Sulfated Polysaccharides Derived from Hypnea valentiae and Their Potential of Antioxidant, Antimicrobial, and Anticoagulant Activities with In Silico Docking

Carrageenan, a sulfated polysaccharide, was produced by certain species of marine red seaweeds, which have been used as a significant source of food, feed, and antibiotic agent throughout history due to their alleged human health benefits. The present study aimed to derive the polysaccharides from Hypnea valentiae and describe the biological applications. Carrageenan was characterized by FT-IR, C-NMR, AFM, and their antimicrobial, antioxidant, and anticoagulant capabilities; furthermore, the larvicidal effect of methanol extract was generated from the seaweed against Aedes aegypti larvae at various concentrations. The molecular docking experiments were carried out computationally for finding the molecular insight of the macromolecules and small molecules' interaction using GLIDE docking by using Schrodinger software. Antibacterial zones of inhibition in different concentrations are compared with the 40 mg/mL higher activity against bacterial pathogens. Carrageenan is strong in all antioxidant activities, with the overall antioxidant (70.1 ± 0.61%) of radical at 250 μg/mL concentration being exhibited. The DPPH scavenging is effective in the inhibition of (65.74 ± 0.58%) radical at a concentration of 160 μg/mL and the hydroxyl scavenging (65.72 ± 0.60%) of activity at a concentration of 125 μg/mL being exhibited. Anticoagulant activities (APPT and PT) of carrageenan fraction were tested. H. valentiae and heparin sulphate shows higher activity of APTT (106.50 IU at 25 μg/mL) in comparison with the PT test (57.86 IU at 25 μg/mL) and the methanol extraction of higher larvicidal activity on A. aegypti (LC50 = 99.675 μg/mL). In this study, the carrageenan was exploited through in vitro and in silico molecular docking studies against antimicrobial, antioxidant, and anticoagulant properties. The results were establishing the potentiality of the carrageenan which is an alternative source to control the mosquitocidal property in the future. Moreover, molecular docking of carrageenan against multiple targets results in −7 to −6 Kcal/mol binding score. Findings of carrageen from in vitro to in silico studies are needed for further validation of clinical pieces of evidence.


Introduction
Marine macroorganisms are a rich source of functionally diversifed bioactive compounds that play an active role in human nutrition and health.Seaweeds are a major source of sulfated polysaccharides, particles which are widely employed in food, feed, and medicine due to their rheological qualities as gelling and stabilizing agents [1,2].Sulfated polysaccharides have numerous biological and physiological activities, including antithrombotic [3], anticoagulant [4], antioxidant [5], antidiabetic [6], antibacterial [7], immunomodulatory [8,9], antiviral [10], antiinfammatory [11], antinociceptive [12,13], antitumor [14], and proinfammatory efects [15,16].Sulfated polysaccharides including agarans, galactans, and carrageenans are also available abundantly.Among these, carrageenan is a generic deviation of linear, sulfated galactans derived from species of red seaweeds [17].Various investigations on the antioxidant and anticoagulant properties of seaweeds or their extracts have been published [18,19].Algal polysaccharides must be shown to serve a signifcant role as free-radical scavengers and antioxidants in the protection of oxidative damage in living organisms in current administration, and anticoagulants have long been used to treat blood during dialysis and surgery, as well as to treat disseminated intravascular coagulation and thrombosis in a variety of disorders and for in vitro blood testing [20,21].Mosquitoes, such as Aedes aegypti, are the deadliest since they act as a vector for a variety of diseases, including dengue fever, malaria, yellow fever, flariasis, and other types of chikungunya as well as Zika virus [22][23][24][25].Furthermore, seaweed sulfated polysaccharide extracts contain primary and secondary metabolites including bioactive chemicals which are biodegradable into harmless products; it may be efective in mosquito larval control [26,27].
Te standard new therapeutic approach is complicated, exhausting, expensive, time-consuming, and laborious.Computational methods such as molecular docking have played a critical role in rationalizing the road to drug discovery in order to overcome these obstacles.A molecular docking feature becomes a promising pharmaceutical research tool for screening candidates from drug libraries efectively [28].In such studies, the ultimate goal was to fnd efective therapeutics for the carrageenan molecule extracted from marine algae.It is evaluated against antimicrobial, anticoagulant, and antioxidant activity using GLIDE docking in the maestro platform of Schrodinger software (Schrödinger Release 2021-2: Maestro, Schrödinger, LLC, New York, NY, 2021).Furthermore, the efciency of the carrageenan compound was exploited with in vitro validation against the foresaid targets.Carrageenan is a sulfated polysaccharide, which is obtained by extraction from red seaweed species.It has been widely utilized in the food industry, agricultural, drug delivery, tissue engineering, and biosensor applications.Terefore, the present study investigated the isolation and biofunctional activities of carrageenan extracted from H. valentiae, characterization of structural properties using Fourier transform infrared (FT-IR) spectroscopy, 13 C nuclear magnetic resonance (NMR), and atomic force microscopy (AFM) spectroscopy and evaluation of the biological properties such as antioxidant, antimicrobial, and anticoagulant activities with in silico molecular docking analysis.

