Anti-inflammatory and Anti-arthritic Effects

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Current Research in Biotechnology 7 (2024) 100220

Available online 6 May 2024

Investigating the anti-inflammatory and anti-arthritis effects of fucoidan from a brown seaweed

Preethy P. Raj

^a

, Rajesh Kanna Gopal

^b^,^*

, Elumalai Sanniyasi

^a^,^*

aDepartment of Biotechnology, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India

bDepartment of Microbiology, Centre for Infectious Diseases, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University (Deemed to be University), Velappanchavadi, PH Road, Chennai-600077, Tamil Nadu, India.

A R T I C L E I N F O Keywords:

Brown Seaweed, Turbinaria decurrens, Fucoidan In vitro anti-inflammatory

In vivo anti-arthritic activity

A B S T R A C T

Severe inflammation in joints caused by the detrimental effects of the immune system is termed Rheumatoid arthritis. The unconstrained proliferation of immune cells and pro-inflammatory cytokines deteriorates Syno- vium which secretes synovial fluid to lubricate joints and cartilage. Non-steroidal anti-inflammatory drugs (NSAIDs) are the only therapeutics for treating rheumatoid arthritis, and long-term intake causes serious side effects on the organs. Fucoidan, a sulfated polysaccharide found on the cell walls of brown algae shows bioactive potential. In our study, fucoidan was extracted from Padina pavonica (PD), Stoechospermum marginatum (StM), Spatolossum macrodontum (SpM), Dictyota bartayresiana (DD), and Turbinaria decurrens (TD) and evaluated for anti-inflammatory and anti-arthritis activities. Fucoidan was extracted and evaluated for anti-inflammatory activity in vitro using RAW 264.7 macrophage cell lines, followed by in vivo anti-arthritis activity on Wistar male rats. Nitric oxide suppression was comparatively high in fucoidan from TD (IC50 − 12.93 µg/mL). Purified fucoidan from TD, significantly reduced inflammation, size of paw edema, downregulated proinflammatory cytokines (IL-6, IL-1β, TNF-α), and upregulated anti-inflammatory cytokine (IL10) in CFA-induced arthritis in Wistar male rats. Biochemical parameters like SOD, CAT, GSH, GPX, and GST and haematological parameters like total-protein, albumin, haemoglobin, and RBC were upregulated, and other parameters like urea, uric acid, creatinine, bilirubin, SGOT, SGPT, ALP, WBC, ESR, RF, and CRP were downregulated. Histopathology of the

Abbreviations: NSAIDs, Non-steroidal anti-inflammatory drugs; %, Percentage; µg, Microgram; µL, Microlitre; 2D, 2-dimensional; AKT, Ak strain transforming (serine/threonine kinase); ALP, Alkaline phosphatase; ALT, Alanine transaminase; AST, Aspartate transaminase; b.w., Body weight; C, Celsius; CAT, Catalase; CD86, Cluster of Differentiation 86; CFA, Complete Freund’s adjuvant; cm, Centimetre; COSY, Correlation spectrometry; COX-2, Cyclooxygenase-2; CRP, C-reactive protein;

DD, Dictyota bartayresiana; DEAE, Diethylaminoethyl cellulose; dL, Decilitre; DMARDs, Disease-modifying anti-rheumatic drugs; DMEM, Dulbecco’s modified Eagle Medium; DPPH, 2,2-diphenyl-1-picrylhydrazyl; EDTA, Ethylenediaminetetraacetic acid; EEZ, Exclusive Economic Zone; ELISA, Enzyme-linked immunoassay; ERK, Extracellular signal-regulated kinase; ESR, Erythrocyte sedimentation rate; FBS, Fetal bovine serum; FT-IR, Fourier transform infrared spectrometry; g, Gram; GC, Glucocorticoids; GPX, Glutathione peroxidase; GSH, Reduced glutathione; GST, Glutathione S-transferase; HD, High-dose; HIV, Human immunodeficiency virus;

HPLC, High-performance liquid chromatography; Hr, Hour; HR-NMR, High-resolution-nuclear magnetic resonance; IFN, Interferon; IL, Interleukin; iNOS, Isoform of nitric oxide synthase; IU, International unit; JEOL, Japan Electron Optics Laboratory Company; JNK, Jun N-terminal kinase; KBr, Potassium bromide; kg, Kilogram;

km, Kilometre; L, Litre; LAF, lymphocyte-activating factor; LD, Low-dose; LPS, Lipopolysaccharide; M, Molar; MAP, Mitogen-activated protein kinases; MDA, Malondialdehyde; mg, Milligram; mL, Millilitre; mM, Millimolar; mm, Millimeter; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; mU, Milli- units; NCCS, National Centre for Cell Science; NF-κB, Nuclear factor kappa B; ng, Nanogram; NHIS, National Health Interview Survey; nm, Nanometre; nM, Nanomolar; NMR, Nuclear magnetic resonance; NO, Nitric oxide; NOS, Nitric oxide synthase; OD, Optical density; ODS-2, Octadecyl-silica-2; PD, Padina pavonica;

PGE-2, Prostaglandin E2; PGE-2, Picogram; pH, Hydrogen potential; PMP, 1-phenyl-3methyl-5-pyrazolone; ppm, Parts per million; RAW, Ralph, rAschke, Watson;

RBC, Red blood cells; RF, Rheumatoid factor; SGOT, Serum glutamic-oxaloacetic transaminase; SGPT, Serum glutamic-pyruvic transaminase; SOD, Superoxide dismutase; SpM, Spatolossum macrodontum; SPSS, Statistical Package for the Social Sciences; STAT, Signal transducer and activator of transcription; StM, Stoecho- spermum marginatum; TB, Tuberculosis; TD, Turbinaria decurrens; TFA, Trifluoroacetic acid; TNF-α, Tumor necrosis factor-α; U, Unit; UV, Ultraviolet; WBC, White blood cells.

* Corresponding authors at: Department of Biotechnology, University of Madras, Guindy Campus, Chennai 600025, India (E. Sanniyasi); Department of Micro- biology, Centre for Infectious Diseases, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University (Deemed to be University), Velappanchavadi, PH Road, Chennai-600077, Tamil Nadu, India (R.K. Gopal).

E-mail addresses: [email protected] (P.P. Raj), [email protected] (R.K. Gopal), [email protected] (E. Sanniyasi).

Contents lists available at ScienceDirect

Current Research in Biotechnology

journal homepage: www.elsevier.com/locate/crbiot

https://doi.org/10.1016/j.crbiot.2024.100220

Received 4 January 2024; Received in revised form 24 April 2024; Accepted 24 April 2024

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Current Research in Biotechnology 7 (2024) 100220 liver, kidney, and ankle joints reveals that fucoidan intake restrained inflammation and tissue damage. There- fore, fucoidan extracted from TD is a potential candidate for the treatment of rheumatoid arthritis.

Introduction

Arthritis is referred to as a joint disorder, in which joints such as ankles, wrists, knees, or knuckles inflammation occurs which later leads to swelling, joint ache, stiffness, and eventually reduced physical activity. From the data obtained from the National Health Interview Sur- vey (NHIS) during the period of 2013 to 2015, about 22.7 % of adults were diagnosed with arthritis and women had a higher prevalence of about 23.5 % when compared with that of men 18.1 % (Barbour et al., 2017). It is estimated that by the year 2040, about 78.4 million adults who are 18 years and above will be diagnosed with arthritis (Hootman et al., 2016).

