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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 6  |  Issue : 1  |  Page : 7-16

Nitrate stress-induced bioactive sulfated polysaccharides from Chlamydomonas reinhardtii


School of Biological Sciences, UM DAE Centre for Excellence in Basic Sciences, Univeristy of Mumbai, Kalina, Santacruz (E), Mumbai, Maharashtra, India

Date of Web Publication24-Apr-2019

Correspondence Address:
Dr. Sirisha L Vavilala
School of Biological Sciences, UM DAE Centre for Excellence in Basic Sciences, Mumbai, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/BMRJ.BMRJ_8_19

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  Abstract 


Sulfated polysaccharides (SPs) are anionic carbohydrate polymers synthesized as extracellular or cell wall components by most of the algae and have potent bioactive properties. In the current study, Chlamydomonas reinhardtii (Cr) cells were attributed to sodium nitrate stress in concentrations such as 5 mM, 10 mM, 20 mM, 30 mM, and a control to determine the productivity and bioactivity of SPs. SPs are extracted by hot water method using 80% ethanol. The percentage yield of SPs increased with an increase in concentration of sodium nitrate as compared to control. Biochemical analysis of the extract showed an increase in carbohydrate content (22%–95%), uronic acid content (23%–60%), and sulfate content from control to 30 mM NaNO3-treated extracts. The amount of reducing and nonreducing sugars was found to be 6.16% and 89.06%, respectively, while the protein content is ~16%. The antioxidant potential of SPs showed increased antioxidant activity with an increase in concentration of NaNO3 stress. The analysis resulted in maximum chelating activity of 83.73% assayed in concentration range of 1–8 μg/ml, total antioxidant activity of 70.36% in concentration 0.05–2μg/ml, and hydroxyl radical scavenging activity of 79.52% in concentration 250–1000 μg/ml; reducing potential was observed with the highest absorbance of 0.87; the 2,2-diphenyl-1-picrylhydrazyl scavenging activity showed the highest activity of 63.61%, while the superoxide scavenging activity was 92% at 0.1–1 μg/ml. Furthermore, Cr-SPs inhibited the growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacterial growth as indicated by clear zones that increased in size with an increasing concentration of NaNO3. These results provide opportunities to develop Cr-SPs as natural antioxidant and antibacterial agents.

Keywords: Algae, antimicrobial activity, antioxidant activity, nitrate stress, sulfated polysaccharides


How to cite this article:
Vishwakarma J, Parmar V, Vavilala SL. Nitrate stress-induced bioactive sulfated polysaccharides from Chlamydomonas reinhardtii. Biomed Res J 2019;6:7-16

How to cite this URL:
Vishwakarma J, Parmar V, Vavilala SL. Nitrate stress-induced bioactive sulfated polysaccharides from Chlamydomonas reinhardtii. Biomed Res J [serial online] 2019 [cited 2019 Jul 20];6:7-16. Available from: http://www.brjnmims.org/text.asp?2019/6/1/7/257037




  Introduction Top


Marine organisms are potentially novel sources of sustainable biologically active secondary metabolites that might exhibit various biotechnological applications. Among marine organisms, marine algae have been identified as a simple unicellular or multicellular organism that includes seaweeds (macroalgae) and many single-celled forms (microalgae) such as blue-green algae, dinoflagellates, and diatoms, belonging to kingdom Protista.[1] Marine algae can be distinguished by the presence of pigment contents in their thallus into three algal groups such as red algae (Rhodophyta), green algae (Chlorophyta), and brown algae (Phaeophyta) in varying forms and sizes.[2]

The metabolites characteristically identified from the cell wall of marine algae under stress conditions represent different structural classes of biologically active compounds. These compounds have been exploited for a wide range of biological activities such as antioxidant, anticoagulant, antimicrobial, antiviral, antifungal, and anticancer activities and immunomodulating activities.[3] The active compounds in algae include proteins, peptides, phenolic compounds, alkaloids, polysaccharides, and pigments.[4] However, there has been an increasing exploitation of the polysaccharides obtained from the marine algae for their potential biological activity. Marine algae have been intensely investigated for sulfated polysaccharides (SPs) for medicinal purpose.[5] The carbohydrate polymers are structurally diverse which makes them valuable material for research and commercial exploitation. The major SPs found in different algae are carrageenans (red algae), fucoidans (brown algae), and ulvans (green algae). Carrageenans have been commercially used as emulsifier, stabilizer, and thickener, whereas fucoidan has been used for medicine and food industries. Ulvan, in turn, has been used in pharmaceutical industries.[6] Green algal polysaccharides are highly heterogeneous and structurally diverse than red and brown algae, which thus hinder studies on their structure. The bioactivity of SPs from green algae was highly influenced by its composition and chemical structure. Various structural factors such as molecular weight, sulfate content, position of sulfate group, degree of sulfation, type of sugar moiety attached, glycosidic bonds, and molecular weight have been attributed to the biological activities of the SPs.[4],[7],[8],[9]