Collection and Extraction of Sample.
Te red seaweed, H. valentiae (Turnur), was collected from Mandapam (Lat.09 °28′177N, Long.79 °18′536E) located on the southeast coastline of Ramanathapuram, Tamil Nadu, India.Te seaweed specimen identifcation number was 1759.Te sample was properly cleaned with seawater to remove all undesired pollutants such as sand particles and epiphytes and then thoroughly cleaned with tap water to discard all salt on the surface.Te water was drained away, and the seaweed was laid out on the blotting paper to absorb any remaining moisture before being shade-dried at room temperature for 3 days and crushed into a fne powder.Following the completion of the sulfated polysaccharide extraction, the procedure was evaluated for 100 g of seaweed powder, which was soaked separately in acetone and methanol solvent in 7:3 for two days in a shaker at 200 rpm for 10 min.Te process was repeated twice to ensure the dry biomass.Tis biomass was dried into a powder and dispersed in 1 L of 0.1 M HCL for 24 h with constant stirring at room temperature.Te pellet was reextracted as above, and the supernatants were pooled.Te supernatant was kept at 4 °C overnight and precipitated with two volumes of absolute 1:1 and stirred continuously for 15 min, and then the precipitate was collected to separate carrageenan gel and distilled water.Carrageenan gel was then completely soaked in 96% alcohol for 1 h and stirred continuously.Te carrageenan gel was separated from alcohol and distilled water by fltration.Subsequently, the mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was neutralized with 1.0 M NaOH and poured in 100 mL of methanol.Te carrageenan was freeze-dried by using a membrane at 70 °C for a 24 h duration which was performed for further processing [29].

Structural Analysis.
Using an alpha FT-IR spectrophotometer and its technique, FT-IR was used to determine the functional groups of the carrageenan polysaccharides [30].Te characterization has been complemented by the 13 C-NMR spectrum.Te 13 C-NMR spectrum of 10 mg of the carrageenan was dissolved in 0.5 mL solution in D 2 O which was recorded at 27 °C on an NMR spectra Bruker model MHz 400 spectrometer.Te proton chemical shift was expressed in parts per million (ppm).Te substance was diluted in distilled water before being mixed with a solution of 200 μg/ mL carrageenan in 0.2 M NaOH.Te carrageenan solutions were allowed to equilibrate for 1 h with NaOH prior to neutralization with HCl (pH∼6-7).Solutions were then passed through a 0.

Antibioflm Activity.
To determine the efect of seaweed methanol extracts against antibioflm activity, about 3.5 mL of nutrient broth and 1.5 mL of bacterial culture were added into sterile test tubes.10 mg of sample was added to diferent concentrations of methanol extract (10,20,30,40, and 50 μg/ mL) and additionally given into each tube.All tubes were incubated in a shaking incubator at 37 °C for 24 h.After incubation, all tubes were washed with distilled water.All the tubes were breeze-dried for a few minutes and added 5 mL of crystal violet and then incubated at 37 °C for 1 h.After incubation, we discarded the crystal violet from all the tubes and washed them with distilled water.About 5 mL of 95.0% ethanol was added into all test tubes and taken OD at 595 nm by UV-visible spectrophotometer [32], respectively.Te antibioflm activity was calculated using the formula of percentage: % of biofilminhibition � controlOD − treatmentOD controlOD × 100. (1)

Antioxidant
where A1 is the absorption of the control and A2 is the absorption of the sample.

