Effect of solvents on bioactive compounds and antioxidant activity of Padina tetrastromatica and Gracilaria tenuistipitata seaweeds collected from Bangladesh

Seaweeds are now recognized as a treasure of bioactive compounds. However, the bioactivity of seaweed originating in Bangladesh is still unexplored. So, this study was designed to explore the secondary metabolites and antioxidant activities of solvent extracts of Padina tetrastromatica and Gracilaria tenuistipitata. Phytochemical screening and FTIR spectra confirm the diverse type of bioactive compounds. Antioxidant activity of extracts were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), reducing power (RP), phosphomolybdenum, hydrogen peroxide and nitric oxide (NO) scavenging assays. Here, methanolic extract of P. tetrastromatica showed highest amount of total phenolic content (85.61 mg of GA/g), total flavonoid content (41.77 mg of quercetin/g), DPPH (77.07%), ABTS (77.65%), RP (53.24 mg AAE/g), phosphomolybdenum (31.58 mg AAE/g), hydrogen peroxide (67.89%) and NO (70.64%) assays compared to its methanolic extracts of G. tenuistipitata. This study concluded that methanol as a solvent extract of brown seaweed (P. tetrastromatica) exhibited bioactivity and antioxidant potentiality which will be useful for pharmacological as well as in functional food application.

FTIR analysis. Different solvent extracts (methanol, ethanol and water) of P. tetrastromatica and G. tenuistipitata showed distant peaks that reported various functional groups in the 4000-450 cm −1 range. The existence of phenols, carboxylic acids, alkoxy, aromatic, alkene, amides/amines, and sulfonate compounds was verified by the findings of the FTIR study, which ensured the presence of O-H, N-H, C-H, C=O, C-C, C-N and S=O bonds at different extracts ( Table 2 Table 1. Preliminary phytochemical screening of different extracts of P. tetrastromatica and G. tenuistipitata. "+" constituent's existence, "−" constituent's non-existence. Quantitative phytochemical analysis. Total phenolic content (TPC). The overall amount of total phenols in different crude extracts was measured using FC reagent and external calibration with Gallic acid at a concentration of 7 mg mL −1 . TPC levels varied significantly among solvent extracts, ranging between 85.61 and 34.11 mg of GA/g (Table 3). Methanolic extract of P. tetrastromatica has the particularly maximum level of TPC (85.61 mg of GA/g), followed by ethanol and water extracts (74.59 and 42.73 mg of GA/g, respectively) (p < 0.05) ( Table 3). Additionally, methanol extracts (68.20 mg of GA/g) have the maximum volume of TPC of G. tenuistipitata, followed by ethanol extract (61.65 mg of GA/g) and water extract (34.11 mg of GA/g) (p < 0.05) ( Table 3).
Total flavonoid content (TFC). The aluminum chloride procedure was used to calculate the concentration of total flavonoid content in different crude extracts at a concentration of 7 mg mL −1 . Methanol extract showed significantly highest amount of TFC in the case of both seaweeds, followed by ethanol and water extracts (p < 0.05) ( Table 3).
Evaluation of total antioxidant capacity. DPPH assay. In this process, nitrogen-free radical in the DPPH is readily scavenged by the antioxidant compounds, and the purple color of DPPH solution is cleared by the antioxidants. The findings show that the antioxidant activity of crude seaweed extracts increases dramatically as the concentration of seaweed extract increases (p < 0.05). The percentage of inhibition of methanolic extracts of P. tetrastromatica and G. tenuistipitata (77.08 and 68.54%, respectively) was slightly higher (p < 0.05) than that of ethanolic and water extracts ( Fig. 2A). Compared to the positive control (i.e., ascorbic acid, IC 50 = 0.00297 mg mL −1 ), the IC 50 values of all crude extracts showed lower DPPH radical scavenging effects (Table 4).
ABTS radical scavenging assay. In vitro antioxidant activity by ABTS radical scavenging assay comprises the reaction that results in the formation of a blue-green ABTS chromophore between ABTS and hydrogen donating oxidizing agent, in this case, potassium persulfate. Among the methanolic extracts, P. tetrastromatica recorded significantly higher ABTS free radical scavenging activity (77.65%, IC 50 = 1.33 mg mL −1 ) followed by G. tenuistipitata (66.09%, IC 50 = 3.01 mg mL −1 ) (Fig. 2B). As shown in Table 4, the IC 50 values exhibited the order (methanol > ethanol > water), comparable to extracts with phenolic and flavonoid content. Compared to the positive control (i.e., ascorbic acid, IC 50 = 0.16 mg mL −1 ), the IC 50 values of all crude extracts showed lower ABTS radical scavenging effects (Table 4).
