#METABOLOMICS WORKBENCH chandrajith_20210413_003516 DATATRACK_ID:2573 STUDY_ID:ST001839 ANALYSIS_ID:AN002982 PROJECT_ID:PR001161 VERSION 1 CREATED_ON June 14, 2021, 9:00 am #PROJECT PR:PROJECT_TYPE Mass spectrometric analyses of natural products PR:PROJECT_SUMMARY With a continuous threat of antimicrobial resistance on human health worldwide, PR:PROJECT_SUMMARY efforts for new alternatives are ongoing for the management of bacterial PR:PROJECT_SUMMARY infectious diseases. Natural products of land and sea, being conceived to be PR:PROJECT_SUMMARY having fewer side effects, pose themselves as a welcome relief. In this respect, PR:PROJECT_SUMMARY we have taken a scaffolded approach to unearthing the almost unexplored chemical PR:PROJECT_SUMMARY constituents of Malaysian red seaweed, Gracilaria edulis. Essentially, a PR:PROJECT_SUMMARY preliminary evaluation of the ethyl acetate and acetone solvent extracts, among PR:PROJECT_SUMMARY a series of six such, revealed potential antibacterial activity against six MDR PR:PROJECT_SUMMARY species namely, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella PR:PROJECT_SUMMARY enterica, methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus PR:PROJECT_SUMMARY pyogenes, and Bacillus subtilis. Detailed analyses of the inlying chemical PR:PROJECT_SUMMARY constituents, through LC-MS and GC-MS chromatographic separation, revealed a PR:PROJECT_SUMMARY library of metabolic compounds. These were led for further virtual screening PR:PROJECT_SUMMARY against selected key role playing proteins in the virulence of the aforesaid PR:PROJECT_SUMMARY bacteria. To this end, detailed predictive pharmacological analyses added up to PR:PROJECT_SUMMARY reinforce Eplerenone as a natural alternative from the plethora of plausible PR:PROJECT_SUMMARY bioactives. Our work adds to the ongoing effort to re-discover and repurpose PR:PROJECT_SUMMARY biochemical compounds to combat the antimicrobial resistance offered by the PR:PROJECT_SUMMARY Gram-positive and the -negative bacterial species. PR:INSTITUTE Sunway University PR:DEPARTMENT Biological Sciences, Sunway University, Selangor, Malaysia PR:LABORATORY Disease Complexity PR:FIRST_NAME Chandrajit PR:ADDRESS Sunway University, No. 5, Jalan Universiti, Bandar Sunway, Petaling Jaya 47500, PR:ADDRESS Selangor, Malaysia PR:EMAIL chandrajitl@sunway.edu.my PR:PHONE +60 3 7491 8622 PR:FUNDING_SOURCE None PR:PROJECT_COMMENTS Ongoing PR:PUBLICATIONS Metabolite profiling of Malaysian Gracilaria edulis reveals Eplerenone as novel PR:PUBLICATIONS antibacterial compound for drug repurposing against MDR Bacteria PR:PROJECT_TITLE Exploring the Antibacterial Potentials of South-East Asian Natural Products PR:PROJECT_TITLE Against Multidrug Resistant Bacteria PR:LAST_NAME Lahiri PR:CONTRIBUTORS Ali Asghar, Syafiq Asnawi #STUDY ST:STUDY_TITLE Metabolite profiling of Malaysian Gracilaria edulis reveals Eplerenone as novel ST:STUDY_TITLE antibacterial compound for drug repurposing against MDR Bacteria ST:STUDY_SUMMARY The current study re-defines a method to reveal bioactive compounds from the ST:STUDY_SUMMARY crude extracts of Malaysian red seaweed Gracilaria edulis, having promising ST:STUDY_SUMMARY antibacterial activities against selected bacterial species. Three species of ST:STUDY_SUMMARY Gram-positive and - negative