Metabolic variations in seaweed, Sargassum polycystum samples subjected to different drying methods via ¹H NMR-based metabolomics and their bioactivity in diverse solvent extracts

2.1 Plant material and samples preparation

S. polycystum samples were collected by hand during low tide (<0.09 m) on reef flats, approximately 100 m from the shore at Teluk Kemang near Port Dickson, Negeri Sembilan, Malaysia (2°26′N, 101°51′E). Collected seaweed samples were cleaned of extraneous materials like epiphytes, sand particles, pebbles and shells using soft bristle brush and thoroughly washed with sea water. The clean samples were then placed in transparent polyethylene bags (inside chilled plastic containers) and transported to the research facilities of the laboratory of Marine Biotechnology, Institute of Bioscience (IBS), Universiti Putra Malaysia (UPM) Serdang, Malaysia. In the laboratory, the samples were further washed thoroughly with tap-, followed by distilled water.

2.2 Drying process

The washed seaweeds were divided into 3 groups for drying process, including air-drying at room temperature; freeze drying and vacuum oven-drying. Throughout the drying procedure, all samples were dried until constant weight was attained. The air-dried (AD) samples were exposed to ambient temperature of 25 °C for 6 days. The vacuum oven dried (OD) samples were prepared by spreading samples evenly in a single layer on trays and placed in a vacuum oven (Memmert USA) and dried at 40 ± 5 °C, maintained for 29 h. The freeze dried (FD) samples were prepared by subjecting fresh samples to over- night freezing at −80 °C in a freezer (Thermo Scientific) followed by lyophilizing in a freeze dryer (Labconco FreeZone, USA) until constant weight was attained. All dry samples were subsequently ground into fine powder with a laboratory-scale blender, and then sieved using a 200 µm-sized sieve. The fine powdered samples were collected in screw-capped bottles and stored in refrigerator until further use.

2.3 NMR measurement and multivariate data analysis

A 500 MHz Varian INOVA NMR spectrometer (Varian Inc., Palo Alto, CA, USA), functioning at a frequency of 499.887 MHz at ambient temperature (25 °C) was employed to determine 1H NMR and J-resolved experiments. Samples were prepared according to the method described by Kim et al. (2011). A 50 mg sample was weighed in a 2.0 mL Eppendorf tube. A total of 0.75 mL of a 1:1 mixture of methanol‑d₄ and potassium dihydrogen phosphate buffer (pH 6.0) in deuterium oxide containing 0.1% trimethylsilylpropionic acid-d4 sodium salt was added. The mixture was vortexed (1 min), ultrasonicated (20 min) and centrifuged (10,000 rpm, 10 min) at ambient temperature. The supernatant (0.6 mL) was transferred to NMR tube to run NMR analysis by subjecting it to 1H NMR measurement. A pre-saturation sequence was applied to eliminate the residual water signal. The resulting spectra were manually phased and baseline corrected using the Chenomx software (v.5.1, Alberta, Canada) with a consistent setting for all spectra. Spectral intensities were binned by equal width (δ 0.04) corresponding to the region of δ 0.50–10.00. The regions of δ 4.70–4.90 representing water and δ 3.23–3.36 representing residual methanol were excluded.

2D J-resolved experiment was determined for additional support in the metabolite identification. Multivariate data analysis by principal component analysis (PCA) was performed with the SIMCA-P software (v. 13.0, Umetrics, Umeå, Sweden) using scaling based on Pareto. Metabolite identification was determined by comparing the identified metabolite peaks with the Chenomx NMR Suite 7.7 library.

2.4 Samples extraction

The dry brown seaweeds powdered was dissolved with 50 mL methanol, and vortexed for 5 min. This solution was extracted by sonication for 30 min using ultrasonic water bath (Power Sonic 505, Korea) at ambient temperature. The solvent extract was then filtered through Whatmann No. 1 filter paper while the dry residue re-extracted with another 50 mL of methanol for a second round of extraction. The extraction process was subsequently repeated in the same manner for a third round of extraction. The filtered extracts were pooled and evaporated to dryness using a rotary evaporator (N-1001S-WD, with EYELA Oil Bath OSB-2000, Japan) at 30 °C, and stored at −20 °C until further analysis. The described seaweed powder extraction procedure was repeated using other solvents i.e., 70% ethanol, acetone, chloroform and hexane. For each solvent tested, the extraction was replicated six times. The concentrated extracts were stored in amber bottles in a freezer for future utilization.

