due to inter-related stressors (heat vs. desiccation or starvation) resulted in possible maintenance of energetic homeostasis in wet or dry season flies. Thus, low or high humidity induced plastic changes in energy metabolites can provide cross-protection to seasonally varying climatic stressors.

Keywords: wet-dry seasons, plastic changes, stress related traits, energy metabolites, cross protection

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Authors: Mohammad Nazri Abdul Bahari, Nurshafika Mohd Sakeh, Siti Nor Akmar Abdullah

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Basal stem rot (BSR) is the most devastating disease in oil palm. Among the oil palm pathogenic fungi, the most prevalent and virulent species associated with BSR is Ganoderma boninense. Early detection of G. boninense attack in oil palm wherein physical symptoms has not yet appeared can offer opportunities to prevent the spread of the necrotrophic fungus. However, poor understanding of molecular defense responses and roles of antifungal metabolites in oil palm against G. boninense has complicated the resolving measures. Hence, characterization of defense-related molecular responses and production of antifungal compounds during early interaction with G. boninense is of utmost important. Four month-old oil palm (Elaeis guineensis) seedlings were artificially infected with G. boninense-inoculated rubber wood block via sitting technique. RNA of samples were extracted from roots and leaves tissues at 0, 3, 7 and 11 days post inoculation (d.p.i) followed with sequencing using RNA-Seq method. Differentially-expressed genes (DEGs) of oil palm-G. boninense interaction were identified, while changes in metabolite profile will be scrutinized related to the DEGs. The RNA-Seq data generated a total of 113,829,376 and 313,293,229 paired-end clean reads from untreated (0 d.p.i) and treated (3, 7, 11 d.p.i) samples respectively, each with two biological replicates. The paired-end reads were mapped to Elaeis guineensis reference genome to screen out non-oil palm genes and subsequently generated 74,794 coding sequences. DEG analysis of phytohormone biosynthetic genes in oil palm roots revealed that at p-value ≤ 0.01, ethylene and jasmonic acid may act in antagonistic manner with salicylic acid to coordinate defense response at early interaction with G. boninense. Findings on metabolite profiling of G. boninense-infected oil palm roots and leaves are hoped to explain the defense-related compounds elicited by Elaeis guineensis in response to G. boninense colonization. The study aims to shed light on molecular defense response of oil palm at early interaction with G. boninense and promote prevention measures against Ganoderma infection.

Keywords: Ganoderma boninense, metabolites, phytohormones, RNA-Seq

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Authors: Jin Young Kim, Kwon Kyoo Kang

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The SGR1 (STAYGREEN1) protein is a critical regulator of plant leaves in chlorophyll degradation and senescence. The functions and mechanisms of tomato SGR1 action are poorly understood and worthy of further investigation. To investigate the function of the SGR1 gene, we generated a SGR1-knockout (KO) null line via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing and conducted RNA sequencing and gas chromatography tandem mass spectrometry (GC-MS/MS) analysis to identify the differentially expressed genes. The SlSGR1 (Solanum lycopersicum SGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid content compared to wild-type (WT) fruit. Differential gene expression analysis revealed 728 differentially expressed genes (DEGs) between WT and sgr1 #1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, for which fold change was >2, and the adjusted p-value was <0.05. Most of the DEGs were related to photosynthesis and chloroplast function. In addition, the pigment, carotenoid changes in sgr1 #1-6 line was accumulated of key primary metabolites such as sucrose and its derivatives (fructose, galactinol, raffinose), glycolytic intermediates (glucose, G6P, Fru6P) and tricarboxylic acid cycle (TCA) intermediates (malate and fumarate). Taken together, the transcriptome and metabolite profiles of SGR1-KO lines presented here provide evidence for the mechanisms underlying the effects of SGR1 and molecular pathways involved in chlorophyll degradation and carotenoid biosynthesis.

Keywords: tomato, CRISPR/Cas9, null line, RNA-sequencing, metabolite profiling

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Authors: Paiwan Buachan, Maneekarn Namsa-Aid, Suchada Jongrungruangchok, Foengchat Jarintanan, Wanlaya Uthaisang-Tanechpongtamb

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Lung cancer or pulmonary carcinoma is the uncontrolled growth of abnormal cells in one or both of the lungs. These abnormal cells can spread to other organs of the body through lymphatic system or bloodstream which is called metastatic stage that leading cause of cancer death. Terrein (C₈H₁₀O₃; MW= 154.06 kDa) is a secondary bioactive fungal metabolite, which was isolated from the Aspergillus terreus. In this study, we investigated the effects of terrein on the inhibition of human lung cancer cell proliferation and metastasis. The A549 human non-small cell lung cancer cell line was used as a model. Terrein significantly inhibited lung cancer cell proliferation measuring by a colorimetric MTT assay (IC₅₀ 0.32 mM) and significantly inhibited metastatic processes including migration, invasion, and adhesion that determined by wound healing assay, transwell assay, and adhesion assay, respectively. These findings indicate that terrein could be a potential therapeutic agent for lung cancer.