Hydroxyl Radical Scavenging Assay.
Te capacity of the seaweed polysaccharides against the scavenging hydroxyl radical was evaluated using Fenton's reaction (Fe 2+ +H 2 O 2 ⟶Fe 3+ +OH − +OH) according to the modifcation method described [35].Te extracts of methanol were evaporated in vials.Te reaction mixture contained 1 mL of (EDTA) solution, 0.5 mL of EDTA (0.018%), and 1 mL of DMSO (0.85% v/v in 0.1 M phosphate bufer, pH 7.4), and 0.5 mL of ascorbic acid (0.22%) was added to each tube.Te solution was incubated at 37 °C for 15 min, and the presence of yellow color was measured spectrophotometrically at 510 nm against the blank sample.Te mixture without the sample was treated as a control.Te scavenging activity was calculated by the following equation: where A0 is the control, A1 is the absorption of the sample, and A2 is absorption without sodium salicylate.

Activated Partial Tromboplastin Time (APTT).
For carrageenan fractions, APTT was calculated using a modifed version of the methodology reported [36].Te positive and negative controls were heparin at a concentration of 5 g/mL and 0.9 percent w/v NaCl, respectively.

Prothrombin Time (PT).
Te methodology of [37] was also used to determine PT, with some modifcations.Te device was also programmed to perform the same procedure in APTT determination.Each poor-platelet plasma and carrageenan solution mixture was incubated for 3 min at 37 °C.Ten, 0.6 mL of prewarmed PT reagent was added, and the time for clot formation was also observed and repeated three times.For positive and negative control, heparin and NaCl were utilized, respectively.Protein preparation: we need to evaluate the antioxidant (2C0D & 5B6M), antimicrobial (1JIJ & 2XCT), and anticoagulant (5E8E) activity against sulfated polysaccharide computationally.Te three-dimensional structure of proteins was retrieved from the database of Protein Data Bank (PDB).Te X-ray crystallographic structures were imported into Maestro using the protein preparation wizard, and this module helps to solve the missing hydrogen bonds, create the disulfde bonds, and optimize (Schrödinger Release 2021-2: Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2021; Impact, Schrödinger, LLC, New York, NY; Prime, Schrödinger, LLC, New York, NY, 2021).

Molecular
Te molecular docking was performed using the Glide package (ligand docking) in the Schrodinger suite.Te standard precision docking method was applied and performed postdocking minimization and analyzed the results in pose viewer Schrödinger Release 2021-2: Glide, Schrödinger, LLC, New York, NY, 2021.
2.9.Mosquito Culture and Larvicidal Activity.A. aegypti mosquito larvae were collected from the Indian Council of Medical Research-Vector Control Research Centre, Madurai, Tamil Nadu, India.Te mosquito larvae were fed a fnely powdered mixture containing a 3 : 1 ratio of dog biscuits and Brewer's yeast for repeated generations at 25-30 °C.According to the procedure used, adult mosquitoes were kept under standard environmental conditions as larvae [38].Following this procedure, a mortality assay was carried out on fourth instar larvae [22,39].Te test for the larvae efect of methanol extract derived from seaweed against mosquito larvae A. aegypti involved 10 mg of the sample in diferent concentrations for 50, 100, 200, and 500 μL.Twenty larvae were added to 250 mL distilled water in triplicates, with 1 mL DMSO serving as a negative control H 2 O. Dead larvae were counted, and the proportion of dead larvae was estimated for the replicates after 24 and 48 h of exposure.Proft analysis was utilized to calculate average LC 50 and LC 90 (lethal concentration) values from the duplicates.