2.5 Phytochemical screening

Phytochemical examination was carried out for all the extracts according to standard protocols. For terpenoids, extract was dissolved in 2 mL of chloroform and evaporated to dryness. To this, 2 mL of concentrated H₂SO₄ was added and heated for about 2 min. A greyish color indicated the presence of terpenoids. The presence of steroids was indicated by development of a greenish coloration by mixing extract with 2 mL of chloroform. Then 2 mL of each of concentrated H₂SO₄ and acetic acid were poured into the mixture. In tests for phenols and tannins, extract was mixed with 2 mL of 2% FeCl₃ solution. A blue-green or black coloration indicated the presence of phenols and tannins. Detection for flavonoids was conducted by dilution of extract with 2 mL of NaOH and the mixture turned to intense yellow coloration. Once HCl was added, the solution became colorless. As for saponins, extract was mixed with 5 mL of distilled water in a test tube. This was shaken vigorously. Formation of stable foam was considered as indication for the presence of saponins.

2.6 Total phenolic content (TPC) assay

The phenolic content in S. polycystum was estimated using Folin-Ciocalteu methods as described by Teoh et al. (2013). Approximately 100 μL of Folin-Ciocalteu’s reagent and 80 μL 7.5% (w/v) of sodium carbonate were added to 20 μL of aqueous extract of various concentrations in a well of a 96-well plate. After 30 min of incubation at ambient temperature in the dark, the absorbance was read at 765 nm against blank (solvent without extract) using ELISA plate reader (Thermo multiskan go). Quercetin was used as positive control and a standard curve was plotted using gallic acid (0–1 mg/mL). The results are expressed as milligram gallic acid equivalent (mg GAE)/100-gram dry weight of samples using the following equation: - $$\text{C = c}\times \text{V/m}$$ where,

C: Total phenolic content (mg GAE/g plant extract)

c: concentration of gallic acid (mg/mL)

V: volume of extract (mL)

m: weight of extract

2.7 Antioxidant activity

2.8 Antibacterial activity

Antibacterial activity was carried out by placing 6 mm diameter of paper disc containing antibiotic onto a Mueller Histon Agar plate where microbes were grown. The bacteria strain was taken from Microbial Culture Collection Unit (UNiCC), Institute of Bioscience, University Putra Malaysia. The microbe culture was standardized to 0.5 McFarland standards, which was approximately 10⁸ cells. Streptomycin was used as standard for each bacterium. The plates were inverted and incubated at 37 °C for 18–24 h, 24–48 h or until sufficient growth had occurred. After incubation, each plate was examined. The diameters of the zones of complete inhibition were measured including the diameter of the disc. Zones were measured to the nearest whole millimeter, using sliding calipers or a ruler, which was held on the back of the inverted petri plate.

2.9 Statistical analysis

All the assays for Antioxidant activities were carried out in six replicates while other assays in triplicates. The values were expressed as mean ± standard deviation. IBM SPSS Statistic 23 was used for the experiment and one-way Analysis of Variances (ANOVA) was used to compare the mean values of intensity. Then the analysis proceeded through Turkey’s multiple range test statistical significance at p ≤ 0.05.

3.1 ¹H NMR spectra of the dried materials and metabolite identification

Discrimination of the metabolite variations among the air, freeze and oven-dried seaweed samples was done by employing ¹H NMR metabolomics analysis. Fig. 1 shows the ¹H NMR spectra of seaweed extracts under different drying conditions whereas Fig. 2 shows the ¹H NMR spectra with identified metabolites. These metabolites were identified based on the 2D J-resolved (Fig. 1) and comparison with the NMR spectra of the reference compounds measured under the same conditions as extracts. All the spectra showed signals in the aliphatic (δ 0.5–3.0), carbohydrate (δ 3.0–5.5) and aromatic region (δ 5.5–8.0).

