High-resolution liquid chromatography mass spectrometry (HR-LCMS) and 1H NMR analysis of methanol extracts from marine seaweed Gracilaria edulis

Abstract A wide variety of bioactive secondary metabolites that seaweeds are claimed to generate can be used by the pharmaceutical industry to create novel medications. The primary goal of the current study was to use the HR-LCMS and NMR approach to analyse the phytochemical content of a methanolic extract of Gracilaria edulis. The HR-LCMS and NMR analysis reveals the presence of bioactive compounds (amino acid, fatty acid, triterpenoids, carotenoid, aromatic compounds, flavonoids, secondary alcohols, diterpenes, lipid, phenolic compounds, sesquiterpenoids, quinolizidine alkaloid, and benzoquinone). The present study’s findings support the existence of significant phytocompounds in G. edulis and are useful for future in-depth investigation to create medications from marine algae to treat a variety of ailments. The goal of current research is to discover all-natural treatments for a wide variety of illnesses and conditions. Graphical abstract


Introduction
Nearly 80% of all plant and animal species in the world are found in the marine ecosystem.The intertidal zone is home to over 150,000 distinct types of seaweed.
Seaweed grows along the coast in places like Mandapam in the Ramanathapuram district.In the intertidal regions of the marine ecosystem, seaweed grows in the shallow water.The seaweeds have a special quality that helps them survive in the saltwater of the aquatic environment (Ragunathan et al. 2019).
The Red Sea is home to many marine algae, which may be classified into three classes: green algae (Chlorophyta), red algae (Rhodophyta), and brown algae (Phaeophyta).Fucoxanthin, a pigment, and the polysaccharides alginates, laminarins, fucans, and cellulose give brown seaweeds their mostly brown color.Ulvan is the main polysaccharide of green seaweeds, which are distinguished by high levels of chlorophyll a and b, while phycoerythrin, phycocyanin and agars are the main polysaccharides in red seaweeds, along the carrageenans as the key polysaccharides.In addition, marine algae are abundant in structurally diversified bioactive chemicals with a variety of biological processes, even though only a few species have been chemically analysed in recent years (Rushdi et al. 2020).
A rich source of naturally occurring bioactive compounds with chemical and structural characteristics not present in terrestrial natural compounds is the marine environment.Several marine creatures create bioactives in reaction to ecological stressors (Shobier et al. 2016).
The pharmaceutical industry has a bright future since marine algae are known to create a large variety of bioactive secondary metabolites, that may be used to make new medicines.The Gracilaria edulis is a significant food and medicine taxon that is a member of the Gracilariaceae family (Agardh et al. 2015).
Polysaccharides, fatty acids, phytosterols and phenolic compounds, and carotenoids are a few chemicals that have been shown to have positive health impacts, such as antioxidant, anticoagulant, or anticancer properties.As a result, it is becoming more important to create novel formulations for the food, cosmetic and pharmaceutical industries that include an algal ingredient (Lourenc ¸o-Lopes et al. 2020).
The aim of the current research was to use HRLC-MS and NMR analysis to examine the phytoconstituents present in G. edulis.The major goal of this study is to find a link between the composition of the substance and its conventional medical use.

High resolution-liquid chromatography-mass spectrometry analysis (HR-LCMS)
There were 35 compounds proven to be present in the methanolic extract of G. edulis based on their molecular formula, mass, and retention time as given in Table S1 (Supplementary Material).
A chromatogram shows the relative quantities of different chemicals that are eluted according to the retention time.The height of the peak served as a measure for the relative concentration of the bioactive chemicals found in the plants.The composition and structure of the compounds are determined by the mass spectrometer's analysis of the compounds eluted at various periods.These mass spectra serve as the data library's unique fingerprint for the molecule.

NMR analysis
Figure S18 (Supplementary material) depicts the NMR spectra of G. edulis.Fatty acids' contribution to the existence of carboxylic -OH at 10.520, Ar-OH absorbs at 5.33, 5.75, 6.45 and 7.09, which indicates the presence of carotenoids, triterpenoids, and phenolic chemicals.The olefinic proton absorption seen at 4.11 and 4.68.In the range of 6-8.5, the typical absorption of the multiplate of the aromatic proton was seen.A methyl group singlet (R-CH3) at positions 0.66 and 0.86 and the singlet of the methylene group at positions 145, 1.61, 1.76, 1.95 and 2.236 indicates the presence of several bioactive substances.

Experimental
This part is described in the Supplementary material.

Conclusion
The effective strategy for drug discovery and development uses secondary metabolites that are generated from natural sources.According to the phytochemical analysis, the current study has shown that G. edulis may be a source of beneficial chemicals.The HR-LCMS approach was used to isolate and characterise bioactive chemicals from G. edulis, and this is the first publication to do so.
Using HR-LCMS high-resolution liquid chromatography-mass spectrometer and NMR analysis, the methanolic extract of G. edulis was found to contain therapeutically significant bioactive compounds such as amino acids, fatty acids, triterpenoids, carotenoid, aromatic compounds, flavonoids, secondary alcohols, diterpenes, lipid, phenolic compounds, sesquiterpenoids and quinolizidine alkaloid.The existence of several bioactive chemicals discovered via this investigation justifies the traditional usage of seaweeds for a variety of diseases.Separating the different components and examining their pharmacological action are still being researched, though.