Keywords: terrein, lung cancer, anticancer, antimetastatic

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Authors: Nacéra Benoussaid, Lehalali Meriem, Benchabane Messaoud

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Our work focuses on the production of compound antibiotic effect of volatile nature namely hydrogen cyanide and the production and identification of molecules phénazinique by some strains of fluorescent Pseudomonas spp isolated from the rhizosphere of some trees for a possible use as bio pesticides antifungal effect and/or antibiotic. We tested the production of hydrogen cyanide of 21 strains of Pseudomonas spp. fluorescent among them 19 strains (90, 47%) showed a positive cyanogenesis.The antagonism test executed in vitro showed that Pseudomonas strains have a higher anti fungal effect relative to their antibacterial effect with diameters of inhibition zones up to 3, 9 cm recorded by the strain F48 against Coleosporiumsp compared with recorded results against bacteria with a maximum inhibition of 1, 26 cm among this antagonistic strain.Three strains were selected by testing for producing phénazines namely PI9, BB9 and F20. The effect of the antimicrobial activity was performed on different culture media (GN, King B, ISP2 and PDA). The results of our study allowed us to retain the King B medium as ideal medium for the production of secondary metabolite. The produced phenazinique compounds was extracted from various organic solvents, and after the results of antibiographie against germs - targets, the extracts of ethyl acetate gave the best results compared to dichloromethane and hexane.The Analysis of these compounds of antibiotic phenazinique effect within layer chromatography (CCM) and high performance liquid chromatography( HPLC) indicate that both strains PI9 and F20 are productive of phenazine-1-carboxylic acid (PCA). The BB9 strain is suspected to be productive of another phenazinique compound.

Keywords: Pseudomonas ssp. fluorescents, antagonism in vitro, secondary metabolite, phenazines, biopesticide.

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Authors: Nasiruddin Khan

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The action of (-)-epicatechin, a cocoa (Theobroma cacao) flavanol that modulates redox/oxidative stress are contributed mainly to their antioxidant properties. The present study investigates the concentration and time dependent effect of (-)-epicatechin metabolites 3MeEc, 4MeEc, and 4SulEc on the production of ROS on BAEC using L-012, Lucigenin as chemiluminescence dye and XO/HX system. Our result demonstrates that 3MeEc shows significant (P <0.05) lowering effect of ROS production in BAEC with increasing concentration of metabolite while L-012 was used as chemiluminescence dye but not in the case of Lucigenin. In XO/HX system, using L-012 as chemiluminescence dye, 3MeEc and 4MeEc showed significant lowering effect on ROS production with increasing concentration from 100-500nM as compared to the positive control (SOD). When Lucigenin was used as chemiluminescence dye, 3MeEc exerted significant lowering effect with increasing concentration when compared to the positive control (SOD) whereas 4MeEc showed significant lowering effect in ROS production from 250 nM on as compared to positive control. For 4SulEc, a significant lowering effect of ROS production was only observed at 100 and 250 nM. Overall, although each metabolite shows considerable effect, 3MeEc exhibited more pronounced effect on decreasing the production of ROS as compared to other two metabolites.

Keywords: epicatechin metabolites, HO-1, Nrf2, ROS

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Authors: Peter C. St. John, Michael F. Crowley, Yannick J. Bomble