In humans, free radicals are uncharged molecules, atoms, or ions with unpaired valence, which play an important role in many biological activities such as transduction and transcription.[10] Humans are constantly exposed to various free radicals such as oxygen-related free radicals and reactive species which comprise superoxide ion, hydroxide ion, hydrogen peroxide, nitric oxide, peroxynitrite, and hypochlorous acid. However, overproduction of free radicals can cause oxidative stress due to imbalance between the production of free radicals and the ability of the body to prevent or detoxify its harmful effects by stabilizing it. Eventually, it may cause many chronic diseases such as atherosclerosis, stroke, cancer, rheumatoid arthritis, diabetes, cardiovascular diseases, chronic inflammation, postischemic perfusion injury, myocardial infarction, and septic shock, aging, and other degenerative diseases in humans.[10] Antioxidants are defense molecules that are capable of neutralizing the unstable free radical species by either inhibiting or delaying the oxidation processes. These molecules have been classified as endogenous (internal) and exogenous (natural or synthetic) sources. Endogenous antioxidant includes glutathione, arginine, creatine, Vitamin E, Vitamin C, Vitamin A, selenium, zinc, and enzymes such as superoxide scavenging, catalase, and glutathione peroxidase. When endogenous antioxidants become unable to neutralize the harmful effects of the free radicals due to stress-induced or diseased condition, the need for an external system of antioxidant rises.[11]

In the recent years, more attention has been focused toward the natural antioxidants from algae. More concern has been given to antioxidant activities with likelihood that it will have a cure to number of diseases caused by “oxidative stress” also owing to side effects and toxicity arising from synthetic antioxidants.[12] The antioxidant ability of alga-derived SPs has been studied by various in vitro antioxidant assays.[13] Earlier reports showed that Chlamydomonas reinhardtii (Cr) has bioactive sulfated polysaccharide compounds, and there is an accumulation of polysaccharides under stress.[3],[14],[15] The aim of the current study is to enhance the production of SPs by sodium nitrate stress and evaluate their antioxidant and antimicrobial properties.


  Materials and Methods Top


Culturing conditions

Algal strain of wild-type CrCC-124 obtained from Chlamydomonas Genetics Center, Duke University, USA, was inoculated from solid to liquid media in inorganic Tris-acetate-phosphate (TAP, pH 7) medium. The culture was maintained thermostatically at 25°C under continuous illumination of 300-μmol photons m−2 s−1 as mentioned by Sirisha et al.[16] The cultures were kept in shaker incubator with a speed of 150 rpm, and growth was monitored regularly by observing the color of the medium from light to dark green. To induce SPs production, cells were grown in Tris Acetate Phosphate medium containing different concentrations of NaNO3 stress ranging from 5 mM, 10 mM, 20 mM, and 30 mM, respectively. Control sample was maintained without any stress.

Extraction and purification of polysaccharides

Extraction and purification were performed as mentioned in Kamble et al.[14] The cells are harvested by centrifuging at 1100 × g for 5 min. The pellets were dissolved in 80% ethanol and macerated. Then, the extract containing the polysaccharides was incubated in hot water bath of 80°C under continuous shaking for 4 h. After 4 h of incubation, the extract was cooled down to room temperature and spin down at 4000 × rpm for 10 min and the supernatant was evaporated using rotary evaporation, carried at 60°C, 114 rpm in 250-mbar pressure for 10 min. The extract is purified by passing through Q-Sepharose™ Fast Flow column, which is equilibrated with water and eluted in NaCl gradient.[14] Further, the dried extract is used for subsequent analysis.

Estimation of total carbohydrates

The total carbohydrate content of extracts was determined by anthrone method as described by Dubois et al.[17] The carbohydrates are hydrolyzed, by treating with acid to form furfurals and hydroxymethylfurfural. These furfurals are then condensed by anthrone reagent to form a blue-green color complex. Glucose (100 μg/ml) is used as standard and prepared in concentrations ranging from 20 to 100 μg/ml, and 5 μl of each extract was allowed to react with anthrone reagent, followed by incubating the tubes in boiling water bath for 8 min. The intensity of the blue-green color complex formed is directly proportional to the amount of carbohydrates present, which was determined spectrometrically at 630 nm.

