Open access peer-reviewed chapter

Seagrass Metabolomics: A New Insight towards Marine Based Drug Discovery

By Danaraj Jeyapragash, Ayyappan Saravanakumar and Mariasingarayan Yosuva

Submitted: November 6th 2020Reviewed: April 26th 2021Published: May 19th 2021

DOI: 10.5772/intechopen.97875

Downloaded: 77

Abstract

Metabolomics is one of the new field of “Omics” approach and the youngest triad of system biology, which provides a broad prospective of how metabolic networks are controlled and indeed emerged as a complementary tool to functional genomics with well-established technologies for genomics, transcriptomics and proteomics. Though, metabolite profiling has been carried out for decades, owing to decisive mechanism of a molecule regulation, the importance of some metabolites in human regimen and their use as diagnostic markers is now being recognized. Plant metabolomics therefore aims to highlight the characterization of metabolite pool of a plant tissue in response to its environment. Seagrassses, a paraphyletic group of marine hydrophilous angiosperms which evolved three to four times from land plants back to the sea. Seagrasses share a number of analogous acquired metabolic adaptations owing to their convergent evolution, but their secondary metabolism varied among the four families that can be considered as true seagrasses. From a chemotaxonomic point of view, numerous specialized metabolites have often been studied in seagrasses. Hence, this chapter focus the metabolome of seagrasses in order to explore their bioactive properties and the recent advancements adopted in analytical technology platforms to study the non-targeted metabolomics of seagrasses using OMICS approach.

Keywords

  • seagrass
  • metabolomics
  • OMICS
  • non-targeted
  • drug discovery

1. Introduction

Over the past decades, metabolomics has emerged as a valuable tool for the comprehensive profiling and metabolic networks in the biological system. Pauling et al. [1] coined the term metabolomics which was first used in 1998 and even up to 2010 metabolomics was considered as an emerging one in the science field. Reports were documented on the complete genome ([2]; Yu et al., 2002), transcriptome [3] and proteome studies [4, 5, 6], but in recent years metabolome analyses using mass spectrometry (MS) - based platforms attracted attention. Even though, metabolite profiling have been carried out for decades, due to ultimate mechanism of a molecule regulation as constituents of metabolic pathways, the prominence of some metabolites in human regimen and their use as diagnostic markers is now being recognized [7].

Currently, metabolomics is a powerful tool for characterizing the metabolites and their metabolic pathways which provides a clear metabolic picture of biological samples. Metabolites are small molecules with diverse structures that are chemically transformed during the cellular metabolism [8]. The number of metabolites is expected to be significantly lowered than the number of genes, mRNAs and proteins which reduce the sample complexity. So far, the total number of metabolites in the plant kingdom is estimated to exist between 100000 to 200000, which make the task more challenging to detect more diverse group of metabolites [9]. Plant metabolomics therefore aims to highlight the characterization of metabolite pool of a plant tissue in response to its environment [10, 11, 12, 13]. Since, metabolomics is a balanced approach that obtains inclusive information on the cell’s, tissues or organisms metabolite content with low molecular weight, their configuration likely to be changed owing to diverse environmental conditions which reproduces different genetic background [14, 15].

Recent reports on the plant metabolome bought huge challenges to analytical technologies that have been used in current plant metabolomics programs. Some analytical approaches comprise metabolite profiling, metabolite target analysis and metabolite fingerprinting which can be employed according to focus of the research and research questions [16, 17]. Metabolite profiling does not certainly determine the absolute concentrations of metabolites; rather their comparative levels within a structurally related predefined group. Targeted metabolite analysis aims to determine the absolute concentration of metabolites using specialized extraction protocols with an adapted separation and detection methods [18]. Metabolite fingerprinting generally not used to detect individual metabolites, but rather it provides a fingerprint of all compounds which can be measured for sample comparison and discrimination analysis by non-specific rapid analysis of crude metabolite mixtures. However, single analytical technology is not enough to cover the whole metabolome owing to the metabolic diversity and their broad dynamic range in cellular abundance. Accordingly, different extraction techniques and combinations of analytical methods are often employed in order to acquire diverse group of metabolite coverage.

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2. Mass spectrometry-based metabolomics analysis

Historically, metabolite concentrations were achieved either by spectrophotometric assays capable of detecting single metabolites or by simple chromatographic separation of mixtures with low complexity. However, over the past decade several methods with high accuracy and sensitivity have been established for the analysis of highly complex mixtures of compounds [19, 20, 21]. These methods include gas chromatography - mass spectrometry (GC–MS), liquid chromatography - mass spectrometry (LC–MS), fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and capillary electrophoresis - mass spectrometry (CE-MS). In addition, NMR coupled with chromatography have found great efficacy in addressing specific issues with respect to medical fields [22, 23] and conceivably more important to the unequivocal determination of metabolite structures [24]. However, NMR shows relatively low sensitivity and hence can be used for profiling the diverse group of metabolites from complex mixtures. The pros and cons of mass-spectrometric based metabolomics is given in Table 1.

