INTRODUCTION:

The role of chitosan in the global market has been getting much attention in the last few decades due to its wide range of uses and its stability with very low toxicity, particularly in the industries such as cosmetics, water treatment, and agrochemicals.¹ When we look at the global inland aquaculture industry, it has been reported that production reached 11.6 million tonnes in 2012, which contributes to 63% of the total farmed fish production. The major contributors to the global production of the inland fisheries sector are 15 countries, notably China, India, Bangladesh, Myanmar, Vietnam, Brazil, Malaysia, and Egypt.² Tilapia (Oreochromis sp.) species significant producers, and the consumers are Asian countries.³ Being the second most farmed fish after carps, the growth rate of Tilapia production is at a steady increase of 10-12% per year. In 2017, the world tilapia production was reported to be at 6,510,700 metric tons, and Malaysia contributed close to 200 metric tons.⁴ The bulk amount of Fish scales from various fish fillet factories disposed of as solid wastes.⁵

Fish scales are made up of hydroxyapatite, calcium carbonate, and collagen type 1, making it a very stable substance that does not degrade quickly.^6-7 There is a dire need to extract value-added biomaterials from these wastes through economically sustainable applications (Seafish.org). Fish scales are a good source of chitin and collagen used in various cosmetic, biomedical, pharmaceutical, and food industries due to their stability and environmental friendliness.⁸

There have been many chitosan applications related to biomedical, food packaging, biomaterials, cosmeceuticals, and nutraceuticals due to its wide range of activities as a stable, non-toxic, antioxidant, antimicrobial biocompatible, and anti-biofilm properties.^9-11Special requirements are needed for products destined for the above-related applications, such as the chitosan samples' specific molecular weight and purity. Considerable attention has been put on low molecular weight chitosan and chito-oligosaccharides with an average molecular weight of 5000 – 10,000 Da.^12-15 In this study, the low molecular weight chitosan and chito-oligosaccharides derived from fish scales were put to the test for their antioxidant capabilities by examining them using five different antioxidant assays.

MATERIALS AND METHOD:

Isolation of Chito-oligosaccharides from fish scales:

Chitosan extraction from fish scales was done using a modified method by Toan (2009).¹⁶ COS was extracted by chemical hydrolysis.¹⁷ Briefly, 10g of chitosan was hydrolyzed in 8M 500 mL of HCl at 72ºC for 3 hours. The solution was then neutralized with 2M Sodium Hydroxide to a final pH of 7. Ultra-pure methanol was added to this solution to precipitate chito-oligosaccharides. Residues rained on the bottom of the flask were filtered through a 0.22-µm nylon membrane.

DPPH free radical scavenging assay:

The antioxidant activities of test samples were studied using the modified Lingnert et al., 1979 method.¹⁸ Firstly the test samples were prepared in 5 different concentrations consisting of 1,2,3,4 and 5 mg/mL in a volume of 1mL. The diluted samples were then mixed in 2mL of 10mM linoleic acid emulsion at pH 6.5 using a vortex mixer. These samples were then incubated at 37°C in total darkness for 15 hours. After incubation, 6mL of methanol (60%) was added to the mixture, and a decrease in absorption was analyzed at 234nm using a spectrophotometer (Shimadzu, UV-1800). All measurements were conducted in triplicates for statistical analysis. The antioxidant activity of the test samples was calculated using the following equation:

% Antioxidant Activity = [(Abs control – Abs sample)/ (Abs control) x 100]

Reducing power activity:

The reducing antioxidant power activities of test samples were determined¹⁹ with modifications. Firstly, the test samples were prepared in 5 different concentrations consisting of 1,2,3,4 and 5mg/mL in a volume of 2.5mL. The diluted samples were then mixed in 2.5mL of 200mmol/L sodium phosphate buffer at pH6.6 and 2.5mL of 10g/L of Potassium Ferricyanide. The mixture was then incubated for 20 minutes at 50°C. After the incubation period, the mixture was added with 2.5mL of 100g/L trichloroacetic acid. The mixture was mixed with a vortex mixer and then centrifuged for 10 minutes at 200pm. The upper layer formed after the centrifugation process was integrated with v/v of deionized water. Finally, 1mL of Added 1g/L ferric chloride was measured at 700nm using a spectrophotometer (Shimadzu, UV-1800). All measurements were conducted in triplicates (n=3) for statistical analysis.