Estimation of reducing sugars

The presence of reducing sugars was quantified using 3,5 dinitrosalicylic (DNSA) method of Miller.[18] 3, 5-Dinitrosalicylic acid (DNSA) detects the presence of free carbonyl group (C=O) of reducing sugars. DNSA is reduced to 3-amino-5-nitrosalicylic acid (ANSA), which under alkaline conditions is converted to a reddish-brown-colored complex. Glucose was used as standard where solutions of concentration 100–1000 μg/ml range were prepared. About 5 μl of each of algal extracts such as control, 5-mM, 10-mM, 20-mM, and 30-mM NaCl stress samples was used for testing. One milliliter of 3,5-dinitrosalicylic acid (DNSA) was added to each of the tubes and incubated in boiling water bath for 10 min. The color change from yellow to red due to the reduction of DNSA to ANSA is observed and its absorbance is measured at 505 nm.

Estimation of sulfate content

The barium chloride-gelatin method was used to determine the amount of sulfate in the extracts according to the method described by Dodgson and Price[19] using sodium sulfate as standard (1 mg/ml) in concentration ranging from 20 to 140 μg/ml. About 1N HCl is used as diluent, and 5 μl of each algal extracts is added to test tubes where the total volume is kept as a 200-μl. This is followed by adding 3% trichloroacetic acid (TCA) to the test solutions and 1 ml of 0.5% barium chloride-gelatin solution to precipitate the barium sulfate. The tubes are incubated at room temperature for 15 min. The turbidity of the barium sulfate formed which indicates the sulfate content present in the extract and its absorbance is determined at 360 nm.

Estimation of protein content

The protein content of the algal extracts containing SPs was determined by Lowry's method (1951).[20] It is based on the reaction of Cu+, produced by the oxidation of peptide bonds, which reacts with the Folin reagent. It involves reduction of phosphomolybdic tungstate to heteropolymolybdenum of the Folin reagent resulting in a strong blue color, which depends partly on the tyrosine and tryptophan contents. The test solutions are diluted in a 1-ml system using distilled water. Bovine serum albumin (1 mg/ml) was used as standard in concentration range of 40–200 μg/ml along with 5 μl of each of the algal extracts as test solutions. Five milliliters of alkaline copper sulfate solution is added, followed by incubation of 15 min at room temperature and later adding 0.5 ml of 1% Folin–Ciocalteu reagent. The tubes are incubated again for 30 min at room temperature and absorbance is taken at 660 nm.

Estimation of uronic acid

The amount of uronic acid is determined according to the Bitter and Muir[21] modified carbazole-sulfuric acid protocol using glucuronic acid as standard. This method involves quantification of chromophore that is formed when the hydrolyzed products of polysaccharides and uronic acid conjugate with the chromogen carbazole. The procedure involves hydrolysis of polysaccharides with mineral acid, dehydration of the uronic acid, and conjugation to a chromogen such as carbazole to form a chromophore that can be quantitated by spectrophotometry. Glucuronic acid is used as standard in concentration range of 4–40 μg/ml prepared from stock solution of 1 mg/ml. The intensity of the color change to purple is measured at 530 nm.

Antioxidant assays

The antioxidant assays were carried out to determine the radical scavenging ability and the reducing potential of the SPs present in the extracts. The antioxidant potential was determined by evaluating the total antioxidant capacity (TAC) and scavenging activity of free radicals such as hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) along with reducing and chelating of ferric and metal ions, respectively. The result of each antioxidant assay is expressed in percentage form.

Total antioxidant assay

The TAC was evaluated using method described by Arivuselvan et al.[22] The free radical scavenging capacity of the sample is used as a measure of TAC. This amount is a measure of antioxidant activity of the sample which is assessed by formation of phosphomolybdenum complex. TAC is the measure of the amount of free radicals scavenged by a test solution, being used to evaluate the antioxidant capacity of biological samples. The antioxidant activity of samples can be evaluated by the phosphomolybdenum complex formation. Ascorbic acid is used as a standard in concentration range of 0.01–0.1 mg/ml from stock solution of 10 mg/ml. Different concentrations of methanol extracts in range of 0.05–2 mg/ml were used as test solutions in 500-μl system using diluent as phosphomolybdic acid. The diluent reagent was prepared in distilled water by mixing 3.26 ml of 0.6-M sulfuric acid and 0.334 g of sodium phosphate (monobasic) along with 0.494 g of ammonium molybdate, making up the volume to 100 ml. The test solutions were incubated in boiling water bath of 90°C for 90 min. The reduction of phosphomolybdic acid by electron transfer coupled with oxidation to phosphomolybdenum results in reduction in blue color. The intensity of the color reduction is measured spectrometrically at 695 nm.