Globally 350 million people have been reported to acquire arthritis.

There are hundred different forms of arthritis and the predominant types are Rheumatoid and Osteoarthritis. Above 22 % of adults beyond the age group of 40, has been reported with osteoarthritis around the world (Kolasinski et al., 2020). Whereas, according to the World Health Or- ganization, 14 million people have rheumatoid arthritis throughout the world (World Health Organization, 2022).

Glucocorticoids (GC) are administered intra-articularly or consumed orally to reduce the swelling and pain instantly but oral consumption is limited to only three or four months due to its severe side effects.

Treatments with disease-modifying anti-rheumatic drugs (DMARDs) have a slowed-down therapeutic effect of six to twelve weeks. Metho- trexate is a DMARD that is effective in controlling the disease. The alternative drugs to methotrexate are sulphasalazine or chloroquine, which could be used in combination to treat the symptoms. Leflunomide is an alternative drug to the aforementioned drugs, it blocks pyrimidine synthesis in vigorously dividing inflammatory cells due to which various hepatic and pulmonary side effects result (Benjamin et al., 2023).

Biological DMARDs apply to about 10–20 % of patients whose response to the above-mentioned drugs is inadequate (K¨ohler et al.,2019). Most of the biological DMARDs have anti-tumor necrosis factor alpha activity such as Infliximab, Etanercept, and Adalimumab but the most significant side effect of these drugs is the susceptibility to tuberculosis (TB). There are other biological agents such as rituximab which reduces the B-cells, abatacept which blocks the T-cell co- stimulation and tocilizumab, an inhibitor of interleukin-6 (Weaver, 2004). Due to several life-threatening side effects such as cardiovascular diseases, pulmonary and hepatic toxicity, etc., associated with the aforementioned arthritis treatment, an alternative natural method of treatment is required with fewer side effects.

The photosynthetic marine macroalgae known as seaweeds are almost copious in most of the regions in the ocean. In India about 8100 km long region is covered with coastline, which is considered to be one among the twelve mega-biodiversity Nations. Nearly 2.17 million km² regions are considered to be the Exclusive Economic Zone (EEZ) which is equal to sixty-six percent of the mainland area (Ganesan et al., 2019).

These seaweeds are classified into three main classes identified as Rhodophyta (red algae), Chlorophyta (green algae), and Phaeophyceae (brown algae) (Guiry and Guiry, 2021; Rindi et al., 2012). There are about 844 species of seaweeds reported in India, among which 434 species belong to red algae, 194 belong to brown algae, and 216 to green algae (Oza and Zaidi, 2001).

The polysaccharides are predominant in seaweeds with greater than eighty percent of their weight, which is dissimilar to the terrestrial plants due to the presence of polyuronides either acetylated, methylated, or sulfated. These polysaccharides are known as dietary fibers which are fermented to different degrees by the gut microbiota but cannot be digested entirely within the body (Nunraksa et al., 2019). It has been reported that Acanthopora, Gracilaria, Ulva, and Codium

comprise about 23 % to 64 % of dietary fibers which is higher when compared with that of the wheat bran (Garcia-Vaquero et al., 2017). The seaweed polysaccharides are predominantly categorized into sulfated and non-sulfated groups. The presence of sulfate moiety in the polysaccharide structure is known as sulfated polysaccharides and they include fucoidans in brown algae, carrageenans in red algae, and ulvans in green algae.

Fucoidan derived from seaweeds exhibits wide range of bioactivities includes anti-inflammatory, immunomodulatory, antiviral, antidiabetic, anticancer, antioxidant, and anticoagulant activities (Cholaraj and Venkatachalam, 2024; Dhahri, 2023; Geng et al., 2024; Hwang et al., 2024; Lee et al., 2024; Li et al., 2024; Nagarajan and Mathaiyan, 2015;

Nguyen et al., 2024; Purwanto et al., 2024; Sanjeewa et al., 2017;

Sanniyasi et al., 2019; Shi et al., 2024; Ummat et al., 2024). These activities are intensively ascribed to the active participation of gut mi- croorganisms and sulfated polysaccharides (Seedevi et al., 2017).

The inflammatory response of the immune system towards various stimulants such as infection, stress, and injury lead to the migration of neutrophils and macrophages to the site of association. This process of migration is known as diapedesis which further steers the increased production of inflammatory mediators such as interleukin-1β (IL-1β), nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2), which are the cause of prolonged inflammation (Jeong et al., 2017; Muralidharan and Mandrekar, 2013).

The fucoidan from brown seaweeds has been reported to interrupt the different stages of the inflammatory process. Firstly, it blocks lymphocyte adhesion and diapedesis, secondly, it inhibits several enzymes involved in the inflammatory process and thirdly it has been reported to induce apoptosis. Several in-vitro and in-vivo studies were carried out to investigate the anti-inflammatory and immunomodulatory properties of the fucoidan from seaweeds. In all the studies, the outcome of the fucoidan treatment was common that is, all the models exhibited reduced phosphorylation of MAPK, ERK, and p38 pathways and downregulation of various signaling pathways such as JNK, NF-κB, STAT1, AKT, MAPK, and ERK; increased levels of cytokines such as IFN-γ and IL-10; declined secretion of TNF-α, IL-1β, IL-6, CD86, IL-8, PGE2, and COX-2 expression; decreased synthesis of nitric oxide and inducible nitric oxide synthesis (Apostolova et al., 2020). Contemporaneously, fucoidan derived from Fucus vesiculosus collected from Barents Sea significantly hampers the cyclooxygenase-2 (COX-2) enzyme with a higher selectivity index (lg(IC80 COX-2/IC80COX-1) of − 1.55 than that of the standard drug indomethacin (− 0.09). It also inhibits the LPS induced MAPK-p38 expression and dose-dependent inhibition on hyal- uronidase enzyme (Pozharitskaya et al., 2020).

Therefore, the objective of the study is to evaluate the in vitro anti- inflammatory and in vivo anti-arthritis potential of fucoidan from a brown seaweed.

Materials and methods Collection of brown seaweeds

Five different brown seaweeds such as Padina pavonica (Linnaeus) (Taylor, 1960) (PD), Stoechospermum marginatum (C. Agardh) (Kützing, 1843) (St.M), Spatoglossum macrodontum (Agardh, 1882) (Sp.M), Tur- binaria decurrens (Duperrey et al., 1825) (TD) and Dictyota bartayresiana (Lamouroux, 1809) (DD) were collected from Olaikuda, and Puduma- dam coastal regions of Ramnad district, Tamil Nadu, India. The collected seaweed samples were shade-dried, homogenized, and stored at room temperature for further studies. The seaweeds were identified by Prof.

Baluswami, Former Professor, Department of Plant Biology and Plant P.P. Raj et al.

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Current Research in Biotechnology 7 (2024) 100220 Biotechnology, Madras Christian College (Autonomous), Chennai. India.

The voucher numbers of the specimens are UnOM-BT-05 (PD), UnOM- BT-07 (St.M), UnOM-BT-08 (Sp.M), UnOM-BT-10 (TD), and UnOM-BT- 13. According to Kaliaperumal and Rao (1975), the reproductive re- ceptacles (high oospore dispersal) in T. decurrens were reportedly greater in the month of November (Kaliaperumal and Rao, 1975). Thus, for in vivo study, the seaweed T. decurrens was again collected during the month of November 2019.