The percent of reducing power activity of the test samples was calculated using the following equation:

% Activity =

[(Abs control – Abs sample)/ (Abs control) x 100]

Chelating ability on ferrous ions:

The chelating ability on ferrous ions of chitosan and chito-oligosaccharides was determined using Dinis et al., 1994²⁰ with slight modifications. Firstly the test samples were prepared in 5 different concentrations consisting of 1, 2, 3, 4 and 5mg/mL in a volume of 1mL. To these samples, 3.7mL of methanol and 0.1mL of 2mM ferrous chloride were added. The reaction commenced as soon as the 0.1mL of 5mM ferrozine was added. The mixtures were agitated using a vortex mixer for 5 minutes and left at room temperature for 10 minutes. After incubation, a decrease in absorption was measured at 562nm using a spectrophotometer (Shimadzu, UV-1800). All measurements were conducted in triplicates (n=3).

The percent of the metal chelating ability of the test samples was calculated using the following equation:

% Activity =

[(Abs control – Abs sample)/ (Abs control) x 100]

Superoxide scavenging activity assay:

Superoxide scavenging activities are determined by using the modified method.²¹ The reaction mixture was prepared, which contain i) 200µL of nicotinamide adenine dinucleotide; ii) the reduced form of NADH, iii) 200µL of nitroblue tetrazolium (NBT), iv) 450uL of phosphate buffer (0.1M); (pH 7.5) and v) 50µL of test compounds. The reaction was initiated by the addition of 100µL of phenazine methosulphate (PMS). The reaction temperature was at 28˚C. The formation of blue-colored formazan dye was measured after three minutes at 560nm. NBT, NADH, and PMS solutions were prepared in phosphate buffer while the test samples were dissolved in 0.5% acetic acid. All measurements were conducted in triplicates. The superoxide scavenging activities (%) were calculated by using the formula:

% Activity = [100- (Abs sample/ Abs control) x 100]

RESULTS:

Total antioxidant activity:

In the total antioxidant activity analysis of chito-oligosaccharide from fish scales, scavenging activity were seen to be dose dependent where scavenging activities increased in higher concentrations (Figure 1). It showed that total antioxidant activities of chito-oligosaccharides to be significantly higher than chitosan (data not shown). Chito-oligosaccharides exhibited antioxidant activities ranging from 48.7% to 70.1%. The positive control used in this study was Ascorbic acid and activities ranged from 52.3% to 68.7%. It was reported that the total antioxidant activities of ascorbic acid compared to chito-oligosaccharide to be insignificant. It is also noteworthy to report that at concentrations of 5mg/mL, chito-oligosaccharides exhibited higher total anti-oxidant activities (70.1%) compared to ascorbic acid (68.7%).

Figure 1: Total antioxidant activity of chito-oligosaccharides from fish scales and ascorbic acid.

Scavenging ability on DPPH radicals:

In the DDPH free radical scavenging analysis of chito-oligosaccharide from fish scales, scavenging activity were seen to be dose dependent where scavenging activities increased in higher concentrations (Figure 2). It exhibited moderate scavenging activities within the range of 33.2-42.1%. However, chito-oligo saccharides were significantly for effective in scavenging activities compared to chitosan (data not shown).

Figure 2: Scavenging activities of chito-oligosaccharides from fish scales and ascorbic acid.

Superoxide anion radical scavenging activity:

In the superoxide anion radical scavenging analysis of chito-oligosaccharide from fish scales, scavenging activity were seen to be dose dependent (Figure 3). Chito-oligosaccharides exhibited scavenging activities in the range of 48.3-71.2%. The scavenging activities of chito-oligosaccharides from Tilapia fish scale high as compare to chitosan (data not shown). However, in previous studies, chito-oligosaccharides did not exhibit significantly higher scavenging activities compared to chitosan. The Ascorbic acid exhibited significantly higher scavenging activities compared to both chito-oligosaccharides and chitosan (P<0.05).