% of mortality �
number of dead larvae number of larvae introduced × 100.(4)

Results
Te FT-IR spectra of the carrageenan were isolated from the red seaweed H. valentiae and the absorption bands typical of carrageenan between the wave numbers 1000 and 3500 cm −1 clearly highlighted the functional groups in all of the analyzed samples (Figure 1, Table 1).Te peaks at 3457.74 cm −1 correlate with O-H stretching H-bonding vibrations which indicated alcohols and phenols.Te existence of alkanes was revealed by the similarity of the peaks found at 2349.36 cm −1 .Furthermore, the peaks observed at 1687.22 cm −1 were attributed to alkenes.Te aliphatic amines had an absorption peak position at 1187.22 cm −1 confrming the -C≡C-stretch.
Te spectral observations at 1222.18 cm −1 indicated asymmetric stretching of S-O, revealing the presence of sulfate [40].Te band at 1222.18-1030.72cm −1 stretching of C-O was attributed to alcohols, carboxylic acids, esters, and ethers.C-H stretching vibrations indicated the existence of alkanes in the band between 1444.61 cm −1 .Te C-O-C of 3, 6-anhydro-L-galactose vibrations has been assigned to the peak at 920.72 cm −1 .Aromatic group C-H stretching vibrations were as described to the peak 845.44 cm −1 .Te last peak which appears to be 650.30cm −1 was related to -C≡C-H: C-H bending the presence of alkanes.Te NMR spectra of the carrageenan provided more structural characterization, which is shown in Figure 2, respectively, and band assignment for a sample of H. valentiae polysaccharide.Te NMR can be used to demonstrate the existence of diferent carrageenan units.Te NMR spectrum was also a complex polymer signal from the anomeric proton at (78.30 ppm), and ring carbons (70.16 ppm) were assigned to 3,6-α-L-anhydrogalactose.Te signal was assigned from the 75.02 ppm which was assigned to H-1 of β-D-galactose linked to α-L-galactose-6-sulfate. Te signal at 102.86 ppm was attributed to carboxyl β-D-galactose.
Te surface topography of the carrageenan was studied with the help of AFM measurements in a contact mode.Te AFM images of the carrageenan soft template and bright spot are shown in (Figure 3(a)).Moreover, the topography of the carrageenan can be observed with peaks and valleys distributed across the surface.Apart from this, huge numbers of deformed shapes with larger sizes can also be seen in the AFM image of the carrageenan (Figure 3(b)).Te AFM is a signifcant source for detecting the morphology and size of the carrageenan macromolecules originating from 0.0 to 0.7 μm.Te carrageenan particles' average height which ranged from 0.00 to 0.72 μm was also noted.
Te antibacterial activity of the methanol extract was evaluated for the resistance to pathogenic bacteria which may cause infection in human beings.Te methanol extract of H. valentiae inhibitory activity against E. faecalis, S. aureus, E. coli, and P. aeruginosa was identifed.Te pathogenic bacterial zone of inhibition in various concentrations of the methanol extract was compared to the 40 mg/ mL higher inhibitory activity against four bacterial pathogens obtained in the results shown in (Figure 4) and Table 2, respectively.Te antibacterial potential of methanol extracts depends upon the ability of permeation to the bacterial cell through the cell wall of bacteria.Moreover, the methanol extract had a minimum inhibitory bacterial efect against the Gram +ve strain rather than the Gram −ve strain.Terefore, the present study focused that both extracts demonstrated high signifcant antibacterial inhibition activity against Gram-negative bacteria rather than Gram-positive bacteria.
