Investigation on bio-properties and in-vivo antioxidant potential of carrageenans against alloxan induced oxidative stress in Wistar albino rats

Journal Pre-proof Investigation on bio-properties and in-vivo antioxidant potential of carrageenans against alloxan induced oxidative stress in Wistar albino rats

Muthusamy Sanjivkumar, Manohar Navin Chandran, Arumugampillai Manimehalai Suganya, Grasian Immanuel PII:

S0141-8130(19)37640-8

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.227

Reference:

BIOMAC 14844

To appear in:

International Journal of Biological Macromolecules

Received date:

20 September 2019

Revised date:

19 February 2020

Accepted date:

20 February 2020

Please cite this article as: M. Sanjivkumar, M.N. Chandran, A.M. Suganya, et al., Investigation on bio-properties and in-vivo antioxidant potential of carrageenans against alloxan induced oxidative stress in Wistar albino rats, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.02.227

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© 2018 Published by Elsevier.

Journal Pre-proof

Title page

Investigation on bio-properties and in-vivo antioxidant potential of carrageenans against alloxan induced oxidative stress in Wistar albino rats

Muthusamy Sanjivkumara, Manohar Navin Chandranb, Arumugampillai Manimehalai Suganyaa and Grasian Immanuela*

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MNP laboratory, Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam-629502, Tamilnadu, India.

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Department of Zoology, S.T. Hindu College, Nagercoil-629501, Tamilnadu, India.

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*Corresponding author e-mail: [email protected]

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Journal Pre-proof Abstract The present investigation was carried out to determine the bio-properties and antioxidant potential of native carrageenan from Kappaphycus alvarezii in comparison with commercial carrageenan under laboratory conditions in Wistar albino rats. In acute toxic study, no toxicity was observed in rats at the dose of 2000mg/kg of both carrageenans, hence three different doses (500, 750 and 1000mg/kg) of native and commercial carrageenans were selected individually for in vivo antioxidant study in rats. The result revealed that, in both native and commercial carrageenans tested animals, the antioxidant enzymes CAT (36.31 & 31.65µmol/mg protein), GPx (5.92& 5.48µg/min/mg protein), SOD (1.91 & 1.55µmol/mg protein), GST (3.09 & 2.97µmol/mg protein),

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GSH (27.56 & 25.31µmol/mg protein) and ascorbic acid (3.76 & 2.35µg/mg protein) levels in the

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kidney samples of the alloxan induced diabetic rats were elevated, but LPO (3.74 & 4.10µmol/min /mg protein) level was decreased in the highest concentration (1000mg/kg) of both native and

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commercial carrageenans treated groups, when compared to standard glibenclamide treated group and control group. In brine shrimp lethality assay, native carrageenan expressed LC50value of

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160.41µg/ml, however commercial carrageenan failed to express LC50 even at 200µg/ml

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concentration. Likewise in hemolytic assay, among the tested concentrations (25 to 250µg/ml) of both carrageenans the highest concentration (250µg/ml) of the same exhibited 38.31 and 65.75% activity on

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red blood cells respectively. Native carrageenan also possessed better anti-inflammatory (84.89%) and anticoagulant activity at the highest concentration (500mg/kg), when compared to commercial carrageenan. Finally both the carrageenans were structurally characterized through UV-Vis, FT-IR,

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FT-Raman and NMR studies. From the results, it was understood that native carrageenan was considered to be a protective antioxidant and anti-therapeutic agent with biological properties. Keywords: Seaweed, Kappaphycus alvarezii, Native carrageenan, Commercial carrageenan, Antioxidant. 1. Introduction In recent years, marine resources (marine algae, sea grasses, sponges, coral reef etc.) have gathered more attention in search for bioactive compounds to develop new drugs and healthy foods. In addition, marine algae are now considered as a rich source of antioxidants [1]. Like other terrestrial plants, marine algae are also showed to a combination of light and high oxygen concentrations, which leads to the formation of free radicals and other strong agents. The absence of such damage in certain species of seaweeds suggests that their cells have some protective 2

Journal Pre-proof antioxidative mechanisms and compounds, which produce various types of antioxidants to counteract environmental stress [2]. Hence, they can be considered as a potential source of novel antioxidants. Antioxidants are beneficial components that neutralize free radicals before they can attack cell proteins, lipids and carbohydrates. The mechanism involves significant inhibition or delay in the oxidative process. Protection against free radicals can be enhanced by ample intake of dietary antioxidants. Enormous studies exposed that antioxidants have unveiled the path to pharmacological activities such as anti-inflammatory, anti-aging, anti-atherosclerosis and anticancer activities [2,3]. The disfunctioning of antioxidant enzymes have been implicated in several disorders including rheumatoid arthritis, reperfusion injury, cardiovascular diseases,

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immune injury as well as diabetes mellitus [4]. There were numerous evidences that complications

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related to diabetes are associated with oxidative stress induced by the generation of free radicals [4, 5]. In diabetes, oxidative stress has been found to be mainly due to an increased production of

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oxygen free radicals and a sharp reduction of antioxidant defenses. Moreover, diabetes also induces changes in the tissue content and the activity of antioxidant enzymes. Antioxidants help to improve

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natural defense mechanisms of the body; hence, compounds with antioxidative properties would be

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useful as an antidiabetic agent [5, 6].

Synthetic antioxidants commonly used such as BHA and BHT have averted lipid

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peroxidation, but they have been restricted because of their toxic and carcinogenic effects. Natural antioxidants are considered safe for use as ingredients in medicine, dietary supplements,

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nutraceuticals and cosmetics with improving consumer health and reducing the effects of harmful diseases and other broader aspects of immune system function [4,5]. So, there has been a considerable interest by the food and pharmaceutical industries to develop nontoxic antioxidant compound that demonstrate measurable health benefits. Marine algal polysaccharides have been demonstrated to play an important role as free-radical scavengers (in vitro) and antioxidants for the prevention of oxidative damage in living organisms. Algal polysaccharide carrageenans from different species of red algal have in-vitro antioxidant activities [3,7]. Furthermore algal carrageenan has a large number of medicinal applications; numerous studies have revealed that carrageenans from red algae are documented to possess a number of biological activities including anticoagulant, antiviral, antitumor and immune-stimulating activities [2]. It is useful in preparation of traditional medicinal teas and cough medicines to combat colds, bronchitis and chronic coughs. It is said to be particularly useful for dislodging mucus and has antiviral properties. They are used as suspension agents and stabilizers in other drugs, lotions and medicinal creams. Other medical 3

Journal Pre-proof applications are as an anticoagulant in blood products and for the treatment of bowel problems such as diarrhea, constipation and dysentery [7]. Considering the importance of the above, the present study was undertaken to evaluate the bio-properties and in vivo antioxidant potential of native carrageenan from red seaweed K. alvarezii in comparison with commercial carrageenan in alloxan induced Wistar albino rats. 2. Materials and Methods 2.1. Carrageenan samples Native carrageenan from Kappaphycus alvarezii was extracted according to the standard

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methodology of Mshigeni and Semesi [8] and commercial carrageenan was obtained from (K.

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alvarezii, C1263, type III κ-carrageenan) Sigma Aldrich, Mumbai were used in this study.

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2.2. Experimental animal

Male adult Wistar albino rats weighing about 160-175g were maintained under controlled

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conditions of 29°±2°C; 12:12h light-dark cycles. The animals were randomized into experimental and control groups and housed individually in sanitized polypropylene cages containing sterile

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paddy husk as bedding. They had free access to standard pellets as basal pellet diet (Sai Enterprise, Chennai) and water ad libitum. All the studies conducted were approved by Ethical Committee

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according to prescribed guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India). Male animals were selected for the

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study, since females are shown to be protected from changes in lipid –induced insulin action, Moreover; female rats may possess counter mechanisms that protect against the adverse effects of fructose such as hyperinsulinemia, insulin resistance and hypertension [9]. 2.3. Acute oral toxicity study

Acute oral toxicity study was performed according to OECD guidelines (Organization for Economic Co-operation and Development). The animals were subjected to fasting for three hours before prior to the experiment. The animals were divided into three groups of three animals each. First group served as a control and received sterile water. While the other two groups received 2000mg/kg doses of native and commercial carrageenans respectively. The carrageenans were suspended in 5ml sterile water individually as they were not easily soluble in other solvents. From each carrageenan suspension 2.5ml was taken and orally fed to the animals for two times at every one hour interval. The animals were observed for every 5 min till 2hrs and then at 4, 8 and 24hrs to 4

Journal Pre-proof detect any changes in the autonomic or behavioral response and also for tremors, convulsion, salivation, diarrhea, lethargy, sleep and coma. Further they were observed daily for fourteen days for mortality [10]. 2.4. Induction of diabetes Diabetes was rendered in overnight fasted male Wistar albino rats by single intraperitoneal injection of Alloxan monohydrate (120mg/kg BW), which was already dissolved in saline solution (0.9% sodium chloride, pH 7). This dose was fixed based on the findings of Mansour et al. [11]. Alloxan is capable of producing fatal hypoglycemia as a result of massive pancreatic insulin

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release. So, rats were supplemented with 10% glucose solution (20ml) for duration of 24h to

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prevent hypoglycemia. Animals were considered as diabetic, if their blood glucose level was above 200mg/dl on the third day after alloxan injection. The treatment was started on the third day of

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2.5. Grouping and treatment schedule

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alloxan injection and it was continued for 45 days.