Optimization of the extraction of fucoidan

For optimization, Padina pavonica (PD) biomass was utilized and the best method of extraction was determined based on the yield of fucoidan. About six different extraction methods were carried out, Method 1 was based on Baky et al. (Abd El Baky et al., 2014); Method 2 was as described by Ji et al. (Ji et al., 2011); Method 3 was defined by Mohamed and Agili, (Mohamed and Agili, 2013); Method 4 was given by Dore et al. (Dore et al., 2013); Method 5 by Immanuel et al. (Immanuel et al., 2012); and Method 6 was carried out based on Garcia-Rios et al.

(García-Ríos et al., 2012) (Table 1).

Characterization of fucoidan

Estimation of carbohydrate in fucoidan

For the estimation of carbohydrates, Du Bois et al. (DuBois et al., 1956) method was followed. A 200 µL of test samples (fucoidan) or the standard was used. The standard used was dextrose, which was serially diluted at 20, 40, 80, 160, 320, and 640 μg/mL concentrations. Then 0.5 mL of phenol (5 %) was added followed by the addition of 2.5 mL of 100

% H2SO4 and incubated for 15 min. and the absorbance values were measured at 480 nm in a spectrophotometer (Hitachi U-2900). The carbohydrate content was determined by the standard graph.

Estimation of sulfate in fucoidan

Terho and Hartiala (Terho and Hartiala, 1971) method was applied for the estimation of sulfate. The sodium sulfate (2 mg/mL) was used as standard which was serially diluted to 20, 40, 60, 80, 100, 120,140, 160, 180, and 200 μg/mL concentrations. The fucoidan samples were hydrolyzed with 2 M of trifluoroacetic acid at 121 ^◦C for 1 hr and it was neutralized (pH =7.0) with 1 N NaOH (Wang et al., 2014), before sulfate content determination. About 100 µL of each sample or standard solution was added to 400 mL of 95 % ethanol followed by 1 mL of barium chloride buffer (Table 2) and 1.5 mL of sodium rhodizonate solution (Table 3). This entire mixture was prepared in the dark condition, and incubated for 10 min. at room temperature, and measured spectrophotometrically at 520 nm. The sulfate content was determined by the standard graph.

Fourier transform infrared spectrometry (FT-IR) analysis

For FT-IR analysis, a pellet was prepared by compressing up to 10,000 psi. The pellet comprises 1 mg of fucoidan (acid-digested) mixed with 100 mg of potassium bromide (KBr). Then, the obtained pellet was placed in the FT-IR sample holder and the samples were analyzed by FT- IR spectrometer in a frequency range between 600 and 4000 cm⁻¹ (Sanniyasi et al., 2019).

In vitro, the anti-inflammatory activity of fucoidan extracted from five different brown seaweeds

DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging assay

DPPH free-radical scavenging assay on the fucoidan samples was carried out using a modified method of Kavitha et al. (Kavitha and Perumal, 2018). The ascorbic acid was used as standard and a series of concentrations of samples or standard (5, 10, 20, 40, 80, 160, and 320 μg/ml) were intermixed with 2.5 mL of DPPH solution (0.135 mM DPPH in methanol). The mixture was then thoroughly vortexed, incubated in dark conditions for 30 min., and spectrophotometrically analyzed at 517 nm. The percentage inhibition of free radical generation was derived from the equation given below.

%DPPHinhibition= [(ODofcontrol− ODoftest)/(ODofcontrol)] ×100 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay (Mosmann, 1983)

The RAW 264.7 cell line (Peter, 2021; Raschke et al., 1978) was procured from the National Centre for Cell Science (NCCS), Pune. The stock cells were cultured in a medium containing 10 % inactivated fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/

mL) and incubated in a carbon dioxide incubator in 5 % CO2 at 37 ^◦C Table 1

Optimization of different procedures for the extraction of sulphated polysaccharide from seaweeds.

Extraction

Methods Sample

quantity Solvents and chemicals used Temperature

maintained Incubation period Centrifugation time References

1. 5 g Water and Absolute

Ethanol. 100 ^◦C 2 hr 10,000 rpm for 10 min (Abd El Baky

et al., 2014) 2. 5 g Water, Ethanol (95 %) and 10 % CaCl2. 80 ^◦C 2 hr, 1 hr, overnight 13,000 rpm for 5 min (Ji et al., 2011)

3. 5 g Petroleum ether and Acetone, 0.1 M

HCl (1:100), 2 % Na2CO3

(1:100) water, 10 % CTAB and 20 % Ethanolic KI.

80 ^◦C, 90 ^◦C, 4 ^◦C Overnight, 1 hr, 2 hr,

15 min, 4 hr 8,000 rpm for 10 min (Mohamed and Agili, 2013)

4. 5 g Acetone, 0.25 M NaCl, Alkaline

protease. 60 ^◦C Overnight, 4 hr 15,000 rpm for 20 min (Dore et al., 2013)

5. 5 g Petroleum ether and Acetone, water, 1

% CaCl2 and ethanol (30 % and 70 %). 80 ^◦C, 4 ^◦C Overnight, 1 hr 10,000 rpm for 10 min and

8,000 rpm for 10 min (Immanuel et al., 2012)

6. 5 g 0.05 M HCl, 0.5 M

NaOH and Ethanol. Room temperature 2 hr, 1 hr 3,500 rpm for 20 min,

8,000 rpm for 10 min (García-Ríos et al., 2012) Table 2

Preparation of barium chloride buffer.

S.NO. Ingredients Quantity

1. 2 M Acetic acid 10 mL

2. 5 mM barium chloride 2 mL

3. 0.02 M sodium hydrogen carbonate 8 mL

Made up of 95 % ethanol to 100 mL

Table 3

Preparation of sodium rhodizonate solution.

S.NO. Ingredients Quantity

1. Sodium rhodizonate 5 mg

2. Distilled water 20 mL

3. L-ascorbic acid 100 mg

Made up with ethanol to 100 mL

P.P. Raj et al.

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Current Research in Biotechnology 7 (2024) 100220 until it is confluent. The trypsinization of the monolayer cell lines was

carried out and 100 μL of the cell suspension which was diluted (50,000 cells/well) was added to each well in the 96-well microtiter plate (Crouch et al., 1993).

When a partial monolayer was formed after 24 hr, the supernatant was removed and the monolayer was once washed with medium and 100 μL of different concentrations (3.125, 6.25, 12.5, 25, 50, 100 μg/mL concentrations) of the test drugs (fucoidan) were added to the partial monolayer in microtiter plates. The plates were then incubated for 24 hr in a 5 % CO2 atmosphere at 37 ^◦C (Gonzalez and Tarloff, 2001).

The test solution present in the wells was then discarded after incubation and to the same wells, 100 μL of MTT reagent (5 mg/mL concentration dissolved in buffer and vortexed thoroughly) was added.

The 96-well plates were then incubated in a carbon dioxide (5 % CO2) incubator at 37 ^◦C for 4 hr. The supernatant formed was then removed from the wells and 100 μL of dimethyl sulfoxide was added and gently agitated the plates to mix the formed formazan. At 570 nm wavelength, the absorbance value was measured using a microplate reader. The percentage of growth inhibition was calculated using the following formula:

Percentageofinhibition(Cellproliferation)

= (ControlOD− (SampleOD/ControlOD)) ×100

The inhibition concentration of the test drug (fucoidan) required to inhibit the cell growth by 50 % and (IC50) values were generated from the dose–response curves on treated cells.