characters were remarkably inhibited by the ST:STUDY_SUMMARY sequential and direct extracts of ethyl acetate and acetone. These were further ST:STUDY_SUMMARY separated through chromatographic methods to reveal a plethora of chemical ST:STUDY_SUMMARY constituents to be considered for a downstream virtual screening against ST:STUDY_SUMMARY selected crucial proteins of the six bacteria. ST:INSTITUTE Sunway University ST:DEPARTMENT Biological Sciences, Sunway University, Selangor, Malaysia ST:LABORATORY Disease Complexity ST:LAST_NAME Lahiri ST:FIRST_NAME Chandrajit ST:ADDRESS Sunway University, No. 5, Jalan Universiti, Bandar Sunway, Petaling Jaya 47500, ST:ADDRESS Selangor, Malaysia ST:EMAIL chandrajitl@sunway.edu.my ST:STUDY_TYPE In vitro antibacterial studies ST:PHONE +60 3 7491 8622 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Gracilaria edulis SU:TAXONOMY_ID 172966 SU:GENDER Not applicable SU:SPECIES_GROUP Rhodophyta #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - GE-LC-EA(s) Solvent:Ethyl acetate | Treatment:Sequential SUBJECT_SAMPLE_FACTORS - GE-LC-AC(s) Solvent:Acetone | Treatment:Sequential SUBJECT_SAMPLE_FACTORS - GE-GC-EA(s) Solvent:Ethyl acetate | Treatment:Sequential SUBJECT_SAMPLE_FACTORS - GE-GC-AC(s) Solvent:Acetone | Treatment:Sequential SUBJECT_SAMPLE_FACTORS - GE-GC-EA(d) Solvent:Ethyl acetate | Treatment:Direct SUBJECT_SAMPLE_FACTORS - GE-GC-AC(d) Solvent:Acetone | Treatment:Direct #COLLECTION CO:COLLECTION_SUMMARY Healthy specimens of Gracilaria edulis were collected from Pantai Morib, CO:COLLECTION_SUMMARY Selangor, Malaysia. Further, for the correct identification of G. edulis, CO:COLLECTION_SUMMARY observation of the characters was carried out according to Guiry & Guiry (2021). CO:SAMPLE_TYPE Seaweed #TREATMENT TR:TREATMENT_SUMMARY Extracts of Gracilaria edulis were prepared through two different approaches, TR:TREATMENT_SUMMARY namely, sequential and direct, following the procedure of Subermaniam et al. TR:TREATMENT_SUMMARY (2020). For the sequential process, the solvents were used in the order of TR:TREATMENT_SUMMARY increasing polarity viz. ethyl acetate < acetone. For the direct extracts, ethyl TR:TREATMENT_SUMMARY acetate and acetone were used separately. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Essentially, for both approaches, the seaweeds were rinsed sequentially with SP:SAMPLEPREP_SUMMARY seawater followed by normal tap and then double distilled water to eradicate SP:SAMPLEPREP_SUMMARY dirt and impurities. Clean samples were then dried using a freeze-dryer and SP:SAMPLEPREP_SUMMARY later crushed into fine granule powder using an electric grinder. Different SP:SAMPLEPREP_SUMMARY fractions of extracts were prepared using 10 grams of each powder to dissolve SP:SAMPLEPREP_SUMMARY them in 100 mL of the mentioned solvents. All the prepared mixtures were made SP:SAMPLEPREP_SUMMARY homogeneous using a rotating shaker (Yihder LM-530D, Shaker, Taiwan) for 24 SP:SAMPLEPREP_SUMMARY hours and finally centrifuged (Eppendorf 5810 R Centrifuge, Germany) at 4000 rpm SP:SAMPLEPREP_SUMMARY for 10 min at 4◦C to separate the supernatant. Each of the clear supernatants SP:SAMPLEPREP_SUMMARY of the extracts was concentrated via a Rotary evaporator (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific EYELA N-1200A Rotary