Metabolomics TechnologyAdvantagesDisadvantages
GC–MS
  • Well established chromatographic-mass spectrometric technology

  • Less expensive

  • Easy to perform

  • Better stability and reproducibility

  • Universal database for metabolite identification is available

  • Time consuming

  • Can only analyze volatile compounds upon derivatization

  • Not able to identify the unknown compounds

LC–MS
  • Highly sensitive

  • Method for sample pre-treatment is simple

  • Wide coverage of metabolite could be identified

  • Scanning range for different ions is usually less

  • Different columns can be used depends on the polarity of compounds

  • Less reproducibility

  • Highly Expensive

  • Difficult to identify highly polar and charged metabolites

FT-ICR-MS
  • High resolution mass spectrometric technique

  • Extemely high mass accuracy

  • High acquisition rates

  • Highly flexible

  • Involves series of steps

  • Use large super conducting magnets

  • Highly expensive than GC–MS and LC–MS

Table 1.

Advantages and disadvantages of mass-spectrometric based metabolomics.

Gas Chromatography - Mass Spectrometry assists the identification and robust quantification of few hundred metabolites in a single plant extracts, which results in inclusive coverage of the central pathways of primary metabolism [25]. GC–MS has a major advantage than other methods that it has long been used for profiling the metabolites and therefore it has stable protocols for machine setup, their maintenance with chromatogram evaluation and interpretation. Though, single analytical system cannot cover the whole metabolome, GC–MS has a quite broad coverage of compounds classes including organic and amino acids, sugars, sugar alcohols, lipophilic compounds and phosphorylated intermediates [26]. During method validation, recovery experiments of all measurable compounds have been done and for unknown compounds, recombination experiments were executed to determine the recovery rates in which the extracts of two plant species are evaluated independently and also with mixtures [27, 28]. Liquid chromatography-based methods offer numerous advantages such as detection of broad range of metabolites, as they suffer from the lower reproducibility of retention time. In addition, they are more susceptible to ion suppression effects due to the predominant use of electrospray ionization, which renders the precise quantification more difficult [29, 30, 31]. FT-ICR-MS and CE-MS has been reported to be worth mentioning, where FT-ICR-MS has unsurpassed mass accuracy thereby allows the researcher to obtain an idea about the chemical composition of the specific compounds. In case of CE-MS, the low-abundance metabolites can be detected and affords good chromatographic separation [32, 33]. Of these techniques, GC–MS is mostly preferred for the separation of low molecular weight metabolites which can be either volatile or can be converted into volatile and thermally stable compounds via chemical derivatization prior to the analysis [34]. The experimental procedure for GC–MS based metabolomics analysis is represented in Figure 1.

Figure 1.

Schematic overview of MS based metabolomics.

3. Derivatization

Derivatization is a process by which a compound is chemically modified to produce a new compound that has properties which are more amenable to specific analytical procedure. Samples analyzed by gas chromatography requires derivatization in order to make them suitable for analysis. Derivatization procedure imparts volatility, decreases the adsorption in the injector, increases the stability of compounds; improve the resolution and detectability between coeluting compounds and overlapping which assist in structure determination [35]. A good derivatizing reagents and the procedure should produce the compound of interest with desired chemical modification and be efficient, reproducible and non-hazardous (www.piercenet.com). For GC, derivatization reaction can be done by three basic types: silylation, acylation and alkylation. Silylating reagents react with compounds containing active hydrogen and are most frequently used in GC. Acylating reagents react with compounds having high polar functional groups such as amino acids or carbohydrates. While alkylating reagents target the active hydrogen’s on amines and acidic hydroxyl group [36].

4. Seagrasses

Seagrasses, a marine hydrophilus angiosperm live entirely in an estuarine or in the marine environment and nowhere else [37]. Seagrass ecosystem act as a breeding and nursery ground for numerous organisms and also help in promoting the commercial fisheries. It is considered to be one of the most productive ecosystems that retain the structural complexity and biodiversity shed light to some researchers to describe seagrass community as marine representation of the tropical rainforests [38]. Currently, seagrasses are assigned to four families Hydrocharitaceae, Cymodoceaceae, Posidoniaceae and Zosteraceae (den [39, 40]). According to angiosperm Group III System, all four families occurred exclusively to monocot order Alismatales [41]; while Les and Tippery [40] favored to treat the same clades as a subclass Alismatidae. The family hydrocharitaceae comprises of three genera namely Enhalus, Halophila and Thalassiaand the Cymodoceaceae encompasses the highest variety of genera includes Amphibolis, Halodule, Syringodium, Cymodoceaand Thalassodendron. Likewise, the Zosteraceae comprises of Zostera, Heterozostera, Nanozostera, Phyllospadixwhich are exclusive marine organisms and the family Posidniaceae are monogeneric [42, 43, 44]. Therefore, seagrasses can be considered as a unique ecological group occurring worldwide in different climatic zones and sharing their metabolic features with their terrestrial counterparts [37]. However, their metabolism must have undergone several adaptations to survive and colonize in shorelines and oceans worldwide [37]. The seagrass ecosystems and their functions were given in Table 2.