Nitric oxide inhibition assay on LPS-induced RAW 264.7 cell lines About 100 μL of the diluted cell suspension of RAW 264.7 cell lines (50,000 cells/well) was added to each well of 96 well microtiter plate.

After 24 hr, when a partial monolayer was formed, the supernatant was removed and the monolayer was washed once with the medium. A serial concentration of fucoidan (3.125, 6.25, 12.5, 25, 50, 100 μg/mL concentrations) was prepared in phenol red-free Dulbecco’s Modified Eagle Medium (DMEM) to give a total volume of 100 μL in each well of a microtiter plate.

After one hour of incubation, the cells were stimulated with 1 μg/mL of LPS. The plates were then incubated at 37 ^◦C for 24 hr in a 5 % CO2

incubator. Following the incubation, about 100 μL of the culture medium with an equal volume of Griess reagent was incubated at room temperature for 10 min, in a 96-well plate. At 540 nm wavelength, the absorbance was measured using a microplate reader (Joo et al., 2014).

From this in vitro study, a single seaweed sample encountered with high anti-inflammatory activity was chosen for the in vivo anti-arthritic studies.

Purification of fucoidan by ion-exchange chromatography

The fucoidan extract of the selected brown alga, Turbinaria decurrens (TD) was further purified using anion exchange chromatography DEAE (Diethylaminoethyl cellulose from Sigma-Aldrich, 15 ×2.5 cm column).

The sample was then eluted with 5 mL of sodium chloride (different molar concentrations ranging from 0 to 3.6 M) with a 0.4 M interval in 50 mM sodium acetate buffer (pH 5.0). Each fraction was collected and dialyzed (Dialysis membrane-70, HiMedia) against Milli-Q water to free the fractions from sodium chloride and conserved for further processes (Sanniyasi et al., 2019).

Monosaccharide composition of fucoidan

Fucoidan (10 mg) was hydrolyzed for an hour at 121 ^◦C in 0.5 mL of 2 M trifluoroacetic acid (TFA). The hydrolyzed fucoidan was then derivatized with PMP (1-phenyl-3methyl-5-pyrazolone) by combining it with the same volume of 0.6 M NaOH, followed by the addition of 100 µL of 0.5 M PMP diluted in methanol, vortexing, and incubating for 1 h at 70 ^◦C in a water bath. A 120 µL of 0.3 M HCl was added to the mixture when it had cooled, and 1 mL of dH2O was used to dissolve it. The

organic phase was obtained after adding 1 mL of chloroform, vortexed, and discarded. After passing through a 0.45 µm membrane filter, the derivatized aqueous phase was eluted using thermo hypersil ODS-2 C18 HPLC columns (250 mm x 4.6 mm, flow rate: 1 mL/ min) at 30 ^◦C.

Fucoidan’s monosaccharide content was determined using UV absorbance at 245 nm.

Two-dimensional nuclear magnetic resonance spectrometric (2D-NMR), correlation spectrometry (COSY)

The purified fucoidan from TD was subjected to ¹H NMR analysis followed by 2D-NMR-

COSY. Both the analyses were done by JEOL (Delta v5.3.2.) The in- strument with spectrometer JNMECZ400S/L1 and D2O (deuterium dioxide) was used as a solvent. Both the 1D NMR and 2D COSY spectrums of pure fucoidan from TD were retrieved, interpreted, and resulted.

In-vivoanti-arthritic activity of fucoidan in rat model Acute toxicity studies

A group of six Wistar male rats were orally administered with fucoidan from TD in graded doses of 1000, 800, and 400 mg/kg body weight. Rats were continuously observed for a period of 48 hr to check for mortality and behavioral changes initially and once daily thereafter till 14 days. The one-tenth and one-fifth of the lethal dose were selected further to carry out the in vivo studies.

Arthritis induction in rat model using complete Freund’s adjuvant Arthritis was induced in the left hind paws of the rats, by intra- peritoneal injection of 0.1 mL of Complete Freund’s Adjuvant (CFA).

The CFA (consists of heat-killed Mycobacterium tuberculosis in sterile paraffin oil (10 mg/mL)). The induction of arthritis was observed within 14 days from the day of administration of CFA. The rats were orally fed with the test compound fucoidan (low- and high-dose) and standard drug (indomethacin) from 14 − 28 days of induction of arthritis.

Anti-arthritic activity of fucoidan

The anti-arthritic potential of fucoidan from TD was determined in vivo in the Wistar male rat model with indomethacin as the standard drug. In this study, five different rat groups were used and each group comprised eight rats. The first group consisted of normal rats (no arthritis induction), the second group was induced with inflammation and treated with a standard drug (indomethacin), the third was a control group (no treatment), the fourth group was treated with the test sample (low-dose – 20 mg/kg) and the fifth group was treated with a high-dose of the test sample (40 mg/kg) (Fig. 1). The rats were euthanized, paw edema in the rat foot, X-ray studies of paw edema, proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-10), biochemical, hematological, and histopathological parameters were recorded and interpreted.

Paw edema on the foot of rats

For analysis of paw edema (inflammation on the foot of a rat), paw volume (Millimetres (mm)) of rat groups was determined using a Ple- thysmometer on the 0th, 4th, 14th^,and 28th days of incubation after induction of arthritis by CFA. The increase or decrease of inflammation on the paw of rats was analyzed for the normal, positive control (indomethacin), negative control, test sample low-dose (20 mg/kg), and test sample high-dose (40 mg/kg) were recorded.

X-ray radiographical imaging on the paw edema of rats

The X-ray radiographical analysis was carried out on the 28th day of incubation of arthritis. The X-ray photographic image depicts the status of inflammation or edema on the paw of rat groups including normal, positive control indomethacin), negative control, test sample low dose (20 mg/kg), and test sample high dose (40 mg/kg).

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Current Research in Biotechnology 7 (2024) 100220

In vivo quantitative expression of cytokines

The serum was segregated from the blood collected from all the rat groups by centrifugation at 3000 rpm for about 10 min and stored at − 20 ^◦C until further evaluation. The expression of different cytokines such as TNF-α, IL-6, IL-1β, and IL-10 were determined by Sandwich-ELISA (Enzyme-Linked Immuno-Sorbent assay) according to manufacturer’s protocol (MyBioSource.Inc) at room temperature. Rat TNF-α (Tumor Necrosis Factor Alpha) ELISA Kit (Catalog No. MBS2507393), Rat Interleukin 6 (IL-6) ELISA Kit (Catalog No. MBS269892), Rat IL-1 beta ELISA Kit (Catalog No. MBS824956), and Rat IL-10 ELISA Kit (Catalog No. MBS355232) were employed for Sandwich-ELISA assay on the expression of cytokines TNF-α, IL-6, IL-1β and IL-10 respectively (Fig. 2).

The absorbance (ABS) at 450 nm was measured with a microplate reader (Robonik, India), and concentration of cytokines were determined with their respective reference standards.