2,2-diphenyl-1-picrylhydrazyl scavenging assay

The assay was carried out according to the method described by Chang et al.[23] with slight modifications. This method is based on decomposing the free radical DPPH. Different concentration of each extract in 0.01–2-mg/ml range is diluted using methanol as diluent in 3-ml system. Ascorbic acid (10 mg/ml) is used as standard in range of 0.01–6 mg/ml. One milliliter of 1:10-diluted DPPH (0.4-mg/ml methanol) solution is added to the test solutions and incubated in dark for 15 min; four milliliters of DPPH was used as reference for control. The fading/disappearance of purple color was observed and measured at 517 nm. The percentage radical scavenging activity of the plant extracts was calculated using the following formula:



Hydroxyl radical scavenging assay

The assay is performed according to a method described by Halliwell et al.[24] The short-lived hydroxyl radical has the potential to hydroxylate proteins, lipids, and DNA and is an extremely reactive species. The putative hydroxyl radical is an extremely reactive and short-lived species that can hydroxylate DNA, proteins, and lipids. The assay uses ascorbic acid-iron-ethylenediaminetetraacetic acid (EDTA) model of hydroxyl radical generating system. The Fe-EDTA solution was prepared as 0.13% by mixing 20 ml of each of 0.26% EDTA and 0.13% ferrous ammonium sulfate. About 0.018% EDTA solution was prepared for the Fenton reaction to occur which combines with Fe+2 ions to form hydroxyl radicals. Ascorbic acid was prepared in concentration of 0.22% and was also used as standard in 10 mg/ml with range of 0.01–0.1 mg/ml. TCA is made as 17.5% and chilled at 4°C. Nash reagent is prepared by mixing 7.5 g of ammonium acetate with 150 μl of acetic acid plus 200 μl of acetone solution in 75-ml distilled water and making up the volume to 100 ml. Different concentrations of each extract (250, 500, 750, and 1000 μg/ml) along with control sample were allowed to react with 1-ml Fe-EDTA, 0.5-ml EDTA, 1 ml of Dimethyl Sulphoxide (DMSO), and 0.5-ml ascorbic acid. The test solutions were incubated in boiling water bath for 15 min at 90°C. The incubation period leads to the formation of formaldehyde by oxidation of hydroxyl radicals by DMSO. On addition of 1-ml ice-cold TCA and 3 ml of Nash reagent to test solutions, the formaldehyde formed in the reaction mixture turns yellow, the intensity of which is measured at 412 nm. The activity is expressed as percentage hydroxyl radical scavenging activity (HRSA) calculated by the following formula,



Reducing assay

This assay was performed by the method mentioned by Zhang et al.[25] The redox reaction ability leading to the inhibition of chain reactions of free radicals is determined whether the sample can reduce ferric ions or not. The basic principle is that potassium ferricyanide (K3 Fe3+(CN)6) reacts with substances that have reducing potential and forms potassium ferrocyanide (K4 Fe2+(CN)6) which further interacts with ferric chloride to give ferric-ferrous complex which is spectrophotometrically measured at 700 nm. It is based on the principle that substances which have reduction potential react with potassium ferricyanide (K3 Fe3+(CN)6) to form potassium ferrocyanide (K4 Fe2+(CN)6), which then reacts with ferric chloride to form a ferric-ferrous complex that has an absorption maximum at 700 nm. The test solution (500-μl system) containing of each algal extract in concentration ranging from 0.05 to 2 mg/ml is diluted in phosphate buffer (0.2 M, pH 6.6). The reaction is initiated by adding 500 μl of 1% potassium ferricyanide and incubating in water bath of 50°C for 20 min to form ferrocyanide. This is followed by adding 1 ml of 10% TCA to stop the reaction. The solutions are treated with 600 μl of 0.1% ferric chloride solution that gives a Perl's Prussian blue color. Ascorbic acid (10 mg/ml) is used as standard in the range of 0.01–2 mg/ml. The absorbance was read at 700 nm in a spectrophotometer, and increasing absorbance indicates an increase in reducing ability.

Metal-chelating assay

This assay was evaluated by the method described by Soler-Rivas et al.[26] protocol. Ascorbic acid is used as standard in concentration of 10 mg/ml and used in range of 0.01–0.1mg/ml. The test solutions of each algal extract were prepared in range of 1–6 mg/ml in 750-μl system using methanol as diluent and water as diluent for standard. This is followed by addition of 50 μl of 2-mM ferrous sulfate (5.56 mg in 10 ml D/W), and tubes are incubated at room temperature for 15 min after vortexing. Ferrozine was prepared as 5 mM by dissolving 24.6 mg in 10-ml water and added about 200 μl after incubation period. The solutions are mixed and again kept for incubation or 10 min at room temperature. The absorbance of solutions is measured at 562 nm.