Te antibioflm activity of methanol extract of H. valentiae has been investigated with bacterial potential against stains    Total antioxidant activity was measured to evaluate the antioxidant capacity of sulfated polysaccharides from H. valentiae extracts.Te carrageenan extract was shown to reduce the total antioxidant scavenging activity of radicals (70.1 ± 0.61%) at 250 μg/mL concentration rather than at the other concentrations of 50-250 μg/mL of the displayed activities.Based on the fndings visualized (Table 4) and noticed, the carrageenan was found to have signifcantly higher total antioxidant activities as compared with L-ascorbic acid (87.22 ± 0.80%) and BHA (81.99 ± 0.75%).Te antiradical assay was determined by measuring the absorbance of the inhibition of DPPH radicals.Te seaweed extract of sulfated polysaccharide showed that higher inhibition activity of these radicals (65.74 ± 0.58%) values for DPPH scavenging at 160 μg/mL concentration rather than at the other concentrations.According to the results shown in (Table 4), carrageenan has the potential for radical scavenging activity when compared to the compounds of L-ascorbic acid (82.05 ± 0.73%) and BHA (79.01 ± 0.70%) that demonstrated efective DPPH neutralizing activity.
Te hydrogen radical scavenging assay was used to determine the inhibition of hydroxyl (OH) radical formation, and the results showed that the scavenging activity of sulfated polysaccharide was signifcant with increasing concentrations.Te extract of carrageenan inhibited the hydrogen peroxide scavenging activity of the OH radical (65.72 ± 0.60%) at 125 μg/mL concentration rather than at the other concentrations of 25-125 μg/mL of the exhibited activities.Furthermore, as shown in Table 4, carrageenan exhibited signifcantly higher hydroxyl radical activities when compared to L-ascorbic acid (81.140.73%) and BHA (77.930.70%).
Te study of anticoagulant activity was analyzed by the APTT and PT assays which demonstrated that the anticoagulant mechanism of carrageenan inhibits plasma coagulation release during the regular phase of the coagulation process blood clotting time (Table 5).Te anticoagulant activity of carrageenan was exhibited higher in APTT (106.50IU at 25 μg/mL) when compared with the PT test (57.86IU at 25 μg/mL) indicating the pathway to inhibition.
Te carrageenan molecule was subjected to small molecular (ligand) docking, the antioxidant targets chosen as mitochondrial 2-cys peroxiredoxin (2C0D) from Plasmodium falciparum and crystal structure of human peroxiredoxin 6 (5B6M).Te docking score results of −7 Kcal/mol indicate strong afnity among the complex molecules, and the interaction diagrams of 3D and 2D are represented in Figure 5. Te antimicrobial target protein chosen as S. aureus tyrosyl-t RNA synthetase (1JIJ) and S. aureus gyrase topoisomerase II (2XCT) against the small molecule showed a strong afnity in their complex molecule with the least binding score of −6.894 Kcal/mol and with their interactive sites represented (Figure 6).Te phytochemical exploited against antioxidant activity using auto dock tool results in −3.3 to −6 Kcal/mol binding score with in vitro validation, wherein in our case, it was shown as −7 Kcal/mol using Glide docking, Schrodinger [41].Te anticoagulant target protein was chosen as the crystal structure of thrombin (5E8E) from humans against carrageenan, showing the amino acid interaction as SER546 forms a hydrogen bond with the NHgroup, and PO4 forms a hydrogen bond with the O-group with a docking score of −6.639.Te structure was illustrated Evidence-Based Complementary and Alternative Medicine with 2D and 3D interaction maps with a pocket site (Figure 7, Table 6).Carrageenan extracted from H. valentiae was found to have the highest larvicidal activity against A. aegypti (LC 50 = 99.675μg/mL; LC 90 = 491.453 mg/L) shown (Figure 8, Table 7).Te larvicidal activity of seaweeds to the larvae of A. aegypti was determined to the inhibition efect of seaweed mortality larvae.Seaweed is a natural product, and particularly, halogenated terpenes might be exploited for the improvement of new larvicidal compounds and its prototype insecticidal agents.