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All the procedures were performed in accordance with the Institutional Animal Ethics Committee (IAEC) constituted as per the direction of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), under Ministry of Animal Welfare Division,

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Government of India, New Delhi. The study was approved by institutional animal ethical

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committee (Ref.No.38001/ME2/2015).

The rats were divided into nine groups with six animals in each group as follows: Group 1- Alloxan induced diabetic rats treated with 500mg/kg of native carrageenan (10ml/kg sterile water) orally, once a day. Group 2- Alloxan induced diabetic rats treated with 750mg/kg of native carrageenan (10ml/kg sterile water) orally, once a day. Group 3- Alloxan induced diabetic rats treated with 1000mg/kg of native carrageenan (10ml/kg sterile water) orally, once a day. Group 4- Alloxan induced diabetic rats treated with 500mg/kg of commercial carrageenan (10ml/kg sterile water) orally, once a day. Group 5- Alloxan induced diabetic rats treated with 750mg/kg of commercial carrageenan (10ml/kg sterile water) orally, once a day.

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Journal Pre-proof Group 6- Alloxan induced diabetic rats treated with 1000mg/kg of commercial carrageenan (10ml/kg sterile water) orally, once a day. Group 7- Normal control, rats orally administered with sterile water (10ml/kg sterile water) orally, once a day. Group 8- Diabetic control- Alloxan induced diabetic rats orally administered with sterile water (10ml/kg sterile water) orally, once a day. Group 9- Alloxan induced diabetic rats treated with 500µg/kg of Glibenclamide (10ml/kg sterile water), once a day.

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Treatment was given to the respective group of animals daily orally for 45 days on fixed

freely available to animals.

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2.6. Collection and processing of kidney samples

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time (11am). During the study period, standard feed (Sai Enterprise, Chennai) and water were made

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After 45th day of experimentation, kidney samples were dissected and quickly removed from each group of rats, washed with cold physiological saline, further washed with distilled water

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2.6.1. Preparation of cell lysate

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to remove adherent lipids and immediately transferred into ice-cold containers.

100mg of kidney samples were taken from each group and they were homogenized

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individually in 2ml sucrose buffer (50mM Tris HCl [pH 7.4], 40mM EDTA [pH 8] and 0.7M sucrose) to give 10% homogenate and centrifuged at 1000rpm for 10min at 4°C. From this, the supernatant was collected and was used for the analysis of various enzymatic and non-enzymatic antioxidant assays by following appropriate methodologies. 2.6.2. Estimation of enzymatic and non-enzymatic antioxidant parameters in kidney samples Homogenized kidney samples were used for the estimation of enzymatic and non-enzymatic antioxidants assays such as catalase [12]; glutathione Peroxidase [13]; SOD [14]; glutathione Stransferase [15] and non-enzymatic antioxidants such as total reduced glutathione [16];lipid peroxidase (LPO)and L-ascorbic acid [17]. 2.8. Biological activities of carrageenans 2.8.1. Brine shrimp lethality assay (Toxicity test)

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Journal Pre-proof The toxic effect of the native and commercial carrageenans was determined by brine shrimp lethality assay [18]. Both carrageenans at different concentrations (50, 100, 150 and 200µg/ml) were individually dissolved in 2.5ml of distilled water and mixed with 10ml of sea water (pH 8.8 and salinity 2.8%), taken in test tubes containing 10 no’s of Artemia. The test tubes were set aside under illumination. The Artemia that survived were counted after 24h. A control was also maintained, without the addition of carrageenan. Simultaneously triplicate tubes were maintained for each concentration. The percentage mortality was calculated by the following formula

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Mortality (%) = (Number of dead Artemia / Initial number of live Artemia) x 100 2.8.2. Hemolytic assay (Cytotoxicity test)

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Hemolytic activity of both native and commercial carrageenans was performed by

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following the method described by Fischer et al. [19] with minor modification. Saline was maintained as a blank solution. A combination of Triton X100 (800µl) and human Red Blood Cell

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(RBC) suspension (200µl) were mixed well and kept as a positive control. A mixture of 27% saline (800µl) and RBC suspension (200µl) were mixed and kept as negative control. The experimental

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set up considered with 200µl each of RBC suspension was mixed with different concentrations (25 - 250µg/ml) of native and commercial carrageenans and they were finally adjusted to 1ml using

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phosphate buffer. Then the tubes were incubated at 37ºC in a water bath for 30min. After incubation, the cells were centrifuged at 3500rpm for 15min. The supernatant was collected and the

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absorbance was measured at 540nm in a UV-vis spectrophotometer. The percentage hemolysis was calculated by the following formula

Hemolysis (%) = (At – Anc / Apc – Anc) x100

At - absorbance of the test sample

Anc- absorbance of the negative control Apc- absorbance of the positive control 2.8.3. Evaluation of in-vitro anti-inflammatory activity Anti-inflammatory activity of different concentrations (100 to 500µg/ml) of native and commercial carrageenans was evaluated by protein (egg albumin) denaturation method [20] with slight modification. Diclofenac sodium, a dominant non-steroidal anti-inflammatory drug was used as a standard. The reaction mixture consisted of 2ml each of different concentrations (100, 200, 7

Journal Pre-proof 300, 400, 500µg/ml) of carrageenan, 2.8ml of phosphate buffer saline (pH 6.4) and 2ml of egg albumin (from fresh hen’s egg). A control was also prepared by the addition of phosphate buffer saline (2.8ml) and egg albumin (2ml).The contents were mixed well and incubated at 27°C for 15min. Denaturation was induced by maintaining the reaction mixture at 70ºC in a water bath for 10min. After cooling to room temperature, the absorbance was measured at 660nm in a UV-vis spectrophotometer by using double distilled water as blank. The percentage inhibition of protein (albumin) denaturation was calculated by the following formula Percentage inhibition (%) = (AT – AC / AC) x 100

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At - absorbance of the test sample

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Ac - absorbance of the control

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2.8.4. Evaluation of in vitro anticoagulation activity

Anticoagulation activity of native and commercial carrageenans was performed by the

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standard methodology [21]. The human blood sample was withdrawn in a syringe and transferred on to a clean white tile (0.5ml each). Native and commercial carrageenans with different

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concentrations (100-500µg/ml) were prepared and added to the blood and mixed well to determine

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anticoagulation.

2.8.4.1. Evaluation of Prothrombin time

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The human blood sample was withdrawn and placed in a tube containing tri sodium citrate to prevent clotting and centrifuged (15min @ 3000rpm) the blood to separate the blood cells from plasma in order to obtain pure platelet plasma (PPP) for prothrombin test. In a clean fusion tube, 0.2ml of plasma, 0.1ml each of native and commercial carrageenans at different concentrations (100-500µg/ml) and 25mM CaCl2 were added and incubated at 37°C in a water bath. Saline water (0.9%) was kept as a control. The clotting time was recorded with stopwatch by tilting the test tubes every 5sec. This time was called as prothrombin time [22]. 2.9. Structural characterization of carrageenans 2.9.1. UV- Vis spectral analysis UV– Vis spectral analysis of native and commercial carrageenan fractions were scanned within the range between 200 and 1000nm in a UV spectrophotometer for determining maximum absorbance (λ max). 8

Journal Pre-proof 2.9.2. FT-IR analysis The FT-IR analysis of native and commercial carrageenans was performed correspondingly by grinding with infrared grade KBr (1:10) and impregnated into the disks under vacuum using a Spectra Lab Pelletizer. FT-IR spectrum in the KBr disc was recorded at the wave number region of 4000–500cm-1 using a Thermo Nicolet Nexus 670 FT-IR spectrophotometer. 2.9.3. FT-Raman analysis The FT-Raman spectrum of both native and commercial carrageenans was recorded on a RFS-100 Bruker FT-spectrometer using a Nd:YAG laser with excitation wavelength of 1064nm. A

