Biochemical and Anti-proliferative activities of seven abundant tropical red seaweeds confirm nutraceutical potential of Grateloupia indica

2.1 Collection of seaweeds samples and preparation of extracts

Tropical seven red seaweeds; Gracilaria corticata (GC; family Gracilariaceae), Grateloupia indica (GI; family Halymeniaceae), Kappaphycus alvarezii (KA; family Solieriaceae), Solieria robusta (SR; family Solieriaceae), Amphiroa anceps (AA; family Lithophyllaceae), Halymenia porphyriformis (HP; family Halymeniaceae), and Sarconema scinaioides (SS; family Solieriaceae) were collected from Saurashtra coast (N. 22° 28′ 8.19″; E. 69° 04′8.24″) of Arabian Sea (Tanna et al., 2019). All collected samples were processed as described previously and stored for further analysis. Dried seaweed powder (10 g) was extracted overnight with aqueous methanol (70%, v/v) and supernatant was collected by centrifugation at 7000 g for 10 min. Supernatant was concentrated using rotary evaporator (100–150 mbar at 37 °C), lyophilized and stored at −20 °C until further use.

2.2 Estimation of total antioxidant activity

Total antioxidant of the red seaweed extracts were analyzed by measuring scavenging capacity of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) free radicals and compared with standard trolox (Hazra et al., 2008; Re et al., 1999). Briefly, ABTS free radicals were generated by mixing ABTS diammonium salt (7 mM) with potassium persulphate (2.45 mM) in dark followed by incubation for 12–16 hrs at room temperature. ABTS free radicals (1 ml; OD₇₃₄ = 0.70 ± 0.02) was mixed with various concentration (200, 400, 600, 800 and 1000 μg ml⁻¹) of seaweed extracts or standard (1–5 μg ml⁻¹ trolox), incubated for 90 min and absorbance was read at 734 nm. ABTS scavenging activity was compared with standard trolox and percentage inhibition was calculated.

2.3 Estimation of radical scavenging activity

DPPH (2,2-diphenyl-1-picrylhydrazyl) is used for the study of scavenging activity (Saeed et al., 2012). About 1 ml of DPPH solution (0.024%, w/v in methanol; OD₅₁₇ = 0.98 ± 0.02) was mixed with different concentration of seaweed extracts (200, 400, 600, 800 and 1000 μg ml⁻¹) or trolox, incubated overnight and absorbance was read at 517 nm. The scavenging activity was determined by comparing the absorbance of extracts with standard trolox (Mishra et al., 2015; Patel et al., 2016).

2.4 Estimation of reducing power

The reducing power was determined using ascorbic acid as standard (Tanna et al., 2018; Tanna et al., 2021). Different concentration of seaweeds extracts (100, 200, 300, 400 and 500 μg ml⁻¹) was mixed with 1 ml of phosphate buffer (0.2 M, pH 6.6) and 1 ml of K₃Fe(CN)₆ (10 mg ml⁻¹). The reaction mixture was incubated at 50 °C for 20 min, 1 ml TCA (100 mg l⁻¹) was added to terminate the reaction. Supernatant was collected by centrifugation at 7000 g for 10 min at room temperature. Freshly prepared FeCl₃ (0.2 ml; 0.1%, w/v) was added to 1 ml of supernatant, incubated for 10 min at room temperature, absorbance was read at 700 nm, and reducing power was calculated.

2.5 Estimation of total phenolic and flavonoid contents

Total phenolic content (TPC) and total flavonoid content (TFC) were estimated with the help of gallic acid and quercetin as a standard, respectively (Tanna et al., 2019; Saeed et al., 2012; Zhishen et al., 1999). Different concentration of seaweed extracts (200, 400, 600, 800 and 1000 μg ml⁻¹) was mixed with Folin-Coicalteu reagents (2.5 ml; 0.2 M), incubated for 90 min, 2 ml sodium carbonate (Na₂CO₃; 75 g l⁻¹) was added, further incubated for 90 min in dark, and the absorbance was read at 760 nm. Total phenolic content was calculated using a standard curve of gallic acid and represented as mg ml⁻¹ gallic acid per 100 mg of extract. Similarly, different concentration of seaweed extracts (200, 400, 600, 800 and 1000 μg ml⁻¹) was mixed with 0.3 ml NaNO₂ (5%, v/v), incubated at room temperature for 5 min, 0.3 ml AlCl₃ (10%, v/v) and 2 ml NaOH (1 M) were added, and absorbance of the reaction mixture was read at 510 nm. TFC was determined as mg ml⁻¹ quercetin per 100 mg of extract from a standard curve.

