INTRODUCTION:

Chitin (β-(1–4)-poly-N-acetyl-D-glucosamine) is the second most abundant biopolymer after cellulose¹. Chitin widely occurs in crustaceans, insect exoskeleton, fungi, mollusc shells, fish scales, algae, bryozoan, sponge and corals². For several biomedical applications, chitin is usually converted into deacetylated form, chitosan³. This natural constituent regarded as a suitable functional material for many applications because of its biocompatibility, biodegradability, non-toxicity and adsorption properties⁴.

As a result of above-cited properties, chitosan can used be as a crucial material in medicine, cosmetics, textiles, wastewater treatment and agriculture³.

A large quantity of chitin and chitosan manufactured from the exoskeleton of crustacean sources (shrimp, crab, lobster and crayfish)⁵. On the other hand, molluscs waste from fish landing centres can become free and rich alternative sources of chitin and chitosan. Marine mollusc can be collected in landing centres throughout the year. Moreover, marine molluscs are relatively consistent in compositions and are not associated with any other contaminants.

In specific, molluscs have a rich amount of CaCO₃ and this results in difficult to extract pure chitosan⁶. Marine mollusc, Murex species is predatory tropical sea snails and are commonly distributed in the Indo-Pacific region. Presently, this waste shell material is extremely underutilized and contributes to major environmental concern due to off-odour and concentration of minerals in a landfill. Recycling shell waste could probably eliminate the disposal problem, and also turn an otherwise useless waste into highly valuable products.
To overcome this challenge, in the present study was conducted to extract the chitosan from M. trapa and physicochemical, structural properties, functional properties, biological applications were analysed.

MATERIALS AND METHODS:

Collection of M. trapa shells:

The M. trapa shells were collected from the Mudasalodai landing centre (11.4831°N, 79.7729° E), Southeast coast of India. The shells were scrapped free to release over tissues, splashed, dried and ground to pass through research laboratory sieve (0.3-0.5mm) and subjected to demineralization, deproteinization and deacetylation for the extraction of chitosan.

Chitosan extraction:

The chitosan extraction was done according to the method of Sangwaranatee et al. (2018) with slight modifications. The shell powder was introduced demineralization process with 1M HCl (1:15 W/V) at room temperature for 48h. The demineralized shell powder was filtered and washed with distilled water until the filtrate becomes neutral pH. The deproteinization process was accomplished by treating demineralized shell powder with 5% NaOH (1:15 W/V) at 70ºC for 48h. The samples were left in the oven at 60ºC for 24h and obtained material is chitin. Chitin was subjected to deacetylation by using 40% NaOH (1:15 W/V) at 90ºC for 6h.

Physiochemical properties:

Ash and Moisture content:

The ash and moisture content was quantified gravimetrically according to the method of Mohan et al. ¹. 0.5gm of chitosan taken in a porcelain crucible was burnt at 600°C for 8h in a muffle furnace. The weight of the crucible residue represented the ash content and the results were given as a percentage. The moisture content of the extracted chitosan was determined through an oven method. 0.5mg of chitosan was dried in a hot air oven at 130ºC for 2h and the weight of chitosan was given as a percentage.

Solubility:

The solubility of prepared chitosan was determined by adding 200mg of extracted chitosan with 200ml of water and the same methodology was followed with 1% acetic acid solution⁷.

Degree of deacetylation:

The Degree of deacetylation (DD) of chitosan was determined by using an FTIR instrument with a frequency of 4,000–400 cm⁻¹. Equation of Mohanasrinivasan et al.⁷ was used, where the absorbance at A1629.85 and A3450.65 cm⁻¹ are the absolute heights of absorption bands of amide and hydroxyl groups, respectively.

DD = 100-(A1629.85 cm^-1 – A3450.65 cm^-1) x 100

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1.33

The factor ‘1.33’ denoted the value of the ratio of A1629.85/A3450.65 for fully N-acetylated chitosan.

Functional and structural analysis:

FT-IR:

The samples of prepared chitosan were characterized in KBr pellets by using an infrared spectrophotometer in the range of 400–4,000 cm⁻¹.

XRD:

The X-ray diffraction patterns of chitosan were analysed by XRD diffractometer. Chitosan sample was placed on a glass slide, and the spectra were recorded using Cu-Kα radiation. A monochromator filtering wave at 40kV and 30mA. The diffraction pattern was obtained at diffraction angles between 2θ=30-80⁰ with a scanning speed of 0.4⁰/min at room temperature.