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few milligrams of both native and commercial carrageenans were individually made into pellets

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and subjected for spectral analysis. Each spectrum was the average of two repeated measurements

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of 150 scans each and 2cm-1 resolution. 2.9.4. 13C and 1H NMR analysis

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Native and commercial carrageenans were individually dissolved in 0.5ml D2O (Deutrium dioxide) and the proton number and carbon number of both carrageenans were identified and 13

C NMR using a Bruker Biospin Avance-400 NMR spectrometer (1H

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confirmed by1H and

frequence = 400.13 MHz and13C frequence = 100.62 MHz) at 298K using 5mm broad band inverse

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probe head equipped with shielded z-gradient and XWIN-NMR software version 3.5 using TMS as an internal reference. One dimensional 1H and 13C spectra were obtained using one pulse sequence. 13

C spectra using Spin Echo Fourier Transform (SEFT) and Quaternary Carbon

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One dimensional

Detection (QCD) 42-sequences were also performed to aid the structure identification. 2.10. Statistical analysis

Before used it in experimental animals, native and commercial carrageenans were screened for acute oral toxicity. The data obtained in the present study were expressed as Mean ± SD and were analyzed using one way ANOVA at 5% level of significance. Further a multiple comparison by Tukey’s test was conducted to compare the significant differences among the parameters using computer software Statistica-6.0 (Statosoft, Bradford, UK). 3. Results and Discussion Toxicological screening is very important for the development of new drugs. The US Food and Drug Administration (FDA) states that, it is essential to screen a new molecules in an animal 9

Journal Pre-proof model to evaluate its pharmacological and toxicological properties (21CFR Part 314) [23]. Intake of pharmacological substances by man has solely increased and this may be in the form of food, medicines and beverages. These substances are capable of eliciting chronic and acute toxicity, which may be mild or severe, depending upon their nature. Acute toxicity is defined as the unwanted effects that occur either immediately or at a short time interval after a single or multiple administration of such substance within 24 hours. The essence of toxicity testing is not only to check how safe a test substance is, but to characterize the possible toxic effects it can produce. Toxicity test may be performed to determine the effect of the test substances on laboratory animals

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and it directly reflected to humans. Polysaccharides isolated from the cell wall of seaweeds possessed immunomodulatory,

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anti-inflammatory, antiviral, antitumor, antithrombotic, anticoagulant and antioxidant bioactivities [24]. Some of these polysaccharides are safe and exhibiting no toxic effects in Wistar albino

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rats.For example, polysaccharide fucoidan from Cladosiphono kamuranus are harmless and

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possessed nontoxic effects in Wistar albino rats even at high concentration of 300 to 4000µg/g of body weight over a period of three months [25]. Likewise, Lim et al. [26] stated that, at the end of

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the acute toxic study no mortality was observed in albino rats treated with highest dosage (2000mg/kg) of fucoidan from Sargassum binderi. But some polysaccharides could be toxic to

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animals even at low concentration, such as the sulfated galactan from the red alga Champia feldmani possessed toxicity at the concentration of 25µg/g itself [27]. Therefore, in the present

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study, before proceed the experiment, both native and commercial carrageenans were subjected for acute oral toxic study in Wistar albino rats. The result revealed that, there was no abnormality or mortality changes observed in Wistar albino rats received 2000mg/kg dose of native and commercial carrageenans till the end of the study (14 days). During acute toxic study (as per the OECD Guidelines 423), the behavior of native and commercial carrageenans treated Wistar albino rats appeared normal and no abnormalities were recorded including changes in skin, fur, eyes, mucous membranes and behavioral pattern. From the result, it was implicated that the experimental animals were found to be safe up to the dose of 2000mg/kg of both carrageenans and therefore, the doses of 500, 750 and 1000mg/kg of carrageenans were fixed for further studies. Experimental induction of diabetes mellitus in animal models is essential for the advancement of our knowledge to know about the various aspects in the pathogenesis of diabetes and finding new drugs to cure [28]. Animal models have historically played a critical role in the exploration and characterization of disease pathophysiology and target, identification and 10

Journal Pre-proof evaluation of novel therapeutic agents or drugs in in-vivo. Alloxan is a diabetic induced cytotoxic agent, causes a cell death and apoptosis by the generation of ROS, superoxide radicals and hydrogen peroxide leads to hyperglycemia [29]. Alloxan induced diabetic animals exhibit oxidative stress due to persistent and chronic hyperglycaemia, which there by depletes the activity of antioxidative defense system and thus promotes de novo free radicals generation [30]. In the present study, alloxan induced diabetic rats were treated with different concentrations (500, 750 and 1000mg/kg) of native carrageenan from red seaweed K. alvarezii and commercial carrageenan to evaluate their antioxidant properties.

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Catalase (CAT) enzyme is an antioxidant enzyme widely distributed in the animal tissues. The increased activities of catalase enzyme was observed in the present investigation suggested that

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native carrageenan from red seaweed K. alvarezii and commercial carrageenan have an in vivo antioxidant activity and are capable of ameliorating the effect of ROS in biological system [31].

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The result of the present study on catalase activity in the kidney samples of normal control rat was

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47.52 ± 1.42µmoles/mg protein, but it was decreased to 16.81 ± 1.56µmoles/mg protein in diabetic control animals. The reduction of catalase enzyme may lead to deleterious effects as a result of

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accumulation of highly reactive free radicals, superoxide and hydrogen peroxide leading to harmful effects such as loss of integrity and function of cell membranes [32]. At the same time lowest

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concentration (500mg/kg) of both native and commercial carrageenans possessed the catalase activity of 26.48 ± 1.73 and 20.41 ± 1.66µmoles/mg protein respectively, but once the

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concentration level of both carrageenans increased (750-1000mg/kg), the CAT activity also significantly increased to 31.23 ± 1.78 and36.31 ± 2.01 & 27.50 ± 1.68 and 31.65 ± 1.63µmoles/mg protein,

respectively.

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was compared with

conventional

oral

antihyperglycemic

drug

glibenclamide (29.61 ± 1.46µmoles/mg protein) treated group (Table 1). Previous reports also suggested that Wistar albino rats treated with aqueous extract of K. alvarezii (200mg kg−1, on alternate days) for 60 days increased the CAT activity in their liver tissue when compared with normal control animals [33]. Hassan et al. [1] also suggested that, supplementation of polysaccharide (250mg/kg) from the green alga Ulva lactuca for a period of four weeks possessed elevated level of CAT enzyme in hypercholesterolemic rats as compared with the hypercholesterolemic control albino rats. Qi and Sun [34] postulated that, hyperlipidemic rats treated with different concentrations (125, 250 and 500mg/kg) of sulphated polysaccharide ulvan from Ulva pertusa an increasing level of CAT activity when compared with the normal control rats at the end of the experimental duration of 30 days. 11

Journal Pre-proof Glutathione peroxidases are the family of key enzymes involved in scavenging oxyradicals in animals. In the present study, both native and commercial carrageenans were tested for their Glutathione peroxidase (GPx) activity. After 45 days of carrageenan treatment, GPx activity was significantly increased in the diabetic animals treated with different concentrations of native and commercial carrageenans (500mg/kg: 3.49 ± 0.20 & 2.91 ± 0.13; 750mg/kg: 4.44 ± 0.35 & 3.51 ± 0.30 and 1000mg/kg: 5.92 ± 0.47 & 5.48 ± 0.65µg of GSH consumed/min/mg protein) as compared with diabetic control animals (2.03 ± 0.13µg of GSH consumed/min/mg protein) (Table 1).The lower GPx activity in untreated diabetic animals may be due to excess hyperglycemia, during this period, GPx enzymes might be functionally impaired due to excess generation of free radicals

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creating oxidative imbalance [35].Treatment of carrageenans would protect the GPX enzyme

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against inactivation by free radicals due to its antioxidant properties. The improved effects on antioxidant enzymes in native and commercial carrageenans were partially due to their immune

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activities [36].