2.6 Cell culture and anti-proliferative activity

The human hepatoma cancer cell line (Huh-7) and human cervical cancer cell line (HeLa) were seeded in 96-well plate with a concentration of 10⁴ cells in each well and incubated at 37 °C under 5% CO₂ in the air for 24 h. Seaweed extracts were added and incubated further for 4–6 h. MTT based assay was performed for the study of anti-proliferative activity following the previously optimized protocol (Patel et al., 2018; Patel et al., 2019; Tanna et al., 2020). Absorbance was taken at 570 nm and following equations were used to calculate the percent anti-proliferative activity: $$\mathit{Anti}-proliferativeactivity\left(\%\right)=100\times \left[1-\frac{({\mathit{abs}}_{570(sample)}-{\mathit{abs}}_{690(blank)})}{({\mathit{abs}}_{570(control)}-{\mathit{abs}}_{690(blank)})}\right]$$

2.7 Extraction and analysis of metabolites

Previously optimized method was used for the extraction and quantification of metabolites (Pandey et al., 2015; Patel et al., 2020; Yadav et al., 2021). Briefly, dried seaweed samples were extracted with methanol (ice-cold) for 10 min at 70 °C. Supernatant was collected by centrifugation 11000 g for 10 min at 4° C, dried and proceed for derivatization (Mishra and Jha, 2009). Metabolites adonitol/ribitol was used as an internal standard for quantification, which was performed by GC–MS. Mass spectra peaks were compared with the library (NIST– National Institute of Standards and Technology) for the identification, whereas quantification was done using internal standard. Metabolite data were analyzed using partial least squares-discriminant analysis (PLS-DA) (Yadav et al., 2021].

2.8 Statistical analysis

All experiments were done in triplicates and three biological replicas were there in each experiment. Analysis of variance (ANOVA) was applied to all data and threshold for statistical significance was considered p < 0.05, all values are represented as mean ± SD (standard deviation of the mean), and significant difference is shown by different letters. Correlation studies and multivariate analysis were performed using principal component analysis and Pearson’s correlation matrix.

3.1 Biological activities of red seaweed–extracts

All red seaweed–extracts showed dose dependent biological activities including total antioxidant, scavenging, and reducing activities (Fig. 1). Seaweeds, GI (70 ± 0.5 %) and AA (62 ± 0.3 %) have high total antioxidant activity at 400 μg concentration. Whereas, GC and HP showed 70 + 0.5 % and 65 ± 0.5 % total antioxidant activity at 1000 μg concentration. Seaweeds, SS and KA showed high antioxidant activity of 64 ± 0.7 % and 56 ± 0.3 % with 400 μg and 1000 μg concentration of extract, respectively (Fig. 1A). High scavenging activity, about 68 ± 0.3 % was observed with GI extract at 1000 μg concentration. Though >50% scavenging activity was noticed with AA (55 ± 1 %) and SS (52 ± 0.6 %) at 600 and 800 μg concentration, respectively. But, both (AA and SS) have shown similar about 58 ± 0.6 % scavenging activity at 1000 μg concentration. Other seaweeds, HP, SR and GC have shown comparatively less activity, about 51 ± 1, 49 ± 0.6 and 46 ± 0.4 %, respectively at 1000 μg concentration (Fig. 1B). Seaweed GI showed the highest reducing activity 40 ± 1 % at 100 μg concentration. Overall, SS has been observed to have the highest reducing activity of 68 ± 0.1 % with 500 μg concentration. The reducing activity was followed by AA, GI, GC and HP with 63 ± 0.2, 57 ± 0.5, 45 ± 0.4 and 43 ± 1%, respectively at 500 μg concentration (Fig. 1C).

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Fig. 1

Different biological activities of selected tropical red seaweeds. (A) Total antioxidant, (B) scavenging, and (C) reducing activity. All activities are shown as mean ± SD (n = 3) and different small letters show a statistically significant difference at p < 0.05 (Tukey test).