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Fig. 1

¹H NMR spectrum of seaweed at different drying methods.

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Fig. 2

¹H NMR spectrum of seaweed.

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A total of 12 metabolites were identified and their characteristic signals are shown in Table 1. Valporic acid, propylene glycol, 3-hydroxyisovaleric acid, acetyl aspartic acid and trimethylamine were presence in aliphatic region between δ 0.92 and δ 2.92. Valporic acid and 3-hydroxyisovaleric acid were detectable at δ 0.92 (s) and δ 1.25 (s), respectively. A doublet peak was observed at δ 1.14 (d, J = 5.0 Hz) suggested appearance of propylene glycol. The presence of N-acetyl aspartic acid and trimethylamine were observed at δ 2.00 (s) and δ 2.92 (s), respectively. Malonic acid, dimethylsulfone, choline, betaine, sarcosine and N-acetylglycine were ascribed based on the signals displayed in the carbohydrate region of δ 3.0 – δ 5.5. The presence of malonic acid was confirmed by the peaks at δ 3.14 (s) whereas peaks of dimethylsulfone was detectable at δ 3.15 (s). Choline and betaine were detectable at δ 3.23 (s) and δ 3.26 (s), respectively. A singlet peaks observed at δ 3.62 suggested the presence of sarcosine. Other metabolite detectable in carbohydrate region was N-acetylglycine, which was detected at δ 3.76 (d, J = 10.0 Hz). Characteristic peaks of formic acid were detected at δ 8.43 (s).

3.2 Discrimination of metabolites among the three drying methods on seaweed samples

Identification of metabolites variations among the three drying methods i.e. air, freeze and oven-dried seaweed samples was done by principal component analysis (PCA). PCA was employed to cluster the features of the samples and analyze the metabolites that contributed to the variations on the seaweed samples. Air, freeze and oven-dried seaweed samples were clearly discriminated as illustrated in Fig. 2a. PC1 shows the most sample variations, followed by PC2. The first two principal components (PC1 and PC2) cumulatively accounted for 79.4% of the total variations. Separation of air, freeze and oven-dried seaweed samples in score plots were achieved by combining PC1 and PC2. PC1 shows the separation between freeze-dried, air, and oven-dried samples whereas PC2 shows the separation between air-dried, freeze and oven-dried samples.

The metabolites in the air, freeze and oven-dried seaweed samples which were illustrated using PCA are shown in the loading line plot for PC1 (Fig. 3b) and PC2 (Fig. 3c). As shown in the loading line plot for PC1 (Fig. 2 b), N-acetylglysine, sarcosine, betaine, choline, dimethylsulfone and malonic acid were the most abundant in freeze-dried samples compared to air and oven-dried. Loading line plot for PC2 (Fig. 3c) illustrated that air-dried seaweed samples contained higher level of betaine, dimethylsulfone and acetyl-aspartic acid compared to oven-dried seaweed samples. Formic acid, trimethylamine, 3-hydroxyisovaleric acid, propylene glycol and valporic acid level were found higher in oven-dried than freeze-dried samples. Sargassum sp. is highly regarded due to its active metabolites that exhibit numerous functions in biological activities. Changes in environmental patterns and processing methods may alter metabolic content. For instance, decreased metabolites level in the air-dried samples was expected due to degradation by air oxidation and thermal effects. Residual oxidative enzymes during the drying process may have influenced the decreased level of metabolites in the samples, as explained by Mediani et al. (2013).

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Fig. 3

(a) PCA score plot (component 1 vs component 2); (b) Loading line plot of component 1 of ¹H NMR data for comparison among the air, freeze and oven-dried seaweed samples and (c) Loading line plot of component 2 of ¹H NMR data for comparison among the air, freeze and oven-dried seaweed samples. For the interpretation of the numbers assigned to the metabolites in the loading line plot, reference is made to Table 1.

