1 Introduction

Polysaccharides derived from seaweeds are important free-radical scavengers and anti-oxidants in the prevention of oxidative stress in living animals [1]. Positive anti-oxidant systems are known to shield marine algae from oxidative stress, and among them, marine algae remain one of the richest potential anti-oxidant sources [2]. Furthermore, byproducts of marine food production can be easily transformed into nutraceuticals and functional food ingredients with antioxidant properties [3]. The majority of research on marine-derived antioxidants has only examined the efficacy of crude extracts, with only a minimal amount of purification and characterization of such antioxidants [4].

Several studies were then conducted to confirm the antioxidant properties of seaweeds [5,6]. However, seaweed polysaccharides are thought to be a moral foundation of anti-oxidants [7]. Sulfated polysaccharides from the marine algae (seaweed) Laminaria japonica [8], Ecklonia kurome [9], Fucus vesiculosus [2], Porphyra haitanesis [10], and Ulva pertusa [11] have been shown to have antioxidant properties over the years. There have been few studies on the relationship amid the structure and antioxidant activity of sulfated polysaccharides derived from seaweeds. Glucan and non-glucan polymer free radical scavenging activities discovered that polyelectrolytes like glucan sulfate or phosphate may boost scavenging activity [12].

Furthermore, seaweed-derived sulfated polysaccharides have been shown to have anticoagulant activity comparable to or greater than heparin [13]. Furthermore, an anticoagulant sulfated polysaccharide from Monostroma nitidum (Chlorophyceae) was reported to have six times the activity of heparin [14]. In comparison, red and green seaweed extracts have lower anticoagulant activity than seaweed extracts [15, 16]. Recently, methanol extract of the red seaweed G. lithophila reported the antibacterial activity against some clinical pathogenic bacteria [17]. In addition, rhamnose-enriched polysaccharide from G. lithophila proved potent anti-diabetic activity against STZ-induced rats [18]. Keeping in mind the importance of seaweed polysaccharide as an antibacterial, antioxidant, anticoagulant, and antidiabetic activity, the current study attempted to determine the in-vitro antioxidant and anticoagulant effects of crude polysaccharide and α-L-Rhamnose derived from G. lithophila.

2 Material and Methods

2.1 Seaweed collection, identification, and pre-treatment

The Seaweed G. lithophila was collected during the low tide period from the intertidal region (up to 0.5 to 2 m depth) from the coastal area of Tranquebar (Lat. 11°01′ N; Long. 79°51′ E) in Tamil Nadu, Southeast coast of India. The seaweed was identified using the standard systematic key described by Umamaheswara rao [19]. The seaweed was washed with seawater followed by tap water, distilled water, and then shadow-dried. The dried seaweed was powdered in a mixer grinder for further extraction.

2.2 Isolation crude polysaccharide

The isolation of crude polysaccharides was carried out following the procedure described by Seedevi et al. [20]. Briefly, 50 g of seaweed powder was dipped in 1:50 volumes (2.5 L) of distilled water and kept at room temperature for 2 h, then homogenized and refluxed at 100 °C for 2 h. After cooling, the mixture was filtered using cheesecloth; further the supernatant was separated from the algal residue by centrifugation at 6000 rpm for 20 min. The residue was further extracted thrice (three times) with double-distilled water at 100 °C for 2 h. All extracts were pooled and concentrated under reduced pressure in a rotary evaporator and dialyzed in cellulose membrane (molecular weight cut off 12,000 kDa) against distilled water for three successive days. The retained fraction was freeze-dried in a lyophilizer (Penguin Plus −4 kg, Lark India) and stored in a refrigerator

2.3 Isolation of α-L-Rhamnose

Seedevi et al. [20] described the isolation of α-L-Rhamnose from G. lithophila. The crude polysaccharide was dissolved in distilled water (10 mg/10ml), centrifuged at 6000 rpm for 10 min, and the supernatant was applied to a Q-Sepharose™ (GE-Heathcare) Fast Flow column (EX 150 mm × 50 cm) equilibrated with distilled water. The column was eluted stepwise with distilled water, with a linear gradient of 0–3 mol/ NaCl at a flow rate of 0.55 ml/min and 15 fractions were pooled together, dialyzed against distilled water, and then freeze-dried. Furthermore, the semi-purified polysaccharide was further purified by gel permeation chromatography using a High-Resolution Sepharose 4-LB Fast Flow column (EX 100 mm × 25 cm), equilibrated with distilled water. The column was eluted stepwise with distilled water; with extend of 0.2 mol/l sodium acetate buffer at an elution rate of 0.33 ml/min. The purified fraction was dialyzed against distilled water and then freeze-dried