Evaporator, Tokyo). A further concentration SP:SAMPLEPREP_SUMMARY using a vacuum concentrator (LaboGene, Brigachtal, Germany) was done to obtain a SP:SAMPLEPREP_SUMMARY viscous liquid for storage at 4◦C and future experiments. SP:PROCESSING_STORAGE_CONDITIONS Room temperature SP:EXTRACT_STORAGE 4℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY GC-MS CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent HP5-MS (30m x 0.25mm, 0.25 um) CH:FLOW_RATE 1.5 mL per minute CH:METHODS_FILENAME GCMS_protocol.txt CH:ANALYTICAL_TIME 64 minutes CH:OVEN_TEMPERATURE 80°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Data processing Comments: NIST Mass Spectral Search Program-2009 version 2. MS:MS_COMMENTS Software/procedures used for feature assignments: Agilent Mass-Hunter MS:MS_COMMENTS Qualitative Analysis B.05.00. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Area % MS_METABOLITE_DATA_START Samples GE-GC-EA(s) GE-GC-AC(s) GE-GC-EA(d) GE-GC-AC(d) Factors Solvent:Ethyl acetate | Treatment:Sequential Solvent:Acetone | Treatment:Sequential Solvent:Ethyl acetate | Treatment:Direct Solvent:Acetone | Treatment:Direct [2,2’-Bifuran]-3-carboxylic acid, 5’-methyl-, methyl ester 7.7157 1,2-Benzenediol bis(trimethylsilyl) ether 2.052 1-Octadecyne 2.2 0.63897 26-Nor-5-cholesten-3á-ol-25-one 1.725 2-Heptyl-1,3-dioxolane 2.882 2-Tridecanone 0.66248 3,8,13,18-Tetraethyl-2,7,12,17-tetramethyl-7, 8-(diacetyl) methylene-7,8-dihydro-21H,23H-porphine copper (II) 4.035 3-Methyl-1,2-diazirine 7.529 4-Penten-2-one, 4-methyl- 1.628 á Carotene 0.9908 Arachidonic acid 1.039 1.4028 Ar-tumerone 1.7063 Cholest-5-en-3-ol 3.4662 Cholesterol 32.831 Decanoic acid, ethyl ester 1.787 5.3174 2.2412 Diisooctyl phthalate 0.914 3.878 0.56258 0.85802 Hexadecanoic acid, ethyl ester 2.074 Molybdenum, bis[(1,2,3,4,5-ü)-1,3-bis(1,1-dimethylethyl)-2,4-cyclopentadien-1-yl] di-æ-carbonyldicarbonyldi-, (mo-mo) 0.646 n-Hexadecanoic acid 5.008 9.078 9.1086 1.2408 Nickel tetracarbonyl 1.797 1.329 Nonadecane 4.034 Oxalic acid, cyclobutyl octadecyl ester 0.15812 Phenol, 2,4-bis(1,1-dimethylethyl) 3.925 0.771 7.7157 4.6668 Phenol, 2-methoxy-3-(2-propenyl)- 6.3257 8.9936 Phytol 2.3 Ribo-ribo disaccharide 0.59025 Silane, tetramethyl 0.18473 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pubchem ID [2,2’-Bifuran]-3-carboxylic acid, 5’-methyl-, methyl ester 609852 1,2-Benzenediol bis(trimethylsilyl) ether 23688911 1-Octadecyne 69425 26-Nor-5-cholesten-3á-ol-25-one 165617 2-Heptyl-1,3-dioxolane 5363203 2-Tridecanone 78067 3,8,13,18-Tetraethyl-2,7,12,17-tetramethyl-7, 8-(diacetyl) methylene-7,8-dihydro-21H,23H-porphine copper (II) 11622 3-Methyl-1,2-diazirine 6420208 4-Penten-2-one, 4-methyl- 19543 á Carotene 42608244 Arachidonic acid 5280489 Ar-tumerone 558221 Cholest-5-en-3-ol 101316738 Cholesterol 5997 Decanoic acid, ethyl ester 12000424 Diisooctyl phthalate 97750 Hexadecanoic acid, ethyl ester 3081462 Molybdenum, bis[(1,2,3,4,5-ü)-1,3-bis(1,1-dimethylethyl)-2,4-cyclopentadien-1-yl] di-æ-carbonyldicarbonyldi-, (mo-mo) 11986227 n-Hexadecanoic acid 628589 Nickel tetracarbonyl 518772 Nonadecane 12401 Oxalic acid, cyclobutyl octadecyl ester 6441116 Phenol, 2,4-bis(1,1-dimethylethyl) 528937 Phenol, 2-methoxy-3-(2-propenyl)- 596373 Phytol 5280435 Ribo-ribo disaccharide 161456 Silane, tetramethyl 6396 METABOLITES_END #END