Indonesian Journal of Chemical Research

http://ojs3.unpatti.ac.id/index.php/ijcr Indo. J. Chem. Res., 10(3), 203-211, 2023

Molecular Networking to Screen Macroalgal Secondary Metabolites: Case for West Timor
Macroalgae

Welem Turupadang1*, Marskel Johanis2

1
Faculty of Maritime Animal Husbandry and Fisheries, Universitas Nusa Cendana, Jalan Adi Sucipto Penfui 85001
Kupang, Nusa Tenggara Timur
2
Office of Maritime Affairs and Fisheries of Kupang Regency, Nusa Tenggara Timur
*
Corresponding author: [email protected]

Received: August 2022 Abstract

Received in revised: October 2022
Accepted: January 2023 The research describes how molecular networking was used as a screening tool to
Available online: January 2023 prioritize the isolation workflow of 40 macroalgae sampled from West Timor waters,
Indonesia, in addition to a Nuclear Magnetic Resonance-based (NMR) spectroscopy
strategy. A mass spectrometry (MS) was employed to generate spectra that later be used
as data to produce the molecular network with the Global Natural Product Social
Molecular Networking (GNPS) website. The screening process used the molecular
network, which assisted in the selection of six samples of macroalgae out of the 40
samples for further examination. Lastly, an NMR-based protocol was employed to choose
the samples of interest to be investigated further. Six samples were shortlisted from the
GNPS molecular network based on specimens, which were then validated with 1H NMR
spectroscopy to finally prioritized three samples.
Keywords: GNPS, Macroalgae, 1H NMR, Molecular networking, Spectra

INTRODUCTION coined in the CRC Handbook of Antibiotic

After an organism has been chosen as a species of Compounds published in 1980 (Ito & Masubuchi,
interest, the next step is preliminary solvent 2014). Dereplication provides rapid identification of
extraction which results in crude extracts. Once the known compounds within crude extracts or semi-
crude extracts are obtained, various screening purified mixtures, facilitating prioritization for
methods are employed to highlight those worthy or further elucidation of only potentially new structures
more in-depth investigations. The traditional and halting an isolation process of known secondary
approach has been bioassay-guided screening, which metabolites (Ito & Masubuchi, 2014; Moser,
usually leads to isolating biologically active Wheeler, & Hayward). Hence, de-replication is a
compounds (Eloff, 1998; Hostettmann, Wolfender, & substantial and vital strategy in natural product
Terreaux, 2001; Koehn & Carter, 2005). Although screening.
the bioassay-guided screening eventually produces a Secondary metabolites are typically amphiphilic,
potential pharmaceutical compound, the frequent re- which allows the compounds to transverse both
isolation of previously known metabolites is a hydrophilic and hydrophobic environments. Their
significant challenge for this strategy (Koehn, 2008; vast polarity and solubility make it challenging to
Koehn & Carter, 2005). analyze and handle the compounds present in the
Alternative approaches have been introduced as initial purification of a crude extract. In particular,
screening tools, namely spectroscopy-guided finding single solvents that will dissolve all the
screening utilizing NMR spectroscopy or MS components of a mixture is difficult, if not
coupled with liquid chromatography (LC-MS). This impossible, without some level of pre-fractionation.
newer strategy, combined with high-quality spectral Preliminary purification steps have commonly been
databases, can help identify known compounds in the done via liquid/liquid partitioning. However, as some
extracts or active substances that have already been extracts may form stable emulsions (John W Blunt et
studied (Ito & Masubuchi, 2014; Koehn, 2008). The al., 1987), leading to the incomplete partitioning of
procedure is known as de-replication, a term first the extract and target compounds and the
requirement of large volumes of solvent, this