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Oven-drying of plant samples at 44.5 °C has been reported as one of the easiest and rapid thermal processing methods that could preserve phytochemicals (Azwanida, 2015; Mediani et al., 2012). Unsurprisingly, formic acid, trimethylamine, 3-hydroxyisovaleric acid, propylene glycol and valporic acid were observed in oven-dried samples. It has been reported that freeze drying method can avoid degradation of metabolites in samples due to the low temperature applied (Kim et al., 2011). Therefore, several metabolites such as N-acetylglysine, sarcosine, betaine, choline, dimethylsulfone and malonic acid were present in large quantities in the freeze dried samples. It was observed therefore that freeze drying was the most efficient process to preserve most of the potentially beneficial metabolites in the samples, hence formed the basis for recommendation as the most suitable processing method.

3.3 Phytochemical screening

By preliminary phytochemical screening, seven different chemical compounds (steroid, phenol, tannins, saponins, flavonoids, terpenoids and glycosides) were investigated in five different extracts (Table 2). Of the (5 × 7 = 35) tests for presence or absence of the above compounds, a total of 27 tested positive, whereas only eight gave negative results. The 27 positive results include steroid, saponins and glycosides to be present in all five solvent extracts tested. Steroid, saponins and terpenoids showed the maximum presence in all five different extracts. Next to this was phenol in 4 extracts. Among the five different extracts, methanol extract revealed the presence of maximum number of 7 compounds followed by chloroform and acetone extracts at 6 compounds each. The 70% ethanol extract revealed five compounds and hexane extract showed only three compounds. The results of phytochemical investigation of various extracts of S. polycystum revealed the presence of various secondary metabolites at varied degrees. Generally, seaweeds are known as medicinal plants, rich in metabolites and have been extensively studied and used in the pharmaceutical industry.

In the present study, the presence of tannins in all extracts of S. polycystum was demonstrated. Many tannin-based drugs are used in medicine as astringent (Jeeva et al., 2012). Tannins are used in the production of leather and ink and also for treating wounds and burns (Tiwari et al., 2011; Oludare and Bamidele, 2015). Tannins have also been found to have antimicrobial properties as they are able to bind to adhesives, play roles in enzyme inhibition, substrate deprivation and membrane distruption (Guo et al., 2018). Saponins have specific biological activities such as anticancer, antiinflammatory, antimicrobial and antioxidant properties (Sidana et al., 2016; Güçlü-Ustündag and Mazza, 2007). Saponin also has the property of precipitating and coagulating red blood cells (Yadav and Agarwala, 2011). Flavonoids are hydroxylated phenolic substances that are reported for their response during antioxidant activity (Sytar et al., 2018). Flavonoid activity is probably due to their ability for scavenging hydroxyl radicals, superoxide anion radicals and lipids peroxy radicals are vital for diseases prevention related to oxidative damage of cell, membrane and protein. Steroids have been reported to possess antibacterial properties and they are very important compounds due to their relationship with compounds like sex hormones (Yadav & Agarwala, 2011). The presence of steroids in all extracts was also shown in the present study. Glycosides are non-carbohydrate moiety known to be used as food additives, prebiotic and biopreservatives (Cuaves et al., 2015). Terpenoids have been reported to possess cytotoxicity against a variety of cancer cells and cancer prophylactic (Huang et al., 2012).

3.4 Total phenolic content

The total phenolic content (TPC) of the 70% ethanol, methanol, acetone, chlorofom and hexane extracts of S. polycystum were determined, expressed in mg GAE/100 g and shown in Fig. 4. TPC for all solvent extracts was highest at concentration 3 mg/mL, ranging from 218 ± 47.93 mg GAE/100 g in chlorofom extract to 627 ± 50.81 mg GAE/100 g in the 70% ethanol extract. The 70% ethanol extract had highest TPC, which was significantly higher than in methanol extract (484 ± 14.74 mg GAE/100 g), followed by hexane extract (357 ± 153.26 mg GAE/100 g) and acetone extract (238 ± 48.66 mg GAE/g extract), while chloroform extract revealed the least TPC. Results of the present study revealed that in terms of the content of total phenolics, the 70% ethanol and methanol were the favourable extraction solvents, in addition to their hygienic features (Moure et al., 2001). Phenolic compounds are commonly found in plants, including seaweeds (Duan et al., 2006) and largely soluble in polar solvents, as they contain polar phenolic hydroxyl groups. Consequently, polar solvents such as ethanol, methanol and water were more efficient to extract phenolic compounds from seaweed, which comprise complex matrix of compounds including sugar, saponins, glycosides, organic acids, tannins, salts, and mucus (Waterman and Mole, 1994; Cho et al., 2007).