Seagrass and Seagrass bedsProperties
Common namesEelgrass, turtle grass, tape grass, shoal grass, and spoon grass
Families: 4
  • Hydrocharitaceae

  • Posidonaceae

  • Zosteraceae

  • Cymodoceaceae

Total Species: 72In India: 14 species exist
HabitatFound in salt and brackish water
Depth1 meter - 58-meter depth
Seagrass Parts
Leaves
  • Chloroplast

  • Lacunae

  • Cuticle

  • Viens

Photosynthesis
Help for buyoncy
Exchange oxygen and carbondioxide in the water column
Transport nutrients throughout the plants
RhizomeStabilize the seagrass beds under wave action
RootsAbsorb nutrients from the soil and transport to the plants
Growth and reproductionSexual reproduction and asexual clonal growth
Biodiversity
  • Small bodied

Small thin leaves
Small rhizome
“Guerilla” strategy
Short lived with fast turnover
Low biomass
Abundant flowering
Many small seeds and seed bank
  • Large bodied

Large thick leaves
Large rhizome
“Phalanx” strategy
Long-lived with slow turnover
High biomass and holds space
Patchy flowering
Few large seeds and seeds germinate rapidly
Ecosystem benefitsLungs of the sea
Creation of Living Habitat
Foundation of Coastal Food Webs
Blue Carbon

Table 2.

Seagrass ecosystem and their benefits.

5. Seagrass metabolome

Seagrasses share a number of analogous acquired metabolic adaptations owing to their convergent evolution, but their secondary metabolism varies among the four families that can be considered as true seagrasses. During the period of ancient Tethys Sea, approximately 90 million years ago surrounded by Africa, Gondwanaland, and Asia, the terrestrial like species returned to the sea and thus explaining the “terrestrial-like” chemical profile of seagrass. From a chemotaxonomic viewpoint, numerous secondary metabolites have been often studied in seagrasses. The metabolome of seagrasses may differ with respect to geographical location, substrates and other physiological factors includes wide fluctuations in the salinity which are prone to synthesize novel metabolites with defined physiological, biochemical, defense and ecological roles [45, 46]. Preliminary suggestions confirmed that seagrasses have pharmaceutically potent bioactive secondary metabolites [47], that are directed to prove to be a lead molecule for drug discovery [48]. The status of metabolomic study in seagrasses reported so far is tabulated in Table 3.

SeagrassMethods usedDerivation methodResultsPotential applicationReference
Zostera marina
Zostera noltii
GC/TOFTrimethyl silylationAdaptivemechanisms are involved through metabolic pathways to dampen the impacts of heat stressSucrose, fructose, and myo-inositol were identified to be the most responsive metabolites of the 29 analyzed organic metabolites.Gu et al. [49]
Cymodocea nodosaGC-QTOF-MSTrimethyl silylationGrowth promoting metabolites (sucrose, fructose, myo-inositol, heptacosane, tetracosane, stigmasterol, catechin and alpha-tocopherol) were lower close to the zone, whereas metabolites involved with stress-response (alanine, serine, proline, putrescine, ornithine, 3,4-dihydroxybenzoic acid and cinnamic acid) were identifiedMetabolomic fingerprinting of seagrass provides opportunities for early detection of environmental degradation in marine ecological studiesKock et al. [50]
Halodule pinifoliaGC–MSTrimethylsilyl etherificationGC–MS analysis revealed the presence of thirty-five compounds which include flavonoids, sugars, amino acids and plant hormonesStudy has explored a newer marine source, H. pinifoliafor RA, which is an emerging potential preclinical chemical entityJeyapragash et al. [51]
Zostera marinaGC–MSTrimethyl silylationDecreased carbohydrate decomposition products and tricarboxylic acid (TCA) cycle intermediate products, indicating that the energy supply of the eelgrass may be insufficient at high temperaturecomposition of the membrane system of eelgrass may change at high temperature and implying that high temperature may cause the membrane system to be unstableGao et al. [52]
Halodule pinifoliaGC–MSTrimethyl silylation98 metabolites in wild and 125 metabolites in SCC were identified. 77 primary and secondary metabolism pathways in wild, while 73 metabolism pathways in SCC were reportedBaseline information on H.pinifoliametabo-lism in the marine and artificial environmentsJeyapragash et al. [53, 54]
Zostera muelleriNMRTrimethyl-silylationSeveral potential bioindicators of low-light stress: a reduction of soluble sugars and their derivatives, glucose, fructose, sucrose and myo-inositol, N-methylnicotinamide, organic acids and various phenolic compounds were identifiedMetabolomics measurements may be useful bio-indicators of low-light stress in seagrassGriffith et al. [55]
Halodule pinifoliaGC–MSTrimethyl-SilylationThree thermo-protective metabolites such as trehalose (sugar), glycine betaine (amino acid) and methyl vinyl ketone (organic acid) were profiled from H. pinifolia(45°C) and is the first report on the occurrence of glycine betaine and methyl vinyl ketone from seagrasses and other aquatic species so farFacilitate the further research on identifying gene to metabolite networks for an effective management of seagrass conservation by genetic manipulationJeyapragash et al. (2021)