DOI: 10.30598//ijcr. 203

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

straightforward technique has been changed to mass-to-charge (m/z) ratio (Bythell, Hendrickson, &
column chromatography. The widely used reversed- Marshall, 2012; Vaiano et al., 2015). An LC-MS
phase chromatography procedure developed by Blunt system includes elements such as high-performance
and Munro has been used for many years to liquid chromatography (HPLC) system; the
overcome problems associated with standard flash ionization source (which interfaces the LC to the
column chromatography (normal phase) (John W MS); and the mass spectrometer. A computer system
is used to control all these elements (Korfmacher,
Blunt et al., 1987). In living systems, most organisms
2005). The HPLC system prepares a mixture of water
have molecules that are either very non-polar
and common hydrophilic solvents (isopropanol,
(lipids/steroids) or very polar (proteins/sugars). methanol, or acetonitrile) as the mobile phase. A
However, most interesting (amphiphilic) natural pumping mechanism in the HPLC system allows
products are frequently not discovered with these pressurized liquid and a sample mixture to pass
molecules but in the intermediate “mass window” through a column filled with the adsorbent stationary
(Woolner, 2017). phase. The adsorbent is typically a granular material
At Victoria University of Wellington (VUW), the (1–50 μm) made of solid particles (typically a silica-
reversed-phase polystyrene divinylbenzene (PSDVB) based bonded phase, e.g., C18) (Niessen, 2003). The
copolymer (HP20) was found to have the advantage ionization source is used as the interface between the
of being a relatively inexpensive adsorbent, stable HPLC eluent and the mass spectrometer. Two
throughout the pH range, and reusable, unlike silica standard atmospheric-pressure ionization (API)
gel (normal phase). Also, acetone and methanol, technique sources are electrospray ionization (ESI)
and atmospheric-pressure chemical ionization
environmentally friendly solvents, are widely used
(APCI). The eluent from the LC is nebulized to
for this technique, giving an additional advantage. produce ions from the evaporating droplets.
This method is known as cyclic loading and has Nebulization is achieved either pneumatically via
benefited the natural product research group at VUW APCI or by a strong electrical field in ESI (Niessen,
for quite some time. The cyclic loading system 2003). Several mass analyzers are available, but
enables crude extracts to be loaded directly onto the nowadays, time-of-flight (TOF) MS is the most
PSDVB column without pre-concentration. widely used system in drug discovery research. In a
Popplewell (Popplewell, 2008), who previously quadrupole time-of-flight (QTOF) instrument, as
studied 34 temperate New Zealand red algae in the used in this study, the quadrupole is used to select
marine natural products group at VUW, developed a precursor ions that will be fragmented later in a
method for algal screening modified from VUW in- collision cell. These generated ions are then
house protocols designed for screening sponge separated by the TOF and detected by a photo-
electron multiplier plate (Loboda, 2014).
extracts (Keyzers, 2003; Popplewell, 2008). The
protocol involves the extraction from 2 g or more
(wet weight) algal material because macroalgae have METHODOLOGY
a more affluent fraction of secondary metabolites
than sponge extracts which typically require ~ 100 g Although a single LC-MS experiment is a
wet weight (Popplewell, 2008). Since the mid-1990s, powerful technique that can collect thousands of
the MNPs group at VUW has been utilizing NMR- spectra relatively briefly, most data is only sitting in
guided isolation of secondary metabolites. the researchers’ drawers or computers. Moreover,
most natural products databases such as the
Mass spectrometry (MS) has become a standard Dictionary of Natural Products
procedure for investigating complex mixtures and (http://dnp.chemnetbase.com/) and MarinLit
molecules. Liquid chromatography coupled with (http://pubs.rsc.org/marinlit) only provide services to
mass spectrometry (LC-MS) is a hyphenated their subscribers. Recognizing this need, the
analytical technique that synergizes the ability to University of California San Diego (UCSD) Centre
perform fractionation via liquid chromatography with for Computational Mass Spectrometry
the mass analysis capability of MS (Korfmacher, (http://proteomics.ucsd.edu) developed Global
2005). This technique works by ionizing a molecule Natural Product Social Molecular Networking
that is smashed and turned into charged fragments, (GNPS) to accommodate the demand for robust
which then would be quantitated based on their
dereplication of natural products that are freely

