3. Qualitative and Quantitative Aspects of Algal-Derived Biocompounds
Phytochemical profiling of algal samples by advanced analytical techniques revealed the presence and relative amounts of different phytochemicals, many with important medicinal properties (e.g., antimicrobial, anti-inflammatory, antioxidant)
[8][24]. Preliminary qualitative phytochemical analysis was carried out to identify the secondary metabolites such as alkaloids, flavonoids, terpenoids, steroids, tannins, phenols, quinones, glycosides, flavanones, flavonols, steroids, and saponins present in the alcoholic/aqueous extracts of marine algae
[68][69][70]. The variation in the antimicrobial and antioxidant activities were due to various parameters at the time the algal samples were collected. These parameters include the (i) presence and relative number of secondary metabolites (of phenolic or free hydroxyl nature) in algae, (ii) method of extraction of the biocompounds and the solvent used in this extraction, (iv) maturity stage of algae, and (v) environmental conditions (e.g., habitats, seasons)
[70][71][72].
Qualitative colorimetric methods were used to evaluate the phytocompounds, and among the different procedures, methanolic extracts were found to have the highest reducing power in comparison with other solvents, such as ethanol, chloroform, and acetone
[70][73]. However, results remain controversial among different studies and seem to be species-specific
[70]. The maximum content of phenolic compounds, such as tannins and flavonoids, has been found in red and brown seaweeds
[68]. Hasan et al. showed that
Hypnea musciformis and
Enteromorpha intestinalis algae collected from the Bay of Bengal possessed high contents of polyphenols associated with high potential of antimicrobial activity
[69].
Other phytochemical screenings of different algal extracts were assessed using standard methods. An FeCl
3 test for tannins in methanolic extracts was assessed for brown seaweeds (i.e.,
Dictyota dichotoma and
Sargassum wightii), green seaweeds (i.e.,
Cladophora glomerata,
Ulva lactuca, and
Ulva reticulata), and red seaweeds (i.e.,
Jania rubens,
Corallina mediterranea, and
Pterocladia capillacea), and the results revealed that tannins are common phytocompounds in seaweeds
[68][70]. These algal species can be used as a drug for gonorrhea and as healing agents, and seem to exert anti-viral, anti-bacterial, and anti-ulcer activities
[50][65]. A Mayer test was used to qualitatively identify the contents of alkaloids in
Dictyota dichotoma,
Jania rubens,
Cystoseira mediterranea, and
Pterocladiella capillacea [68]. These are important as antimicrobial agents to inhibit the growth of both Gram-positive and Gram-negative bacteria
[70]. Flavonoids, flavonols, quinones and glycosides, flavanones, saponins, and steroids were evaluated qualitatively using the Shinoda test, NaOH test, foam test, and Liebermann–Burchard test, respectively, in different algal species to analyze their therapeutic values
[73][74]. In addition, an NaOH test was employed to detect the higher quantity of coumarins in Rhodophyta species (i.e.,
Gracilaria salicornia and
Mastophora rosea), which, because of their peculiar physicochemical features, were found to display an anticoagulant activity to treat lymphedema
[75]. Moreover, saponins and steroids were analyzed through this method in Chlorophyta species (i.e.,
Halimeda cuneata and
Pseudocodium devriesii) and Phaeophyta (i.e.,
Pelvetia wrightii and
Dictyota dichotoma)
[68][70].
Quantitative analysis of flavonoids, tannins, and phenolics are usually carried out using aluminum chloride assay, 2,2-azinobis 3-ethylbenzothiazoline-6-sulfonate (ABTS) radical scavenging assay, hydroxyl radical scavenging assay, Fe
2+ chelation assay, and Folin–Ciocalteu reagent (FCR) methods
[69][73][76].
As evoked earlier, marine algae also possess a range of macro- and micro-elements required by humans and animals, such as Ca, Na, Mg, K, P, I, Fe, and Zn
[72][77]. Semiquantitative and discriminant analyses were used to calculate different percentages of such elements (e.g., Ca, Mg, Na, and K), even within the same group of seaweeds, to differentiate the type of seaweed according to their quantitative mineral levels
[77]. For instance, K is known to be present in high proportions in some Phaeophyta species (e.g.,
Padina arborescens,
Hizikia fusiforme, and
Sargassum thunbergia), while Ca was in high proportion in other Phaeophyta species (e.g.,
Scytosiphon lomentaria and
Sargassum tortile). In addition, Mg was found in relatively high quantities in Chlorophyta (e.g.,
Ulva conglobata,
Ulva pertusa, and
Enteromorpha compressa), and Chlorine (Cl) was predominantly found in
Pseudocodium devriesii,
Gracilaria Salicornia, and
Mastophora rosea [72][77][78].