Estimation of biochemical parameters

The biochemical parameters were quantitatively determined based on the method illustrated in the respective Assay Kit manufacturer’s protocol. Superoxide dismutase (SOD) Activity Assay Kit (Colorimetric) from BioVision − Catalogue No. K335-100; Catalase Activity Colori- metric/Fluorometric Assay Kit from BioVision – Catalogue No. K773- 100; Reduced Glutathione (GSH) Colorimetric Assay Kit from Elabs- cience – Catalogue No. E-BC-K030-M; Glutathione peroxidase (GPX) Assay Kit (Colorimetric) from Abcam – Catalogue No. ab102530;

Glutathione S-Transferase Assay Kit (Colorimetric) from Abcam – Catalogue No. ab65326; Lipid Peroxidation (MDA) Assay Kit (Colori- metric/Fluorometric) from Abcam – Catalogue No. ab118970; Urea Colorimetric Assay Kit from BioVision – Catalogue No. K375-100; Uric Acid Colorimetric/Fluorometric Assay Kit from BioVision – Catalogue No. K608-100; Rat Total Protein Elisa Kit from MyBioSource.Inc – Fig. 1.The illustration of an in vivo anti-arthritis study of fucoidan on Wistar male rats.

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Catalogue No. MBS3808613; Creatinine (Serum) Colorimetric Assay Kit from Cayman Chemical – Catalogue No. 700460; Bilirubin (Total and Direct) Colorimetric Assay Kit from BioVision – Catalogue No. K553- 100; Alanine Transaminase.

Activity Assay Kit (SGPT) (Colorimetric/Fluorometric) from Abcam – Catalogue No. ab105134; Aspartate Aminotransferase Activity Assay Kit (SGOT) from Abcam – Catalogue No. ab105135; Alkaline Phosphatase (ALP) Assay Kit (Colorimetric) from Abcam – Catalogue No. ab83369;

Rat Albumin ELISA Kit from CrystalChem – Catalogue No. 80662.

Estimation of hematological parameters

Hematological parameters including hemoglobin (Cyanmethemo- globin Method) described by Bhaskaram et al. (Bhaskaram et al., 2003);

erythrocyte sedimentation rate (ESR) by Miller et al. (Miller et al., 1983); rheumatoid factor (RF) by Rat rheumatoid factor ELISA kit from MyBioSource.Inc – Catalogue No. MBS702417; and C-reactive Protein (CRP) by rat C-reactive protein ELISA kit from Abcam – Catalogue No.

ab108827 were determined quantitatively. For RBC and WBC counts, about 500 µL of blood sample was collected from the left ventricle of the heart of rats using a syringe and transferred to capillary pipettes containing anticoagulant EDTA. Then the RBC and WBC counts were analyzed using an automated hematology analyzer (Sysmex XE-5000 hematology analyzer, Sysmex Kobe, Japan). RBCs and WBCs were counted and recorded as 1 x 10⁶cells/µL, and 1 x 10³cells/µL respectively.

Histopathological study

The organs such as the liver, kidney, and ankle joints were isolated from the rats after euthanized and stored in 10 % buffered formalin solution (pH – 7.0) for 24 hr and washed with ethanol (70 %). Then the organs were dehydrated using 100 % ethanol and embedded in paraffin and thin sections were obtained by a rotary ultra-microtome. The obtained thin sections were stained using hematoxylin and eosin dyes, mounted on a glass slide, and observed under a light microscope and photographs were taken and recorded.

Statistical analysis

The data obtained from in vitro and in vivo studies were evaluated with SPSS 20.0. The values expressed were the means of triplicate data.

Results

Collection of brown seaweeds

The marine seaweed samples were collected from two different locations on the East Coast Region namely Pudumadam (9^◦16

′

21.34

″

N, 78^◦59

′

_32.33

″

E), and Olaikuda (9^◦18

′

_52.06

″

_{N, 79}^◦₂₀

′

_04.33

″

_{E) of Man-}

dapam, and Rameswaram respectively in the Ramnad District of Tamil Nadu, India (Fig. 3). Turbinaria decurrens (TD) represents the family Sargassaceae, whereas other seaweeds (PD, St.M, Sp.M, and DD) belong to the family Dictyotaceae (Fig. 4).

Optimization of the extraction of fucoidan

Among the different extraction methods involved in this study, method M5 was found efficient in yielding about 9.58 % of crude fucoidan from the dry biomass of seaweeds (Fig. 5A).

Characterization of fucoidan

The total crude fucoidan yield was found quantitatively higher in Dictyota bartayresiana (DD) and Turbinaria decurrens (TD) with 0.937 g and 0.716 g respectively from about 10 g of dry seaweed biomass (Fig. 5B). Carbohydrate content was determined higher in PD (63.45 %) and low sulfate content of 19.43 %), whereas, Sp.M had low carbohydrate content of 54.34 % and low sulfate content of 21.18 % (Fig. 5C).

TD and DD have a significant carbohydrate and sulfate contents of 58.32

% and 32.34 %, and 59.28 % and 25.64 % respectively. However, St.M constitutes of 56.2 % and 24.58 % of carbohydrate and sulfate (Fig. 5C).

Nevertheless, the estimated fucoidan content was found greater in TD (90.66 %), followed by DD (84.92 %), PD (82.86 %), and lower in St.M (80.78 %) and Sp.M (75.52 %).

Fourier transform infrared spectrometry (FT-IR) analysis

In the FT-IR spectrum, down peaks at frequency wavelengths 1411.89 cm⁻¹(R-O-SO3-), and 1116.78 cm⁻¹(R-SO3-) in PD; 1409.96 cm⁻¹(R-O-SO₃^-), 1296.16 cm⁻¹, and 1124.50 cm⁻¹(R-SO₃^-) in St.M;

1409.96 cm⁻¹, 1232.51 cm⁻¹, and 1124.50 cm⁻¹in Sp.M; 1407 cm⁻¹, 1200.98 cm⁻¹in DD; and 1201.82 cm⁻¹, and 1133.07 cm-¹in TD represents both the organic sulfate and sulphonate groups. Similarly, peaks Fig. 2. The illustration of sandwich-ELISA assay for the quantitative expression of cytokines.

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at 812.03 cm⁻¹, 812.03 cm⁻¹, 810.03 cm⁻¹, 799.60 cm⁻¹, and 780 cm⁻¹ depict the presence of C-O-S stretch to sulfate group. Consequently, the C–H stretch (2916.37 cm⁻¹, and 2850 cm⁻¹in PD; 2922.16 cm⁻¹, and 2852.72 cm⁻¹in St.M; 2922.16 cm⁻¹, and 2852.72 cm⁻¹in Sp.M;

2915.03 cm⁻¹, and 2849.14 cm⁻¹in DD; and 2915.39 cm⁻¹, and 2849.08 cm⁻¹in TD found in all the fucoidan samples of five different seaweeds highlights the carbohydrate content.

Therefore, based on the results obtained from the FT-IR analysis, the presence of functional groups such as organic sulfates (R-O-SO3-),

sulphonates (R-SO3-), C-O-S stretch to a sulfate group, and C–H stretch to carbohydrate together constitute the presence of fucoidan (sulfated polysaccharide) in all the five different seaweed samples (Fig. 6).

In vitro anti-inflammatory study of fucoidan

The in vitro anti-inflammatory study constitutes, a DPPH free radical scavenging assay, MTT cytotoxicity assay, and LPS-induced nitric oxide inhibition assay.