DOI: 10.30598//ijcr. 204

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

available to the global research community (Wang et A cluster is formed when edges connect nodes
al., 2016). GNPS is an open database that can and comprise a unique set of related molecules as
analyze, organize, and create networks from tandem structurally similar compounds tend to have similar
mass spectra data (Wang et al., 2016). Moreover, the properties and belong to the same group (Morrow,
publicly available GNPS database (known as Tian, & Zhang, 2010; Nguyen et al., 2013). GNPS
MassIVE) is used to compare experimental data with also enables the annotation of putative nodes;
the known spectra library, which is helpful for therefore, the mass difference can be used to annotate
dereplication in natural products. other nodes (Sumner et al., 2007). GNPS provides
network visualization on their website
(https://gnps.ucsd.edu/ProteoSAFe/static/gnps-
splash2.jsp). However, a more sophisticated third-
party software, called Cytoscape, is available to
visualize the network (http://www.cytoscape.org)
(Shannon et al., 2003; Smoot, Ono, Ruscheinski,
Wang, & Ideker, 2010). This open-source freeware
visualizes critical defining attribute values in the
network in different shapes, colors, and sizes (Cline
et al., 2007). Cytoscape is a reliable tool for
displaying large data sets in other areas such as
metabolomics, biochemical pathways, population
networks, and even social science research (Boya P
et al., 2017; Kofia, Isserlin, Buchan, & Bader, 2015;
Watrous et al., 2012; Zhou, Shaverdian, Jagadish, &
Michailidis, 2009). The current freeware version is
Figure 1. A scheme shows how molecular networks are Cytoscape v3.9.1 (released in January 2022).
created from LC-MS data (Modified from Watrous The research was carried out in Timor Island and
(Watrous et al., 2012) used by permission) Semau Island coastal waters, East Nusa Tenggara
Province, Indonesia. Four locations were visited to
Instead of a linear comparison, GNPS uses a collect fresh macroalgae samples (Figure 2). Three of
vector-based approach to match two or more the sites are in Timor Island i.e., Sulamu Beach (10°
different MS/MS spectra (see Figure 1 for details). 3' 5.463'' S and 123° 36' 52.29'' E), Pasir Panjang
Experimental mass spectra with unique Beach (10° 8' 55.464'' S and 123° 36' 13.356'' E), and
fragmentation patterns are combined into Tablolong Beach (10° 19' 2.136'' S and 123° 28'
multidimensional vectors. Vectorization in GNPS 13.728'' E). While another collection site, Akle
happens by taking not only the peak intensity but Beach, is on Semau Island (10° 19' 20.7048'' S and
also the ion's mass-to-charge ratio (m/z). An overall 123° 20' 12.444'' E). Fresh macroalgae were sampled
vector is generated by plotting the m/z ratio of in the intertidal area during the low tide period.
multiple peaks (n) in n-dimensional space. Each Samples were collected in water no deeper than knee
overall vector represents a compound or potentially height. A total of 40 samples of different species of
isomers and will be shown as nodes. The overall brown, red, and green macroalgae were collected
vectors of different compounds can be aligned and from all four sites (Table 1). The number of green
compared with each other; thus, the cosine of the algae (Chlorophyta) and red alga (Rhodophyta)
angle between two or more vectors can be used to collected were the same, while fewer brown alga
measure their similarity. The cosine score represents (Phaeophyta) was sampled. Interestingly, Akle Beach
how closely related two nodes (hence compounds) in Semau Island provided more species than the other
are, which varies from 0 (completely unrelated) to 1 three beaches.
(identical spectra). In the network, the cosine score is Prior to extraction, samples were dried under the
usually represented by the thickness of a line (edge) sun for two to three days, labelled and stored. The
that connects two nodes. extraction was done in the Integrated Science
Laboratory of Universitas Nusa Cendana, Kupang,