Table 3.

Status of metabolomics studies in seagrasses.

Primary metabolites from seagrasses reported to be similar, to that of any other terrestrial angiosperms [56]. Despite the higher phenol content, seagrasses found to be rich source of protein which alleviates the chronic problem of protein deficiency in developing countries like India [47]. In addition, seagrasses are a rich source of secondary metabolites such as simple phenolic compounds, phenylmethane and phenylethane derivatives, flavonoid and volatile derivatives with high commercial value [38]. Jeyapragash et al., reported that the plant growth regulators enhance the production of flavonoid production in the callus and cellular suspension cultured cells of seagrass H. pinifolia(Figure 2) Though, quantum of research has been published with respect to metabolome of seagrasses, there is still a dearth of knowledge as compared to the terrestrial plants. Seagrass offers remarkable opportunities to derive new commercially valuable phytochemicals when compared to algae [57, 58]. Therefore, the metabolite content from seagrasses constitutes another treasure of the ocean. Henceforth, the knowledge on metabolomics analysis from seagrass is decisive for understanding the complete metabolite picture and to explore their bioactive properties.

Figure 2.

Growth regulators mediated flavonoid production in callus and cellular siuspenison ofH. pinifolia.

Seagrasses, the only higher plants solely living in the marine habitats and are ultimate importance for marine ecological systems close to the shorelines. Several studies dealt with the function of seagrasses as primary producers, shelter and food for fish, turtles and invertebrates as well as spawning areas for these organisms [59, 60, 61]. The reviews existing on seagrasses with different focus than the present one deal in more detail with other aspects of the ecological role of seagrasses, particulary the metabolite classes which are very few and primitive. Seagrasses reported to share the most features of primary and secondary metabolites with respect from the Alismatales order which live in land and freshwater habitats [62]. Kannan and Kannal [63] and Pradheeba et al. [56] reported that primary metabolites such as carbohydrate, protein and lipid content from seagrasses acts as a rich source of nutritional value and was eveidenced by the obvious increase in the carbohydrate content of E. acoroides, T. hemporichii[63], C. rotundata[56] and lipid content of C. nodosa[64]. Higher protein content form seagrasses have also been reported in E. acoroides[63], Ruppia cirrhosa[64], C. serrulata, S. isoetifolium, H. ovalis, H. pinifolia[47], C. rotundataand H. uninervis[56] that are found to accumulate manximum concentration than the seaweeds [47].

Secondary metabolism occurs in seagrasses depends on the season and environmental conditions and was reported as a rich source of diverse natural products from simple to conjugated phenolic compounds such as phenolic acids, flavones, tannins and lignins [65, 66]. It was also reported that P. oceanicaharbors compounds ranging from simple phenol derivatives, phenylmethane, phenyethane and phenypropane derivatives [67]. Athiperumalsami et al. [47] reported the occurrence and absence of alkaloids, antroquinones, catechins, coumarins, flavonoids, phenols, saponins, quinones, tannins and secondary metabolites in the tropical seagrass Cymodocea serrulata, Halodule pinifoliaand Syringodium isoetifolium. Heglmier and Zindron [62] reported 51 natural products in P. oceanicathat includes phenols, flavones, phenylmethane, ethane derivatives. Though reports availed on the seagrass derivatives, limited study dealt with the chemistry of secondary metabolites of seagrasses [62, 68]. The list of metabolites profiled and the metabolic pathways from wild seagrasses are tabulated in Table 4 and Figure 3.