Fig. 3.A) Topo-geographical view of the seaweed sample collection site showing Pudumadam and Olaikuda located in the State of Tamil Nadu, India; B) Green pinpoint showing both the locations are situated in the Ramnad District of Tamil Nadu, where Olaikuda is situated in the Rameswaram Island; C) and D) shows the exact location where seaweed samples were collected (Google Earth Pro 7.3.3.7699). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.Five brown seaweeds collected for the study from Pudumadam, and Olaikuda coastal area; PD: Padina pavonica, St.M: Stoechospermum marginatum, Sp.M:

Spatoglossum macrodontum, DD: Dictyota bartayresiana, and TD: Turbinaria decurrens.

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DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging assay The DPPH free radical scavenging assay infers the antioxidant nature of the compounds. Therefore, fucoidan extracts were subjected to analysis, for their antioxidant activity by comparing them with a standard drug ascorbic acid. The oxidation of DPPH turns it into a purple color. Whereas, it reduces when it reacts with a strong antioxidant and remains yellow. It resulted that, the fucoidan from the five different seaweeds exhibited no antioxidant activity and remained purple when the concentrations were increased (Supplementary figure 1).

The calculated percentage of inhibition of oxidation of DPPH was also found very least with <30 % for all the fucoidan samples, but the standard drug ascorbic acid had >90 % inhibition when the concentration was gradually increased (Fig. 7). The IC50 values were also determined and found >320 µg/mL for all the fucoidan extracts. The IC50 value of the standard (Ascorbic acid) was only 28.42 µg/ml.

Therefore, fucoidan extracts of all five seaweeds exert no antioxidant properties.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay

The cytotoxicity of fucoidan extracted from the five different seaweeds was analyzed on the RAW 264.7 macrophage cell lines. The percentage of cell viability persists even though the concentration of fucoidan increased. The conversion of MTT into formazan was seen in all the concentrations tested and revealed the metabolic activity of live cells (Supplementary figure 2). At a higher concentration of fucoidan (100 µg/mL), the cell viability exists with 72.7 % for PD, 71.8 % for St.M, 84.3 % for Sp.M, 81.6 % for DD, and 83.2 % for TD (Supplementary

figure 2). Therefore, the IC50 value of fucoidan from all the five different seaweeds was found as >100 µg/mL. Hence, fucoidan extracted from Padina pavonica (PD), Stoechospermum marginatum (St.M), Spatoglossum macrodontum (Sp.M), Dictyota bartayresiana (DD), and Turbinaria decur- rens (TD) exhibits less cytotoxicity on macrophage cell lines.

Nitric oxide inhibition assay on lipopolysaccharide (LPS) induced RAW 264.7 Macrophage cell lines

The lipopolysaccharide (LPS) mediates unrestrained expression of nitric oxide synthase (NOS), and the inducible isoform of nitric oxide synthase (iNOS), results in exacerbated production of nitric oxide in RAW 264.7 macrophage cells and thus leads to inflammation. When the Griess reagent reacts with available nitric oxide, the solution turns pinkish-red in color and diminishes when the concentration of nitric oxide is low. The nitric oxide inhibition was analyzed for the fucoidan extracts of all five different brown seaweeds. It resulted that, the nitric oxide inhibition was found higher in 50 µg/mL, and 100 µg/mL concentrations of fucoidan extracts of both Dictyota bartayresiana (DD) and Turbinaria decurrens (TD) when compared with other fucoidan extracts (Supplementary figure 3). Consequently, it has been revealed that the fucoidan extracts of all the five different seaweeds do not inhibit the nitric oxide content directly, whereas, it inhibits the synthesis of nitric oxide when treated with RAW 264.7 macrophage cells.

Intriguingly, at a maximum concentration of fucoidan (100 µg/ mL), the lowest production of nitric oxide was evident in RAW 264.7 macrophage cells. However, among five different seaweeds, Dictyota bartayresiana (DD), and Turbinaria decurrens (TD) showed relatively greater inhibition of nitric oxide generation with the IC50 value of 29.70 µg/mL, and 21.93 µg/mL (Fig. 8). The IC50 values for Padina pavonica Fig. 5. A) Quantitative yield of fucoidan obtained from different extraction methods; B) Total fucoidan obtained from five brown seaweeds; C) Carbohydrate and sulfate content in five brown seaweeds.

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Fig. 6. FT-IR spectrum of fucoidan of their respective brown seaweeds.

Fig. 7.Graphical representation of the antioxidant activity of fucoidan (DPPH free radical scavenging assay) from different brown seaweeds.

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(PD), Stoechospermum marginatum (StM), and Spatoglossum macrodontum (SpM) were 34.13 µg/mL, 339.48 µg/mL, 178.63 µg/mL. Therefore, fucoidan from Turbinaria decurrens (TD) was found as a potential anti- inflammatory agent than the other fucoidan samples extracted from four different seaweeds studied in this investigation. Hence, fucoidan from Turbinaria decurrens (TD) was selected and forwarded for the in vivo Anti-arthritis studies.

Purification of fucoidan by ion-exchange chromatography

For characterization and in vivo anti-arthritis studies, about 200 g of shade-dried and homogenized Turbinaria decurrens biomass was subjected to hot-water extraction, followed by the removal of alginate and partial purification by membrane dialysis. The crude fucoidan was purified by anion exchange chromatography using DEAE Cellulose at different concentration gradients of sodium chloride (NaCl). Hence, comparatively 0.8 M NaCl concentration was found effective in eluting pure fucoidan among another concentration gradient. The total yield of pure fucoidan was estimated as 19.453 g, which was about 9.726 % of the total biomass of Turbinaria decurrens utilized (200 g).

Monosaccharide composition of fucoidan

The fucoidan isolated from TD constitutes 58.2 % of fucose, 13.4 % of galactose, 11.9 % of xylose, 10.6 % of mannose, and 5.4 % of rhamnose.

Two-dimensional nuclear magnetic resonance spectrometric (2D-NMR), correlation spectrometry (COSY)

A standard fucoidan from Fucus vesiculosus (Sigma-Aldrich) was also characterized by 2D-NMR-COSY in addition to the fucoidan of TD. The

1H NMR spectrum of standard Fucoidan consists of peaks at 5.337 ppm, 5.279 ppm represents the presence of Anomeric proton in the compound, peak at 3.886 ppm corresponds to ring protons with sulfate groups, and finally, peaks at 2.216 ppm, 1.661 ppm, 1.610 ppm, 1.386 ppm, and 1.242 ppm corresponds to the methylated protons of the compound (Fig. 9A). Similarly, the ¹H NMR spectrum of fucoidan isolated from Turbinaria decurrens also showed anomeric protons (5.334 ppm, 5.197 ppm), ring protons (4.440 ppm, 4.111 ppm, 3.829 ppm, 3.764 ppm), and methylated protons (2.161 ppm, 2.063 ppm, 1.435 ppm, 1.377 ppm, 1.22 ppm) (Fig. 9B).

Results from 2D-COSY NMR reveal that the proton density around 4.0 ppm and between 1.0 ppm and 2.0 ppm represents the ring protons with sulfate groups and methylated protons respectively found in both Fig. 8. Graphical representation of nitric oxide inhibition of fucoidan at different gradient concentrations.