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Earlier reports also suggested that, significant increase in GPx activity was observed in the serum sample of the animal treated with 200mg kg−1 of K. alvarezii extract when compared with

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control rats [33]. Regular administration of seaweed supplement (48g) also increased the GPx enzyme level in the patients with type 2 diabetes mellitus [37]. Likewise, Kang et al. [38] reported

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that, intraperitonial administration of fucoidan (100mg/kg) from brown seaweed Undaria pinnatifida to the rats for 14 days possessed elevated level of GPx against CCL4- induced oxidative

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stress. Previous reports also suggested that the administration of sulphated polysaccharide ulvan from green seaweed U. pertusa at the dose of 125mg/kg and 500mg/kg in hyperlipidemic rats could increase the activities of GPx as compared with hyperlipidemic group of animals. Low dose (125mg/kg) of ulvan treated animals showed stronger GPx activity than normal control group [34]. Superoxide dismutase (SOD) has been reported as one of the most important enzyme in the enzymatic antioxidant defense system [39]. SOD is considered to be a stress protein which is synthesized in response to oxidative stress. In the present study SOD activities in the kidney samples of the normal control and diabetic control animals was 0.38 ± 0.04 and 0.26 ± 0.02µmol/mg protein, respectively. Alloxan induced diabetic animals possessed decreased level of SOD activity when compared to normal control rats. In the present study, treatment with different concentrations of native and commercial carrageenans significantly increased the SOD activity (500mg/kg: 1.07 ± 0.01 & 0.89 ± 0.03;750mg/kg: 1.45 ± 0.02 & 1.15 ± 0.03 and 1000mg/kg: 1.91 ± 0.04 & 1.55 ± 0.06µmol/mg protein), which was comparable to the standard drug glibenclamide 12

Journal Pre-proof (1.51 ± 0.04µmol/mg protein) (Table 1). The elevated level of SOD activity in the kidney samples of carrageenan treated animals might be due to the removal of oxidative stress caused by hyperglycemia. SOD plays an important role in the elimination of ROS and protects cells against the deleterious effects of super oxide anions derived from the peroxidative process in kidney tissues [40]. Likewise Mahmoud et al. [41] reported that SOD activity was significantly decreased in the liver and heart tissues of untreated fructose induced diabetic rats when compared with normal control animals (four weeks). They also pointed out that treatment with green seaweed E. flexuosa extract (50mg/kg) on the diabetic rats significantly alleviated the activities of SOD in the liver, kidney and heart. Lin et al.[42] found out that, different concentrations (150, 300 and 600mg/kg)

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of green algal (E. prolifera) polysaccharide treated streptozotocin induced high-sugar, high-fat

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diet (65% basal feed, 20% sucrose, 10% lard, 2.5% egg yolk powder and 2.5% cholesterol) diabetic groups possessed elevated level of SOD in a dose-dependent manner when compared with the

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untreated high-sugar, high-fat diet (received equivalent distilled water) diabetic group at the ends of the experiment (four weeks). Further they suggested that SOD activity exhibited a decreasing

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trend in the untreated diabetic group throughout the experimentation when compared with the control group. Bhateja and Singh [43] reported that treatment with different concentrations (250,

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500 and 1000mg/kg) of Acacia tortilis polysaccharide in streptozotocin-nicotinamide induced diabetic rats for six weeks increased the dismutation of oxygen radicals and eliminating peroxides

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study too.

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by increasing level of SOD, catalase and GPx enzymes. The same trend was obtained in the present

Glutathione-S-transferases (GSTs) are a group of enzymes that are important in the detoxication of many different xenobiotics in mammals. These enzymes protect the cells against toxicants by conjugating the thiol group of the glutathione to electrophilic xenobiotics and thereby defend cells against the mutagenic, carcinogenic and toxic effects. Meena et al.[44] reported that GST level was decreased in the liver tissue of galactosamine-induced hepatitis rats. This may be due to the increased oxidative stress in galactosamine-induced hepatitis rats when compared with normal control rats. The same authors pointed out those animals pretreated orally with brown seaweed S. polycystum extract (125mg/kg) for 15 days showed the elevated level of GST. Mahmoud and Hussein [45] proposed that GST level in nitrosodiethylamine (NDEA) induced (two weeks and 24weeks) albino rats was decreased due to renal toxicity. However, the aqueous extract (50mg/kg) and the sulphated polysaccharides (50mg/kg) of green algae U. lactuca elevated the GST level at the entire period of the study, which might be one of the important reno protective 13

Journal Pre-proof mechanisms of polysaccharides against NDEA-induced renal toxicity [46]. Similarly, in the present study GST activity in the kidney samples of the normal control animals was recorded as 3.68 ± 0.07µmol/mg protein. GST activity was significantly lowered in diabetic control animals (1.49 ± 0.03µmol/mg protein), when compared with normal control animals, this may be due to the increased oxidative stress caused by alloxan. Oral administration of different concentrations of native and commercial carrageenans (500, 750 and 1000mg/kg) resulted in increasing activity of GST (500mg/kg: 2.16 ± 0.01 & 2.07 ± 0.06; 750mg/kg: 2.72 ± 0.05 & 2.40 ± 0.04 and1000mg/kg: 3.09 ± 0.08& 2.97 ± 0.06µmol/mg protein). Glibenclamide treated diabetic rats also exhibited the GST (3.37 ± 0.06 µmol/mg protein) activity nearer to the commercial carrageenan treated animals

of

(Table 1).Supplementation with native and commercial carrageenans was found to increase the

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activity of these GST enzyme. Free radical scavenging enzyme GST in both carrageenans had evolved to prevent excessive oxidation stress.

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Glutathione (GSH) is an important non enzymatic antioxidant present in the kidney. GSH

re

is capable of preventing damage to important cellular components caused by reactive oxygen species such as free radicals, peroxides, lipid peroxides and heavy metals [47]. It protects cellular

lP

proteins against reactive oxygen species generated from exposure to carbon tetrachloride. Bhateja and Singh [43] stated that, GSH is significantly increased in diabetic rats when treated with Acacia

na

tortilis polysaccharide for 6 weeks. Previous reports have also exhibited that, pretreatment of sulfated-polysaccharide fraction (3, 10, 30 and 90mgkg−1) from red algae Gracilaria caudate

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possessed a resultant increase in the gastric gut GSH (within one hour) on ethanol-induced (gastric damage) rats [48]. The possibility may be due to an increase in GSH level could be secondary to a decrease in the free radical production. Nagy [49] reported that, oral administration of brown seaweed Hydroclathrus clathratus extract causes significant enhancement in the level of hepatic GSH on alloxan-induced diabetes mellitus in albino rats at the end of the 30 days experiment [49]. Likewise in the present study, at the end of 45th day of experiment, GSH activity was significantly increased in the kidney samples of the diabetic rats treated with different concentrations of native and commercial carrageenans (500mg/kg: 20.51 ± 1.99 & 20.47 ± 1.46; 750mg/kg: 24.50 ± 1.62 & 22.66 ± 1.81 and 1000mg/kg: 27.56 ± 1.42 & 25.31 ± 1.33µmol/mg protein), however in glibenclamide treated animals, the GST level was found to be 26.67 ± 1.52µmol/mg protein (Table 1). Increased level of GSH is associated with decreased level of lipid peroxidation, which was confirmed in this study. The GSH activity in the kidney samples of the normal control rats was observed as 30.80 ± 1.65µmol/mg protein. At the same time, GSH activity was decreased (16.21 ± 14

Journal Pre-proof 1.41µmol/mg protein) in the untreated diabetic control rats at the end of the experiment. This may be due to increased oxidative stress caused by alloxan induced diabetes [50]. Lipid

peroxidation (LPO)

is

the oxidative degradation

of lipids

by

the

enzyme

lipoxygenases. It is the process in which free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain reaction mechanism. The chemical products of this oxidation are known as lipid peroxides or lipid oxidation products [51]. Seaweed supplementation (48g Pills with equal parts of dry powdered sea tangle and sea mustard) in diabetic patients significantly lowered the level of LPO in erythrocytes

of

as compared to non-treated diabetic individuals [37]. Similarly in the present study, LPO level in the kidney sample of the control animal was recorded as 3.7 ± 0.31µmoles/min/mg protein. At the

ro

same time LPO activity in the kidney sample of diabetic control animals was 5.34 ± 0.48µmoles/ min/mg protein. LPO activity was reduced significantly in the animals treated with different

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concentrations of native and commercial carrageenans (500mg/kg: 4.35 ± 0.20 & 4.76 ± 0.71;

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750mg/kg: 4.06 ± 0.36 & 4.42 ± 0.62 and 1000mg/kg: 3.74 ± 0.25 & 4.10 ± 0.44µmoles/min/mg protein). Dose dependent fall in LPO activity was observed and the finding was compared with the

lP

standard drug glibenclamide, treated animals and the result was found to be 4.7 ± 0.24µmoles/ min/mg protein (Table 1). These results suggested that seaweed polysaccharide supplementation

na

decreases oxidative stress in vivo due to a combination of improved antioxidant activities and decreased peroxidation processes. Bhateja and Singh [43] stated that treatment of Acacia tortilis