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For all above biological activities, half maximal concentration (EC₅₀) was calculated for each red seaweed–extract (Fig. 2). Seaweed GI showed the highest antioxidant activity with the lowest EC₅₀ values of 120 ± 4 μg ml⁻¹ followed by SS (190 ± 10 μg ml⁻¹), whereas the lowest activity was observed with SR as it showed the highest EC₅₀ (770 ± 6 μg ml⁻¹). Seaweed, GI also showed the highest scavenging activity with EC₅₀ 656 ± 1 μg ml⁻¹, followed by AA (672 ± 4 μg ml⁻¹). The lowest scavenging activity was found with KA extract (EC₅₀ 1235 ± 20 μg ml⁻¹). In case of reducing power capacity, the lowest EC₅₀ was observed with SS (237 ± 3 μg ml⁻¹), followed by GI (294 ± 4 μg ml⁻¹) and AA (306 ± 2 μg ml⁻¹). Seaweed KA also showed the lowest reducing activity with the highest EC₅₀ (938 ± 18 μg ml⁻¹).

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Fig. 2

Half maximal effective concentration (EC₅₀; μg ml^-1) of red seaweeds for different biological activities. All activities are shown as mean ± SD (n = 3) and different small letters show a statistically significant difference at p < 0.05 (Tukey test).

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3.2 Total phenolic and flavonoids contents

The maximum amount of total phenolic content was found with seaweed GI (80 ± 10 mg ml⁻¹ per g extract), followed by AA and SS which contained 20 ± 2 and 15 ± 2 mg TPC per ml per g of extract, respectively (Fig. 3A). Similarly, the highest flavonoid content is also detected with GI (617 ± 72 mg ml⁻¹ per g extract) followed by AA and SS, in which 472 ± 113 and 408 ± 103 mg TFC per ml per g of extract were determined (Fig. 3B). The lowest TPC (8–10 mg ml⁻¹ per g extract) was found in SR, HP and KA; whereas KA also have the lowest TFC (352 ± 110 mg ml⁻¹ per g extract).

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Fig. 3

TPC and TFC of tropical red seaweeds. (A) TPC is shown as GAE (mg ml^-1) per gram of extract. (B) TFC is shown as quercetin equivalent (mg ml^-1) per gram of extract. All data are shown as mean ± SD (n = 3) and different small letters show a statistically significant difference at p < 0.05 (Tukey test).

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3.3 Correlation analysis

A correlation analysis was performed individually for all seven red seaweeds (Table S1). A very strong (0.9–1.0; R² > 0.9) significant (p < 0.05) correlation was observed among all biological activities (total antioxidant, scavenging and reducing) and contents (TPC and TFC) for red seaweeds AA, GC, GI, HP and SR. Seaweed SS also showed strong significant correlations (0.9–1.0; R² > 0.9; p < 0.05) among biological activities and content, except TFC, which showed moderate (0.5–0.7; R² = 0.3–0.6) insignificant (p > 0.05) correlation with other biological activities including TPC. Seaweed KA showed strong significant correlation (0.9–1.0; R² > 0.9; p < 0.05) amongst TPC, total antioxidant and scavenging activities. However, reducing activity showed week (0.35–0.45) while TFC showed strong (0.6–0.7) correlations with other biological activities.

3.4 Anti-proliferative activity

Seaweeds, HP and GI were found to have the highest proliferation-inhibition activities (about 40 ± 0.5%) on HeLa cells. Similarly, GI and KA showed the maximum 44 ± 2% proliferation-inhibition on Huh-7 cells (Fig. 4). Other red seaweeds showed low anti-proliferation activities; GC and AA showed 32–35%, and SR and KA showed 26–28% proliferation-inhibition on HeLa cells. Similarly, seaweeds, AA, GC, HP, SR and SS showed<40% proliferation-inhibition on Huh-7 cells.

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Fig. 4

Proliferation–inhibition of tropical red seaweed extracts on carcinoma cell (HeLa and Huh-7) lines using MTT assay. All data are shown as mean ± SD (n = 3) percent inhibition and different small letters show a statistically significant difference at p < 0.05 (Tukey test).