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the standard fucoidan (Sigma-Aldrich) and fucoidan from Turbinaria decurrens (Fig. 9A and B). Therefore, the 2D-NMR COSY results evidently revealed the purity of fucoidan from TD.

In vivo anti-arthritic studies of fucoidan Acute toxicity study

At the dose of 400 mg/kg b.w., there were no abnormal clinical signs including skin color and behavioral changes like alertness, grooming, restlessness, tremors, convulsion, and writhing at any time during the observation period. The effect of fucoidan on touch response, torch

response, pain response, righting reflex, gripping strength, pinna reflex, corneal reflex, pupils, urination, salivation, and lacrimation were also found normal. These results indicated that the fucoidan was quite safe even at a high dose of 400 mg/kg b.w. and had no acute toxicity at this dose. Therefore, as a result of the acute toxicity study, the fucoidan from TD, 20 mg/kg and 40 mg/kg b.w. dosages were fixed as low-dose and high-dose respectively for carrying out the in vivo anti-arthritic study.

Paw edema on the foot of rats

As a result, the paw edema was not developed in the normal rat group (arthritis not induced) (Fig. 10A, Group 1). In the case of the Fig. 9. A) The ¹H NMR spectrum of the standard fucoidan (Sigma Aldrich) of Fucus vesiculosus (inside 2D-COSY spectrum); and B) pure fucoidan of Turbinaria decurrens (TD) (inside 2D-COSY spectrum).

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standard drug (indomethacin) supplemented rat group, paw edema was under control (Fig. 10A, Group 2). However, in the control rat group, no test compound or standard drug was supplemented, therefore, huge paw edema was developed (Fig. 10A, Group 3). Intriguingly, the size of paw edema was comparatively smaller than the control rat group in the fucoidan-supplemented dose rat group (Fig. 10A, Group 4), and paw

edema was completely cured and reduced in the fucoidan-supplemented high-dose rat group (Fig. 10A, Group 5). Therefore, paw edema developed by the induction of the arthritis was cured completely by the test compound fucoidan in vivo.

Based on the paw edema volume determined by the Plethysmometer, the paw volume was around 3 mm on the 0th day of induction of Fig. 10. A) Photographs showing the paw edema on Wistar male rats, Group 1: Normal rat; Group 2: Indomethacin treated rat; Group 3: Control rat; Group 4:

Fucoidan, Low-dose treated rat; Group 5: Fucoidan, high-dose treated rat. B) The estimated volume of paw edema on the 4th, 14th, and 28th days of induction of arthritis in rats on various rat groups. C) Percentage reduction of paw edema on the 4th, 14th, and 28th days of induction of arthritis in rats supplemented with fucoidan and standard drug.

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Current Research in Biotechnology 7 (2024) 100220 arthritis, whereas, the paw edema was developed in all the rat groups

except the normal rat group (arthritis not induced). The paw edema volume was 6.23 mm, 5.03 mm, and 3.9 mm in the indomethacin- supplemented rat group, for the 4th, 14th, and 28th days of induction respectively in which, the paw edema volume was decreased gradually (Fig. 10B). Similarly, the paw edema volume was 7.66 mm and 7.23 mm for low-dose (20 mg/kg) and high-dose (40 mg/kg) fucoidan supplemented rat groups on the 4th day of induction, which was gradually decreased on the 14th day and 28th days of induction into 6.46 mm and 5.63 mm and 5.33 mm and 4.8 mm respectively for low-dose and high- dose (Fig. 10B). Intriguingly, it is clear, that the fucoidan supplemented rat groups showed a decrease in the volume of paw edema (inflammation) in arthritis induced rats. There was no change in the paw volume of control rat groups on the 4th, 14th, and 28th day of induction with 8.13 mm, 7.43 mm, and 7.56 mm respectively (Fig. 10B). Percentage reduction in paw edema volume on fucoidan supplementation was about 5.78 %, 13.06 %, and 29.50 % in LD (Low-dose: 20 mg/kg) for 4th, 14th and 28th days of arthritis induction respectively (Fig. 10C). Whereas, it was 11.07 %, 24.23 %, and 36.51 % in HD (High-dose: 40 mg/kg) for the same consecutive days of arthritis induction respectively (Fig. 10C).

X-ray imaging study on the paw edema of rats

The X-ray photographic study serves as additional evidence of the anti-arthritic potential of fucoidan. The normal rat group showed no inflammation or edema in the foot ankle joints (Fig. 11, Group 1).

Similarly, no paw edema or inflammation was recorded in the standard drug (indomethacin) supplemented rat group (Fig. 11, Group 2).

However, huge paw edema or inflammation (See arrow marks in Fig. 11, Group 3) in the ankle joint was evident in the control rat group. But the inflammation (Paw edema) in the ankle joints was slightly seen in the fucoidan low-dose rat group (see arrow marks in Fig. 11, Group 4) and the same was completely cured and absent in the fucoidan high-dose rat group (Fig. 11, Group 5). Therefore, it is clear that fucoidan derived from TD is a potential compound for the treatment of arthritis.

In vivo quantitative expression of cytokines

Four different cytokines including Interleukin 10 (IL-10), Interleukin 1β (IL-1β), Interleukin 6 (IL-6), and Tumor necrosis factor-α (TNF-α), and their expression were studied in vivo. Interleukin 10 (IL-10) is

otherwise termed a cytokine synthesis inhibitory factor (CSIF) an anti- inflammatory cytokine that affects inflammation and immunoregula- tion. IL-10 serves in the inhibition of tumor metastasis. So, the expression of IL-10 results in the suppression of inflammation. In this in vivo study, IL-10 gene expression was found very low in the control rat group (41.54 pg/mL). However, it was high in normal rat group (87.3 pg/mL), followed by low-dose rat group (50.03 pg/mL), high-dose rat group (63.96 pg/mL), and indomethacin rat group (73.66 pg/mL) (Supple- mentary figure 4). Moreover, the IL-10 gene expression was significantly enhanced in low-dose and high-dose fucoidan treated rat groups (Fig. 12).

Interleukin 1 beta (IL-1β) is a major proinflammatory cytokine and a leukocytic pyrogen termed a lymphocyte-activating factor (LAF) generated by activated macrophages or inflammasome. IL-1β gene expression was found high in control rat group (112.84 pg/mL), very low in normal rat group (19.21 pg/mL), and suppressed in low-dose rat group (66.18 pg/mL), high-dose rat group (54.96 pg/mL), and indomethacin rat group (42.54 pg/mL) (Supplementary figure 4). Hence, the gene expression of IL-1β was found suppressed in both the fucoidan treated rat groups (Fig. 12).

Like other proinflammatory cytokines, interleukin 6 (IL-6) also in- duces inflammation in several autoimmune diseases including Alz- heimer’s disease, Diabetes, Multiple sclerosis, and Rheumatoid arthritis.

Thus, pharmaceutical agents that inhibit the IL-6 are the best choice for autoimmune disease treatment. Tocilizumab is a humanized monoclonal antibody and an immunosuppressive drug that suppresses the interleukin IL-6 for the treatment of rheumatoid arthritis. Therefore, the gene expression of IL-6 was chosen for this in vivo study. The IL-6 gene expression was extremely high in the control rat group (271.59 pg/mL), whereas, it was extremely low in the normal rat group (27.54 pg/mL).