Compound NameMolecular FormulaMolecular weight (g/ mol)Exact Mass (g/mol)
D-GlucoseC6H12O6180.156180.063
MaltoseC12H22O11342.297342.116
D-FructoseC6H12O6180.156180.063
SucroseC12H22O11342.297342.116
InositolC6H12O6180.156180.063
Methyl alpha-D-
Glucopyranose
C7H14O6194.183194.079
D-GalactoseC6H12O6180.156180.063
LactoseC12H22O11342.297342.116
L-RhamnoseC6H12O5164.157164.068
D-RiboseC5H10O5150.13150.053
Adenosine-2′:3′- cyclic
monophosphate
C10H14N5O7P347.224347.063
N-Acetyl-Î-D-glucosamineC8H15NO6221.209221.09
Aspartyl-LeucineC10H18N2O5246.263246.122
GlycineC2H5NO275.06775.032
ThreonineC4H9NO3119.12119.058
ValineC5H11NO2117.148117.079
ProlineC5H9NO2115.132115.063
AlanineC3H7NO289.09489.048
ThiamineC12H17N4OS+265.355265.112
MethionineC5H11NO2S149.208149.051
PhenylanalineC9H11NO2165.192165.079
TyrosineC9H11NO3181.191181.074
Methyl PyroglutamateC6H9NO3143.142143.058
Glutamic acidC5H9NO4147.13147.053
Vanillic acidC8H8O4168.148168.042
Oxalic acidC2H2O490.03489.995
gamma-Aminobutyric acidC4H9NO2103.121103.121
CitrateC6H5O7−3189.099189.004
Stearic acidC18H36O2284.484284.272
Hexadecanoic acidC16H32O2257.422257.244
Potassium GluconateC6H11KO7234.245234.014
Nicotinic acidC6H5NO2123.111123.032
Phosphoric acidH3PO497.99497.977
Sodium PyrophosphateNa4P2O7265.9265.871
AcetamideC2H5NO59.06859.037
Decanedioic acidC12H22O4230.304230.152
Indoleaceteic acidC10H9NO2175.187175.063
1-Napthaleneacetic acidC12H10O2186.21186.068
4-HydroxybenzaldehydeC7H6O2122.123122.037
2,4-dihydroxybenzaldehydeC7H6O3138.122138.032
3,4-DihydroxybenzoicC7H6O4154.121154.027

Table 4.

List of metabolites identified from wild seagrasses.

Figure 3.

Distribution of metabolic pathways of differential metabolites derived from wildseagrass(A) and their active networks (B) (adapted from [53,54]).

The presence of sulphated flavones was reported to be accumulated in Halophilaand Thalassiaspecies [69] and in Z. marina[70], but not found in Syringodiumspp. and P. oceanica. Mc Millan [71] found the presence of either flavones or phenolic acid in 43 segrass species while the sulphated flavones found specifically in five species namely, Zostera, Phyllospadix, Enhallus, Thallasiaand Halophilaspecies. The sulphated flavonoids have also been traced out in Halophila ovalisand Thalassia testudinum[72]. It was found that the flavonoid glycosides and acyl derivatives in P. oceanicaand 15 flavonoid derivatives in Halophila johnsonii[73]. Similar to flavonoids and phenolic compounds, the sterol composition has also been reported from temperate seagrasses than the tropical seagrass. The presence of long cahin fatty acids in P. oceanica[74] and α-hydroxy fatty acids in Z. mulleri[75]. The polar lipids and fatty acids from Z. marinaand Phyllospadix iwatensis [76], phospholipids and glycolipids from Z. marina[77] and steroids, fatty acids from Z. japonica[78] confirms the prevalence of volatile compounds in segrasses.

Jeyapragash et al. [53] investigated the systematic identification and characterization of metabolic changes in wild and SCC of H. pinifoliausing GC–MS based metabolomics approach. It was found that the wild sample accumulated 98 metabolites, while SCC with 125 metabolites along with their relative abundance. The metabolites profiled from wild and SCC was used to map their biochemical pathways. Interestingly, the accumulated metabolites in wild were spanned with 77 primary and secondary metabolism pathways and 73 pathways in SCC. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis confirmed insightful biochemical alterations occurred in wild and SCC such as glutathione metabolism, glutamate metabolism, phenylalanine and tyrosine metabolism in wild with significant variations in citric acid metabolism, glycolysis, gluconeogenesis, oxidative phosphorylation related pathways of SCC respectively. Random forest analysis helped to group top 15 metabolites and their correct classification. The data obtained will provide baseline information on H. pinifoliametabolism in the marine and artificial environments. Furthermore, it helps to study the stress responsive mechanisms of seagrasses in the marine environment which would further aid in the dissection stress tolerance mechanism.