Antimicrobial assay

Agar well diffusion method

One hundred microliters of overnight grown culture of  Neisseria More Details mucosa and Staphylococcus aureus were plated in GM3 agar plate. Nitrate stress (0–30 mM) subjected Cr-SPs ranging from different concentrations 0–2 mg/ml were loaded in the wells punched in the agar plates along with control, and the plates were incubated at 37°C for 24 h. The ability of control and nitrate stress induced Cr-SPs to inhibit the growth of these bacteria were compared by measuring the diameter of the zones of inhibition (millimeters).[27],[28]

Statistical analysis

All the experiments are performed in triplicates maintaining experimental duplicates each time. The analysis was done using the Statistical Analysis System OriginPro 8.0.


  Results Top


Biochemical analysis of the extract

The total carbohydrate content determined by anthrone method using glucose as standard was found to be increasing from 22.41% to 94.57% with increased NaNO3 concentration [Figure 1]. The amount of reducing sugar content present in the total carbohydrates was measured by DNSA method and was found to be around ~ 3%–7% [Figure 1]. The nonreducing content was in range from 16% to 89% which was calculated by subtracting the reducing sugar content from total carbohydrate content. The total amount of sulfate determined by barium chloride-gelatin method was found to be increasing from 79% to 90% from control to 30-mM NaNO3 stress-induced samples [Figure 1], indicating that the polysaccharides are indeed sulfated. As SPs are known to have uronic acid associated with them, Bitter and Muir method[21] with modified carbazole reaction was performed to estimate the uronic acid content present in the extract. Results clearly showed steady increase in uronic acid content ranging from 23% to 60% with the highest uronic acid content showed by 20-mM NaNO3-stressed samples [Figure 1]. Total protein content was measured by Lowry's method, which ranged from 7% to 13% for control to 30-mM NaCl-stressed samples, indicating that total amount of protein is very less in the extract. All the biochemical assays clearly indicate that the extract is enriched with polysaccharides that are sulfated and have high uronic acid content [Figure 1].
Figure 1: Total biochemical constituents of the extracts attributed to various concentrations of NaNO3 stress (data presented as mean ± standard deviation of three independent experiments)

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Analysis of antioxidant potential of Cr-SPs induced by NaNO3 stress

With the increasing damage caused by oxidative stress and the pressure of availability of potential antioxidant agent, it has been important to find natural antioxidant with no harmful effects. Algal SPs are known to have significant antioxidant potential.[29] Various factors such as sulfate content, distribution of sulfate groups, method of extraction, and presence of electrophilic groups such as keto or aldehyde group in uronic acids can facilitate the liberation of hydrogen from O-H bond have been attributed to the antioxidant potential of SPs.[14],[30] As the extract was found to be enriched with SPs, their antioxidant potential was then assayed by various in vitro biochemical assays.

Total antioxidant assay of NaNO3 stress-induced Cr-SPs

The total antioxidant assay depends on electron-transfer mechanism leading to the reduction of phosphomolybdic acid to phosphomolybdenum. The total antioxidant activity evaluates the ability of the SPs to reduce Mo (VI) to Mo (V) at different concentrations. The control samples showed total antioxidant potential of 1.6%–58%. While the nitrate-stress induced SPs showed an enhanced total antioxidant activity of 12%–70% from 0.05- to 2-mg/ml concentration [Figure 2]. About 2-mg/ml concentration of 30-mM NaNO3-treated samples showed a maximum total antioxidant ability of 70.3%, indicating a 1.2-fold increased activity induced by nitrate stress as compared to control.
Figure 2: Total antioxidant activity of NaNO3-induced sulfated polysaccharides and control (data presented as mean ± standard deviation of three independent experiments)

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Reducing potential of NaNO3 stress-induced Cr-SPs

The electron-transfer mechanism of antioxidants can be assessed by its ability to reduce ferric ions. Reducing potential of the SPs was determined on their ability to reduce Fe (III) to Fe (II). The reducing potential was measured in terms of absorbance units at 700 nm. The reducing potential of control and Nitrate-induced Cr-SPs were determined using ascorbic acid as standard. It was observed that with increasing concentrations of the extract from 0.05 to 2 mg/ml, there was an increase in absorbance units. The results showed a significant increase in reducing potential for controland nitrate stress induced SPs with values ranging from 0.07–0.167, 0.082–0.19, 0.086–0.23, 0.09–0.32, 0.1–0.4, 0.11–0.48, 0.13–0.58, and 0.44–0.87, respectively. Thus, the SPs has prominent reducing potential probably due to the presence of anionic sulfate cation that might be responsible to reduce ferricyanide complex to ferrous form [Figure 3].[14]
Figure 3: Iron-chelating ability of NaNO3-induced sulfated polysaccharides and control (data presented as mean ± standard deviation of three independent experiments)