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Fig. 4

Total phenolic content for Sargassum polycystum crude extract from different solvents expressed in mg GAE/100 g. Data presented are based on the mean of three replicates of each of the solvent systems (acetone, chloroform, hexane, methanol, 70% ethanol) ± standard deviation (SD).

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The results of this study clearly demonstrated that different extraction solvents from polar to non polar yield diverse total phenolics content due to differences in their chemical composition and strucutures. Besides, total phenolics content in the present study was much higher compared to previous study on green and red seaweed (Chew et al., 2008). This could be due to phlorotannins, which are mostly found in brown seaweeds (Machu et al., 2015; Chew et al., 2008). Phlorotannins in brown seaweed mainly consist of phloroglucinol, eckol and dieckol with the presence of multiple phenolic groups (Suleria et al., 2015). They are integral structural components of cell walls which play reproductive roles in algal reproduction and exhibit therapeutic properties such as antioxidant, anticancer, anti-aging and anti-inflammatory (Machu et al., 2015).

3.5 Antioxidant activity

3.5.1

3.5.1 DPPH radical scavenging activity assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging antioxidant assay has been used extensively as a free radical to evaluate the antioxidant potentials in plant extracts (Duan et al., 2006; Farasat et al., 2014). DPPH assay is considered a valid, accurate, easy, and economical method to evaluate radical scavenging activity of antioxidants, since the radical compound is stable and needs not be generated. The new methodology for evaluation of antiradical efficiency towards DPPH was proposed, which is advantageous over other methods (Sanchez-Moreno et al., 1998). The DPPH method considers not only the antioxidant concentration, but also the time taken for the scavenging reaction to reach a plateau. The results of the evaluation are highly reproducible and comparable to other free radical scavenging methods (Gil et al., 2000). Seaweed with antioxidant molecules are able to reduce the DPPH free radicals by attacking the molecules to add hydrogen atom to it or donate to it an electron, which results in the DPPH solution changing colour from purple to yellow (Indu and Seenivasan, 2013). It has been reported that brown seaweed rich in natural bioactive compounds like carotenoid, fucoxanthin, phenolic and flavonoids have higher antioxidant potentials in comparison with red and green seaweeds (Leelavathi and Prasad, 2014; Cox et al., 2010; Kindleysides et al., 2012). The observation of increase in free radical scavenging activities in a dose-respont manner in the 70% ethanol, methanol, acetone, chloroform and hexane extract from S. polycystum as shown in Fig. 4. The determination of free radical scavenging activity of S. polycystum extract at different concentrations is shown in Fig. 5, where ascorbic acid was used as the standard. All sample tested show antioxidant activities and possessed prominent antioxidant properties.

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Fig. 5

DPPH scavenging activities of S. polycystum at different concentrations (0–3.0 mg/mL) determined spectrophotometrically at 517 nm. Data presented are based on the mean of three replicates in each of the solvent systems (acetone, chloroform, hexane, methanol, 70% ethanol) ± standard deviation (SD).