In addition, Jeyapragash et al. [79] reported the heat stress responsive metabolomics analysis of seagrass H. pinifoliain the marine environment. Since, ocean warming is a major global concern in the marine environment, the study focussed to understand the tolerance mechanism of seagrass Halodule pinifoliaunder temperature stress (24, 29, 37, and 45°C) using OMICS approach. Ecophysiologiacal responses such as net photosynthesis (ΔF/F’m) and dark respiration (Fv/Fm) were also studied. Results found that photosynthetic efficiency (ΔF/F’m) significantly reduced due to heat stress, while the rate of dark respiration rate increased as compared to the control (24°C), respectively. Metabolomics study revealed that heat stress could cause huge metabolic alterations with respect to sugar, amino acids and organic acids. Interestingly, thermo-protective compounds such as trehalose (sugar), glycine betaine (amino acid) and methyl vinyl ketone (organic acid) were accumulated from H. pinifolia(45°C) which was the first report on the occurrence of glycine betaine and methyl vinyl ketone from seagrasses and other aquatic species so far. These findings would help the research groups to focus on the gene to metabolite networks mechanism for an effective management of seagrass conservation by genetic manipulation.

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6. Seagrass cell suspension culture

Plants are considered as the factories of chemical compounds produced in order to carry out their biochemical pathways for survival and propagation [80]. All plants produce secondary metabolites which gained importance in pharmaceutical applications since ancient periods. The plant-based drug discovery gained importance with the development of anti-infectious and anti-cancer drugs which contributes to new bioactive molecules that are being isolated for the treatment of other diseases such as diabetes and obesity [81]. However, the important plant derived drugs are obtained commercially by the extraction from their respective plants. Currently, the natural plant habitats are vanishing due to environmental and geopolitical instabilities and so making it very difficult to procure important secondary metabolites and in the process many potential bioactive compounds have been left undiscovered. Plant cell culture is considered as a promising alternative approach for producing the bioactive compounds that are challenging to be obtained by chemical synthesis or plant extraction [82]. Plant cell culture studies have been carried out on the basis of the totipotent nature, in which the cell has the full set of genes necessary for secondary metabolisms [83]. The production of secondary metabolite via plant tissue culture have been commercialized sincelate 1950s, when atropine from the roots of Atropa belladonnawas accumulated in roots and callus [84]. The list of metabolites profiled and their metabolic pathways from SCC of seagrasses were shown in Figure 4 and tabulated in Table 5.

Figure 4.

Distribution of metabolic pathways of differential metabolites derived from SCC ofseagrass(A) and their active networks (B) (adapted from [53,54]).

QueryMatchHMDBPubChem
Rosmarinic acidC18H16O8360.318360.085
Caffeic acidC9H8O4180.159180.042
p-Coumaric acidC9H8O3164.16164.047
Protocatacheuic acidC7H6O4154.121154.027
p-Anisic acidC8H8O3152.149152.047
Vanillic acidC8H8O4168.148168.042
NaringeninC15H12O5272.256272.068
4-hydroxybenzoic acidC7H6O3138.122138.032
Fructose-6-phosphateC6H13O9P260.135260.03
Glucose-6-phosphateC6H13O9P260.135260.03
GlucoseC6H12O6180.156180.063
Phosphoenol pyruvic acidC3H5O6P168.041167.982
Pyruvic acidC3H4O388.06288.016
Citric acidC6H5O7−3189.099189.004
Fumaric acidC4H4O4116.072116.011
3-PGAC3H7O7P186.056185.993
Ketoglutaric acidC5H6O5146.098146.022
Malic acidC4H6O5134.087134.022
Succinic acidC4H6O4118.088118.027
MannoseC6H12O6180.156180.063
Oxaloacetic acidC4H4O5132.071132.071
SucroseC12H22O11342.297342.116
D-FructoseC6H12O6180.156180.063
RaffinoseC18H32O16504.438504.169
TrehaloseC12H22O11342.297342.116
TuranoseC12H22O11342.297342.116
MannitolC6H14O6182.172182.079
InositolC12H22O11342.297342.116
XylitolC5H12O5152.146152.146
AlanineC3H7NO289.09489.048
AspargineC4H8N2O3132.119132.053
Aspartic acidC4H7NO4133.103133.038
Glutamic acidC5H9NO4147.13147.053
GlycineC2H5NO275.06775.032
ProlineC5H9NO2115.132115.063
SerineC3H7NO3105.093105.043
ThreonineC4H9NO3119.12119.058
ValineC5H11NO2117.148117.079
2,4-dihydroxybenzoic acidC7H6O3138.122138.032
2-hydroxybutyric acidC4H8O3104.105104.047
Gamma-aminobutyric acidC4H9NO2103.121103.121
Dimethylamine(CH3)2NH45.08545.058
EthanolamineC2H7NO61.08461.053
ThiamineC12H17N4OS+265.355265.112
Nicotinic acidC6H5NO2123.111123.032
PyridoxineC8H11NO3169.18169.074
PhenylanalineC9H11NO2165.192165.079
TryrosineC9H11NO3181.191181.074
Shikimic acidC7H10O5174.152174.053
Acotinic acidC6H6O6174.108174.016
Xylonic acidC5H10O6166.129166.048
Ascorbic acidC6H8O6176.124176.032
Guanine-2′3’-cyclic
monophosphate
C10H12N5O7P345.208345.047
PantothenateC9H16NO5218.229218.103
SphingosineC18H37NO2299.499299.28
N-acetylglucosamineC8H15NO6221.209221.09
Aspartyl leucineC10H18N2O5246.263246.122
2-hydroxy glutaric acidC5H8O5148.114148.037
Glyceric acidC3H6O4106.077106.027
Chlorogenic acidC16H18O9354.311354.095
RhamnoseC6H12O5164.157164.068
Guanosine monophosphateC10H15N5O11P2443.202443.024
RiboseC5H10O5150.13150.053
Adenosine-2′3’-cyclic
Monophosphate
C10H14N5O7P347.224347.063
Dihydroquercetic acidC15H12O7304.254304.058
Adenosine-2-
Monophosphate
C10H14N5O7P347.224347.063
p-hydroxybenzoic acidC7H6O3138.122138.032
Quinic acidC7H12O6192.167192.063
TryptophanC11H12N2O2204.229204.09
Pyroglutamic acidC5H7NO3129.115129.043
Salicylic acidC7H6O3138.122138.032
MethionineC5H11NO2S149.208149.051
Lactic acidC3H6O390.07890.032
Isovaleric acidC5H10O2102.133102.068
2-oxyglutaric acidC5H6O5146.098146.022
2-hydroxyisobutyric acidC4H8O3104.105104.047
1-methylnicotinic acidC7H8NO2+138.146138.056
HypoxanthineC5H4N4O136.114136.039
Indole Acetic acidC10H9NO2175.187175.063
Naptheline acetic acidC12H10O2186.21186.068

