#METABOLOMICS WORKBENCH chandrajith_20210413_003516 DATATRACK_ID:2573 STUDY_ID:ST001839 ANALYSIS_ID:AN002981 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        	LC-MS
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity
CH:COLUMN_NAME                   	Agilent Zorbax Eclipse Plus C18 (100 x 2.1mm, 1.8 um)
CH:FLOW_RATE                     	0.5 mL per minute
CH:COLUMN_TEMPERATURE            	25 °C
CH:METHODS_FILENAME              	LCMS_protocol.txt
CH:SOLVENT_A                     	0.1% formic acid in Milli-Q water
CH:SOLVENT_B                     	0.1% formic acid in acetonitrile
CH:COLUMN_PRESSURE               	45 psi
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	LCMS Laboratory (Monash University Malaysia)
#MS
MS:INSTRUMENT_NAME               	Agilent 6520 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
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	m/z
MS_METABOLITE_DATA_START
Samples	GE-LC-EA(s)	GE-LC-AC(s)
Factors	Solvent:Ethyl acetate | Treatment:Sequential	Solvent:Acetone | Treatment:Sequential
(-)-Isoamijiol		305.247
(+)-3-hydroxy pelargonic acid	197.1143	
(22S)-1α,22,25-trihydroxy-23,24-tetradehydro-24a-homo-20-epivitamin D3 / (22S)-1α,22,25-trihydroxy-23,24-tetradehydro-24a-homo-20-epicholecalciferol		460.343
(25S)-5alpha-cholestan-3beta,6alpha,7beta,8beta,15alpha,16beta,26-heptol		502.374
10- [5]-ladderane-decanoic acid	331.2643	331.264
13-cis-retinal	285.2226	285.222
1-Hydroxyvitamin D3 3-D-glucopyranoside	599.3558	599.356
2E-Heptenyl acetate	157.1221	
2-Hydroxyhexadecanoic acid		290.269
3-Deoxy-D-arabinitol	137.0807	
3-Oxo-5β-chola-7,9(11)-dien-24-oic Acid	371.2583	371.258
3-tert-Butyl-5-methylcatechol	181.1222	181.122
3Z,6Z,9Z,12Z,15Z-octadecapentaenoic acid	275.2013	275.201
4-(2-hydroxypropoxy)-3,5-dimethyl-Phenol	197.1169	197.117
4-Vinylcyclohexene diepoxide	141.091	
5(S)-HETE lactone	303.2317	303.232
6,8-nonadienal	139.1113	
6-Paradol		296.222
7beta-Hydroxy-lathyrol	335.2218	
C16 Sphinganine	274.2736	274.274
Carindone		530.349
cis-1,2-dimethylcyclohexane	113.1324	113.133
Cyrneine A	317.2116	317.211
Diethyl Oxalpropionate		203.091
Discodermolide	594.3997	594.401
Emmotin A	279.1596	279.16
Eplerenone		415.212
Gingerol	295.1913	
Haplophytine		653.297
Harderoporphyrin	609.2709	609.271
Nonoxynol-9		634.452
N-propyl α, α-dimethylarachidonoyl amine		374.341
Onchidal		277.179
Pheophorbide a	593.2756	593.277
Pirimiphos-methyl		306.103
Pyropheophorbide a		535.271
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Pubchem ID
(-)-Isoamijiol	13969915
(+)-3-hydroxy pelargonic acid	36599
(22S)-1α,22,25-trihydroxy-23,24-tetradehydro-24a-homo-20-epivitamin D3 / (22S)-1α,22,25-trihydroxy-23,24-tetradehydro-24a-homo-20-epicholecalciferol	52931337
(25S)-5alpha-cholestan-3beta,6alpha,7beta,8beta,15alpha,16beta,26-heptol	52931337
10- [5]-ladderane-decanoic acid	42607294
13-cis-retinal	42607294
1-Hydroxyvitamin D3 3-D-glucopyranoside	6436079
2E-Heptenyl acetate	24779645
2-Hydroxyhexadecanoic acid	92836
3-Deoxy-D-arabinitol	270738
3-Oxo-5β-chola-7,9(11)-dien-24-oic Acid	5284079
3-tert-Butyl-5-methylcatechol	66095
3Z,6Z,9Z,12Z,15Z-octadecapentaenoic acid	5312511
4-(2-hydroxypropoxy)-3,5-dimethyl-Phenol	136125637
4-Vinylcyclohexene diepoxide	7833
5(S)-HETE lactone	6439592
6,8-nonadienal	60060292
6-Paradol	5283342
7beta-Hydroxy-lathyrol	94378
C16 Sphinganine	91486 
Carindone	101316738
cis-1,2-dimethylcyclohexane	16628
Cyrneine A	16628
Diethyl Oxalpropionate	97750
Discodermolide	643668
Emmotin A	42608142
Eplerenone	20779789
Gingerol	643668
Haplophytine	442793
Harderoporphyrin	442103
Nonoxynol-9	12401
N-propyl α, α-dimethylarachidonoyl amine	72385
Onchidal	5283417
Pheophorbide a	253193
Pirimiphos-methyl	34526
Pyropheophorbide a	34526
METABOLITES_END
#END