SEM:

The structure of extracted chitosan was observed using a scanning electron microscope (JEOL-JSM 5610LV, JEOL, Ltd, Tokyo, Japan) at an acceleration voltage of 10kV and a current of 10mA after sputter coating the samples with gold.

TG/DTA:

Thermal stability of the chitosan was analysed by a simultaneous thermal analyser (NETZSCH-STA 449 F3 JUPITER Instrument, Germany). The measured amount of chitosan sample and heated to temperature ranged from 30 to 600⁰C with a heating range of 20⁰C/min under a nitrogen flow of 50cm³/min.

Water and fat binding capacity:

Water and fat binding capacity of the extracted chitosan were analysed by the method described by Cho et al.⁸ with minor modifications. Water or fat binding capacity was carried out by weighing a 15ml centrifuge tube containing 0.5gm of extracted chitosan, further adding 10ml of water or coconut oil and mixed in a laboratory vortex for a minute. The content was kept in the room temperature for 30min with shaking every 5min. After incubation period centrifuged at 4000rpm for 20min. After the supernatant was discarded, the tube with water or oil absorbed chitosan was weighed again. WBC and FBC were calculated using the following formula:

WBC (%) = [water bound (g)/sample weight (g)] ×100;

FBC (%) = [Fat bound (g)/sample weight (g)] ×100.

Animal Housing:

The experiment was performed in accordance with the regulation specified by the Institutional Animal Ethics Committee (Reg. No: AU-IEAC/1186/1/18), Annamalai University. Healthy Male rat was used in the current study. Animals were fed on a standard diet with water ad libitum.

Hemocomtability assays:

Procoagulant ability:

Procoagulant ability was analysed by the method described by Hu, Lu, et al.,⁹ with minor modifications. Wistar rat blood was collected with sodium citrate anticoagulant tubes and kept in 4ºC for further uses. The measured amount of sample was added to tubes, taking commercial chitosan as the positive control and a black tube as the negative control. Tubes were kept in a water bath at 37ºC, the anticoagulated blood was added to the tubes (1ml per tube), and one tube was slowly tilted once every 30s, whereas the second tube was kept undisturbed. When a blood clot appeared in the first tube, the second tube began to tilt every 30s. Timing stopped when the blood in the second tube began to coagulate. The time from the start of blood added to the blood coagulation in the second tube was recorded as coagulation time.

Plasma recalcification assay:

The plasma recalcification time was determined according to the method described in the literature of Hu et al (2018a) with minor modifications. Briefly, Wistar rat blood was collected in an anticoagulant tube and centrifuged at 4000rpm for 10min. Supernatant was separated to obtain platelet-poor plasma (PPP). In the 2ml eppendorf tube, 100µl of PPP was added then 100mg of isolated chitosan has added as the experimental group. Commercial chitosan and blank tube were used as a positive and negative control group respectively. All the tubes were incubated in a water bath at 37ºC for 3 min and then 0.1mL of 0.025mol/L calcium chloride solution was added, well mixed, and placed in a 37ºC water bath again while the stopwatch was started until white jelly began to occur in the mixed solution.

Minimum bactericidal activity:

The minimum bactericidal activity was performed on the extracted chitosan according to the method described in the literature of Khalili et al. (2012) with minor modifications. Antibacterial activity was performed against two pathogenic bacteria’s Bacillus subtilis (gram-positive) and Vibrio cholerae (gram-negative). The sterile disk was impregnated with 20µl of different concentration of chitosan were air-dried was placed on the agar plate. Agar plates were incubated at 37ºC for 24h after incubation period zones inhibition were measured in mm including the diameter of the disc¹⁰.

Statistical analysis:

Values are mean±SD for three rats in each group and the significance of the differences between mean values were determined by using one-way analysis of variance (ANOVA). Probability (p) value less than 0.05 were considered significant.

RESULTS AND DISCUSSION:

Yield:

The yield of the chitosan from the M. trapa was 17% on a dry weight basis (Table.1).

The yield of chitosan content from mollusc species, such as golden apple snail shell was found to be at 42.56%. The yield of chitosan from two different oysters Mytilus edulis and Laevicardium attenuatum was 51.80% and 43.80% respectively¹¹. On the other hand, the yield of chitosan from cuttlebone of Sepia kobiensis was 29.87% ¹². In addition, the yield of chitosan from the squid pen 50.54% and 28% respectively^13,14.

Table.1

Ash and Moisture content:

The ash content of the mollusc chitosan was found to be 0.954% (Table.1). Low ash content in the prepared chitosan could be one reason for the solubility of isolated chitosan. The amount of ash content in chitosan quantification is an important pointer for the demineralization step and efficiency for the elimination of calcium carbonate¹⁵. High-value chitosan must have <1% ash content¹⁶, while the moisture content of mollusc chitosan in this current study was 4.2% (Table.1) and this explains that the high-quality chitosan was isolated.