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Metal-chelating ability of NaNO3 stress-induced Cr-SPs

The chelating capacity analyses the ability of the antioxidant agent to form stable complexes with the metal ions. The iron-chelating ability is carried out to check whether Cr-SPs can bind to ferrous ions or not.[31] In this study, the chelating capacity of the SPs was observed for 1–8-μg/ml concentration range against ascorbic acid with concentration range from 0.01 to 0.1 μg/ml. All the samples exhibited chelating activity in increasing order for increasing concentration. Similarly, the SPs obtained from the concentrations confined to NaNO3 stress showed increasing chelating activity with increasing concentration of the assay. The 5-mM, 10-mM, 20-mM, and 30-mM NaNO3 stress extract showed increasing chelating effect from 26%–76%, 42%–79.96%, 47%–80%, and 54%–83%, respectively, as compared to control (8%–79.51%) [Figure 4]. Therefore, the present study indicates that the high percentage chelating activity in higher concentrations may attribute to its high sulfate and carbohydrate content.
Figure 4: Iron reducing capacity of sulfated polysaccharides under increasing NaNO3 conditions (data presented as mean ± standard deviation of three independent experiments)

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Hydroxyl radical scavenging ability of NaNO3 stress-induced of Cr-SPs

The hydroxyl radical scavenging ability of SPs has been determined against ascorbic acid standard with concentration range from 250 to 1000 mg/ml [Figure 5]. It was observed that the hydroxyl radical scavenging potential of the samples is dose dependent. Control sample showed hydroxyl radical scavenging ability of 3%–64%. SPs induced by increasing concentration of nitrate stress, showed linear increase in the hydroxyl radical scavenging ability. The maximum activity was observed by 30-mM NaNO3-treated sample at 1000 μg/ml, which is ~80% that is 1.25-fold more than the control samples [Figure 5]. This enhanced activity is mainly attributed to enhanced SPs produced by NaNO3 stress.
Figure 5: Hydroxyl radical scavenging activity of sulfated polysaccharides under increasing NaNO3 concentrations (data presented as mean ± standard deviation of three independent experiments)

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Determination of superoxide radical scavenging activity of NaNO3 stress-induced Cr-SPs

The superoxide radical scavenging activity was determined using Phenazine Methosulphate/ Nicotinamide Adenine Dinucleotide (PMS/NADH) system. The SPs extracted showed promising superoxide radical scavenging activity ranging from 60% to 92% in an increasing manner with increasing concentration (0.1–1 μg/ml). The 5-mM sample showed maximum activity of all samples, while 10-mM and 30-mM extract expressed almost activity as of control and 20 mM showed slightly greater activity [Figure 6]. Such high percentage of superoxide radical scavenging activity along the increasing concentration range could be due to high sulfate and carbohydrate content.[32]
Figure 6: Superoxide scavenging activity of the sulfated polysaccharides under increasing NaNO3 concentrations (data presented as mean ± standard deviation of three independent experiments)

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2,2-diphenyl-1-picrylhydrazyl radical scavenging ability of NaNO3 stress-induced Cr-SPs

In this study, the DPPH activity had been determined with different concentrations ranging from 0.05 to 2 mg/ml. Control samples showed 1%–64% DPPH radical scavenging ability while NaNO3-treated samples showed 5%–63% [Figure 7]. These results clearly indicate that with increased NaNO3 concentration, there is enhanced Cr-SPs production that showed promising hydrogen radical scavenging, DPPH radical scavenging, chelating ability, reducing potential, and total antioxidant potential. Further purification and understanding their structural characteristics would help them develop as good antioxidant agents.
Figure 7: 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity of sulfated polysaccharides under increasing NaNO3 concentrations (data presented as mean ± standard deviation of three independent experiments)

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Antimicrobial activity of control and NaNO3 stress-induced Chlamydomonas reinhardtii sulfated polysaccharides

The Cr-SPs obtained after giving nitrate stress were assessed for antimicrobial activity using agar well diffusion assay along with control against Gram-negative bacteria N. mucosa andGram-positive S. aureus. Clear zones up to 22.33 mm and 29.67 mm were obtained in case of S. aureus and N. mucosa, respectively. Nitrate-stressed Cr-SPs clearly inhibited the growth of both the bacteria [Table 1] and [Figure 8].
Table 1: Antimicrobial activity of sulfated polysaccharides against Staphylococcus aureus and Neisseria mucosa (diameter of inhibition zone in mm)

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Figure 8: Antibacterial potential of Chlamydomonas reinhardtii sulfated polysaccharides under different nitrogen conditions against Staphylococcus aureus and Neisseria mucosa