Discussion
Te present study on methanol extract of H. valentiae was tested for yield, antioxidant, antimicrobial, anticoagulant with in-silico molecular docking, and larvicidal activity properties.Te extract efect is an important parameter for the chemical compounds which have been used in the screening of bioactive substances from red seaweeds enriched with secondary metabolites and have potential antioxidant, antimicrobial, and anticoagulant activities [42].
Te FT-IR spectrum is one of the most important tools for polysaccharides and their spectroscopic assignments of functional groups.Te O-H stretching of alcohol and phenol vibration from the intramolecular hydrogen bond corresponded to the absorption peak position at 3457.74 cm −1 [43].Te absorption peaks at 2349.36-1687.22 cm −1 is attributable to C-H stretch and -C≡C-stretch alkane functional groups, which probably confrmed the polymerbound water which is a characteristic feature of red seaweed polysaccharides [44].Tese absorption bands were described as enhancing the activation of molecular chain movements.Furthermore, the absorption peaks at 1222.18-1030.8 Evidence-Based Complementary and Alternative Medicine molecular weight skeleton of sulfated D-galactans and carrageenan bands in H. valentiae [46].Tese absorption bounds of oxygen, nitrogen, and biofunctional groups confrmed the presence of polyphenol compounds, polysaccharides, and protein, primary, and secondary metabolites.
Te NMR spectroscopy is one of the efcient techniques for determining the structural characters of seaweed polysaccharides [47].Recently, the report has demonstrated that the NMR spectrum used in the presence of diferent carrageenan components was observed to indicate the conversion of 3,6-α-L-anhydrogalactose units into its alditol  Evidence-Based Complementary and Alternative Medicine derivatives anomeric protons containing μ and ] carrageenan [48].Previous studies illustrate the occurrence of β-D-galactospectroscopic functional group of sulphated in the sugar residues, which are in conform that the trisaccharide branches on bacterial polysaccharide [49,50].Collectively, the literature has reported that Gracilaria caudata analyzes the structural features of β-D-galactos linked to α-L-galactose-6-sulfate, and methyl or pyruvate molecules are substituted by carrageenan polysaccharide [47,51].Tese reports indicated that the α-glycosidic peaks in the carrageenan backbone were partially broken [52].NMR spectroscopy is also useful for recognizing the conformations of polysaccharides.
Te AFM is single molecular spectroscopic technology to detect the conformation of morphological structures of polysaccharides in AFM images which provides a perfect observation of molecular assembling.Te AFM provides visualization of the diferent types of sulfated polysaccharides such as curdlan [53], xanthan [54], carrageenan [55], xyloglucan [56], pectin [57], arabinoxylan [58], and starch [59].Te previous literature has functionalized the AFM based on the purpose of many fundamental food systems of carrageenan polysaccharides [48].Similar reports have identifed the height range in the structure of the food particles [49].Based on the fndings, the fbrous height of carrageenan was topographically similar to sulfated polysaccharides derived from the red seaweed [44].Te majority of the results from AFM analysis have reviewed the macromolecules of functional food polymers [60].Terefore, AFM is nanostructure characteristics of polysaccharides   Te potential antibacterial activity of carrageenan polysaccharides from H. valentiae derivatives was evaluated to afect microbial pathogens of (E.faecalis and S. aureus (Gram +ve) and E. coli and P. aeruginosa (Gram −ve) organisms.Te antibacterial activity was revealed to be strong against biologically multidrug-resistant pathogens S. aureus and E. faecalis [52].In addition, numerous reports have described the antibacterial potential using algae extracts' strong efect on various bacterial pathogens such as E. coli, S. aureus, and E. faecalis.Tese pathogenic bacterial activities to enhancing the biostimulants of medicinal properties valuable for enhancing the mint and basil antimicrobial activity against bacteria [62].Previous reports have analyzed the antibacterial activity against bacteria Spatoglossum asperum biological and pharmaceutical useful for valuable drugs [50].Te antibacterial activities of highly potential biomedical applications such as drug delivery, wound healing, and tissue engineering.Te antibacterial capabilities could improve rapid healing by making a barrier against microbial contamination [63].
In the present study, H. valentiae were tested for antibioflm activity potential against pathogenic strains that the reports revealed the methanol extract inhibited bioflm formation.Based on the reports, we have identifed the bioflm activity inhibition of Sargassum wightii and Halimeda gracillis seaweed activity against antimicrobial pathogens and antibioflm activity against various clinically signifcant pathogenic microorganisms [64].Te previous literature reported that the bioflm activities could be applied in diferent medical treatments to the prevention of various bioflm-related infections [65].Te bioflm pathogens are eco-friendly to presently used metal-based antifouling agents [66].Collectively, the results have indicated the signifcant antibioflm potential of the Ulva lactuca microbiocidal efect of diferent microorganisms that lead to the formation of cariogenic bioflm against the environment.It has been useful to the need for clinical studies to completely enhance the antibioflm and mechanical properties of the new product [67].