DOI: 10.30598//ijcr. 205

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

Indonesia. The samples were macerated and employed to get fragments of the samples based on
extracted twice in methanol for 24-hour periods. their m/z ratio to maximize the amount of data
Both extracts were then combined. The methanol obtained since some compounds only ionized under
extract was subsequently concentrated in vacuo to one mode. Spectra data from LC-MS/MS were
dry samples completely or remove the methanol. As converted and exported as mgf files to the GNPS
the last step, the remaining solution (presumably website along with text files of meta-data attributes
water) was removed in a freeze dryer at -100oC to - to produce the network. On the GNPS website, each
121oC. The dried samples were finally obtained and positive or negative ion mode dataset was run
sent to Victoria University of Wellington for further separately. All essential parameters were set to create
analysis. consensus spectra: parent mass tolerance was set to
0.02 Da, and MS/MS fragment ion tolerance was set
Table 1. Number of macroalgal collected on each site at 0.02 Da. Also, the consens5us spectra that
based on phylum contained less than two spectra were discarded. The
Number of Number of Number of
Minimum Pairs Cosines score was set to 0.7, and the
Location Phaeophyta Rhodophyta Chlorophyta Total
(brown) (red) (green) minimum fragment ions matched was set to six data
Sulamu 4 3 2 9 to produce the network. The results were then
Beach exported and later visualized in the Cytoscape
Pasir - 4 2 6 application (version 3.7) and displayed in “preferred
Panjang layout” settings. Positive mode data showed 574
Beach nodes and 857 edges (Figure 3), while negative mode
Akle 4 6 10 20 showed fewer nodes and edges, 120 and 182,
Beach respectively (Figure 4).
Tablolong 2 2 1 5
The positive ion GNPS network shows that
Beach
Total 10 15 15 40
macroalgae collected from Akle Beach have the
potential for further study (Figure 3). The network is
dominated by clusters formed from algae collected at
Akle Beach (more than 10 clusters), followed by two
clusters from Sulamu Beach and Tablolong Beach
(which connect to a Sulamu Beach cluster), with no
clusters from Pasir Panjang Beach. Akle Beach has
the most clusters since 20 out of 40 macroalgae
samples were collected from this site. Conversely, in
the negative ion mode, no single cluster was formed
from algae collected from a specific location as they
have nodes that also belong to extracts from other
sites (Figure 4).
Figure 2. Sampling locations of macroalgae from West
Timor waters, Indonesia. The map retrieved from
However, the negative ion network also
https://www.google.com/maps/@- confirms that potential macroalgae for screening
10.1345414,123.5736125,70500m/data=!3m1!1e3 (used come from similar beaches, as shown in the positive
by permission) ion mode (i.e., Akle Beach, Sulamu Beach, and
Tablolong Beach). This may occur due to the
RESULTS AND DISCUSSION production of secondary metabolites from
The 40 macroalgae samples collected from West macroalgae as chemical defenses against herbivores.
Timor waters in Indonesia were extracted using As Pasir Panjang Beach is located in urban areas, the
methanol (MeOH) at room temperature and were absence of herbivory fish in this site might slightly
dried afterward to obtain a crude extract weight. alter the function of secondary metabolites on this
Before LC-MS analysis, all samples were diluted in particular beach (Pereira & Da Gama, 2008; Schmitt,
MeOH to a set of concentrations (0.1 µg µL-1). In the Hay, & Lindquist, 1995). When the phylum of
macroalgae is considered for grouping the GNPS
LC-MS, both positive and negative modes were
network (Figures 5 and 6), green macroalgae

DOI: 10.30598//ijcr. 206

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

(Chlorophyta) were shown as the most prolific (mainly between Oct-Dec each year)(Ormond &
source of compounds, followed by brown Banaimoon, 1994) The samples collected from West
(Phaeophyta) and red macroalgae (Rhodophyta). Timor waters found more green and red macroalgal
Thirteen clusters were formed from green algae, species than brown during the Oct-Nov sampling.
while brown and red algae generated nine and seven This result suggested brown algae may be an
clusters, respectively. These results contradict the understudied resource for finding new marine natural
products.
trend in macroalgae secondary metabolites research
since the most prolific source of macroalga natural
products is red algae which account for more than
50%, followed by brown for almost 40%, and the rest
are from green seaweed (Hasanela, N., & Souhoka,
F. A., 2022; Telussa, I., Hattu, N., & Sahalessy, A.,
2022; Bijang, C. M., Tehubijuluw, H., & Kaihatu, T.
G., 2018; John W Blunt, Copp, Keyzers, Munro, &
Prinsep, 2014; John W Blunt, Copp, Keyzers,
Munro, & Prinsep, 2013, 2015, 2016; John W. Blunt,
Copp, Keyzers, Munro, & Prinsep, 2017; Cabrita,
Vale, & Rauter, 2010; Leal et al., 2013).