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  Discussion Top


There is an increasing need for natural antioxidants for neutralizing the oxidative stress caused by reactive species or free radicals formed due to endogenous metabolic process, leading to various diseases and aging of cells.[33] The excess production of reactive oxygen species (ROS) can trigger apoptosis and cause various degenerative diseases such as atherosclerosis, cancer, cardiovascular diseases, diabetes, hypertension, rheumatoid arthritis, and aging and also neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.[10] Research reports had suggested that synthetic antioxidants available commercially such as butylhydroxyanisole and butylhydroxytoluene are known to cause severe side effects on the liver and can act as toxic molecules after long-term exposure.[14] Therefore, with this respect, the antioxidant effect of natural materials such as plants and algae has been implemented. Recently, much focus has been driven to marine algae due to the presence of diverse secondary metabolites which can act as natural antioxidants, by neutralizing the ROS causing oxidative damage against various diseases and aging processes.[34] Marine algae have been prolific sources of SPs, which have potential bioactivities. In the current study, Chlamydomonas reinhardtii was used to induce sulphated polysaccharides with nitrate stress. Earlier research on this algae showed to secrete SPs when subjected to abiotic stress.[14],[15] In the present study, the algaehas been exposed to sodium nitrate stress to determine its effect on the production of SPs, and it was found that the extract was found enriched with carbohydrate content ranging from 22% to 95% for control to 30-mM stress with high sulfate content [Figure 1], thus indicating a positive effect of sodium nitrate stress on the production of SPs. Similarly, earlier the study with Spirulina platensis alsoreported high values of carbohydrate and sulfate content with increased nitrogen stress. The carbohydrate and sulfate content was found significantly greater than S. platensis, which reported maximum 17.81%.[5] The estimation of reducing sugars and nonreducing sugars with the highest concentration of 6.16% and 89.06%, respectively, in 10-mM and 30-mM NaNO3 stress was observed, indicating that high concentration of NaNO3 may support high concentration of nonreducing sugars over reducing sugars. The SPs have been associated with uronic acid content which confers it slightly acidic nature.[35] From the results obtained, it was observed that 20-mM and 30-mM NaNO3 stress indeed increased the uronic acid content in the extract by 60% and 43%, respectively. The estimation of protein showed a very minute quantity of protein concentration present in the SPs.

In the present work, various in vitro antioxidant assays have been used to determine the antioxidant potential of SPs attributed to sodium nitrate stress. From the results, it can be said that the SPs have potentially significant radical scavenging and chelating potential. The total antioxidant activity determined the ability of a molecule to neutralize the ROS by donating an electron to protect damage from oxidative stress.[36] In the current study, the SPs induced with NaNO3 showed enhanced total antioxidant activity as compared to control [Figure 2]. The results showed that SPs extracted from 5-mM and 10-mM NaNO3 stress showed an increase in total antioxidant ability and then the extracts were attributed to 20-mM and 30-mM NaNO3 stress. Earlier reports also showed efficient total antioxidant activities by SPs of various green and brown algal species.[37],[38]

Iron molecules are known to enzymatically catalyze lipid oxidation causing the formation of oxygen-free radicals. Which are attributed to its ability of transferring single electron.[39] Iron chelators thus effectively stabilized the Fe (II) ions into reduced Fe (III) state, which is then pass out through urine or feces.[31] In earlier studies, the iron-chelating ability of SPs was determined to be dose dependent.[40],[41],[42] Similarly, the chelating ability of Cr-SPs was concentration dependent with the range of 4–8 mg/ml with a linear increase in chelating activity with an increase in concentration with the highest activity of 64%. The chelating compounds are also confined to acquire two or more binding sites within the same molecule, with a single central atom and polysaccharides showing this specificity.[14] Moreover, the chelating ability of the Cr-SPs under increasing NaNO3 concentrations [Figure 4] was found to be more than SPs of brown marine algae Dictyopteris justii having activity of 27%.[42]

The hydroxyl radicals are highly active and unstable ion. It is formed by Fenton reaction between transition-metal ions, but in the absence of these transition ions, it is formed by oxidation of hydrogen peroxide. Therefore, the antioxidant mechanism generated includes either suppression or removal of hydroxide ion.[43] From the results obtained, the radical scavenging activity was found increasing in 5-mM and 10-mM NaNO3 stress extracts whereas 20 mM and 30 mM showed lesser scavenging activity as compared to that of 5-mM and 10-mM samples [Figure 5]. Therefore, with high nitrate concentration, it might have restricted the scavenging activity to certain limit. However, there was an increase in activity for increasing concentration of the sample. From reports of previous studies, the hydroxyl radical scavenging activity of different fucoidans showed maximum 80% activity, while seaweed Sargassum tenerrimum extract had 61.56%, thus indicating that SPs have potential hydroxyl radical scavenging activity and can be commercially exploited for the same.[37],[43]