2.4 Chemical and structural characterization of crude polysaccharide and α-L-Rhamnose

The chemical and structural characterization of crude polysaccharide and α-L-Rhamnose of G. lithophila were described previously by Seedevi et al. [20]

2.5 Antioxidant activity

2.5.1 Total antioxidant activity

Total antioxidant activity was assessed using the described method of Arivuselvan et al. [21]. In a flash, 2.0 ml of crude polysaccharide and α-L-Rhamnose at various concentrations (50–250 μg/ml) were combined with 1.0 ml of 0.6 M sulfuric acid, 28mM sodium phosphate, and 4mM ammonium molybdate. The mixture was incubated in a water bath at 95 °C for 90 min. Then the mixture was cooled to room temperature and the absorbance of each solution was measured at 695 nm. As standards, L-ascorbic acid and BHT were used.

\({\displaystyle \begin{array}{c}\Delta \textrm{A}\ \mathrm{sample}\hbox{--} \Delta \textrm{A}\ \mathrm{blank}\\ {}\textrm{Total}\ \mathrm{antioxidant}\ \left(\%\right)=1\hbox{--} -----------------------------------\times 100\\ {}\Delta \textrm{A}\ \textrm{control}\end{array}}\)

2.5.2 Scavenging ability on DPPH radicals

The DPPH free radical scavenging activity of crude and α-L-Rhamnose was inspected using the Zhang et al. [22] method. In brief, a 0.1mM DPPH solution in 100% methanol was prepared, and 1ml of this solution was added to 4 ml of sample in 40% methanol at various concentrations (10–160 μg/ml). The mixture was vigorously shaken and incubated at 30 °C in the dark for 15 min. The reduction of the DPPH radical was measured by continuously monitoring the decrease in absorption at 517 nm. As standards, L-ascorbic acid and BHT were used.

$${\displaystyle \begin{array}{c}\Delta \textrm{A}\ \mathrm{sample}\hbox{--} \Delta \textrm{A}\ \mathrm{blank}\\ {}\textrm{DPPH}\ \textrm{scavenging}\ \mathrm{effect}\ \left(\%\right)=1\hbox{--} -----------------------------------\times 100\\ {}\Delta \textrm{A}\ \mathrm{control}\end{array}}$$

2.5.3 Scavenging ability on ABTS radicals

The ABTS radical scavenging activity of crude polysaccharide and α-L-Rhamnose was determined using the method of Giao et al. [23]. This method is based on antioxidant molecules' ability to satisfy the long-lived ABTS+ species, a blue-green chromophore with a typical absorption wavelength of 734 nm. The addition of antioxidants to the preformed radical cation reduces it to ABTS, resulting in color loss. The results were expressed as a percentage of inhibition and the L-ascorbic acid and BHT serving as standards.

$${\displaystyle \begin{array}{c}\Delta \textrm{A}\ \mathrm{sample}\hbox{--} \Delta \textrm{A}\ \mathrm{blank}\\ {}\textrm{ABTS}\ \mathrm{scavenging}\ \textrm{effect}\ \left(\%\right)=1\hbox{--} -----------------------------------\times 100\\ {}\Delta \textrm{A}\ \mathrm{control}\end{array}}$$

2.5.4 Reducing power assay

The reducing power was calculated using the method described by Zhang et al. [22]. In a nutshell, 1ml of sample solution (50–400 μg/ml) in phosphate buffer (0.2 M pH 6.6) was combined with 1ml of potassium ferricyanide (1%, w/v) and incubated at 50 °C for 20 min. The reaction was then stopped by adding 2.0 ml of TCA (10% w/v) and the solution was mixed with 1.25 ml of ferric chloride (0.1% w/v) before measuring at 700 nm. As standards, L-ascorbic acid and BHT were used.

2.6 Anticoagulant activity

2.6.1 APTT assay

The anticoagulant activity of the crude polysaccharide and α-L-Rhamnose was determined using the activated partial thromboplastin time (APTT) coagulation assay. In brief, citrated normal human plasma (90 μl) was mixed with 10 μl of sample separately and incubated for 1 min at 37 °C in separate glass vials. Then, 100 μl of bovine cephalin was added and incubated at 37 °C for 3 min. After the incubation, 100 μl of pre-warmed 0.25 M CaCl2 solution was added, and the clotting time was measured and compared to the standard; the activity was expressed as IU/mg.

2.6.2 PT assay

For crude polysaccharide and α-L-Rhamnose, the Prothrombin Time (PT) assay was used. 90 μl of citrated normal human plasma was mixed with 10 μl of sample separately and incubated at 37 °C for 10 min. The pre-incubated PT assay reagent (200 μl) was added after the incubation period, and the clotting time was recorded.