Figure 4. GNPS network of macroalgae based

upon sampling locations in negative ion mode.
Colors are arbitrarily assigned to collection site

Molecular networking based on individual

species enabled a selection of samples to be
prioritized in the isolation workflow. Six samples
were selected from both positive and negative mode
networks to be processed further for NMR screening
(Figure 7). The samples are Amphiroa sp1
(WEM_01_005) from Sulamu Beach; Amphiroa sp2
Figure 3. GNPS network of macroalgae based upon (WEM_03_002), Ulva sp1 (WEM_03_004), and
sampling locations in positive ion mode. Colors are Ulva sp2 (WEM_03_005) from Pasir Panjang Beach;
arbitrarily assigned to collection site Padina sp2 (WEM_04_019) from Semau Island,
Laurencia snackeyi (WEM_05_001) from Tablolong
However, the seasonal assemblage of intertidal Beach (Figure 9). These samples were selected based
macroalgae in the tropics usually shifts to more green on clusters formed where the clusters were
and red algae in late summer/the rainy season dominated by one of the six specimens (Figure 7).

DOI: 10.30598//ijcr. 207

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

Figure 7. GNPS network of macroalgae based on

specimens. Colors are arbitrarily assigned to 40
different samples of species

Six extracts that had been prioritized through

molecular networking (previous section), were then
Figure 5. GNPS network of macroalgae based upon fractionated before using 1H NMR to verify the
phylum in positive ion mode. Colors are arbitrarily presence of interesting secondary metabolites.
assigned to collection site Although NMR spectroscopy does not offer
biological information, it is perceived that novel
structures often lead to interesting biological activity
(Keyzers, 2003). As argued before, the intermediate
75% fraction (mass window) showed the most
interesting peaks compared to the 30% and 100%
Me2CO fractions.

Figure 8. 1H NMR spectra from 75% semi-purified

fractions of six prioritized samples of macroalgae
Figure 6. GNPS network of macroalgae based upon (600 MHz, CD3OD)
phylum in negative ion mode. Colors are arbitrarily
assigned to collection site

DOI: 10.30598//ijcr. 208

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

Out of six samples, three were initially chosen molecular network; Dr. Helen Woolner and Dr. Joe
as having interesting peaks between 3.5-5 ppm Bracegirdle both for their NMR knowledge.
(Figure 8); namely WEM_01_005 (Amphiroa sp1),
WEM_04_019 (Padina sp2), and WEM_05_001 REFERENCES
(Laurencia snackeyi). The resonances in these
Bijang, C. M., Tehubijuluw, H., & Kaihatu, T. G.
downfield correspond to oxymethine protons,
(2018). Biosorpsi Ion Logam Kadmium (Cd2+)
predominantly sugars signals (Figure 8).
Pada Biosorben Rumput Laut Coklat (Padina
australis) Asal Pantai Liti Pulau Kisar. Indo. J.
Chem. Res., 6(1), 51-58.
Blunt, J. W., Calder, V. L., Fenwick, G. D., Lake, R.
J., McCombs, J. D., Munro, M. H., & Perry, N.
B. (1987). Reverse Phase Flash
Chromatography: A Method for The Rapid
Partitioning of Natural Product Extracts.
Journal of Natural Products, 50(2), 290-292.
Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M.
H., & Prinsep, M. R. (2013). Marine Natural
Products. Nat. Prod. Rep., 30(2), 237-323.
Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M.
H., & Prinsep, M. R. (2015). Marine Natural
Methanol extractions from West Timor Employing Validating Products. Nat. Prod. Rep., 32(2), 116-211.
Macroalgae samples subsampled for GNPS algorithm screening process Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M.
untargeted LC-MS/MS (40 samples). based on MS/MS with 1H NMR
[Notes: Wem_01 are from Sulamu Beach; spectra to spectroscopy (3 H., & Prinsep, M. R. (2016). Marine Natural
Wem_02 and Wem_03 are from Pasir prioritize the samples)
Panjang Beach; Wem_04 are from Semau isolation
Products. Nat. Prod. Rep., 33, 382-431
Island; Wem_05 are from Tablolong Beach] workflow (6 Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M.
samples)
H. G., & Prinsep, M. R. (2017). Marine Natural
Products. Nat. Prod. Rep., 34(3), 235-294.
Figure 9. Prioritization process that showing how
Boya P, C. A., Fernández-Marín, H., Mejía, L. C.,
dereplication by GNPS has helped in prioritizing
Spadafora, C., Dorrestein, P. C., & Gutiérrez,
isolation workflow
M. (2017). Imaging Mass Spectrometry and
MS/MS Molecular Networking Reveals
CONCLUSION Chemical Interactions Among Cuticular
Bacteria and Pathogenic Fungi Associated with
This study began with the sampling of 40
Fungus-Growing Ants. Scientific Reports, 7(1),
specimens of tropical Indonesian macroalgae from
5604.
West Timor waters. A molecular networking
Bythell, B. J., Hendrickson, C. L., & Marshall, A. G.
screening was employed to priorities the isolation
(2012). Relative Stability of Peptide Sequence
workflow. The crude extracts of the samples were
Ions Generated by Tandem Mass Spectrometry.
analyzed using liquid chromatography-mass
Journal of The American Society for Mass
spectrometry (LC-MS)/mass spectrometry (MS) to
Spectrometry, 23(4), 644-654.
generate data for creating the molecular network
Cabrita, M. T., Vale, C., & Rauter, A. P. (2010).
through the Global Natural Product Social Molecular
Halogenated Compounds from Marine Algae.
Networking (GNPS) platform. Based on the clusters
Marine drugs, 8(8), 2301-2317.
formed, six samples were prioritized from the
Cline, M. S., Smoot, M., Cerami, E., Kuchinsky, A.,
molecular network. The suggestions from the
Landys, N., Workman, C., . . . Gross, B. (2007).
screening process were then validated with 1H NMR
Integration of Biological Networks and Gene
spectroscopy, which finally prioritised three samples.
Expression Data Using Cytoscape. Nature
protocols, 2(10), 2366.
ACKNOWLEDGMENT
Eloff, J. N. (1998). A Sensitive and Quick
I indebt Associate Prof. Rob Keyzers for his Microplate Method to Determine The Minimal
guidance and support for this research, also a thank Inhibitory Concentration of Plant Extracts for
you to Sarah Andreassend for her help on the Bacteria. Planta Med, 64(8), 711-713.