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Production of chemicals from engineered organisms in a batch culture typically involves a trade-off between productivity, yield, and titer. However, strategies for strain design typically involve designing mutations to achieve the highest yield possible while maintaining growth viability. Such approaches tend to follow the principle of designing static networks with minimum metabolic functionality to achieve desired yields. While these methods are computationally tractable, optimum productivity is likely achieved by a dynamic strategy, in which intracellular fluxes change their distribution over time. One can use multi-stage fermentations to increase either productivity or yield. Such strategies would range from simple manipulations (aerobic growth phase, anaerobic production phase), to more complex genetic toggle switches. Additionally, some computational methods can also be developed to aid in optimizing two-stage fermentation systems. One can assume an initial control strategy (i.e., a single reaction target) in maximizing productivity - but it is unclear how close this productivity would come to a global optimum. The calculation of maximum theoretical yield in metabolic engineering can help guide strain and pathway selection for static strain design efforts. Here, we present a method for the calculation of a maximum theoretical productivity of a batch culture system. This method follows the traditional assumptions of dynamic flux balance analysis: that internal metabolite fluxes are governed by a pseudo-steady state and external metabolite fluxes are represented by dynamic system including Michealis-Menten or hill-type regulation. The productivity optimization is achieved via dynamic programming, and accounts explicitly for an arbitrary number of fermentation stages and flux variable changes. We have applied our method to succinate production in two common microbial hosts: E. coli and A. succinogenes. The method can be further extended to calculate the complete productivity versus yield Pareto surface. Our results demonstrate that nearly optimal yields and productivities can indeed be achieved with only two discrete flux stages.

Keywords: A. succinogenes, E. coli, metabolic engineering, metabolite fluxes, multi-stage fermentations, succinate

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Authors: Zakia Fatima, Liu Lu, Donghao Li

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The analysis of biological fluid metabolites holds significant importance in various areas, such as medical research, food science, and public health. Investigating the levels and distribution of nutrients and their metabolites in biological samples allows researchers and healthcare professionals to determine nutritional status, find hypovitaminosis or hypervitaminosis, and monitor the effectiveness of interventions such as dietary supplementation. Moreover, analysis of nutrient metabolites provides insight into their metabolism, bioavailability, and physiological processes, aiding in the clarification of their health roles. Hence, the exploration of a distinct, efficient, eco-friendly, and simpler methodology is of great importance to evaluate the metabolic content of complex biological samples. In this work, a green and rapid analytical method based on an automated online two-dimensional microscale carbon fiber/activated carbon fiber fractionation system and time-of-flight mass spectrometry (2DμCFs-TOF-MS) was used to evaluate metabolites of urine samples after oral administration of food supplements. The automated 2DμCFs instrument consisted of a microcolumn system with bare carbon fibers and modified carbon fiber coatings. Carbon fibers and modified carbon fibers exhibit different surface characteristics and retain different compounds accordingly. Three kinds of mobile-phase solvents were used to elute the compounds of varied chemical heterogeneities. The 2DμCFs separation system has the ability to effectively separate different compounds based on their polarity and solubility characteristics. No complicated sample preparation method was used prior to analysis, which makes the strategy more eco-friendly, practical, and faster than traditional analysis methods. For optimum analysis results, mobile phase composition, flow rate, and sample diluent were optimized. Water-soluble vitamins, fat-soluble vitamins, and amino acids, as well as 22 vitamin metabolites and 11 vitamin metabolic pathway-related metabolites, were found in urine samples. All water-soluble vitamins except vitamin B12 and vitamin B9 were detected in urine samples. However, no fat-soluble vitamin was detected, and only one metabolite of Vitamin A was found. The comparison with a blank urine sample showed a considerable difference in metabolite content. For example, vitamin metabolites and three related metabolites were not detected in blank urine. The complete single-run screening was carried out in 5.5 minutes with the minimum consumption of toxic organic solvent (0.5 ml). The analytical method was evaluated in terms of greenness, with an analytical greenness (AGREE) score of 0.72. The method’s practicality has been investigated using the Blue Applicability Grade Index (BAGI) tool, obtaining a score of 77. The findings in this work illustrated that the 2DµCFs-TOF-MS approach could emerge as a fast, sustainable, practical, high-throughput, and promising analytical tool for screening and accurate detection of various metabolites, pharmaceuticals, and ingredients in dietary supplements as well as biological fluids.

Keywords: metabolite analysis, sustainability, carbon fibers, urine.