Te antioxidant potential of polysaccharides for various free radical scavenging ability of higher polyphenol and favonoids content was extracted, and they could have excellent antioxidant activity [68].Te antioxidant activity having higher polyphenol content in soluble fraction is observed to precipitate.Te highly efective of polyphenol and favonoids compounds with their hydroxyl group for scavenging free radicals [64].Te antioxidant activity of polysaccharides could protect the human system from reactive oxygen species which afect such macromolecules as membrane lipids, proteins, and DNA and lead to many functional disorders of the human body.In the current study, phenolic content was higher in methanol extract from H. valentiae which were screened for total antioxidant activity at 250 μg/ml concentration.Te highest total antioxidant activity is present in the methanol precipitate from H. valentiae (70.1 ± 0.61%).Previous literature reports have indicated the highest total antioxidant activity in the fractionated polysaccharides from Turbinaria conoides (246.6 mg AscAE/g) Gracilaria fliforms (353.3AscAE mg/ g), and Enteromorpha compressa (326.6 mg AscAE/g) [27].Similar results have reported the highest total antioxidant activity occurred in extract sulfated polysaccharides Sargassum tenerrimum (22.0 ± 06) [69].Similarly, the study reported the total antioxidant activity in aqueous extract of codium sp which showed the activity of 85.53 ± 0.25% [70].Te results revealed that the polysaccharides conformed that the total antioxidant activity which was the highest inhibition in the methanol extract from the red seaweed.Te potential antioxidant activity screened for DPPH radical scavenging was due to their hydrogen donating ability.Te free radical scavenging activity indicated a higher value in methanol extract from H. valentiae (65.74 ± 0.58%).Based on the results, the highest scavenging ability was present in the ethanolic precipitate of S. wightii (78.3 ± 0.25%) [64].Te antioxidant activity of polysaccharides extracted from Undaria pinnatifda showed that the highest inhibition of hydrogen peroxide screening ability [71].In our study, the occurrence of the sulfate group to activate the hydrogen atom of the anomeric carbon through the sulfate content to adsorb the antioxidant exhibited the scavenging ability of Sargassum thunbergii [72].Te same other results demonstrated that the sulfated polysaccharide efect on inhibiting the formation of these radicals was higher in the extract from Laminaria japonica [73].
Te results showed that the exhibited hydroxyl radical formations determine the scavenging activity of sulfated polysaccharides was signifcantly higher than the extract from H. valentiae (65.72 ± 0.60%).Te hydroxyl radical result reveals the damage to the biomolecules including protein, deoxyribonucleic acid, and polyunsaturated fatty acids in the human cell membrane.Tese costs must lead to the expansion of various infections including cancer (Undaria pinnatifda) [71].Previous literature reports have demonstrated that sulfate groups of algal polysaccharides are various kinds of biological activities as antioxidant activity in radicals scavenging (Sargassum thunbergii) [72].Tese results indicate the antioxidant activity of sulfate content had a signifcant source of hydroxyl radical scavenging ability [20].
Te present investigation continues to the methanol extract screening anticoagulant activity.Anticoagulant activity has evaluated the APTTand PTassay that showed higher inhibition of the coagulant in the soluble fraction.Te APTT and PT assay demonstrate the anticoagulant mechanism of the carrageenan as blood coagulation.Te methanol precipitate of H. valentiae inhibited higher APTT (106.50IU at 25 μg/mL) and PT (57.86 IU at 25 μg/mL).Te anticoagulant property of the carrageenan was assessed using human plasma from healthy donors.Te results found in the APTT assay showed that the carrageenan had higher coagulation properties.Te main source of the anticoagulant property of the carrageenan appears could be antithrombic activity [16].Te anticoagulant pathway indicated the potentiality of the sulfated polysaccharides in spreading the coagulation time that paves the technique for more possibility for their application in pharmaceutical industry for drugs [74].Te ray Evidence-Based Complementary and Alternative Medicine skin dermatan sulfated DS showed higher anticoagulant activity of the skin DS as shown by APTT and PT anticoagulant drug of potentially useful in therapy.Te report suggested the higher content of the sulfate group which presented higher coagulant property [75].
Sulfated polysaccharides are heterogeneous natural polymers found in massive quantities in marine algae with a wide range of therapeutic applications due to their chemical structure and composition.Te identifcation of chemicals that reduce infammation is of importance because infammation plays a role in illnesses.Antiinfammation activity has been determined using a variety of approaches.With results of in vitro assays, several derivatives of carrageenan oligosaccharides had better antioxidant activity than carrageenan oligosaccharides, indicating that chemical modifcation of carrageenan oligosaccharides could improve their antioxidant activity [76].Te investigations of these studies showed noticeable signifcance, and the resultant complex molecules amino acid interaction with bond length are depicted in Table 7. Carrageenan had been exploited with antimicrobial, antiviral, and anticancer activity.Te current research observed a signifcant interaction of the carrageenan with multiple targets resulting majorly around −7 to −6 Kcal/mol binding score [28,77].
Te mosquito larvicidal activity of methanol extract from H. valentiae has been found in the highest larvicidal activity than the control A. aegypti (LC 50 = 99.675μg/mL; LC 90 = 491.453mg/L).Te present investigation focused on the larvicidal efcacy of the potential efect on the development of H. valentiae against A. aegypti.Based on the results observed in Padina australis (LC 50 = 82.55mg/mL), Sargassum binderi (LC 50 = 160.07)mg/mL, Bryopsis pennata (LC 50 = 192.43mg/mL), the methanol extract was much efective on A. aegypti larvicidal activities.Te report provided information on numerous efects using diferent seaweed extracts of A. aegypti [78].Gracilaria fliforms has been reviewed in the literature larvicidal activity against the larvae Anopheles stephensi in which the higher inhibition is LC 50 = 0.255867 and LC 90 = 3.434589 mg/L [27].Te mosquito activity of the current research reported damage and shrinkage of cells from the midgut epithelium of the seaweed treated larvae Anopheles stephensi and A. aegypti which is (LC 50 : 58.34; LC 90 : 114.91).Te application of the seaweed extracts is derived from terrestrial aromatic and medicinal values [64].Halimeda macroloba has been reported in the literature in the past (LC 50 -1119.0;LC 90 -1890.3)and similarly Ulva lactuca (LC 50 -952.9;LC 90 -1830.4)and Caulerpa racemosa (LC 50 -886.0;LC 90 -1790.1)[79].Te same was reported to show the activity against the larvicidal activity of Hypnea muciformis and Padina gymnospora [80].Based on the results responsible for the larvicidal action, demonstrating the use of seaweed extracts has high potential as a mosquito control strategy.