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polysaccharide (500 and 1000mg/kg) in streptozotocin-nicotinamide induced diabetic rats for a period of six weeks showed the decline level of LPO in various tissue homogenates (liver, kidney and pancreas) when compared with non-treated diabetic rats. Pretreatment of sulfatedpolysaccharide of red seaweed G. caudate on ethanol-induced Swiss mice exhibited significant decrease in the LPO level in gut gastropathy [48]. TBARS or LPO levels in erythrocytes of type 2 diabetic patients received seaweed supplementation were significantly lower than non-treated diabetic individuals [37]. Compared with the non-treated diabetic model group, streptozotocin induced diabetic rats treated with different concentration (150, 300 and 600) of polysaccharide from seaweed E. prolifera possessed lower LPO activity [42]. Ascorbic acid (Vit. C) is acting as a chain breaking antioxidant impairs with the formation of free radicals in the process of formation of intracellular substances throughout the body, including collagen, bone matrix and tooth dentine [52]. Vitamin C prevents the increased production of free radicals induced by oxidative damage to lipids and lipoproteins in various 15

Journal Pre-proof cellular compartments and tissues as found in diabetes mellitus [53]. Yildirim and Buyukbingol [54] stated that, vitamin C level was decreased in the kidney tissues of the streptozotocin induced diabetic rats at the end of the sixth week of experiment, when compared to those of controls. But diabetic rats treated with Vitamin C and cobalt (1g/l vitamin C with 0.5mM COCl2 in drinking water) resulted in partial restoration of vitamin C level in the kidney throughout the experiment. In an another report, pretreatment of alcoholic extract from brown seaweed S. polycystum (200mg/kg) for a period of fifteen days acted as a natural antioxidant and increased level of Vit. C, when compared with non-treated acetaminophen induced toxic rats [55]. Similarly, in the present study, the ascorbic acid level was lower in diabetic rats (1.20 ± 0.03µg/mg protein) as compared to normal

of

control non diabetic rats (1.06 ± 0.02µg/mg protein) at the end of the experimentation. Treatment

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with different concentrations (500, 750 and 1000 mg/kg) of native and commercial carrageenans resulted significantly increase dascorbic acid level (500mg/kg: 1.85 ± 0.05 & 1.46 ± 0.03; 750

-p

mg/kg: 2.44 ± 0.08 & 1.80 ± 0.02 and 1000mg/kg: 3.76 ± 0.06 & 2.35 ± 0.04µg/mg protein) in the kidney samples of the experimental animals, this may be due to the presence of abundant Vit. C, a

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key antioxidant observed in carrageenans. Invariably, glibenclamide treated diabetic animals resulted lower (3.66 ± 0.04µg/mg protein) ascorbic acid level, compared with native carrageenan

lP

treated animals (Table 1). No much significant difference was noted in the ascorbic acid level, between carrageenans and glibenclamide treated animals. Thus the present study confirmed that,

na

the level of (in vivo) antioxidant capacity was increased in alloxan induced diabetic animals treated with different concentrations of native carrageenan in comparison with commercial carrageenan at

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the end of the experiment. However, in our previous study we found that in-vitro antioxidant potential of native carrageenan was better, when compared with commercial carrageenan [3]. Polysaccharides extracted from marine macro algae have demonstrated various biological activities, such as antibacterial, antioxidant, anti-inflammatory, anti-coagulant, anti-viral along with apoptotic activity [56]. Brine shrimp assay implies an easy, inexpensive and rapid bioassay has been successfully used as a preliminary study to find out the cytotoxic and antitumor products. Many scientists had reported the cytotoxicity and antitumor activities of terrestrial plants and algae using brine shrimp as a model organism [57]. In the present study, native carrageenan from K. alvarezii and commercial carrageenan were screened for cytotoxic activity using the brine shrimp lethality assay. At the doses of 50, 100 and 150µg/ml, both native and commercial carrageenans showed non cytotoxic effect against the brine shrimp. Mortality was directly related to the cytotoxicity of the extract and a dose-dependent activity was also observed in both tested carrageenans. The highest 16

Journal Pre-proof mortality was noticed at the maximum concentration (200mg/kg) of both native (70.0 ± 1.16%) and commercial (40.8 ± 0.860%) carrageenans. This clearly indicated that native carrageenan possessed better cytotoxic activity than commercial carrageenan. The cytotoxicity may be related to the antioxidant activity of the native carrageenan [6]. Relleve et al. [58] reported that commercial kappa and lambda carrageenans from Copenhagen Pectin A/S (Denmark and Shemberg Corporation, Philippines) possessed potential pharmacological value with the respective LC50 value of 1300µg/ml and 400µg/ml respectively against brine shrimp. Invariably, in the present study commercial kappa carrageenan failed to show

of

LC50 even at 200µg/ml concentration. But native carrageenan from K. alvarezii exhibited the LC50 at 160.40µg/ml concentration. It indicated that bioactive components are abundantly presented in

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native carrageenan; therefore it could be accounted for its pharmacological effects. Therefore, native carrageenan can be considered as a promising candidate for marine algal derived anticancer

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compound with antioxidant activity. Relleve et al. [58] documented brine shrimp lethality assay of

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commercial iota, kappa and lambda carrageenans, but none has focused on brine shrimp lethality assay using native carrageenan from K. alvarezii. Thus the present findings clearly emphasized that

lP

the native carrageenan from K. alvarezii can be considered as an effective drug for anti-cancer therapy to destroy the abnormal cell mass or cancer cells in comparison with commercial

na

carrageenan due to its cytotoxic activity (Table 2a). Our previous study also proved that native carrageenan possessed highest anticancer activity against human carcinoma cell lines such as colon

[3].

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(HT-29), liver (Hep G2) and osteosarcoma (MG63), when compared with commercial carrageenans

Hemolytic activity is an important indicator of cell toxicity; it gives a quantitative measure of the hemoglobin release. It is useful to establish whether the cytotoxic activity is related to direct damage on the cell membrane or not. Limitation of extracts to be used as a therapeutic agent is their potential to cause damage to mammalian cells. Baker et al. [59] also pointed out that, the interaction between complement and carrageenan has been widely reported that it inhibits hemolytic complement activity in in vivo and in vitro. Intravenous administration of 5mg/kg carrageenan has been reported to reduce hemolytic complement by 99%. In the present study, the hemolytic activity of both native and commercial carrageenans was assessed against normal human erythrocytes. It revealed that at the lowest concentration (25µg/ml) of both carrageenans, the percentage hemolytic activity was very less (6.94 ± 0.19 and 16.04 ± 0.26% respectively). However, when the concentration of carrageenan increased, the hemolytic activity was also significantly (P<0.05) increased in both the tested carrageenans. It was observed 17

Journal Pre-proof that at the highest concentration of 250µg/ml, both native and commercial carrageenans showed 38.31 ± 0.24 and 65.75 ± 0.10% of hemolytic activity on red blood cells, respectively. Comparatively, the lysis of human red blood cells was much higher in commercial carrageenan treated group than native carrageenan treated group. Invariably, Shanmuga et al. [60] exhibited that the carrageenan-magnetic nano particles possessed anti hemolytic activity even at the highest concentration of 4100µg/ml. The results of the present study indicated that native carrageenan possessed non hemolytic activity in comparison with commercial carrageenan (Table 2b). Considering these, it could be suggested that native carrageenan is non toxic to normal cells, hence it can be considered to be a protective anti-therapeutic agent with

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biological properties. One of the interests in algal sulfated polysaccharides as anti-inflammatory agents is the

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growing body of evidence illustrating their ability to interfere with the migration of leukocytes to sites of inflammation. Rodrigues et al. [61] pointed out that red algal sulphated polysaccharides

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were widely possessed anti-inflammatory activity. Likewise, Coura et al. [62] also stated anti-

re

inflammatory effects of sulphated polysaccharides from the red seaweed Gracilaria cornea. In the present study, in-vitro anti-inflammatory activity of native and commercial carrageenans was

lP

evaluated against the denaturation of egg albumin protein. Denaturation of protein is a welldocumented cause of inflammation. Here, both native and commercial carrageenans exhibited a

na

concentration dependent denaturation of egg albumin protein. The result obtained was compared to the diclofenac sodium, a reference drug. At the lowest concentration (100μg/ml) of both native and