Reduction is the process of accepting an electron or donating a hydrogen ion to stabilize the other molecule. Such property is determined in the presence of reductones, which attributes to the antioxidant effect by donating hydrogen to free radicals and thus breaks the chain. They are also known to act on certain peroxidase precursors to prevent lipid peroxidation by hydrogen peroxide.[43] In the present study, the results obtained almost a linear relationship between reducing potential and samples (control, 5 mM, 10 mM, 20 mM, and 30 mM) at increasing concentration (0.05–2 mg/ml). Thus, it can be stated that the SPs obtained have prolific reducing potential [Figure 3].

The superoxide radical is a dioxygen one-electron ion formed compressing high concentration oxygen, while superoxide dismutase catalyzes the breakdown of such harmful oxygen radicals into hydrogen peroxide and molecular oxygen.[44] The results clearly showed that the SPs have superoxide radical scavenging ability in the range of 60%–86% [Figure 6].

DPPH is a stable nitrogen-free radical that readily stabilized upon accepting hydrogen ion.[31] The DPPH scavenging activity in the present study reported a significant increase in the scavenging activity in the initial concentration from 0.05 to 0.5 mg/ml, while the concentrations 1 mg/ml and 2 mg/ml had similar effects as of control that is around 31.94% and 55.81%, respectively [Figure 7]. The 5 mM and 10 mM had similar DPPH activity as that of control while 20 mM and 30 mM NaNO3 stress induced SPs showed a significant increase in the activity than control. The DPPH scavenging activity of SPs obtained that Sargassum tenerrimum extract was found to be 64.66%, which is greater than found in the present study involving sodium nitrate stress. Therefore, it can be said that SPs may not inflict strong DPPH scavenging activity.[37] However, the DPPH has been known to determine antioxidant potential of phenolic compounds and flavonoids.[45] In the present study, the DPPH radical scavenging ability of Cr-SPs could be linearly proportional to phenolic content, which is an efficient ROS scavenger.[46]

Moreover, Cr-SPs in the present study showed an efficient antibacterial activity. With increased NaNO3 concentration, there is enhanced production of SPs that efficiently inhibited the growth of both Gram-positive and Gram-negative bacteria such as N. mucosa and S. aureus. Cr-SPs showed increased zones of inhibition with increased NaNO3 concentration, the diameter of which ranges from 18- to 30-mm diameter zones [Figure 8]. SPs are known to play a key role in preventing bacterial and viral infections. Earlier reports showed that S. platensis fluid extracts were tested against Gram-negative bacterium ( Escherichia More Details coli), a Gram-positive bacterium (S. aureus), a yeast (Candida albicans), and a fungus (Aspergillus niger) and found to be effective against C. albicans and A. niger.[5],[47],[48],[49],[50],[51] However, in the current study, Cr-SPs are effective against both Gram-positive and Gram-negative bacteria. There is little information available about the factors that affect the antioxidant and antimicrobial abilities of SPs under stress conditions; besides, reports have suggested that compositions and ratios of monosaccharide as well as types of glycosyl linkages can be involved in modulating the bioactive properties.[30]


  Conclusion Top


SPs were induced and extracted from fresh water alga under different NaNO3 concentrations (5, 10, 20, and 30 mM) by hot water extraction method. The various biochemical assays performed indicated that the extracts are enriched with polysaccharides which are sulfated. Various in vitro antioxidant assays have been used to determine its antioxidant capacity in a concentration-dependent manner. All the assays showed an increasing antioxidant potential of NaNO3-induced SPs over control indicating its significant antioxidant ability. Hydroxyl radical scavenging assay, superoxide radical scavenging activity, and reducing potential assay showed promising results with percentage activities up to 80% and 92% and absorbance about 0.8935, respectively. Therefore, the high sulfate content can be subjected to high antioxidant potential in scavenging hydroxyl radical, reducing ability, and scavenging superoxide activity. Moreover, Cr-SPs effectively inhibited the growth of both N. mucosa and S. aureus growth as indicated by clear zones ranging from 18 to 23 mm. Further investigations into the molecular mechanisms and structure–function relationship of these Cr-SPs would help develop them as natural therapeutics against various oxidative stress-related diseases and also as novel antibiotics.

Acknowledgments

This work is supported by the Department of Atomic Energy, India.

Financial support and sponsorship

UM DAE Centre for Excellence in Basic Sciences.

Conflicts of interest

There are no conflicts of interest.



 
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