DOI: 10.30598//ijcr. 209

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

Hasanela, N., & Souhoka, F. A. (2022). Effect of Molecule and Gene Cluster Families.
Heating Coarse Extract of Brown Macroalgae Proceedings of the National Academy of
(Padina australis) from Tial Waters, Salahutu Sciences, 110(28), E2611-E2620.
District, Central Maluku Regency on Niessen, W. (2003). Progress in Liquid
Antioxidant Activity. Indo. J. Chem. Res., 10(2), Chromatography–Mass Spectrometry
102-109. Instrumentation and Its Impact on High-
Hostettmann, K., Wolfender, J.-L., & Terreaux, C. Throughput Screening. Journal of
(2001). Modern Screening Techniques for Plant chromatography A, 1000(1), 413-436.
Extracts. Pharmaceutical biology, 39(sup1), 18- Ormond, R., & Banaimoon, S. (1994). Ecology of
32. Intertidal Macroalgal Assemblages on the
Ito, T., & Masubuchi, M. (2014). Dereplication of Hadramout Coast Of Southern Yemen, an Area
Microbial Extracts and Related Analytical of Seasonal Upwelling. Marine Ecology-
Technologies. The Journal of antibiotics, 67(5), Progress Series, 105, 105-105.
353. Pereira, R. C., & Da Gama, B. (2008). Macroalgal
Keyzers, R. A. (2003). The Isolation of Biologically Chemical Defenses and Their Roles in
Active Secondary Metabolites From New Structuring Tropical Marine Communities. In
Zealand Marine Organisms. Dissertation, Algal chemical ecology (pp. 25-55): Springer.
Victoria University of Wellington, Popplewell, W. L. (2008). Isolation and Structure
Koehn, F. E. (2008). High impact Technologies For Elucidation of New Secondary Metabolites from
Natural Products Screening. In Natural New Zealand Marine Red Algae.Dissertation,
Compounds as Drugs Volume I (pp. 175-210): Victoria University of Wellington,
Springer. Schmitt, T. M., Hay, M. E., & Lindquist, N. (1995).
Koehn, F. E., & Carter, G. T. (2005). The Evolving Constraints on Chemically Mediated
Role of Natural Products in Drug Discovery. Coevolution: Multiple Functions for Seaweed
Nature Reviews Drug Discovery, 4(3), 206-220. Secondary Metabolites. Ecology, 76(1), 107-
Kofia, V., Isserlin, R., Buchan, A. M. J., & Bader, G. 123.
D. (2015). Social Network: a Cytoscape app for Shannon, P., Markiel, A., Ozier, O., Baliga, N. S.,
visualizing co-authorship networks. Wang, J. T., Ramage, D., . . . Ideker, T. (2003).
F1000Research, 4, 481-481. Cytoscape: A Software Environment for
Korfmacher, W. A. (2005). Foundation Review: Integrated Models of Biomolecular Interaction
Principles and Applications for LC-MS in New Networks. Genome research, 13(11), 2498-
Drug Discovery. Drug discovery today, 10(20), 2504.
1357-1367. Smoot, M. E., Ono, K., Ruscheinski, J., Wang, P.-L.,