Table 1: Physicochemical properties of chitosan from M.trapa shell

Properties	Value
Yield (%)	17
Ash (%)	0.954
Moisture (%)	4.2
Degree of deacetylation (DD %)	73
Solubility (%)	1% CH₃COOH
Appearance	White

Degree of Acetylation:

In the present study, the degree of acetylation value was observed as 73% (Table.1) for M. trapa chitosan. This result has been confirmed in previous works (Boudouaia et al. 2019).

FT-IR:

The FTIR spectra of the mollusc chitosan samples obtained and commercial chitosan is shown in Fig.1A. FTIR investigations showed a stretching vibration band in mollusc chitosan in the range of 3450–3200cm⁻¹ corresponding to NH and OH functional group. The NHCOCH3 group in pyranose ring were observed in the range of 2880cm⁻¹. Major absorption bands were observed between 1200 and 1000cm-1 were attributed to free amine group present in mollusc chitosan. During the deacetylation process, all the bands with an overlap of the peaks between 1600 to 1660cm-1 (amine) were observed to a single large peak, which indicates a decrease in acetyl groups and hydrogen bonds. The previous study done by Kumari and Rath, ¹⁷, suggested that the band at 1597cm⁻¹ has a larger intensity than at 1655 cm⁻¹ which suggested that the deacetylation process was effective.

Solubility:

In the present study, the solubility of the mollusc chitosan was treated with 1% CH₃COOH and results were presented in Table.1. Based on the result, M. trapa mollusc chitosan demonstrated blameless solubility (absolute). Mohanasrinivasan et al., (2014) reported that the solubility of shrimp shell waste chitosan was absolute⁷. The solubility variation of chitosan and its derivatives based on the solvent system to be used for the extraction method¹.

Crystalline structure of chitosan:

The X-ray diffractogram results for the M. trapa chitosan and commercial chitosan are presented in Fig.1B and C. The diffractogram of mollusc chitosan and commercial chitosan showed three major peaks around at 2θ- 10º and 20º those are corresponding to the crystallographic planes respectively, which are present in the diffractograms of all samples. In the current study, the peaks observed in chitosan isolated from mollusc shell was found similar to previous studies^16,18.

Fig.1 (A) FT-IR spectrum of M.trapa shell chitosan and commercial chitosan. (B) Powder X-ray diffraction patterns of M. trapa shell chitosan and commercial chitosan.

Surface morphology of chitosan:

The morphology of the isolated chitosan from M. trapa was inspected by scanning electron microscopy. These images are shown in Fig.2. Mollusc shell exhibits a homogenous morphology characterized by a compact structure with non-pours and non-fibrous structure.

TG-DTA:

TG/DTA of isolated chitosan and commercial chitosan were recorded in the temperature range of 30 to 500ºC. The TG/DTA analysis results indicated that the mass loss of isolated chitosan occurred in two different steps (Fig.3). The mass loss of mollusc chitosan was 5% in the first step and reduced to 53% in the second step. Mohan et al., (2019) suggested that mass loss in the first step and second steps are the processes of water evaporation and chitosan polymer degradation.

The mass losses in the isolated chitosan from the molluscs in the present study occurred in two steps similar to the previous studies¹⁹. The maximum thermal degradation temperature was 360ºC for mollusc chitosan (Fig.3A). The DTA max value of isolated chitosan differed from 290 to 390ºC that has been reported in previous studies^18,20,21. The TG/DTA analysis results of commercial chitosan also showed that mass loss occurred in two different steps. 5% mass loss observed in the first step and 50% mass loss occurred in the second step. The DTG max value of commercial chitosan 360ºC was close to the mollusc chitosan (Fig.3B), In addition, thermal stabilities of isolated chitosan were close to the commercial chitosan.

Fig.2: SEM micrographs of (A) M.trapa shell chitosan and (B) commercial chitosan.

Fig.3: TG/DTA of (A) M.trapa chitosan and (B) commercial chitosan.

Binding capacity:

Fat binding capacity:

The fat binding capacity of mollusc chitosan and commercial chitosan was measured using coconut oil. Fat binding capacity of mollusc chitosan and commercial chitosan was investigated and the values were found to be 252% and 350% (Table.2) respectively. Fat binding capacity is lower than the commercial chitosan because it depends on the chitosan processing order (demineralization and deproteinization°° processes)²¹. Deproteinization process was carried first in the current study and it resulted in reduced fat binding capacity of chitosan which is similar to the previous studies. Kumari et al., (2017) Stated that changing the order of chitosan isolation steps would vary fat binding capacity²¹. Cho et al., (1998) reported the fat binding capacity vales of chitosan were ranging from 319 to 560%⁸.