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commercial carrageenans possessed minimum (80.45 ± 1.26 % and 79.53 ± 1.15%, respectively) level of protein denaturation, whereas, when the concentration level increased, the denaturation level was also significantly (P<0.05) increased (Table 3). At the highest concentration of 500µg/ml, the denaturation level was observed to be 84.89 ± 1.03% in native and 83.28 ± 1.03% in commercial carrageenans. The anti-inflammatory activity of the standard drug diclofenac sodium exhibited the protein denaturation level in between 66.50 and 78.77% at the lowest concentrations of 100 to 300μg/ml, respectively and it was found to be quite low when compared to both native and commercial carrageenans. But the highest concentration (500µg/ml) of drug showed the denaturation level of 90.64%. It was comparatively higher than both native and commercial carrageenans. Thus the present findings exhibited a concentration dependent percentage inhibition of protein (albumin) denaturation by native carrageenan from K. alvarezii and commercial carrageenan. These findings revealed that, both native and commercial carrageenans exhibited excellent anti-inflammatory activity in in vitro model. This is the first attempt to determine the anti18

Journal Pre-proof inflammatory activity of carrageenans, because there are no evidences available regarding antiinflammatory activity of carrageenans. Invariably, few earlier reports suggested that carrageenans have shown to possess immunomodulatory activities that may help in triggering the immune responses, but sometimes may cause negative effects such as inflammation [63]. However, some indirect evidences showed that the exposure to carrageenan can cause occurrence of ulcerative colitis and intestinal neoplasms in people belonging to similar geographic distribution. Further, higher consumption of carrageenan leads to a greater incidence of inflammatory bowel disease and cancer [64]. Opposed to this, many evidences also suggested that lifelong administration of kappa/lambda-carrageenan from C. crispus or G. mamillosa at the concentrations of 0, 0.1, 5, 15, or

of

25% in the diet to groups of five male and five female mice had no adverse effects [65]. Similarly,

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Ranganayaki et al. [66] stated that the metabolic compounds from K. alvarezii possess antiinflammatory activity probably due to its inhibition of hyaluronidase enzyme. This may be the

-p

reason for the anti-inflammatory properties of carrageenans from K. alvarezii.

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Coagulation is the process by which the blood to form solid masses, or clots. Anticoagulants are used to treat and prevent blood clots that may occur in the blood vessels. Marine

lP

sulfated polysaccharides have also been shown to possess anticoagulant activities. In the present study, the anti-coagulant activity of native and commercial carrageenans was carried out and it was

na

proved that both the carrageenans have remarkable anti-coagulant activity against human blood samples when compared to control (Fig. 1). Anderson and Duncan [4] proposed that λ-carrageenans

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and κ-carrageenans from the seaweeds of Chondrus crispus and Polyides rotundus and also degraded carrageenan from seaweed Eucheuma spinosum have anticoagulant activity on intravenous injection in rabbits. But still now, no studies have reported about the comparison on anticoagulant activity of native and commercial carrageenans. The mechanism underlying the anticoagulant activity of carrageenans involved thrombin inhibition. In the present study, both native and commercial carrageenans showed the strongest anticoagulant activity by prolonged thrombin formation. There was no detectable clot formation was recorded for the time duration of 5min in both native and commercial carrageenans. At the lowest concentration (100µg/ml), plasma thrombination was observed at 5.24 ± 0.44min in native and 5.21 ± 0.04min in commercial carrageenans. When the concentration level increased, prothrombin time was also significantly (P<0.05) increased in both carrageenans. At the highest concentration of 500µg/ml, both native and commercial carrageenans extended the plasma prothrombin time at a higher rate and it was noted as 10.03 ± 0.05 and 9.98 ± 0.05 min, respectively, whereas, the plasma thrombination in control sample 19

Journal Pre-proof was recorded within 70sec (Fig. 2).The time taken for thrombin inhibition was dependent on the concentration of the carrageenans. At the highest concentration of 500µg/ml, prolonged prothrombin time was observed in both carrageenans. From the present observation, it can be suggested that native carrageenan is considerably better when compared to commercial carrageenan, because high degree of sulfation could be at the origin of the anticoagulant effect [67]. The structural analysis of a bioactive compound is very essential to know about the chemical groups observed in the particular compound. In the present study, the native and commercial carrageenans were subjected for structural characterization analysis. UV-Visible spectrum showed a

of

broad band between the region of 300 and 340 nm at the absorption maxima of 318 - 325nm in native carrageenan and 314 - 327nm in commercial carrageenan. This may be due to the presence of

ro

uronic acid and glycosidic linkages. Likewise, Arti Srivastava and Rajesh Kumar [68] stated that carrageenan polymer showed a broad band between the region of 300 and 340nm may be due to the

-p

presence of uronic acid and glycosidic linkages. The FT-IR spectra of native carrageenan from K.

re

alvarezii and commercial carrageenan are shown in Fig. 3. The spectral results depicted that the functional groups of both carrageenans were found in between the wave number 563.01 -

lP

1540.91cm–1. In native carrageenan, the functional groups like sulphate (563.01& 1147.88cm–1), alkynes (588.15cm–1), β,D-galactopyranoside (610.27cm–1), 2,4-disulphate (657.68cm–1), bending of

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pyranose ring (707.31cm–1), α-D-galactoyranosyl (738.49cm–1), α-galactose residues (760.24cm–1), sulphate ester (780.24 & 1026.60cm–1), 3-linked β,D-galactose (794.09cm–1), D-galactose-4-sulfate

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(847.01cm–1), 3,6-anhydrogalactose residue (888.58cm–1), anhydro-D-galactose (923.73cm–1), sulfate ester & glycosidic linkage (1058.22cm–1), covalent sulfate (1213.80cm–1) and uronic acid (1461.32cm–1), were observed. Similarly, the commercial carrageenan exhibited majority of the above functional groups such as sulphate (577.29cm–1), β-D-galactopyranosyl (600.14cm–1), 3,6anhydrogalactose (635.08cm–1) residues, β-D-galactopyranosyl-4-sulphate (645.48cm–1) and carbolic acid & carbolic ester (1540.91cm–1), etc. Based on the spectral result, it could be confirmed that both native and commercial carrageenans belong to kappa (κ) type. In accordance with these, Manuhara et al. [69] stated that several absorption peaks were observed in the above pointed area of carrageenan from K. alvareziito the presence of sulphate ester (1234cm–1), glycosidic linkage (1072cm–1), 3,6- anhydro-d-galactose (926cm–1) and D-galactose-4-sulphate (849cm–1), which indicated that the carrageenan was κ-type. Sekkal and Legrand [70] suggested that the bands observed in the region between 580 and 608cm−1 are due to the sulphate group of κ-carrageenan. They have also observed the bands at the regions of 719 (pyranose ring), 734 (α-D-galactoyranosyl) 20

Journal Pre-proof and 743cm−1(α-galactose) in the FTIR spectrum revealed the presence of κ- carrageenan, among these, the band obtained in the region 719cm-1 was being the stronger one. Pereira et al. [71] and Dewi et al. [72] were pointed out that particularly intense signal observed at 840 - 850cm-1 attributed to D-galactose-4-sulfate as seen in the spectra indicating the simultaneous presence of κcarrageenan. Likewise, Nivedita Sahu and Ganesh Kumar [73] characterized the carrageenan from G. filicina through FTIR analysis. They recognized the peaks at similar regions at or around 1030 (sulphate ester), 1070 (glycoside linkage), 1150 (galacto-pyranosyl groups), 1240 (covalent sulphate groups) and 1380cm−1 (carbolic acids). Nasima et al. [74] were observed the bands at 1158, 1030 and 1010cm-1 regions assigned to C-O, O-H and C-C stretching vibrations of pyranose ring and they

of

are common to all polysaccharides.

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The FT-Raman spectrum has many benefits in the analytical laboratory, including speed, easy sample preparation and spectral information that are interpreted in the same way as FT-IR

-p

spectra. FT-Raman allows the samples to be analyzed in their original form, which is a major

re

advantage when the sample structure is subjected to change with heat or pressure. In the present study, the FT-Raman spectrum of native carrageenan from K. alvarezii was compared with FT-

lP

Raman spectrum of commercial carrageenan (Fig. 4). It was noticed that more bands in lowfrequency range was observed in Raman spectra, when compared to IR spectroscopy within the

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spectral range between 3000 – 100cm-1.The spectra of both native and commercial carrageenans showed the bands at the regions of 380, 360 and 418cm-1 indicating the presence of glycosidic

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linkage belongs to κ- carrageenan. Both the carrageenans spectra exhibited the bands at the region of 462 and 487cm-1, which may be due to the presence of C2 group of β-galactose residues. The bands observed at the regions of 583, 610 and 975cm-1 in both carrageenans may be due to the presence of sulfate content belonged to kappa carrageenan and the peaks at the region of 732cm -1 indicated the presence of 3,6-anhydrogalactose residues. Further the native and commercial carrageenans showed the strong Raman band at approximately 846cm-1 region that was assigned to D-galactose-4-sulphate (G4S) and vibrations observed at around 809, 816 and 817cm-1 regions indicated the presence of sulphate ester, a characteristic band of carrageenan. Peaks observed in both carrageenans at the spectral regions of 1000, 1127 and 1155cm-1 were also similar to that of many other polysaccharides and vibrations observed in the region between 1500 and 1270cm-1 (1273, 1297, 1300, 1322, 1387, 1467, 1494 and 1487cm-1) were mainly due to the coupling of sulphate ester (CCH) bending motions. Peaks observed in the spectrum of both native (2898, 2826, 2797 and 2717cm-1) and commercial (2883, 2837, 2760, 2746 and 2713cm-1) carrageenans may be due to C-H stretching 21