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Authors: Giancarlo La Marca, Engy Shokry, Fabio Villanelli

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Epilepsy is one of the most common neurological disorders, with an estimated prevalence of 50 million people worldwide. Twenty five percent of the epilepsy population is represented in children under the age of 15 years. For antiepileptic drugs (AED), there is a poor correlation between plasma concentration and dose especially in children. This was attributed to greater pharmacokinetic variability than adults. Hence, therapeutic drug monitoring (TDM) is recommended in controlling toxicity while drug exposure is maintained. Carbamazepine (CBZ) is a first-line AED and the drug of first choice in trigeminal neuralgia. CBZ is metabolised in the liver into carbamazepine-10,11-epoxide (CBZE), its major metabolite which is equipotent. This develops the need for an assay able to monitor the levels of both CBZ and CBZE. The aim of the present study was to develop and validate a LC-MS/MS method for simultaneous quantification of CBZ and CBZE in dried blood spots (DBS). DBS technique overcomes many logistical problems, ethical issues and technical challenges faced by classical plasma sampling. LC-MS/MS has been regarded as superior technique over immunoassays and HPLC/UV methods owing to its better specificity and sensitivity, lack of interference or matrix effects. Our method combines advantages of DBS technique and LC-MS/MS in clinical practice. The extraction process was done using methanol-water-formic acid (80:20:0.1, v/v/v). The chromatographic elution was achieved by using a linear gradient with a mobile phase consisting of acetonitrile-water-0.1% formic acid at a flow rate of 0.50 mL/min. The method was linear over the range 1-40 mg/L and 0.25-20 mg/L for CBZ and CBZE respectively. The limit of quantification was 1.00 mg/L and 0.25 mg/L for CBZ and CBZE, respectively. Intra-day and inter-day assay precisions were found to be less than 6.5% and 11.8%. An evaluation of DBS technique was performed, including effect of extraction solvent, spot homogeneity and stability in DBS. Results from a comparison with the plasma assay are also presented. The novelty of the present work lies in being the first to quantify CBZ and its metabolite from only one 3.2 mm DBS disc finger-prick sample (3.3-3.4 µl blood) by LC-MS/MS in a 10 min. chromatographic run.

Keywords: carbamazepine, carbamazepine-10, 11-epoxide, dried blood spots, LC-MS/MS, therapeutic drug monitoring

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Authors: Patricia M. B. Saint-Vincent, Anna Furches, Stephanie Galanie, Erica Teixeira Prates, Piet Jones, Nancy Engle, David Kainer, Wellington Muchero, Daniel Jacobson, Timothy J. Tschaplinski

Abstract:

Uridine diphosphate-glycosyltransferases (UGTs) are enzymes that catalyze sugar transfer to a variety of plant metabolites. UGT substrates, which include plant secondary metabolites involved in lignification, demonstrate new activities and incorporation when glycosylated. Knowledge of UGT function, substrate specificity, and enzyme products is important for plant engineering efforts, especially related to increasing plant biomass through lignification. UGTs in Populus trichocarpa, a biofuel feedstock, and model woody plant, were selected from a pool of gene candidates using rapid prioritization strategies. A functional genomics workflow, consisting of a metabolite genome-wide association study (mGWAS), expression of synthetic codon-optimized genes, and high-throughput biochemical assays with mass spectrometry-based analysis, was developed for determining the substrates and products of previously-uncharacterized enzymes. A total of 40 UGTs from P. trichocarpa were profiled, and the biochemical assay results were compared to predicted mGWAS connections. Assay results confirmed seven of 11 leaf mGWAS associations and demonstrated varying levels of substrate specificity among candidate UGTs. P. trichocarpa UGT substrate processing confirms the role of these newly-characterized enzymes in lignan, flavonoid, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress responses.

Keywords: Populus, metabolite-gene associations, GWAS, bio feedstocks, glycosyltransferase

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Authors: Selisha A. Sooklal, Phelelani Mpangase, Shaun Aron, Karl Rumbold

Abstract:

Organically bound fluorine (C-F bond) is extremely rare in nature. Despite this, the first fluorinated secondary metabolite, fluoroacetate, was isolated from the plant Dichapetalum cymosum (commonly known as Gifblaar). However, the enzyme responsible for fluorination (fluorinase) in Gifblaar was never isolated and very little progress has been achieved in understanding this process in higher plants. Fluorinated compounds have vast applications in the pharmaceutical, agrochemical and fine chemicals industries. Consequently, an enzyme capable of catalysing a C-F bond has great potential as a biocatalyst in the industry considering that the field of fluorination is virtually synthetic. As with any biocatalyst, a range of these enzymes are required. Therefore, it is imperative to expand the exploration for novel fluorinases. This study aimed to gain molecular insights into secondary metabolite biosynthesis in Gifblaar using a high-throughput sequencing-based approach. Mechanical wounding studies were performed using Gifblaar leaf tissue in order to induce expression of the fluorinase. The transcriptome of the wounded and unwounded plant was then sequenced on the Illumina HiSeq platform. A total of 26.4 million short sequence reads were assembled into 77 845 transcripts using Trinity. Overall, 68.6 % of transcripts were annotated with gene identities using public databases (SwissProt, TrEMBL, GO, COG, Pfam, EC) with an E-value threshold of 1E-05. Sequences exhibited the greatest homology to the model plant, Arabidopsis thaliana (27 %). A total of 244 annotated transcripts were found to be differentially expressed between the wounded and unwounded plant. In addition, secondary metabolic pathways present in Gifblaar were successfully reconstructed using Pathway tools. Due to lack of genetic information for plant fluorinases, a transcript failed to be annotated as a fluorinating enzyme. Thus, a local database containing the 5 existing bacterial fluorinases was created. Fifteen transcripts having homology to partial regions of existing fluorinases were found. In efforts to obtain the full coding sequence of the Gifblaar fluorinase, primers were designed targeting the regions of homology and genome walking will be performed to amplify the unknown regions. This is the first genetic data available for Gifblaar. It has provided novel insights into the mechanisms of metabolite biosynthesis and will allow for the discovery of the first eukaryotic fluorinase.

Keywords: biocatalyst, fluorinase, gifblaar, transcriptome

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Authors: Changde Zhang, Qiang Zhang, Shilong Zheng, Jiawang Liu, Shanchun Guo, Qiu Zhong, Guangdi Wang

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Fulvestrant was approved by FDA to treat breast cancer as a selective estrogen receptor downregulator (SERD) with intramuscular injection administration. ZB716, a fulvestarnt-3 boronic acid, is an SERD with comparable anticancer effect to fulvestrant, but could produce good pharmacokinetic properties under oral administration with mice or rat models. To understand why ZB716 produced much better oral bioavailability, it was proposed that the boronic acid blocked the phase II direct biotransformation with the hydroxyl group on the 3 position of the aromatic ring on fulvestrant. In this study, ZB716 or fulvestrant was incubated with human liver microsome and oxidation cofactor NADPH in vitro. Their metabolites after oxidation were profiled with the Q-Exactive, a high-resolution mass spectrometer. The result showed that ZB716 blocked the forming of hydroxyl groups on its benzene ring except for the oxidation of C-B bond forming fulvestrant in its metabolites, and the concentration of fulvestrant with one more hydroxyl group found in the metabolites from incubation with fulvestrant was about 34 fold high as that formed from incubation with ZB716. Compared to fulvestrant, ZB716 is expected to be much difficult to be further bio-transformed into more hydrophilic compounds, to be difficult excreted out of blood system, and to have longer residence time in blood, which can lead to higher oral bioavailability. This study provided evidence to explain the high bioavailability of ZB716 after oral administration from the perspective of its difficulty of oxidation, a phase I biotransformation, on positions on its aromatic ring.

Keywords: biotransformation, fulvestrant, metabolite profiling, ZB716

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Authors: Nahid H. Hajrah, Jamal S. M. Sabir, Neil Hall

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The jasmonic pathway is ubiquitous in plants and is crucial to plant development. It Is involved in fertility, ripening, and sex determination as well as in response to environmental stresses such as herbivory, pathogen drought or temperature shock. Essentially the jasmonic pathway acts to shut down growth in order to induce defence pathways. These pathways include the production of secondary metabolites which have evolved to defend against herbivores and pathogens but are of increasing interest due to their roll in medicine and biotechnology. Here we describe the transcriptional response of Rhazia stricta (a poisonous shrub widely used in traditional medicine) to jasmonic acid, in order to better characterize the genes involved in secondary metabolite production and its response to stress. We observe coordinated upregulation of flavonoid biosynthesis pathway leading to flavonols, flavones and anthocyanins but no similar coordination of the monoterpene indole alkaloid pathway.