Reducing power assay. The antioxidant activity of altered crude extracts was evaluated using the reducing power assay. This assay is dependent on the hydrogen ion in antioxidants reducing ferric (Fe 3+ ) to ferrous (Fe 2+ ) product, changing the color of the substance to different shades of green to blue depending on the antioxidant function. Here P. tetrastromatica showed significantly higher (p < 0.05) absorbance (A 700nm 0.885-2.927) compared to the absorbance of G. tenuistipitata (A 700nm 0.678-2.047) (Fig. 2C). Ascorbic acid was used as a reference compound to determine the reduction ability of different crude extracts from the seaweed species. The crude methanolic extract had the highest reducing power of all the samples tested of P. tetrastromatica and G. tenuistipitata, and the data were 53.24 and 46.81 mg of AAE/g, respectively.
Phosphomolybdenum assay. To determine the antioxidant ability of extracts, the phosphomolybdenum method is widely utilized. In this method, converting Mo (VI) to Mo (V) forms phosphomolybdenum (V) complex, a bluish-green colored compound in the presence of antioxidant-containing substances. Here P. tetrastromatica showed significantly higher (p < 0.05) absorbance (A 695nm 4.071) compared to the absorbance of G. tenuistipitata (A 695nm 3.369) (Fig. 2D). However, in the context of water extract, G. tenuistipitata showed better absorbance at Table 2. Major functional groups of active components based on the peak value of Fourier transform infrared. "+" constituent's existence, "−" constituent's non-existence.
NO scavenging assay. The antioxidant activity of crude extracts was evaluated using the nitric oxide scavenging assay. Here, methanolic extracts of P. tetrastromatica (70.64%) and G. tenuistipitata (62.18%) had slightly higher (p < 0.05) scavenging efficacy than ethanol and water extracts (Fig. 2F). The scavenging activity showed concen- www.nature.com/scientificreports/ tration-dependent manner for the all the crude extracts of both seaweed. As shown in Table 4, the IC 50 values of NO scavenging ability exhibited the order (methanol > ethanol > water) which is similar to the DPPH, ABTS and H 2 O 2 scavenging activity. Compared to the positive control (i.e., ascorbic acid, IC 50 = 0.0824 mg mL −1 ), the IC 50 values of all crude extracts showed lower NO scavenging effects (Table 4). Correlations between total phenolic contents and total flavonoid contents. The Pearson correlation analysis approach revealed a solid and positive linear correlation between total phenolic content (TPC) and total flavonoid content (TFC) of various seaweed extracts [TPC-TFC: R 2 = 0.9263 ( Fig. 3G)]. Prospective studies show that total phenolic and flavonoid content are significant antioxidant activity determinants in different crude extracts of seaweed.

Discussion
The physiological and mechanical capabilities of marine living beings that permit them to endure in multifaceted living forms give an extraordinary impending generation of secondary metabolites (phytochemicals), which are not observed in earthborn circumstances. Hence, crude extracts of seaweed are amongst the foremost excessive fountainheads of unique, exceptional, and identified bioactive compounds 30 . And the fact that only a few studies have been conducted on Bangladeshi seaweed assets emphasizing its bioactivity or secondary metabolites existences. Hence, it becomes time demanding to be familiar with almost completely unexplored Bangladeshi seaweed assets for way more excellent knowledge of its bio-functional activity as the abundance and accessibility of bioactive compounds of seaweeds are to a significant extent changes concurring to geographic area, natural condition, season, development and fair as the profundity of inundation 29 . In Bangladesh, however, there is a lack of information on available secondary metabolites and antioxidant properties of seaweed. However, other researchers explored Bangladeshi seaweeds, and a variety of phytochemicals and promising antioxidant properties were discovered 16,28 . Hence, in the present study, we used a combination of qualitative and quantitative tests (Phytochemical analysis and FTIR) to screen out for functionally bioactive compounds and determine antioxidant activities