Journal Pre-proof vibrations of 3,6-anhydro-D-galactose residues of κ- carrageenan. In an another study, Neilsen et al. [75] reported the interaction of H2O with κ- carrageenan has been subjected to the Raman domain in low frequency (400 - 20cm-1) region expressed the presence of β-galactose residues. Sekkal and Legrand [70] pointed out that a band observed between 380 and 360cm-1 in the spectrum of κcarrageenan extracted from E. cottoni has been assigned to a mode of a glycosidic linkage. They also reported the band at 415cm-1 region of Raman spectrum denoted the presence of 3-D-galactose, where the potential energy distribution of 3-D galactose indicated that this band was due to a skeletal mode. Again they pointed out that the peaks aroused in the region of 480 - 460cm-1 was probably due to a contribution of a bending or torsional vibration of the C2 group, the occurrence of

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sulfate content in the region between 580 and 973cm-1 and also the signals noted in the region 732cm-1 was due to the presence of 3,6-anhydrogalactose residue. Malfait et al. [76] reported the

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signals noted in the regions between 810 and 824cm-1 were probably due to the C-O-S

-p

pseudosymmetric vibrations. Sekkal and Legrand [70] reported that, vibrations observed in the region between 1500 and 1270cm-1 indicated the presence of α- and ?? -D-galactose and it was due

re

to the coupling of CCH bending motions and more intense band noticed in the regions between 1467

lP

and 1009cm-1 indicated the presence of carrageenan belonged to kappa fraction. NMR spectral study can be used very effectively to identify the type, number and the

na

proportions of different sugar residues in a polysaccharide as well as different linkage configurations and positions in the polymer can also be determined [77]. In the present 13

C NMR spectrum of native carrageenan from K. alvarezii expressed an anomeric

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investigation

carbon of disulphate group in the region of 21.04ppm (Fig 5 a1). Other than this region, the spectrum didn’t show any signals in the mentioned regions, this may be due to its superfluous purity or the type and seasonal variation of seaweed used for carrageenan extraction or carrageenan may lose their valuable compounds at the time of extraction. In commercial carrageenan, signals obtained within the region of 92 and 95ppm probably due to the presence of anomeric carbon of 3,6-anhydro-D-galactose residues of k-carrageenan and also the galactose-4-sulfate (G4S) vibrations in the exact region between 71 and 76ppm (Fig 5 b1). Likewise, Montolalu et al. [78] portrayed that the chemical composition of carrageenan from K. alvarezii can be significantly affected by different extraction processes, like extraction temperature and time. They also pointed out that carrageenan used for industrial purposes may always obtained without alkaline treatment and alcoholic precipitation. This could be the reason why commercial carrageenan has certain chemical elements which was lack in native carrageenan. In an another report, Henares et al. [79] 22

Journal Pre-proof stated that in

13

C NMR spectrum of hydrolyzed iota-carrageenan of Pseudoalteromonas

carrageenovora, revealed that within the regions between 105 to 90ppm anomeric carbon signals were present. Similarly, Van de Velde et al. [80] reported that the signals observed in the commercial kappa carrageenan between the region 104 and 76ppm indicated the presence of Galactose-4-sulfate (G4S) and the peaks noted in the regions of 97 to 72ppm showed the presence of 4 linked 3,6-anhydrogalactose (DA- unit). In 1H NMR spectrum of this study, both native and commercial carrageenans showed the peaks at the region between 5.11ppm and 4.7ppm respectively indicating the presence of chemical elements that belonged to κ- carrageenan (Fig. 5 a2 and b2). In accordance with these, Van de Velde et al. [80] pointed out that 1H NMR spectrum of

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native carrageenan extracted from K. alvarezii showed the peaks at the region 5.11ppm may be due

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to the presence of κ-monomer. Knutsen et al. [81] also protruded that 1H NMR spectrum of alkali extracted carrageenan from the gametophytes of Ahnfeltiopsis devoniensis showed two main signals

-p

(5.29 and 5.09ppm) at the region of anomeric proton. These signals corresponded to the anomeric proton of iota (DA2S) and kappa (DA) carrageenans. Campo et al. [82] reported that 1H NMR

re

spectroscopy of different carrageenan types (iota, kappa, lambda, mu, nu and theta) from different species of red seaweeds Gigartina sp., Chondrus crispus, Eucheuma and Hypnea spp. were based

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on the position and intensity of the resonances of the α-anomeric hydrogens of the repeating D- and

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DA units in the region from 5.1 to 5.7ppm. 4. Conclusion

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The overall study stated that native carrageenan extracted from K. alvarezii and commercial carrageenan offers a promising therapeutic value in prevention of oxidative stress that developed in diabetes. These effects could be mainly attributed to their antioxidant properties and act as a potential antioxidant bioactive compound. Both carrageenans also exhibited anticoagulant and antiinflammatory activities and could be relatively considered that the carrageenan from K. alvarezii possessed better pharmacological activities than commercial carrageenan. Further the structural analysis revealed that both carrageenans belong to kappa type. Acknowledgement The authors are gratefully acknowledge the DST-SERB, New Delhi, Govt. of India, for its financial support in the form of a Research grant (Grant No: EMR/2017/001453). References

23

Journal Pre-proof [1] S. Hassan, S. Abd El-Twab, M. Hetta, B. Mahmoud, Improvement of lipid profile and antioxidant of hypercholesterolemic albino rats by polysaccharides extracted from the green alga Ulva lactuca Linnaeus, Saudi J. Biol. Sci. 18 (2011) 333–340. [2] L. Pereia, A Review of the nutrient composition of selected edible seaweeds, in seaweed: ecology, nutrient composition and medicinal uses. V. H. Pomin edn. 15-47, Nova Science, New York, NY, USA. (2011) 11. [3] A.M. Suganya, M. Sanjivkumar, M. Navin Chandran, A. Palavesam, G. Immanuel, Pharmacological importance of sulphated polysaccharide carrageenan from red seaweed Kappaphycus alvarezii in comparison with commercial carrageenan, Biomed. Pharmacother.

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84 (2016) 1300-1312.

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[4] W. Anderson, J.G.C. Duncan, The anticoagulant activity of carrageenan, J. Pharm. Pharmacol. 17 (1965) 647-654.

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[5] T. Hu, D. Liu, Y. Chen, J. Wu, S. Wang, Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro, Int. J. Biol. Macromol. 46 (2010) 193–

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Antinociceptive and anti-inflammatory activities of sulphated polysaccharides from the red seaweed Gracilaria cornea, Basic Clin. Pharmacol. Toxicol. 110 (2012) 335 - 341. [63] D. Chen, X.Z. Wu, Z.Y. Wen, Sulfated polysaccharides and immune response: promoter or inhibitor, Panminerva medica, 50 (2008) 177-183. 28

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[72] E.N. Dewi, R. Ibrahim, S. Suharto, Morphological structure characteristic and quality of semi refined carrageenan processed by different drying methods, in “ICTCRED 2014”. In: H. Hadiyanto, S. Heru, K.R. Ocky, (Eds.), Procedia Environ. Sci. 23 (2015) 116–122. [73] C. Nivedita Sahu, M. Ganesh Kumar, Characterization of isolated polysaccharide and biochemical attributes of red algae Grateloupia filicina, Int. J. Sci. Res. 3 (2014) 2319-7064. [74] A. Nasima, R. Shahidan, M. Faizal, B.R. Nazaruddin, R.P. Edy-Giri, S.R. Ari, Investigation of the gelation mechanism between amino acid surfactant based micro-emulsion and kappacarrageenan gel network, Sains Malays. 43 (2014) 203-209. [75] F. Neilsen, P.A. Land, F.M. Nicolaisen, A low- frequency Raman study (20-400cm1) of aqueous agarose and k-carrageenan gels, A contribution to the study of the structure of water, Acta Chem. Scand. 34 (1980) 749. [76] T. Malfait, H. Van Dael, F. Van Cauwelaert, Molecular structure of carrageenans and kappa oligo-mers: Raman spectroscopic study, Int. J. Biol. Macromol. 11 (1989) 259-264. 29

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carrageenans, Carbohydr. Res. 13 (2004) 2309-13.