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3.5 Multivariate correlation analysis

A cumulative multivariate correlation analysis was performed between biological activities and phytochemical composition (Table S2). Overall total antioxidant of tropical red seaweeds showed a significant (p < 0.5) and very strong (0.9–1.0) correlation with scavenging activity (0.940; R² = 0.833), TFC (0.870; R² = 0.757) and proliferation–inhibition of Huh-7 cells (0.708; R² = 0.501). Overall total antioxidant activity showed a significant strong (0.7–0.9) correlation with reducing activities (0.792; R² = 0.627), and a moderate (0.4–0.7) correlation with TPC (0.628; R² = 0.395) and proliferation–inhibition of HeLa cells (0.538; R² = 0.290). Similarly, overall scavenging activity was strongly correlated with reducing activity (0.763; R² = 0.582), TFC (0.897; R² = 0.804) and proliferation–inhibition of Huh-7 cells (0.801; R² = 0.642). Within phytochemical composition, TPC showed a strong correlation with TFC; whereas TFC not only showed strong correlation with TPC but also with antioxidant and scavenging activities.

A cumulative principal component analysis of different biological activities and contents were performed for red seaweeds (Fig. 5). The biplot inferred from PCA showed 82.42% variation (PC1), and categorized red seaweeds based on their biological activities. The analysis revealed that GI has nutraceutical potential as it showed high biological activities including total antioxidant, total scavenging, anti-proliferative activity of HeLa cells and also have maximum TPC.

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Fig. 5

A cumulative principal component analysis. A Bi-plot of different biological activitiesof red seaweeds deduced from the Pearson correlation matrix.

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3.6 Metabolite profiling

A total of 57 different metabolites were detected in red seaweeds (Table 1). These metabolites were grouped in the different categories; about 25 types of sugars, 15 fatty acids, 9 sugar derivatives, 7 different amino acids and one polyol were quantified. Maximum 39 different metabolites were detected in SS followed by 23 in GI, 22 in HP, 21 in GC and AA, 19 in KA, and lowest 13 was found in SR. It was also observed that seaweed SS have maximum 12 exclusive metabolites, followed by 4 unique metabolites owned by GI, HP contained 2 while GC have 1 specific metabolites. No exclusive metabolites were detected in other seaweeds AA and SR. Sugar mannobiose was abundantly detected in seaweeds GI (5600 ± 500 µg g⁻¹ DW) followed by AA (4000 ± 250 µg g⁻¹ DW) and SS (2500 ± 150 µg g⁻¹ DW). Seaweed GC contained maximum glucose (11160 ± 185 µg g⁻¹ DW), and KA contained maximum galactose (1275 ± 260 µg g⁻¹ DW), while no abundance of sugars were found in HP and SR seaweeds. Amino acid valine was detected in all seaweeds in the range of 0.5–7 µg g⁻¹ DW. Among fatty acids, palmitic acids were detected in all seaweeds with maximum amount 83 ± 7 µg g⁻¹ DW in GI, followed by SS (40 ± 5 µg g⁻¹ DW). About 20 µg g⁻¹ DW palmitic acids were estimated in seaweeds HP and KA, 17 ± 6 µg g⁻¹ DW palmitic acids was found in AA. Comparatively low amount of palmitic acid was detected in seaweeds SR (10 ± 2 µg g⁻¹ DW) and GC (5 ± 2 µg g⁻¹ DW). Interestingly, GC contained high amount of heptanoic acid (252 ± 43 µg g⁻¹ DW) and octanoic acid (123 ± 7 µg g⁻¹ DW). Metabolites allose, galactonic acid, glucuronic acid and pinitol were exclusively detected in GI. Similarly, glucose and tagatofuranose were found exclusively in GC and KA, respectively. Two sugar-metabolites lyxose and ribofuranose were detected exclusively in HP. About 12 different metabolites, decanoic acid, dodecanoic acid, lignoceric acid, octadecenoic acid, allopyranose, mannitol, sucrose, tagatose, talose, trehalose, xylose and arabinitol were observed exclusively in seaweed SS.

A score plot was inferred using PLS-DA (Partial Least Squares Discriminant Analysis) for metabolites which were commonly detected in all seaweeds (Fig. 6). PLS-DA plot showed two important key measures; one is variable importance in projection (VIP) and the second is the weighted sum of absolute regression coefficients. The colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each red seaweed group. A hierarchical cluster analysis was done and a heat map was inferred using Spearman’s rank correlation coefficients (Supplementary Figure S1). The deduced heat map showed differential accumulation of metabolites in different red seaweeds.

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Fig. 6

A score plot inferred by partial least squares discriminant analysis for metabolites commonly detected in all red seaweeds. AA: Amphiroa anceps, GC: Gracilaria corticata, GI: Grateloupia indica, HP: Halymenia porphyriformis, KA: Kappaphycus alvarezii, SS: Sarconema scinaioides, and SR: Solieria robusta.

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