using various in vitro spectroscopic assays of different crude extracts of two significant Bangladeshi seaweeds (P. tetrastromatica and G. tenuistipitata). Furthermore, a correlation between TPC, TFC, and antioxidant activity was investigated in order to well appreciative the role of phenols and flavonoids in antioxidant activity. The presence of any phytoconstituents primarily influenced by the dissolvable solvent used for extraction and the seaweed physicochemical properties. The essential bioactive compounds in seaweed can be screened using various methods while keeping different solvents and situations in consideration 28 . In this study, we used methanol, ethanol, and water extracts having a dielectric constant of about 33, 25, and 80, respectively 31 . The phytochemical screening indicates active secondary metabolites such as saponin, terpenoid, cardiac glycoside, phlobatannin, phenolic, and flavonoid in various extracts at different concentrations (Table 1). Among them, terpenoids were absent in the aqueous extract in both seaweed because they are non-polar compounds and required non-polar solvents for extraction 32 . Besides these, all components showed positive results in the methanolic extract of both seaweeds. Our present finding coincides with the findings of other authors; who also found several phytochemicals in case of altered solvents from brown seaweed P. tetrastromatica 20,33 . But in the case of G. tenuistipitata there has been no prior research on preliminary phytochemical screening was found in the literature. However, some scholars also identify several phytoconstituents from red algae G. corticata and G. verrucosa respectively, which the current results validate 34,35 . As indicated by the relevant studies, several phytochemicals in red algae H. musciformis collected in Bangladesh, linked to the current observation 16 .
Fourier transformed infrared spectroscopy (FTIR) can be intended to qualitatively analyze different functional groups in seaweed crude extracts (Fig. 1). FTIR analysis ensured phenols, carboxylic acids, alkoxy, aromatic, www.nature.com/scientificreports/ alkene, amides/amines, and sulfonates in the crude extracts of seaweed (Table 2). Previous researcher also used FTIR to identify several phytochemicals from brown seaweed P. tetrastromatica, associated with our current observation 33,36 . They noticed a different category of compounds in other extracts, which may be attributed to differences in extraction methods and seaweed origin. Also, some scholars identify different functional groups from red seaweed G. rubra and H. musciformis, respectively, which is almost similar to the present findings 16,37 . The incidence of specific fatty acids in various extracts has been observed, determining each extract's antioxidant activity. www.nature.com/scientificreports/ The polarity of any solvent plays a significant role in the extraction of phenolic compounds from some plant or fruit 38 . Since it can suppress polyphenol oxidase activity, methanol is usually the most effective solvent for polyphenolic extraction 39 . Our present study found that methanolic extract contained a significant amount of phenolics, 85.61 mg of GA/g for P. tetrastromatica and 68.20 mg of GA/g for G. tenuistipitata. In contrast, ethanol and water extract contain fewer amounts (Table 3). TPC's current observation is underpinned by the findings of other scholars [40][41][42] , who also reported that methanolic extract when compared to other extracts; extract has the most incredible volume of TPC. Similar results in red and brown seaweed obtained from the Bangladeshi coast 28 . Some academics reported 69.5 and 25.29 mg GAE/g, respectively in the methanolic extracts of P. tetrastromatica, which is a more petite figure than the one we have now 43,44 . This wider variety of results may be attributed to environmental conditions, the origin of the seaweed, or the varietal extraction method. Similarly, methanol extract has the most significant percentage of total flavonoid content for both seaweed species (Table 3). Our results obtained are similar to the case of other researchers, who also found that methanolic extracts showed the maximum quantity of TFC compared to other solvents 16,21,43 .