Conclusion
Te fndings of this study suggested that the natural carrageenan derivatives with high sulfate content have greater radical scavenging antioxidant and antibacterial properties.
Carrageenan from red seaweeds has also been shown to have in vitro anticoagulant functions, confrming its explicit role in the coagulation pathway.However, more research is needed to confrm that seaweeds have high mosquitocidal activity in the future.In addition, the carrageenan was evaluated computationally using glide ligand-molecular docking with multiple targets of antimicrobial, antioxidant, and anticoagulant activity.Te minimal binding score explicits the strong afnity among the complex molecules.However, our potential candidate needs to further validate the in vivo model and extend it to clinical trials.
22μm flter and deposited onto 1 cm of a freshly cleaned glass plate surface (sample volume ∼60 μL).

Table 1 :
Interpretation of functional groups in the carrageenan using FT-IR.
Figure 1: FT-IR spectrum of the carrageenan from Hypnea valentiae.

Table 3
(10,20,30,40ly.Te diferent concentrations of methanol extract(10,20,30,40, and 50 μg/mL) were tested.Te highest activity against antibioflm activity (11.1 ± 0.885 at 50 μg/mL) methanol extract was recorded for E. faecalis.In P. aeruginosa, the inhibition of the bioflm activity rate as 14.3 ± 0.979 at 50 μg/ mL concentration of methanol extract was found.Te percentage of inhibition in E. faecalis activity efect methanol extract was signifcantly higher than that in P. aeruginosa.Te current study investigates the antibioflm activity of H. valentiae extracts bacterial activity against two diferent bacterial strains.

Table 6 :
Molecular docking of the carrageenan molecule against three diferent activities of 5 multiple targets, the bond length in which the range below 3 Å is hydrogen bond interaction.

Table 7 :
Te efect of aqueous and methanol extract of the Hypnea valentiae against Aedes aegypti larvae.