2.7 Statistical analysis

One-way analysis of variance (ANOVA) was performed on all experimental data, and Dunnett’s multiple comparison test (GraphPad Software, USA) was also employed to find differences in means at the level of p < 0.05.

3 Results and discussion

3.1 Chemical and structural characterization of crude polysaccharide and α-L-Rhamnose

The chemical and structural characterization of the crude polysaccharide and α-L-Rhamnose from G. lithophila were reported in our previous study by Seedevi et al. [18], following, the yield (70% and 39.3% (w/w)), total carbohydrate (75.7 and 89.7%), protein (0 and 0%), ash (18.2 and 3.2 %), moisture (14.8 and 1.3 %), molecular weight (37 kDa and 24 kDa) and the monosaccharide composition of the crude polysaccharide reported rhamnose (79.82%), fructose (8.38%), galactose (3.95%), xylose (3.31%), and glucose (1.48%) and the α-L-Rhamnose reported 95.88% of rhamnose, 1.13% of xylose, and 2.21% of fructose. Furthermore, the crude polysaccharide and α-L-Rhamnose were structurally characterized via UV-vis, HPLC, FT-IR, NMR, DSC, and Polarimeter analysis [18].

3.2 Antioxidant activity

3.2.1 Total antioxidant activity

The total antioxidant capacity (TAC) assay determines the antioxidant capacity of natural compounds. At different concentrations (50–250 μg/ml), crude polysaccharide and α-L-Rhamnose demonstrated total antioxidant activity ranging from 31.1 to 64.22% and 39 to 74.11%, respectively (Fig. 1). At a concentration of 250 μg/ml of α-L-Rhamnose, the maximum inhibition was 74.11%. The total antioxidant activity was found to increase with increasing concentration. In comparison, at the highest concentration of 250 μg/ml, the standards BHT and L-ascorbic acid stated 91.11 and 89.6% total antioxidant activity, respectively. Similarly, at 1000 μg/ml, the total antioxidant activity of purified sulfated polysaccharide from S. tenerrimum was found to be 62.55% [24]. The crude polysaccharide of G. lithophila demonstrated maximum total antioxidant activity at the lowest concentration than the crude polysaccharide of S. tenerrimum was 62.55% at 1000 μg/ml [24]. Similarly, at a concentration of 1000 μg/ml, T. ornata crude polysaccharide showed 88.17% activity [24]. However, Costa et al. [13] reported that of the five heterofucans such as SF-0.5v, SF-0.7v, SF-1.0v, SF-1.0v, and SF-2.0v, SF-0.7v and SF1.0v from S. filipendula reported 77.3% and 90.7% antioxidant activity at 0.5mg/ml, respectively. In the current study, however, α-L-Rhamnose from G. lithophila demonstrated 74.11% total antioxidant activity even at a lower concentration of 250 μg/ml. Thus, the total antioxidant activity of α-L-Rhamnose from G. Lithophila was higher than in all of the previous studies.

Fig. 1
figure 1

Total antioxidant activity of crude polysaccharide and α-L-Rhamnose

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3.2.2 DPPH radical scavenging assay

The inhibitory effect of crude polysaccharide and α-L-Rhamnose on DPPH radical was found to be significant and concentration-dependent, with inhibition levels ranging from 22.5 to 69.91% and 29.89 to 83.31% (Fig. 2). In 160 μg/ml concentration of α-L-Rhamnose noted the highest activity as 83.31%. The antioxidant activity of the standards BHT and L-ascorbic acid at their highest concentrations was 79.42% and 83.95%, respectively. In the present study a maximum DPPH scavenging activity was shown by the crude polysaccharide (69.91%) at the concentration of 160 μg/ml, which is higher when compared to the results of previous study on two acid protein-containing crude heteropolysaccharides from Ampelopsis from, i.e., at the concentration range of 0.25 to 8 mg/ml, the scavenging effort of BHT, ALPS and ASPS ranging from 24 to 99%, 3.9 to 19.3%, and 5.1 to 56.7%, respectively [25]. In an earlier study, the crude polysaccharide from S. tenerrimum reported the DPPH scavenging activity of 64.66% at the maximum concentration of 1000 μg/ml [24]. Likewise, the DPPH scavenging activity of three crude polysaccharides from the brown seaweed S. pallidum was found to be 17.8%, 19.1%, and 10.2% at the concentration of 3.8 mg/ml [26]. In the present study, α-L-Rhamnose showed 83.31% of DPPH radical scavenging activity at 160 μg/ml concentration. The DPPH scavenging activity may be considered that α-L-Rhamnose which radical the hydrazine on reacting with the hydrogen donors in it. In addition to that DPPH scavenging activity may be attributed the occurrence of monosugars. The DPPH scavenging activity of the two high rhamnose containing purified sulfated polysaccharides (S1, S2) and de-sulfated polysaccharides (DS-1, DS-2) from Undaria pinnitafida showed 30.87%, 35.01%, 56.12%, and 60.88% at 1.4mg/ml concentration [27]. Similarly, the DPPH antioxidant activity of four purified polysaccharides from the brown seaweed S. pallidum was 3.55%, 3.26%, 6.60%, and 4.94% at the concentration of 5 mg/ml, respectively [26]. Likewise, the DPPH scavenging activity of the four polysaccharides such as SMP-1, SMP-2, SMP-3, and SMP-4 extracted from Salvia miltiorrhiza showed 86.7%, 89.4%, 76.8%, and 81.9% at 4mg/ml concentration [28]. The difference in antioxidant activity of the polysaccharides could be attributed to a variety of factors, including molecular weight (MW) and chemical structure [22].