Leal, M. C., Munro, M. H., Blunt, J. W., Puga, J., & Ideker, T. (2010). Cytoscape 2.8: New
Jesus, B., Calado, R., . . . Madeira, C. (2013). Features for Data Integration and Network
Biogeography and Biodiscovery Hotspots of Visualization. Bioinformatics, 27(3), 431-432.
Macroalgal Marine Natural Products. Natural Sumner, L. W., Amberg, A., Barrett, D., Beale, M.
Product Reports, 30(11), 1380-1390. H., Beger, R., Daykin, C. A., . . . Viant, M. R.
Loboda, A. (2014). Method and System for (2007). Proposed Minimum Reporting
Operating A Time of Flight Mass Spectrometer Standards for Chemical Analysis Chemical
Detection System. In: Google Patents. Analysis Working Group (CAWG)
Morrow, J. K., Tian, L., & Zhang, S. (2010). Metabolomics Standards Initiative (MSI).
Molecular Networks in Drug Discovery. Metabolomics: Official journal of the
Critical Reviews™ in Biomedical Engineering, Metabolomic Society, 3(3), 211-221.
38(2), 143-156 Telussa, I., Hattu, N., & Sahalessy, A. (2022).
Moser, A., Wheeler, P., & Hayward, S. (2005). Morphological Observation, Identification and
Dereplication of Natural Products by NMR: A Isolation of Tropical Marine Microalgae from
Three-Stage Approach ACD/Structure Ambon Bay, Maluku. Indo. J. Chem. Res., 9(3),
Elucidator Suite and ACD/Labs’ Content 137-143.
Databases. Vaiano, F., Serpelloni, G., Focardi, M., Fioravanti,
Nguyen, D. D., Wu, C.-H., Moree, W. J., Lamsa, A., A., Mari, F., & Bertol, E. (2015). LC–MS/MS
Medema, M. H., Zhao, X., . . . Jackson, C. and GC–MS Methods in Propofol Detection:
(2013). MS/MS Networking Guided Analysis of

DOI: 10.30598//ijcr. 210

Welem Turupadang et al. Indo. J. Chem. Res., 10(3), 203-211, 2023

Evaluation of the Two Analytical Procedures. Zhou, H., Shaverdian, A. A., Jagadish, H. V., &
Forensic science international, 256, 1-6. Michailidis, G. (2009). Multiple step social
Wang, M., Carver, J. J., Phelan, V. V., Sanchez, L. structure analysis with Cytoscape. Paper
M., Garg, N., Peng, Y., . . . Bandeira, N. (2016). presented at the 2009 IEEE Symposium on
Sharing and Community Curation of Mass Visual Analytics Science and Technology.
Spectrometry Data with Global Natural Products
Social Molecular Networking. Nature
biotechnology, 34, 828–837.

https://www.nature.com/articles/nbt.3597#supplemen
tary-information
Watrous, J., Roach, P., Alexandrov, T., Heath, B. S.,
Yang, J. Y., Kersten, R. D., . . . Raaijmakers, J.
M. (2012). Mass spectral molecular networking
of living microbial colonies. Proceedings of the
National Academy of Sciences, 109(26), E1743-
E1752.
Woolner, V. H. (2017). New Halogenated
SecondaryMetabolites from Red Algae of the
South Pacific. (PhD), Victoria University of
Wellington,

DOI: 10.30598//ijcr. 211