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Journal Pre-proof Author statements MS. Title: Investigation on bio-properties and in-vivo antioxidant potential of carrageenans against alloxan induced oxidative stress in Wistar albino rats

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MS. Id. No: IJBIOMAC_2019_7410

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Authors

preparation

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Muthusamy Sanjivkumar = Conceptualization, Methodology, software, Writing- original draft

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Manohar Navin Chandran = Visualization, Investigation, Data curation

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Arumugampillai Manimehalai Suganya = Software, Validation, Reviewing & Editing

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Grasian Immanuel = Project administration, Supervision

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Journal Pre-proof Fig. 1 . Anticoagulation activity of native carrageenan (NC) and commercial carrageenan (CC)

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against human blood samples

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Fig. 2. Antithrombotic activity of different concentrations of native and commercial carrageenans

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Journal Pre-proof Fig. 3. FT-IR spectrum of native (a) and commercial (b) carrageenans 1/1/2009 1:56:01 AM

100

D:\NIU\Deiva Kumari 06.11.14\N.0

2500 2000 Wavenumber cm-1 Page 1 of 1

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3000

1461.32 1213.80 1147.88 1058.22 1026.60 923.73 888.58 847.01 794.09 780.24 760.24 738.49 707.31 657.68 610.27 588.15 563.01 1500

1000

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3500

-p

96

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Transmittance [%] 97 98

99

(a)

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(b)

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1/1/2009 1:55:08 AM

3500

3000

2500 2000 Wavenumber cm-1 Page 1 of 1

33

1500

645.48 635.08 600.14 577.29

1540.91

97

98

Transmittance [%] 99

100

101

D:\NIU\Deiva Kumari 06.11.14\C.0

1000

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Fig. 4. FT-Raman spectrum of native (a) and commercial (b) carrageenans

(b)

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(a)

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Fig. 5. 13C NMR analysis of native (a1) and commercial (b1) and 1H NMR analysis of native (a2) and (b2) and commercial carrageenans (a1)

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(b1)

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(b2)

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Journal Pre-proof Table 1. Antioxidant markers of kidney in diabetic induced Wistar albino rats treated with native and commercial carrageenans GPx CAT Sample

(µmoles/mg protein)

(µg of GSH consumed/ min/ mg protein)

SOD

GST

GSH

LPO

(µmol/mg protein)

(µmol/mg protein)

(µmol/mg protein)

(TBARS)

20.51 ± 1.99

(µmoles/min /mg protein)

Ascorbic acid (Vit C) (µg/mg protein)

G1 NC 4.35 ± 0.20

1.85 ± 0.05

24.50 ± 1.62

4.06 ± 0.36

2.44 ± 0.08

27.56 ± 1.42

3.74 ± 0.25

3.76 ± 0.06

2.07 ± 0.06

20.47 ± 1.46

4.76 ± 0.71

1.46 ± 0.03

1.15 ± 0.03

2.40 ± 0.04

22.66 ± 1.81

4.42 ± 0.62

1.80 ± 0.02

3.49 ± 0.20

1.07 ± 0.01

2.16 ± 0.01

31.23 ± 1.78

4.44 ± 0.35

1.45 ± 0.02

2.72 ± 0.05

36.31 ± 2.01

5.92 ± 0.47

1.91 ± 0.04

20.41 ± 1.66

2.91 ± 0.13

27.50 ± 1.68

3.51 ± 0.30

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26.48 ± 1.73

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(500mg/kg)

(750mg/kg)

(1000mg/kg)

G6 CC (1000mg/kg) G7 (Normal control)

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(750mg/kg)

0.89 ± 0.03

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G5 CC

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G4 CC (500mg/kg)

3.09 ± 0.08

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G3 NC

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G2 NC

31.65 ± 1.63

5.48 ± 0.65

1.55 ± 0.06

2.97 ± 0.06

25.31 ± 1.33

4.10 ± 0.44

2.35 ± 0.04

47.52 ± 1.42

6.33 ± 0.52

0.38 ± 0.04

3.68 ± 0.07

30.80 ± 1.65

3.7 ± 0.31

1.06 ± 0.02

16.81 ± 1.56

2.03 ± 0.13

0.26 ± 0.02

1.49 ± 0.03

16.21 ± 1.41

5.34 ± 0.48

1.20 ± 0.03

29.61 ± 1.46

5.41 ± 0.28

1.51 ± 0.04

3.37 ± 0.06

26.67 ± 1.52

4.7 ± 0.24

3.66 ± 0.04

G8 (Diabetic control) G9 Glibenclamide (500µg/kg)

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Journal Pre-proof Each value is the Mean ± SD of nine individual data (n=9) within each column, means with different superscript letters are statistically significant (one way ANOVA; P<0.05 and subsequently post hoc multiple comparison with Tukey’s test).

CAT- Catalase

G2 NC: Group 2-Native carrageenan

GPx- Glutathione peroxidase

G3 NC: Group 3-Native carrageenan

SOD- Superoxide dismutase

G4 CC: Group 4-Commercial carrageenan

GST- Glutathione S transferase

G5 CC: Group 5-Commercial carrageenan

GSH - glutathione

G6 CC: Group 6-Commercial carrageenan

LPO- Lipid peroxidase

G7 Group 7-Normal control

Ascorbic acid - Vitamin C

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G1 NC: Group 1-Native carrageenan

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G8 Group 8-Diabetic control

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G9 Group 9-Standard drug Glibenclamide

Table 2. Cytoyoxic and toxic effect of different concentrations of native and commercial

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carrageenans against brine shrimp Artemia (a) and red blood cells (b)

150 200

LC 50

Commercial carrageenan

% Inhibition

Probit

% Inhibition

Probit

1.6989

6.6 ± 0.314a

0.0628

6.6 ± 0.470a

0.079

2.000

23.3 ± 0.206a

0.2673

16.6 ± 0.714b

0.1692

2.1760

43.3 ± 0.414a

0.4649

30.0 ± 0.600b

0.2446

2.3010

70.0 ± 1.16a

0.6141

40.8 ± 0.860b

0.3076

LC 50

Nil

100

Native carrageenan

160.40 µg/ml

50

Log C

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Concentration of carrageenans (µg/ml)

na

(a)

Each value is the Mean ± SD of triplicate analysis; within each row, means with different superscript letters are statistically significant (One way ANOVA test, P<0.05 and subsequently post hoc multiple comparison with SNK test)

(b) Inhibition (%) Concentration of

Native carrageenan

Commercial carrageenan

6.94±0.19a

16.04±0.26b

carrageenans (µg/ml) 25

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Journal Pre-proof 50

8.76 ±0.12a

20.63±0.17b

75

9.47±0.13a

31.56±0.05b

100

10.01±0.05a

36.46±0.09b

125

12.18±0.03a

37.50±0.08b

150

15.67±0.16a

39.92±0.45b

175

19.87±0.14a

45.48±0.06b

200

26.49±0.22a

49.89±0.07b

225

31.08±0.21a

55.07±0.09b

250

38.31±0.24a

65.75±0.10b

Each value is the Mean ± SD of triplicate analysis; within each row, means with different superscript letters are statistically significant (One way ANOVA test, P<0.05 and subsequently

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post hoc multiple comparison with SNK test)

Table. 3. Anti-inflammatory activity of different concentrations of native and commercial

Concentrationof

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carrageenans in comparison with a standard drug

Anti inflammatory activity (%) Commercial Standard Drug

Native

drug(µg/ml)

Carrageenan

carrageenan

(Diclofenacsodium)

80.45 ± 1.26a

79.53 ± 1.15a

66.50± 1.40b

81.0 ± 1.08a

80.84 ± 1.17a

71.60± 1.30b

81.20 ± 1.07a

81.28 ± 1.07a

78.77±1.95a

83.06 ± 1.13a

82.78 ± 1.09a

83.38± 1.69a

84.89 ± 1.03a

83.28 ± 1.03a

90.64± 1.02b

200 300 400 500

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100

na

carrageenans/

Each value is the Mean ± SD of triplicate analysis; within each row, means with different superscript letters are statistically significant (One way ANOVA test, P<0.05 and subsequently post hoc multiple comparison with SNK test)

39

Journal Pre-proof Highlights 

Extracted the polysaccharide k- carrageenan from red seaweed Kappaphycus alvarezii



Compared the toxicity effect of both native and commercial k-carrageenans in Wistar albino rats



Assessed the bio-properties of both native and commercial k-carrageenans in Wistar albino rats Determined the structural characteristics of both k-carrageenans through standard

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techniques

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

40