However, the IL-6 gene expression was suppressed in fucoidan-treated low-dose (91.11 pg/mL) and high-dose rat groups (62.54 pg/mL) (Supplementary figure 4 and figure 12) when compared with the indomethacin rat group (53.26 pg/mL). Therefore, it is clear that fucoidan inhibits the IL-6 gene expression in arthritis-induced rats.

Tumor necrosis factor alpha (TNF-α) is a cytokine (endogenous pyrogen), involved in the cell signaling to induce other proinflammatory cytokines by the immune system during the invasion of pathogens in the host as an inflammatory response. Drugs that inhibit TNF-α are often

Fig. 11. X-ray photographs of paw edema showing the incidence of inflammation in different rat groups; Group 1: Normal rat; Group 2: Indomethacin treated rat;

Group 3: Control rat; Group 4: Fucoidan, low-dose treated rat; Group 5: Fucoidan, high-dose treated rat.

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prescribed for autoimmune diseases and most commonly rheumatoid arthritis. In this present investigation, the TNF-α expression level was found high in the control rat group (52.13 pg/mL), and low in the normal rat group (7.06 pg/mL). Moreover, the TNF-α level was found suppressed in the low-dose rat group (26.53 pg/mL), the high-dose rat group (13.46 pg/mL), and the indomethacin rat group (11.73 pg/mL) (Supplementary figure 4 and figure 12). Thus, fucoidan supplementation suppresses the expression of TNF-α in arthritis-induced rats.

From the results obtained from the cytokine gene expression study, it is very clear that the anti-inflammatory cytokine, IL-10 expression was enhanced by 20.43 % and 53.97 % in low-dose and high-dose rat groups.

Contrastingly, the proinflammatory cytokines were suppressed, IL-1β by 41.35 % and 51.29 %, IL-6 by 66.45 % and 76.97 %, and finally, TNF-α by 49.1 % and 74.17 % in low-dose and high-dose rat groups respectively.

Estimation of biochemical parameters

The antioxidant enzymes such as Superoxide dismutase (SOD), Catalase (CAT), Glutathione (GSH), Glutathione peroxidase (GPX), and Glutathione S-transferase (GST) were estimated in the blood samples of all rat groups. In addition to this, lipid peroxidation (deteriorating effect of free radicals) was also estimated. The SOD level was found very low (only 53 % when compared with the normal rat group) in the control rat group (64.63 U/mL), 121.56 U/mL in the normal group. But, found greater in low-dose (85.43 U/mL), high-dose (93.96 U/mL), and indomethacin (97.46 U/mL) rat groups (Fig. 13A). Likewise, the CAT level was also found higher in normal rat group (34.36 mU/mL) and lower in control rat group (17.86 mU/mL) (only 51 % when compared with normal rat group) and found elevated in low-dose (25.63 mU/mL), high- dose (27.6 mU/mL), and indomethacin (30.16 mU/mL) rat groups (Fig. 13A).

The estimated concentration of GSH was lesser in the control rat group (29.06 µM/L) (only 57 % in comparison to the normal rat group) and greater in the normal rat group (50.43 µM/L). However, the GSH level in low-dose (41.16 µM/L), and high-dose rat groups (37.56 µM/L), was found closer to the indomethacin rat group (39.03 µM/L) (Fig. 13A).

Similarly, GPX level was found high in normal rat group (18.83 mU/

mL), followed by indomethacin rat group (15.73 mU/mL), high-dose rat group (14.23 mU/mL), low-dose rat group (11.06 mU/mL), and very low in control rat group (8.16 mU/mL) (only 43 % when compared to normal rat group). Therefore, GPX was found enhanced in fucoidan- supplemented (low-dose and high-dose rat groups) rats (Fig. 13A).

Consequently, the GST level was also found very low in the control rat group (0.26 U/mL) (only 29 % in comparison to normal rat group) than

the normal rat group (0.89 U/mL). Moreover, the level of GST enzyme was found induced in low-dose (0.49 U/mL), high-dose (0.67 U/mL), and Indomethacin (0.66 U/mL) rat groups (Fig. 13A).

In contrast to the above-studied antioxidant enzymes, lipid peroxidation was higher in the control rat group (0.17 nM/mL), and low in the normal rat group (0.08 nM/mL) (only 47 % when compared to the control rat group). Interestingly, the lipid peroxidation was inhibited in low-dose (0.15 nM/mL), high-dose (0.13 nM/mL), and indomethacin (0.12 nM/mL) rat groups (Fig. 13A). As a result, the antioxidant enzymes were enhanced and similarly lipid peroxidation was found hampered in the fucoidan supplemented rats. The SOD and catalase levels were enhanced by 45.38 % and 54.53 % in fucoidan supplemented rat group. Similarly, fucoidan supplementation induced 29.24 % of GSH, 74.38 % of GPX, and 157.69 % of GST in rats.

Blood urea ((NH2)2CO) and uric acid (C5H4N4O3) concentrations determine the regular function of the kidney. In this current investigation, urea and uric acid levels were lower in the normal rat group (12.13 mg/dL and 1.66 mg/dL) respectively. However, the urea and uric acid levels were higher in the control rat group (39.4 mg/dL, and 4.33 mg/

dL), whereas, both were found to decrease in low-dose (33.66 mg/dL, and 3.66 mg/dL)), high dose (28.76 mg/dL, and 3.53 mg/dL)), and indomethacin (27.16 mg/dL, and 2.63 mg/dL) rat groups (Fig. 13A).

Urea and uric acid levels were suppressed by 36.99 % and 22.66 % in fucoidan supplemented rats. Therefore, both the urea and uric acid levels in blood were controlled in fucoidan-supplemented rats than in control rats and it does not affect the function of the kidneys in rats.

The total protein and albumin contents in blood were found lower in the control rat group with 7.08 µg/mL, and 2.53 ng/mL respectively compared to the normal rat group (9.16 µg/mL, and 5.76 ng/mL), whereas, both the parameters were enhanced in low-dose (7.93 µg/mL, and 3.56 ng/mL), high-dose (8.46 µg/mL, and 3.93 ng/mL), and indomethacin (8.53 µg/mL, and 4.46 ng/mL) rat groups (Fig. 13B). Both total protein and albumin are the most important parameters in blood, and were found induced in fucoidan supplemented rats (19.83 % and 55.33 % respectively).

Contrastingly, creatinine and bilirubin levels in blood were found induced in the control rat group respectively (2.66 mg/dL, and 5.6 mg/

dL) when compared with the normal rat group (1.86 mg/dL, and 2.73 mg/dL). At the same time, both creatinine and bilirubin were suppressed in low-dose (2.16 mg/dL, and 4.76 mg/dL), high-dose (1.73 mg/dL, and 3.96 mg/dL), and indomethacin (1.66 mg/dL, and 3.73 mg/dL) rat groups respectively (Fig. 13B). Therefore, creatinine and bilirubin levels were controlled in fucoidan supplemented rats with 53.75 % and 41.41

% respectively.

Fig. 12.Graphical representation of the IL-10, IL-1β, IL-6, and TNF-α cytokine expression in the fucoidan-fed rats’ group.

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Fig. 13. A) Graphical representation of different biochemical parameters expressed among different rat groups (inside Urea and Uric acid); B) Other important biochemical parameters among rat groups. C) Graphical representation of Haematological parameters among rat groups.

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