Keywords: medicinal plants, Rhazia stricta, jasmonic acid, transcriptional analysis

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Authors: Sultan Z. Mahmud, Emily C. Graff, Adil Bashir

Abstract:

Phosphoprotein enriched in astrocytes 15 kDa (PEA-15) is a multifunctional adapter protein which is associated with the regulation of apoptotic cell death. Recently it has been discovered that PEA-15 is crucial in normal neurodevelopment of domestic cats, a gyrencephalic animal model, although the exact function of PEA-15 in neurodevelopment is unknown. This study investigates how PEA-15 affects the blood-brain barrier (BBB) permeability in cat brain, which can cause abnormalities in tissue metabolite and energy supplies. Severe polymicrogyria and microcephaly have been observed in cats with a loss of function PEA-15 mutation, affecting the normal neurodevelopment of the cat. This suggests that the vital role of PEA-15 in neurodevelopment is associated with gyrification. Neurodevelopment is a highly energy demanding process. The mammalian brain depends on glucose as its main energy source. PEA-15 plays a very important role in glucose uptake and utilization by interacting with phospholipase D1 (PLD1). Mitochondria also plays a critical role in bioenergetics and essential to supply adequate energy needed for neurodevelopment. Cerebral blood flow regulates adequate metabolite supply and recent findings also showed that blood plasma contains mitochondria as well. So the BBB can play a very important role in regulating metabolite and energy supply in the brain. In this study the blood-brain permeability in cat brain was measured using MRI magnetization transfer (MT) effect on the perfusion signal. Perfusion is the tissue mass normalized supply of blood to the capillary bed. Perfusion also accommodates the supply of oxygen and other metabolites to the tissue. A fraction of the arterial blood can diffuse to the tissue, which depends on the BBB permeability. This fraction is known as water extraction fraction (EF). MT is a process of saturating the macromolecules, which has an effect on the blood that has been diffused into the tissue while having minimal effect on intravascular blood water that has not been exchanged with the tissue. Measurement of perfusion signal with and without MT enables to estimate the microvascular blood flow, EF and permeability surface area product (PS) in the brain. All the experiments were performed with Siemens 7T Magnetom with 32 channel head coil. Three control cats and three PEA-15 mutant cats were used for the study. Average EF in white and gray matter was 0.9±0.1 and 0.86±0.15 respectively, perfusion in white and gray matter was 85±15 mL/100g/min and 97±20 mL/100g/min respectively, PS in white and gray matter was 201±25 mL/100g/min and 225±35 mL/100g/min respectively for control cats. For PEA-15 mutant cats, average EF in white and gray matter was 0.81±0.15 and 0.77±0.2 respectively, perfusion in white and gray matter was 140±25 mL/100g/min and 165±18 mL/100g/min respectively, PS in white and gray matter was 240±30 mL/100g/min and 259±21 mL/100g/min respectively. This results show that BBB is compromised in PEA-15 mutant cat brain, where EF is decreased and perfusion as well as PS are increased in the mutant cats compared to the control cats. This findings might further explain the function of PEA-15 in neurodevelopment.

Keywords: BBB, cat brain, magnetization transfer, PEA-15

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Authors: Elif Coskun Daggecen, Seyma Dokucu, Yekta Gezginc, Ismail Akyol

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Fermented dairy food quality is mainly determined by the sensory perception and influenced by many factors. Today, starter cultures for fermented foods are being developed to have a constant quality in these foods. Streptococcus thermophilus is one of the main species of most a starter cultures of yogurt fermentation. This species produces lactate by lactose fermentation from pyruvate. On the other hand, a small amount of pyruvate can alternatively be converted to various typical yoghurt flavor compounds such as diacetyl, acetoin, acetaldehyde, or acetic acid, for which the activity of three genes are shown to be especially important; ldh, nox and als. Up to date, commercially produced yoghurts have not yet met the desired aromatic properties that Turkish consumers find in traditional homemade yoghurts. Therefore, it is important to select starters carrying favorable metabolic characteristics from natural isolates. In this study, 30 strains of Str. Thermophilus were isolated from traditional Turkish yoghurts obtained from different regions of the country. In these strains, transcriptional levels of ldh, nox and als genes were determined via a newly developed qPCR protocol, which is a more reliable and precision method for analyzing the quantitative and qualitative expression of specific genes in different experimental conditions or in different organisms compared to conventional analytical methods. Additionally, the metabolite production potentials of the isolates were measured. Of all the strains examined, 60% were found to carry the metabolite production potential and the gene activity which appeared to be suitable to be used as a starter culture. Probable starter cultures were determined according to real-time PCR results.