Water binding capacity:

Water binding capacity of chitosan isolated from mollusc M.trapa and commercial chitosan was found to be 280% and 451% (Table. 2) respectively. It was observed that water binding capacity of commercial chitosan was significantly better than water-binding capacity of mollusc chitosan. Water-binding capacity mainly depends on the demineralization and deproteinization steps. Cho et al., (1998) reported that water-binding capacity of shrimp chitosan is in the range of 805%. Mohanasrinivasan et al., (2014) suggested that water-binding capacity are functional properties of chitosan, which may vary based on the method of preparation⁷.

Table: 2: Fat and water binding capacity of M.trapa chitosan and commercial chitosan.

Properties	FBC	WBC
*M.trapa* chitosan	252%	280%
Commercial chitosan	350%	451%

Hemocompatibility:

Procoagulant ability:

The effect on blood coagulation time of isolated chitosan was investigated in vitro, as shown in Fig.4A. The current investigation revealed that chitosan isolated from M. trapa and commercial chitosan has significant procoagulant effects (***p<0.001) when compared with the negative control. Both chitosan enhanced the procoagulant effects, It was probably because water-soluble substitution improved the protonation ability of amino groups in the molecular chain of chitosan⁹. Although, the haemostatic mechanisms of chitosan is not yet fully understood many studies explained that the procoagulant activity of the chitosan majorly involved the agglutination of red blood cells²².

Plasma recalcification time:

Fig.4B shows the concluded results of the plasma recalcification time test. The plasma recalcification time of the commercial chitosan and mollusc chitosan was significantly lower than the negative control group. Some research evidenced that chitosan facilitated hemostasis through interaction with erythrocytes, linking them together to establish a cellular net structure²³. Cho et al., (1998) demonstrated that chitosan enhanced the rabbit platelet adhesion and aggregation by increasing of platelet intracellular calcium mobilization and enhancing expression of glycoprotein IIb/IIIa complex on platelet membrane surfaces, which might account for its haemostatic effects⁸. Although various possible mechanism has been reported, still the mechanism of chitosan on haemostatic effects has not been fully understood. Hu, Lu, et al., (2018) reported the Plasma recalcification time of chitosan were ranging from 120 Seconds⁹.

Minimum bactericidal concentration:

Minimum bactericidal concentration (MBC) of mollusc chitosan was determined, which are presented in Fig.4C. The current study shows that in 10mg/mL and 20mg/mL concentrations of chitosan, doesn’t have any effects on growth of V.cholerae and B.subtilis and in 30 mg/mL of chitosan the growth of V.cholerae and B.subtilis decreased (Zone of inhibition- 0.1 mm and 0.2 mm correspondingly). In addition, in 40mg/mL concentrations of chitosan, the growth of V.cholerae and B.subtilis and is inhibited by 0.1 mm and 0.3 mm respectively. Growth of bacteria’s inhibited 0.3 mm and 0.5 mm at the concentration of 50mg/ml. Decreases in concentrations of chitosan resulted in a decrease in the zone of inhibition, indicating concentration depended on action of isolated chitosan. In general, bacterial inhibition activity of chitosan is considered based on its chemical and structural properties²⁴. The exact antibacterial mechanism of chitosan and its derivatives is still unknown. In the current study scenario, high solubility nature of isolated chitosan increasing the permeability of cell membrane and ultimately disrupted bacterial cell membrane with the release of cellular contents²⁵.

CONCLUSION:

Chitosan from M.trapa has been extracted by the chemical treatment method. XRD, FT-IR, SEM and TG/DTA were used to analyse the structural characterization of chitosan. M. trapa shell waste was proven to be one of the important untouched sources for the preparation of chitosan and this was supported by all the properties such as high degree of deacetylation, solubility and colour. Further purification and improving the method of chitosan can improve the physicochemical properties of the M. trapa chitosan. The results of this present study suggest that Murex trapa shell chitosan could be an alternative source of heparin with low manufacturing cost.

CONFLICTS OF INTEREST /COMPETING INTERESTS:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Received on 16.10.2020 Modified on 10.07.2021

Research J. Pharm. and Tech 2022; 15(12):5729-5735.

DOI: 10.52711/0974-360X.2022.00966