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In the present study, all the extracts depicted higher tendency to scavenge the DPPH radicals in a dose dependent manner and steadily increased with increase in extract concentrations with DPPH reduction ranging from 8.2 to 61.4% up to concentration of 3 mg/mL except for acetone and hexane extract. At the concentration of 3 mg/mL, the 70% ethanol extract show the highest activity compared to other extract (61.4 ± 0.171%) which suggested that the compound extracted by ethanol possess more potential to scavenge DPPH free radicals due to the higher concentration of phenolic content present in the extract as discussed above. Previous studies reported that ethanolic and aqueous extracts of Sargassum spp. including S. horneri, S. macrocarpum and S. siliquastrum produced more than 60% of DPPH radical scavenging activity (Boonchum et al., 2011; Matsukawa et al., 1997). Meanwhile, chloroform extract (37.3 ± 0.089%) exhibit slightly higher activity than methanol extract (34.2 ± 0.250%) followed by hexane extract which was obtained at 16.6 ± 0.194%). Acetone extract exhibited the least DPPH inhibition (8.2 ± 0.007%) at that concentration while the control (ascorbic acid) attained the half maximal inhibitory concentration (IC₅₀) at concentration of 0.028 mg/mL. The variance in the DPPH radical scavenging activity of each sample studied suggests that the different extracting solvents used may have been affected the radical scavenging activity as the different polarities of each antioxidant compound groups existing in the seaweeds (Marinova and Yanishlieva, 1997). Methanol is the best choices of solvent in the determination of free radical scavenging activity via DPPH assay (Marinova and Batchvarov, 2011), where polyphenols from water were best extracted using polar solvents (Moure et al., 2001). However, the solubility of the antioxidant compound group and phytochemical composition of the solvent used for the seaweed extraction also played crucial roles as demonstrated in the antioxidant activities (Naczk and Shahidi, 2006; Rattaya et al., 2015).

3.6 Antibacterial activity

Seaweed contains epiphytic bacteria that are excellent source of natural antimicrobial and antioxidant compounds (Horta, et al., 2014). Many researches have been reported on the antimicrobial activity of Sargassum sp. extract against gram-positive as well as gram-negative bacteria (Patra et al., 2008; Zubia et al., 2008; Yoon et al., 2010). However, differences between results of the other studies could be due to production of bioactive compounds related to the seasons and area, drying methods, organic solvents used for extraction of bioactive compounds and differences in assay methods. The drying stage is crucial because fresh seaweed contains volatile antimicrobials (terpenoid and bromo-ether compounds, hydrogen peroxide and volatile fatty-acids) that may be lost due to high temperature (Mendes et al., 2013). In the present study, antibacterial study of the 70% ethanol, methanol, acetone, chloroform and hexane extract from S. polycystum was tested against Gram-positive bacteria (Staphylococcus aureus (ATCC 43300) and Bacillus subtilis (B29)) and Gram-negative bacteria (Pseudomonas aeruginosa (ATCC 15442) and Escherichia coli (UPMC 25922). The inhibition zone towards selected bacteria were presented in Fig. 6 and Table 4. The 70% of ethanol, acetone, chloroform, and hexane extract showed inhibition zone towards Staphylococcus aureus (ATCC 43300) with moderate inhibition zone between 10 mm and 15 mm while hexane showed antibacterial activity with <10 mm. It was found that 70% of ethanol extract showed inhibition zone towards all bacteria tested compared to other extraction solvents, which suggested that ethanol was the best solvent for extracting the antibacterial active constituents from the S. polycystum. Meanwhile, the absence of zone of inhibition in other extraction solvents (methanol, acetone, chloroform and hexane) may be due to low concentration of antibacterial active metabolites, which cause the resistance towards microorganisms to the seaweed extract.

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Fig. 6

Antibacterial activity of Sargassum sp. with different solvent, M = methanol, H = hexane, A = acetone, C = chloroform and E = 70% ethanol.

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The antimicrobial inhibitory activity rely and depends on the solvent used to extract antibacterial compounds within the seaweed. The influence of antioxidant activity of phenolic compounds prevents microbial growth effectively by binding to their cell surfaces and affect metabolism of the bacteria (Glombitza, 1979; Reguant et al., 2000). It is believed that other phytochemicals from S. Polycystum such as flavonoids, tannins and terpenoid which can be extracted by polar solvents, may play crucial roles in antibacterial activity. However, the activity may also depend on the concentration of antibacterial active metabolites.