Keywords: gene expression, RT-PCR, starter culture, Streptococcus thermophilus

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Authors: Marianna Ciaccia, Gennaro Agrimi, Isabella Pisano, Maurizio Bettiga, Silvia Rapacioli, Giulia Mensa, Monica Marzagalli

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Objective: Untargeted metabolomic analysis of extracellular metabolites is a powerful approach that focuses on comprehensively profiling in the extracellular space. In this study, we applied extracellular metabolomic analysis to investigate the metabolism of two probiotic microorganisms with health benefits that extend far beyond the digestive tract and the immune system. Methods: Analytical techniques employed in extracellular metabolomic analysis encompass various technologies, including mass spectrometry (MS), which enables the identification of metabolites present in the fermentation media, as well as the comparison of metabolic profiles under different experimental conditions. Multivariate statistical analysis techniques like principal component analysis (PCA) or partial least squares-discriminant analysis (PLS-DA) play a crucial role in uncovering metabolic signatures and understanding the dynamics of metabolic networks. Results: Different types of supernatants from fermentation processes, such as dairy-free, not dairy-free media and media with no cells or pasteurized, were subjected to metabolite profiling, which contained a complex mixture of metabolites, including substrates, intermediates, and end-products. This profiling provided insights into the metabolic activity of the microorganisms. The integration of advanced software tools has facilitated the identification and characterization of metabolites in different fermentation conditions and microorganism strains. Conclusions: In conclusion, untargeted extracellular metabolomic analysis, combined with software tools, allowed the study of the metabolites consumed and produced during the fermentation processes of probiotic microorganisms. Ongoing advancements in data analysis methods will further enhance the application of extracellular metabolomic analysis in fermentation research, leading to improved bioproduction and the advancement of sustainable manufacturing processes.

Keywords: biotechnology, metabolomics, lactic bacteria, probiotics, postbiotics

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Authors: Lalita Subedi, Jae Kyoung Chae, Yong Un Park, Cho Kyo Hee, Lee Jae Hyuk, Kang Min Cheol, Sun Yeou Kim

Abstract:

Neuroinflammation may mediate the relationship between low levels of estrogens and neurodegenerative disease. Estrogens are neuroprotective and anti-inflammatory in neurodegenerative disease models. Due to the long term side effects of estrogens, researches have been focused on finding an effective phytoestrogens for biological activities. Daidzein present in soybeans and its active metabolite equol (7-hydroxy-3-(4'-hydroxyphenyl)-chroman) bears strong antioxidant and anticancer showed more potent anti-inflammatory and neuroprotective role in neuroinflammatory model confirmed its in vitro activity with molecular mechanism through NF-κB pathway. Three major CNS cells Microglia (BV-2), Astrocyte (C6), Neuron (N2a) were used to find the effect of equol in inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), MAPKs signaling proteins, apoptosis related proteins by western blot analysis. Nitric oxide (NO) and prostaglandin E2 (PGE2) was measured by the Gries method and ELISA, respectively. Cytokines like tumor necrosis factor-α (TNF-α) and IL-6 were also measured in the conditioned medium of LPS activated cells with or without equol. Equol inhibited the NO production, PGE-2 production and expression of COX-2 and iNOS in LPS-stimulated microglial cells at a dose dependent without any cellular toxicity. At the same time Equol also showed promising effect in modulation of MAPK’s and nuclear factor kappa B (NF-κB) expression with significant inhibition of the production of proinflammatory cytokine like interleukin -6 (IL-6), and tumor necrosis factor -α (TNF-α). Additionally, it inhibited the LPS activated microglia-induced neuronal cell death by downregulating the apoptotic phenomenon in neuronal cells. Furthermore, equol increases the production of neurotrophins like NGF and increase the neurite outgrowth as well. In conclusion the natural daidzein metabolite equol are more active than daidzein, which showed a promising effectiveness as an anti-neuroinflammatory and neuroprotective agent via downregulating the LPS stimulated microglial activation and neuronal apoptosis. This work was supported by Brain Korea 21 Plus project and High Value-added Food Technology Development Program 114006-4, Ministry of Agriculture, Food and Rural Affairs.

Keywords: apoptosis, equol, neuroinflammation, phytoestrogen

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Authors: Miroslava Cuperlovic-Culf, Lipu Wang, Ketty Boyle, Nadine Makley, Ian Burton, Anissa Belkaid, Mohamed Touaibia, Marc E. Surrette

Abstract:

Metabolic changes are one of the major factors in the development of a variety of diseases in various species. Metabolism of agricultural plants is altered the following infection with pathogens sometimes contributing to resistance. At the same time, pathogens use metabolites for infection and progression. In humans, metabolism is a hallmark of cancer development for example. Quantified metabolomics data combined with other omics or clinical data and analyzed using various unsupervised and supervised methods can lead to better diagnosis and prognosis. It can also provide information about resistance as well as contribute knowledge of compounds significant for disease progression or prevention. In this work, different methods for metabolomics quantification and analysis from Nuclear Magnetic Resonance (NMR) measurements that are used for investigation of disease development in wheat and human cells will be presented. One-dimensional 1H NMR spectra are used extensively for metabolic profiling due to their high reliability, wide range of applicability, speed, trivial sample preparation and low cost. This presentation will describe a new method for metabolite quantification from NMR data that combines alignment of spectra of standards to sample spectra followed by multivariate linear regression optimization of spectra of assigned metabolites to samples’ spectra. Several different alignment methods were tested and multivariate linear regression result has been compared with other quantification methods. Quantified metabolomics data can be analyzed in the variety of ways and we will present different clustering methods used for phenotype determination, network analysis providing knowledge about the relationships between metabolites through metabolic network as well as biomarker selection providing novel markers. These analysis methods have been utilized for the investigation of fusarium head blight resistance in wheat cultivars as well as analysis of the effect of estrogen receptor and carbonic anhydrase activation and inhibition on breast cancer cell metabolism. Metabolic changes in spikelet’s of wheat cultivars FL62R1, Stettler, MuchMore and Sumai3 following fusarium graminearum infection were explored. Extensive 1D 1H and 2D NMR measurements provided information for detailed metabolite assignment and quantification leading to possible metabolic markers discriminating resistance level in wheat subtypes. Quantification data is compared to results obtained using other published methods. Fusarium infection induced metabolic changes in different wheat varieties are discussed in the context of metabolic network and resistance. Quantitative metabolomics has been used for the investigation of the effect of targeted enzyme inhibition in cancer. In this work, the effect of 17 β -estradiol and ferulic acid on metabolism of ER+ breast cancer cells has been compared to their effect on ER- control cells. The effect of the inhibitors of carbonic anhydrase on the observed metabolic changes resulting from ER activation has also been determined. Metabolic profiles were studied using 1D and 2D metabolomic NMR experiments, combined with the identification and quantification of metabolites, and the annotation of the results is provided in the context of biochemical pathways.

Keywords: metabolic biomarkers, metabolic network, metabolomics, multivariate linear regression, NMR quantification, quantified metabolomics, spectral alignment

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Authors: Jamilah Syafawati Yaacob, Muhammad Aiman Ramli

Abstract:

Cili padi or Capsicum frutescens is one of capsicum species from nightshade family, Solanaceae. It is famous in Malaysia and is widely used as a food ingredient. Capsicum frutescens also possess vast medicinal properties. The objectives of this study are to determine the most optimum 2,4-D hormone concentration for callus induction from stem explants C. frutescens and the effects of different 2,4-D concentrations on expression of compounds from C. frutescens. Seeds were cultured on MS media without hormones (MS basal media) to yield aseptic seedlings of this species, which were then used to supply explant source for subsequent tissue culture experiments. Stem explants were excised from aseptic seedlings and cultured on MS media supplemented with various concentrations (0.1, 0.3 and 0.5 mg/L) of 2,4-D to induce formation of callus. Fresh weight, dry weight and callus growth percentage in all samples were recorded. The highest mean of dry weight was observed in MS media supplemented with 0.5 mg/L 2,4-D, where 0.4499 ± 0.106 g of callus was produced. The highest percentage of callus growth (16.4%) was also observed in cultures supplemented with 0.5 mg/L 2,4-D. The callus samples were also subjected to HPLC-MS to evaluate the effect of hormone concentration on expression of bio active compounds in different samples. Results showed that caffeoylferuloylquinic acids were present in all samples, but was most abundant in callus cells supplemented with 0.3 & 0.5 mg/L 2,4-D. Interestingly, there was an unknown compound observed to be highly expressed in callus cells supplemented with 0.1 mg/L 2,4-D, but its presence was less significant in callus cells supplemented with 0.3 and 0.5 mg/L 2,4-D. Furthermore, there was also a compound identified as octadecadienoic acid, which was uniquely expressed in callus supplemented with 0.5 mg/L 2,4-D, but absent in callus cells supplemented with 0.1 and 0.3 mg/L 2,4-D. The results obtained in this study indicated that plant growth regulators played a role in expression of secondary metabolites in plants. The increase or decrease of these growth regulators may have triggered a change in the secondary metabolite biosynthesis pathways, thus causing differential expression of compounds in this plant.

Keywords: callus, in vitro, secondary metabolite, 2, 4-Dichlorophenoxyacetic acid

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