Each algal extract obtained is generally mixed with impurities and consists of one or multiple components; therefore, analysis using separation techniques is very important
[74]. Different analytical techniques such as high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), and one or more combined techniques, such as high performance liquid chromatography–mass spectrometer (HPLC–MS), gas chromatography–mass spectrometry (GC–MS), and high performance liquid chromatography–diode array detection (HPLC–DAD) were used for the identification of bioactive compounds from algal extracts
[8][35][36][63][79][80].
Carotenoids and chlorophylls are the most exploited fraction of algae pigments. Due to the lipid peroxidation ability of carotenoids in tissues, in-vivo studies of different biomass extracts were important
[65][80]. Furthermore, the total antioxidant activity of carotenoid extracts has been evaluated by UV–Visible (UV–Vis) spectrophotometric methods and/or enzymatic assays
[36]. In addition, carotenoids and chlorophylls were quantified by HPLC–photodiode array (HPLC–PDA), identifying all-trans-zeaxanthin, all-trans-lutein, all-trans-β-carotene, all-trans-α-carotene, chlorophyll-α, chlorophyll-β, pheophytin-α, and hydroxychlorophyll-α in the green microalgae
Chlorella sorokiniana and
Scenedesmus bijuga [36][80]. Furthermore, HPLC-PDA-MS/MS, HPLC equipped with UV detectors, and MS/MS were used for identification and/or quantification of the carotenoids from algal biomass spectrometry
[19][79][81]. Liquid chromatography–mass spectrometry (LC–MS) coupled with PDA and MS showed a high sensitivity for carotenoids and carotenoid esters detection
[19]. To investigate antioxidant and anti-cancer properties, the analysis of carotenoids (e.g., β-carotene) has been performed by HPLC–UV/Vis or HPLC–DAD
[79][81]. Moreover, for liquid-liquid extracts (analysis done by dissolving the dry extract in the compatible solvents) and the identification of compounds (e.g., astaxanthin, canthaxanthin), HPLC–DAD represents a powerful technique
[51].
HPLC is the most sensitive method and is extensively used to separately identify a wide range of compounds like flavonoids and lipids
[10][36][81]. Thus, to obtain an adequate measure of the antioxidant potential of individual molecules, pre-column reaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with ultra-HPLC (UHPLC) separation was used
[62][82][83]. Thereby, isoflavonoids, a class of flavonoids, can be structurally distinguished from other flavonoids using HPLC. Isoflavonoids present in brown (e.g.,
Undaria pinnatifida, Sargassum muticum, and
Sargassum vulgare) and red (e.g.,
Hypnea spinella, Halopytis incurvus, Chondrus crispus, and
Porphyra sp.) seaweed species were analyzed using modified methodologies of UHPLC–MS/MS
[73][81]. In addition, the DPPH free radical scavenging method in cooperation with UHPLC–PDA analysis revealed the presence of two radical scavenging xanthophyll fragments, namely diadinoxanthin and diatoxanthin
[84]. Furthermore, HPLC was found to be an alternative method for lipid analysis because it can potentially resolve all the various classes of lipids in crude lipid extracts
[81]. Furthermore, HPLC–MS can be used to obtain a more detailed picture of lipid species within each class
[79][81]. When using HPLC, sample pretreatment is important; therefore, methanol was used for dissolving the residue, while fat-soluble impurities were extracted with hexane
[85]. In some cases, normal phase HPLC coupled in parallel to an evaporative light-scattering detector (ESLD) and quadrupole MS was used to detect a large amount of saturated hydrocarbon in crude lipid extracts
[19][81].
In most cases, especially for analytical research and the development of nutraceuticals, it is necessary to evaluate the suitability of the analytical techniques. Algal lipid quantification is generally carried out based on indirect methods, such as Nile red fluorescence or related dye-partition assays, gravimetric measurement of crude lipid extracts, or GC analysis of lipid-derived fatty acid methyl ester (FAME)
[51][81][85]. Numerous anomalies can affect neutral lipid quantification, including distortions due to β-carotene, complex kinetics of the fluorescent signal, and issues with sensitivity or specificity. Nile red fluorescence is visibly specific for lipid droplets, and is used as one of the most popular methods of algal lipid analysis
[81].
GC/MS and NMR techniques are also used for lipid analysis
[81]. GC is a popular method used on its own and/or in combination with various detection techniques such as PDA, UV, MS, MS/MS, HPLC, electron capture detector (ECD), and flame ionization detector (FID)
[79]. With GC analysis, acyl constituents and FAME, derived from both neutral and polar lipids, can be selectively analyzed in each lipid extract
[85]. Algal-derived FAs, as methyl or ethyl esters, could be then analyzed by LC–MS and/or GC–FID
[85]. Moreover, post-methylated lipid analyses can be carried out using GC–MS. Reversed-Phase HPLC (RP–HPLC) was a widely applied analysis method, but this technique fails to separate highly polar compounds from the less polar ones
[36]. Therefore, capillary electrophoresis (CE) using DAD (CE–DAD), which shows shorter application time, higher efficiency, and selectivity, is used as a substitute method to RP–HPLC for fast SFE extracts characterization
[36].