Flavonoids are natural phenolic compounds having a unique structural characteristic which leads them to a wide range of biofunctional properties, like free radical scavenging and antioxidant properties 45 . It is often difficult to quantify the antioxidant efficacy of any natural extracts as individual studies work through unique specific mechanisms 46 . More specifically, different methods such as PTIO and nitroprusside-Griess reagent are being used for performing NO scavenging assay 47 . In our present study, different in vitro antioxidant assays including DPPH, ABTS, reducing power assay, phosphomolybdenum, H 2 O 2 scavenging and NO scavenging assay were performed to evaluate antioxidative properties of the crude extracts of two seaweeds. The antioxidant efficacy of altered crude extracts increased with increasing concentration, showing that these properties are dose dependent. The influence of the amount of bioactive phytochemicals might be responsible for higher antioxidant activity with the increase of concentration. In the case of all antioxidant assays, it was observed that brown seaweed (P. tetrastromatica) showed higher activity compared with the red seaweed (G. tenuistipitata). An approach similar to ours has been presented earlier 28,48 . The antioxidant capacity of the crude extracts ordered in the rank methanol, ethanol and water extract ( Fig. 2A-F), which is similar with the findings of other researchers 16,[49][50][51] . This is due to the fact that methanol extracts can have an H-donating property, allowing them to stop the oxidation process by transforming free radicals to stable compounds. However, the highest effect was observed for ethyl acetate fraction in the case of P. pavonica 52 , ethyl acetate and petroleum ether fraction in the case of G. verrucosa 53 and aqueous extract in the case of P. boergesenii 54 which are contradictory to the present findings. These differences might be due to the variation in solvent used for analysis and the differences in the analytical method. Here, TPC and various antioxidant assays of seaweed extracts shown a strong positive linear correlation. Other scholars also documented a similar positive linear correlation amongst TPC and various antioxidant activities of seaweed extract 16,28,55 . Though, other researcher observed a negative correlation between TPC and antioxidant activity (activity of lipid peroxidation inhibition) in the case of red seaweed which test was not performed in our current research 56 . Furthermore, total phenolics and flavonoid content of various seaweed extracts had a similar positive correlation which is in agreement with the results of other researchers 28,57 . Our present finding evidently recommends the existence of phenolic or flavonoid compounds in methanolic extracts may be primarily responsible for the result of the highest antioxidant activity in the crude extracts.

Methods
Seaweed sample collection and preparation. Mature G. tenuistipitata and P. tetrastromatica sample were collected from the wild source at Saint Martin's Island (92° 28′ 40.12″ E and 20° 65′ 51.43″ N) of Bay of Bengal of Bangladesh in March 2020. Saint Martin's Island is still considered a biologically diverse ecosystem free of external pollutants, with a dense growth of various seaweeds. Permission of sample collection was gained from the local government before harvesting seaweed following local and national regulations. In this experiment, samples of two different species of seaweeds (one red and one brown) were commonly found in the rocky surfaces during low tide. Dr. Md. Enamul Hoq, Former Director of BFRI, authenticated the botanical identification of seaweed species as the voucher specimen has been previously deposited at BFRI herbarium [BFRI (MFTS-RS-18/19-034) and BFRI (MFTS-BS-18/19-048)]. The entire plant was collected from the exposed rock www.nature.com/scientificreports/ to ensure that the holdfast would not be left out. The collected thallus was washed thoroughly with clean seawater to remove dirt, sand, and other impurities. The specimen was preserved in an icebox at 4 ℃ and transported to the laboratory to maintain the fresh quality. Fresh samples were then washed thoroughly with distilled water for further removal of any other remaining impurities. Cleaned seaweed was then kept in a freeze dryer (VaCo 2, Zirbus, UK) for 48 h at − 83 ℃ to remove the moisture. Dried samples were sealed in plastic bags and stored in a refrigerator at 4 ℃ for further analysis in the laboratory. Qualitative analysis of phytochemical substances. Newly prepared all crude extracts of seaweed were subjected to qualitative assessments for the identification of various classes of active phytochemical constituents such as saponin 58 , terpenoid 59 , cardiac glycosides 60 and phlobatannin 16 following standard methods. General reactions in these analyses exposed the presence or absence of these compounds in the crude extracts tested.
FTIR spectroscopy. Different crude extracts of P. tetrastromatica and G. tenuistipitata were used to determine the presence of characteristic peaks and their functional groups using FTIR spectroscopy (Perkin Elmer Spectrum 2) [61][62][63] . FTIR spectra were recorded within the wavelengths of 450 and 4000 cm -1 . Analysis was done in triplicate and confirmed the spectrum in case of all extracts.
Quantitative analysis of phytochemicals. Total phenolic content (TPC). This parameter was carried out in the crude extracts using Folin-Ciocalteu Phenol reagents and external calibration with Gallic acid following by 41 with slight modification. Briefly, 0.5 mL extract solution was added with 0.1 mL of FC reagent solution. After 15 min, 2.5 mL of saturated Na 2 CO 3 (75 g L −1 ) was added in the solution and allowed to stand for 30 min at RT and absorbance was measured at 760 nm using the spectrophotometer (T80 + UV/Vis Spectrophotometer, UK). The concentration of total phenolics was calculated as mg of Gallic acid equivalent per gram. The calibration equation for Gallic acid was Total flavonoid content (TFC). This parameter was computed in the crude extracts using the aluminum chloride colorimetric method with minor modifications 43   www.nature.com/scientificreports/ by 50%. Ascorbic acid was used as a positive control. The percent radical scavenging activity of the crude extracts was calculated using the following formula: where: A 0 is the absorbance of DPPH radicals + methanol and A 1 is the absorbance of DPPH radicals + sample extract.