Figure 3: Superoxide anion radical scavenging activity of chito-oligosaccharides from fish scales and ascorbic acid.

Ferrous ions chelating activity:

In the metal chelating analysis of chito-oligosaccharide from fish scales, activities were seen to be dose dependent (Figure 4). Chelating agents can be considered as secondary antioxidants they act as an oxidised metal stabiliser. Chito-oligosaccharides exhibited activities in the range of 48.7-61.3%. Chito-oligosaccharide exhibited significantly higher activities compared to chitosan (data not shown). The EDTA used as positive control used in this study, exhibited metal chelating activity (P<0.05).

Figure 4: Metal chelating activities of chito-oligosaccharides from fish scales and EDTA

Reducing Power:

In the reducing power analysis of chito-oligosaccharide time dependent increased (Figure 5). The reducing power assay is measured based on the conversion of Fe3+ to Fe2+. The reducing capacity serves as a good indicator of a potential antioxidant candidate to donate electrons. Chito-oligosachharide exhibited activities ranging from 0.3-2.8%, higher than chitosan (data not shown).

Figure 5: Reducing power of chito-oligosaccharides from fish scales and Ascorbic acid.

DISCUSSION:

In this study, we evaluated the antioxidant activities of chito-oligosaccharides derived from fish scales. Several marine spices, along with chitosan derivatives such as seaweed, possessed potential antioxidant activity measured by DPPH free radical scavenging activity.^22-31Here, we analyze the antioxidant activities via various methods, i.e., DPPH, superoxide anion radical scavenging activity, ferrous ions chelating activity, and reducing power analysis. As above, the assay stated that that COS could scavenge radicals to a certain extent. Many studies have concluded that among the significant factors influencing the scavenging capacity of any chitosan and its derivatives are the degree of deacetylation (DD) and the molecular weight (MW).³² This can be confirmed in our study that chitooligosaccharides derived from fish scales had a molecular weight of 4.6 kDa, respectively, with a degree of deacetylation of 94.65%, which can be considered low molecular weight chitosan with a high degree of deacetylation. Many recent studies are being done to modify the chitosan molecules to improve the antioxidant capabilities by grafting functional groups into the molecular structure, such as polyphenols. This modified polyphenol-chitosan possesses increased antioxidant activities and is now widely used in nutraceuticals, food manufacturing, pharmaceuticals, and medicals.^33-35

Previous studies showed that chitosan with a molecular weight of 307 kDa with a degree of deacetylation of 80% showed no scavenging activity when tested against DPPH assay but showed a significantly increased activity when the chitosan was grafted with gallic acid.³⁶ The reducing power increased from 9.4% to 89.5%, which agrees with previously reported studies.³⁷ The chitosan concentration tested in their study was 200-5000 µg/mL with a molecular weight of 121 and degree of deacetylation of 805. They reported DPPH scavenging activities to be enhanced when chitosan was treated with epigallocatechin. In another study, the team grafted chitosan with a molecular weight of 200 kDa and a degree of deacetylation of 90% with ferulic acid. The improvement in DPPH scavenging activity is from 41.4% to 96.6%.³⁸ This could be attributed to the intra and intermolecular hydrogen bonds, which are regarded as standard in higher molecular weight chitosan—the DPPH scavenging activities. Results obtained from our study for the chitosan samples were in the range of 33.2-42.1% and 39.5-52.3% for COS samples at a concentration of 1-5mg/mL.

CONCLUSION:

The chitosan and its derivatives as a candidate for scavenging related activities. The various chitosan derivatives play an essential role in human health and nutrition in general. The present work results indicated that COS derived from fish scales exhibited good antioxidant activities if compared against other sources of chitosan found in the literature. This study identifies opportunities to develop value-added products from fish processing by-products, especially fish scales with biological activity such as antioxidant properties.

ACKNOWLEDGMENT:

The authors would like to acknowledge the Institute of Marine Biotechnology, Universiti Malaysia Terengganu for the research funding.

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Received on 05.06.2021 Modified on 30.10.2021

Research J. Pharm. and Tech 2023; 16(1):8-12.

DOI: 10.52711/0974-360X.2023.00002