NMR, MS, HPLC–MS, HPLC–UV–MS, and GC–MS have been applied to perform a pharmaceutical-grade analysis of biocompounds. For terpenes, GC–MS or NMR were found to be applied for structural determination. GC coupled to an electrospray ionization (GC–ESI) and GC–MS analyses are very selective for identification of heat-labile components (e.g., volatile materials, hydrocarbons, and FAs) in phytoextracts
[36][79]. 1D- and 2D-NMR, MS/MS, HPLC, and chiral GC–MS analyses are preferred for structure evaluation
[63][84]. Proton NMR (
1H NMR) spectroscopy has gained attention as a good analytical tool for structural analysis of polysaccharides (including determination of monosaccharide constituents, partial depolymerization by reductive hydrolysis, identification of disaccharide repeating units) and sequence analysis by enzymatic degradation due to its advantages of simple calibration, easy application, and fast optimization of the experiment
[36][63][79]. However, this technique was only suggested for chemical identification and not quantification, due to possible structural irregularities, which could lead to misleading and complex signals. The linkage positions of carbohydrates and the linking relationships are determined concomitantly with heteronuclear single-quantum correlation spectroscopy (HSQC) and heteronuclear multiple bond correlation spectroscopy (HMBC)
[36][84][86]. Globally, hydrocarbons characterization is mainly done by GC/MS and NMR
[87].
Thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA) were used to analyze thermal properties of polysaccharides, lipids (from supercritical extracts), and algal proteins, which can be quantified by determining the nitrogen content using Kjedhal analysis
[36][86].
Infrared (IR) spectrometry is a common analysis technique used to identify functional groups present in algal extracts
[79]. Thereby, glycoprotein structural details (e.g., sugars attached to the protein via (1→4)-linked β-galactose residues and β-linked glucose residues) have been elucidated using Fourier-transform infrared (FTIR) and NMR spectra
[54][80]. Furthermore, glycoproteins obtained from
Codium decorticatum were purified and characterized using HPLC, IR, NMR, and Circular Dichroism (CD)
[15]. Generally, IR-KBr plate (mixing the powder sample with potassium bromide (KBr) and then pressing it into a disc mode) helped to identify algae’s (e.g., Ulvan’s) chemical components
[36][86]. Further, attenuated total reflectance-FTIR (ATR-FTIR) and Raman spectroscopy techniques are used to identify agar and other polysaccharides sources of seaweeds
[63]. Spirulina is an important edible alga with increasing commercial interest, and a faster and more highly efficient analytical platform was introduced to qualitatively and quantitatively characterize
Spirulina pigments in different dietary supplements
[87]. Thereby, analysis of the
Spirulina pigment fraction was possible through a highly complex and developed analytical strategy, consisting of Fourier-transform ion cyclotron (FT–ICR) in both direct infusion (DIMS) mode or coupled with UHPLC. This strategy was used to accurately identify and overcome failures of conventional LC–MS-based methods (e.g., low separation efficiency, long analysis time, and low mass accuracy)
[79][87].
TLC can be employed to elute extracts of chlorophyll α and multiple carotenoids, such as β-carotene, oscillaxanthin, zeaxanthin, β-cryptoxanthin, echinenone, and myxoxanthophyll
[87]. The TLC method evaluates both quantitatively and qualitatively extracted algal components (e.g., hydrocarbons) among different solvents (mobile phases such as acetic acid/hexane/acetone/diethylamine/diethyl ether) and temperatures
[36][87].
Several chromatographic methods, such as TLC, HPLC, GC, high-performance anion-exchange chromatography-pulsed amperometric detector (HPAEC–PAD), and CE, have been used for the separation and selective analysis of agaro-oligosaccharides (AOS)
[35][84].
ESI and matrix-assisted laser desorption/ionization (MALDI) have advanced the structural analysis of AOS and carrageenan oligosaccharides (COS). Different fragmentation patterns were obtained by ESI-tandem MS due to sulfation substitution allowing researchers to selectively detect COS among other polysaccharides
[35][63]. Thereby, detailed oligosaccharide information, such as accurate molecular weight, chain length distribution, fragments information, monosaccharide compositions, linkages, and location of various modifications, has been identified
[35]. Recently, MS has been used as a powerful detection tool for elucidating the oligosaccharide structure due to its sensitivity
[79][84].
For the quantitative analysis of toxins, LC–MS/MS methods have proven their efficiency, although they are limited for multi-component analyses (MCA)
[36].