ABTS radical scavenging assay. The antioxidant activities of different extracts were evaluated through the ABTS radical scavenging by the extracts ability to scavenge ABTS with slight modification 49 . Aliquot concentrations (1, 3, 5 and 7 mg mL −1 ) of extracts (50 µL) was added with 950 µL of ABTS solution (7 mM ABTS solution and 2.45 mM Potassium persulfate) followed by incubation at RT for 16 h in dark. Spectrophotometer (T80 + UV/ Vis Spectrophotometer, UK) was applied to evaluate the absorbance at 734 nm. IC 50 values were tested for each sample at each concentration. Ascorbic acid was used as a positive control. The percentage of inhibition was calculated using the following formula, where: A control is the absorbance of ABTS radicals + solvent and A sample is the absorbance of ABTS radicals + sample extract.
Reducing power assay. Antioxidant activity of different crude extracts reducing power at various concentrations with insignificant modification 16 . Briefly, 1.5 µL of extracts was mixed with 1.5 µL of phosphate buffer (0.2 M, pH 6.6) and 1.5 µL of potassium hexacyanoferrate (1%, w/v). After incubation at 50 °C in a water bath for 20 min, 1.5 µL of trichloroacetic acid solution (10%) was added and centrifuge at 800×g for 10 min. The supernatant was collected and mixed with 3 mL of DW and 200 µL of ferric chloride solution (0.1%, w/v) and incubated at RT for 10 min for stable absorbance at 700 nm; as the more absorbance of the reaction mixture more the reducing power of the extracts will be. Here ascorbic acid was used as a positive control. Antioxidant activity was also expressed as equivalents of ascorbic acid.
Phosphomolybdenum assay. The antioxidant activity of different extract solution (water, ethanol and methanol) was evaluated by the green phosphomolybdenum complex formation with slight modification 15 . A reagent solution was prepared with 0.6 M H 2 SO 4 , 28 mM Sodium phosphate and 4 mM Ammonium molybdate. Further, 1.8 mL reagent solution was mixed with 0.2 mL of dilute extract solution and placed in a boiling water bath for 90 min at 95 °C. After cooling down, the absorbance of each sample was measured at 695 nm using spectrophotometer (T80 + UV/Vis Spectrophotometer, UK). Blank was run same procedure just replacing the extract with the equivalent solvent. Antioxidant activity was also expressed as equivalents of ascorbic acid.
Hydrogen peroxide scavenging activity. Extracts antioxidant activities were evaluated by the hydrogen peroxide scavenging activity with slight modification 28 . Briefly, aliquot extracts at various concentrations was added 0.3 mL hydrogen peroxide solution (40 mM) and 1.2 mL phosphate buffer (40 mM; pH 7.4) and vortexes well. Different concentrations were tested for each sample to get IC 50 value. Ascorbic acid was used as a positive control. The percentage of inhibition of the crude extracts was calculated using the following formula: where: A 0 is the Absorbance of control and A 1 is the Absorbance of sample solution.
NO scavenging assay. Different crude extracts antioxidant activity was evaluated by the NO scavenging ability with slight modification 14 . Briefly, 50 µL of extracts was mixed with 450 µL of sodium nitroprusside (SNP, 10 mM) and incubated the mixture at room temperature for 4 h. Further, 450 µL of griess reagent was added to the mixtures. After 10 min the absorbance was measured at 546 nm using spectrophotometer (T80 + UV/Vis Spectrophotometer, UK). IC 50 values were tested for each sample at each concentration. Ascorbic acid was used as a positive control. The percentage of inhibition was calculated using the following formula, where: A 0 is the Absorbance of control and A 1 is the Absorbance of sample solution.
Statistical analysis. The obtained experimental data was analyzed through the standard statistical procedure. Data were analyzed using SPSS software (IBM Co., Chicago, IL). Analysis of variance (ANOVA) and Duncan's multiple range method were used to compare solvents and samples. Values were expressed as means ± standard deviations. Differences were considered significant at p < 0.05. All analyses were performed in triplicate.