Table 5.

List of metabolites identified from suspension cultured cells seagrasses.

Improved plant cell culture techniques made possible to increase the target metabolite production under in-vitroconditions. However, agronomically focussed biotechnology is capable to make use of plant tissue culture; such methods are not usually available to marine biologist and notably, in-vitroculture of seagrasses has been far more problematic due to lack of the suitable culturing conditions [85]. Available literature has brought that 14% of seagrasses are at an elevated risk of extinction under IUCN red list of threatened species [86]. Though numerous studies have been addressed the in-vitro culture of seagrass [38, 87, 88, 89, 90, 91, 92, 93], no report exist on the metabolomics analysis from suspension cultured cells of seagrass. In-vitropropagation techniques of different seagrass species for restoration and protocol for seagrass protoplast isolation are prevailing [94, 95, 96, 97, 98]. Among all these protocols, Carpeneto et al. [96] was successful in the cell wall regeneration of protoplast from Posidonia oceanicaand Cymodocea nodosa.Recently, establishment of cell suspension culture has been achieved in C. nodosa[99], Halodule pinifolia, C. rotundataand C. serrulata[38]. Jeyapragash et al., [53, 54] reported that the metabolites synthesized from seagrass H. pinifoliain the marine and artificial environment will be highly similar with a total of 98 metabolites in wild and 125 metabolites in SCC respectively and however the cellular suspension accumulated the higher content. The study suggested that the use of seagrass cellular suspension for metabolomics engineering will provide a new facet for novel metabolite identification and characterization. Nevertheless, there lay famine knowledge in the metabolite accumulation pattern and their different biotechnological and pharmaceutical applications. Enhancement of metabolite biosynthesis can also be achieved via the precursors or elicitor treatments of plant cells (Figure 5). Precursors are the compound which act as intermediate in or at the beginning of biosynthetic route, treatments using the same stands a good chance of increasing the yield of the desired product. Exogenous supply of biosynthetic precursors to the culture medium induces the high yield of targeted products (Whitmer et al., 1998; [100]).

Figure 5.

Elicitor induced comparative metabolomics of wild and cellular suspension of seagrass.

Plant also synthesizes the secondary metabolites to protect themselves in response to various environmental stresses. It might be physical, chemical or a biological factor which induces the higher secondary metabolism known as elicitors. The use of elicitors in cell suspension cultures has been developed to enhance the yield of secondary metabolites, wherein elicitation of target compounds can be induced by the addition of trace number of elicitors [101]. Biotic and abiotic elicitors are available which depends on the target compounds that need to be synthesized.