Fig. 2
figure 2

DPPH radical scavenging activity of crude polysaccharide and α-L-Rhamnose

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3.2.3 ABTS scavenging assay

The crude polysaccharide and α-L-Rhamnose showed excellent ABTS scavenging activity in a concentration-dependent manner. The scavenging ability was stated to be 25.04–74.46% and 33.01–87.32% at the numerous concentrations of 25–125 μg/ml used in the present study for crude polysaccharides and α-L-Rhamnose (Fig. 3). The maximum of 87.32% scavenging capacity was detected at the concentration of 125 μg/ml of α-L-Rhamnose. It showed owing ABTS scavenging capability, with increased concentration; whereas the BHT and L-ascorbic acid presented 83.29% and 84.99% of scavenging ability at 125 μg/ml concentration. In the present study, the ABTS free radical activity of crude polysaccharide was maximum when compared to the previous crude polysaccharide from S. tenerrimum stated 70.33% at the concentration of 1000 μg/ml [24]. The ABTS radical scavenging ability of sulfated polysaccharide from brown alga S. tenerrimum was calculated as 70.33% at 1000 μg/ml [24]. The methanolic extract from the red alga Compsopogon helwanii reported an increasing trend in its antioxidant activity, i.e., 55.8% and 74.3% at 50 and 100 μg/ml concentration respectively [29]. Similarly, sulfated fucoidan from L. japonica, agar-like sulfated galactan from Nori sp., and sulfated polysaccharide from Fucus vesiculosus showed ABTS antioxidant activity of 81%, 75.8%, and 66.9% at the concentration of 100 μg/ml [30,31,32, – 33].

Fig. 3
figure 3

ABTS scavenging activity of crude polysaccharide and α-L-Rhamnose

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3.2.4 Reducing power

Reducing properties are frequently associated with the presence of reductones, which have been shown to exhibit antioxidant effects by breaking the free radical chain by donating a hydrogen atom [34]. According to reports, reductones interact with specific peroxide precursors to stop the generation of peroxide [35, 36]. The reducing power of crude polysaccharide and α-L-Rhamnose from G. lithophila was assessed based on the measurement of Fe3+–Fe2+ transformation. At concentrations of 50–400 μg/ml, the crude polysaccharide and α-L-Rhamnose showed reducing powers of 1.23–16.11% and 2.54–21.92%, respectively (Fig. 4). The compound α-L-Rhamnose had the greatest recorded reducing the power of 21.9% and shown a consistent rise in reducing power through increasing concentration. At the highest concentration of 400 μg/ml, the (BHT and L-ascorbic acid) standards contributed 18.32% and 20.67% of the reducing power, respectively.