7. Seagrasses—a source for marine based drug discovery

Ravn et al. [102] reported that phenolic acids such as p-coumaric acid, caffeic acid, ferulic acid, protocatacheuic acid, p-hydroxybenzoic acid, vannillic acid, gentisic acid found predominant in Halophila ovalis, Thalassia hemprichii, Halodulesp., Cymodoceaspp., Enhalus acoroides, Syriingodium isoetifoliumand other seagrass species. In addition, the pharmaceutically potent rosmarinic acid has been isolated from Z. marina[102] Z. noltii[103], H. pinifolia[51] and also from the detritus of Z. noltiiand Z. marina[57]. Along with rosmarinic acid, caffeic acid and chlorogenic acid found to be present in some trace amounts, since caffeic acid acts as a precursor for roamarinic acid in the shikimic acid biosynthesis. It was also been reported that the predominance of caffeic acid was accumulated in the leaves of P. oceanica[104] and Thalassodendron ciliatum[105]. Jeyapragash et al. [51], profiled 45 metabolites from Halodule pinifolia, which includes caffeic acid, coumaric acid, chlorogenic acid and rosmarinic acid which found predominant than other compounds. Recently, biofilm associated, multidrug resistant Pseudomonas aeruginosainfection remain a challenging problem in the clinical field since the conventional antibiotic therapy are largely inefficient and new approaches are needed. Inactivating the QS virulence mechanism with anti-infective agent is an attractive approach to prevent bacterial infections without resistance development [54]. Seagrass Halodule pinifolia(Miki) Hartog has been shown to exhibit potential antimicrobial activities against P. aerugonasaPAO1. Preliminary screening on antibiofilm activity showed that the methanolic extract of H. pinifoliaexhibited potential inhibition of biofilm formation (96%) as compared to the control respectively. Eight bioactive compounds such as 4-hydroxybenzoic acid, rosmarinic acid, 4-methoxybenzoic acid, p-coumaric acid, protocatacheuic acid, caffeic acid, naringenin, vanillic acid, were profiled. Of these compounds, 4-methoxybenzoic acid (4-MBA) showed maximum bacterial growth inhibition that act as a lead molecule with minimum inhibitory concentration (MIC). Furthermore, 4-MBA at MIC concentration reduced the virulence factors and down regulated the level of QS mediated virulence transcripts. The study suggests that seagrasses may act as a newer source for the marine based drug discovery and may act as anti-infective agent against biofilm-mediated harmful pathogens.

8. Conclusion

To summarize, experiments in seagrass metabolomics to date helped us to validate a vast array of metabolites and their alterations in response to various stress mechanisms. This approach has previously enabled to recognize a large number of metabolites whose accumulation is affected upon the exposure of organisms under stress conditions. Nevertheless, despite the many advancements that have been achieved in this field, much work is still needed to identify the seagrass metabolites and their novel metabolic pathways connected to stress response and their tolerance mechanism and to interpret the extensive organization and interaction among gene to metabolite networks. This chapter provides knowledge on the systematic identification and metabolic characterization of seagrass metabolites using metabolomics approach. The bioactive potential of compounds derived from seagrasses paves a way to lead as potential inhibitors of many harmful pathogens in the pharmaceutical sectors and therefore, seagrass explored as newer marine source for the development of plant-based drugs. Further, in-vitro cultures of seagrass afford an alternate model for the up-regulation of enhanced bioactive compound synthesis. Moreover, various stress related metabolomics approach of wild seagrasses should be studied in order to derive diverse group of bioactive metabolites as much as possible, so as to fill the knowledge gap of seagrass metabolites and step forward towards the commercialization of bioactive natural products from seagrasses.

Acknowledgments

The authors would be grateful to University Grants Commission - Basic Scientific Research (UGC-BSR) for their funding and thankful to the Centre of Advanced Study in Marine Biology, Annamalai University for their research facility. Our sincere gratitude to Department of Biotechnology, Karpagam Academy of Higher Education for their scientific interactions to complete the work.

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Conflict of interest

The authors declare that there is no conflict of interest.

Abbreviations

GC–MSGas Chromatography–Mass Spectrometry
LC–MSLiquid Chromatography Mass Spectrometry
FT-ICR-MSFourier Transform- Ion Cyclotron Resonance-Mass Spectrometry
SCCsuspension cultured cells
NMRNuclear Magnetic Resonance
4-MBA4-methoxy benzoic acid
MICminimum inhibitory concentration
KEGGKyoto Encyclopaedia of Genes and Genomes
IUCNInternational Union for conservation of Nature
CE-MSCapillary Electrophoresis-Mass Spectrometry

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Danaraj Jeyapragash, Ayyappan Saravanakumar and Mariasingarayan Yosuva (May 19th 2021). Seagrass Metabolomics: A New Insight towards Marine Based Drug Discovery, Metabolomics - Methodology and Applications in Medical Sciences and Life Sciences, Xianquan Zhan, IntechOpen, DOI: 10.5772/intechopen.97875. Available from:

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