Fig. 4
figure 4

Reducing power ability of crude polysaccharide and α-L-Rhamnose

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The crude polysaccharide in the current investigation demonstrated a maximum reducing power capability than the crude sulfated polysaccharide from S. Tenerrimum reported a 0.99% concentration at 1000 μg/ml [24]. The α-L-Rhamnose showed higher reducing power ability than that of the purified polysaccharide, obtained from fresh persimmon fruit Diospyros kaki namely PFP and its derivatives (PF-SI, PF-SII, and PF-SIII) which showed the reducing power of 0.981, 0.987, 0.871, and 0.966% at 400 μg/ml concentration [36]. Similarly, the reducing power of polysaccharide from the brown seaweed S. graminifolium was calculated as 0.21, 0.37, 0.42, 0.57, and 0.89% at the concentrations of 0.2, 0.4, 0.8, 1.6, and 3.2 mg/ml, respectively [37]. Furthermore, the reducing power capacity of two sulfated polysaccharide fractions (F1 and F2) and de-sulfated polysaccharide fractions (DF-1 and DF-2) from Corallina officinalis showed moderate reducing power (0.424%, 0.303%, 0.53%, and 0.348%) at higher concentration of 1.4 mg/ml [38]. When compared to other studies, the reducing power of the crude polysaccharide and α-L-Rhamnose was higher in the current investigation, since the antioxidant activity of the polysaccharide derivatives with different molecular weights varied. The molecular weight was found to boost antioxidant activity. However, Zhao et al. [39] discovered that in terms of antioxidant activity, low molecular weight compounds outperform high molecular weight products. Regardless, the findings of all of the preceding studies indicate that the molecular weight plays an important role in the antioxidant properties of polysaccharides. Along with molecular weight, sulfur group and mono-sugar composition influence the antioxidant activity.

3.3 Anticoagulant activity

3.3.1 APTT and TT anticoagulant activity

The many problems of blood coagulation and fibrinolysis have made it a high priority in recent years biomedical research to examine anticoagulant, thrombolytic, and antithrombic reagents from various sources [40]. The APTT test, which precisely identifies the anticoagulant potency of the substance, is the most popular [41]. The anticoagulant properties of crude polysaccharide and α-Rhamnose are discussed. Through the use of APTT and PT assays, G. lithophila effects on human blood coagulation were assessed. The APTT anticoagulant activity of crude polysaccharide and α-L-Rhamnose proved 23 IU and 28.2 IU at 25 μg/ml; whereas the heparan sulfate exposed highest clotting time at 170 IU. Following the, PT assay of the crude polysaccharide and α-L-Rhamnose disclosed the clotting time of 1.92 IU and 3.58 IU at 25 μg/ml. Heparan sulfate comparison revealed 124 IU (Table 1).

Table 1 Anticoagulant activity of crude polysaccharide and α-L-Rhamnose from G. lithophila on human blood
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Nardella et al. [42] well-defined the anticoagulant activity of low molecular weight fraction (F21, F22, D21, and D22) from brown seaweed Ascophyllum nodosum which showed 2.2, 25.3, 0.2, and 7.6 IU/mg. In the present study, the α-L-Rhamnose showed the APTT activity of 28.20 IU/mg which coincides with the result of APTT activity of A. nodosum polysaccharide fraction F22. In the present study, much higher activity exhibited by crude polysaccharide of APTT at 506 (s) and α-L-Rhamnose 620 (s) and for PT showed 26.88 and 50.12 s, respectively. Similarly, Mao et al. [43] reported the sulfated polysaccharide from Monostroma latissimum showed anticoagulant capacity as APTT > 200 s and PT 17.6 s at highest concentration of 50 μg/ml. Providentially, in the present study α-L-Rhamnose showed better anticoagulant activity in lower concentrations. The variation in the anticoagulant potential may be due to the difference in the sugar residue and the position of sulfate group in the polysaccharides [41, 44]. While the PT test revealed that the anticoagulant polysaccharide derived from Caulerpa cupressoides did not affect extrinsic pathways on the coagulation fall, the APTT test suggested that the drug functioned on intrinsic and common routes [45, 46].

The current study results show that α-L-Rhamnose has the highest anticoagulant activity, though it is weaker than heparin. Matsubara et al. [37] observed similar results for the anticoagulant proteoglycans purified from Codium pugniformis, a marine green alga. Besides, the different molecular weights of sulfated polysaccharides showed different anticoagulant activities, in which higher molecular weight (216.4–61.9 kDa) had difficult anticoagulant actions [47]. These results propose that the crude polysaccharide and α-L-Rhamnose from G. lithophila have a reflective outcome on the anticoagulant activity, and the molecular weight plays a significant role in the anticoagulant action.

4 Conclusion

In conclusion, crude polysaccharide and α-L-Rhamnose from G. lithophila established strong antioxidant activities such as total antioxidant activity, DPPH radicals, ABTS radicals and reducing power, and good anticoagulant activity, particularly APTT, and PT. This biological activity of the crude polysaccharide and α-L-Rhamnose had higher portion of rhamnose and their molecular weight may possess the antioxidant and anticoagulant activity. So, the crude polysaccharide and α-L-Rhamnose from G. lithophila could be used as a potential natural source of antioxidant and anticoagulant, as a food supplement, or as a feature in the pharmaceutical industries.