Marine resources, such as crab, shrimp and squid are tapped for food production, which results in coastal waste from canning industries. Management of coastal waste is one of the major problems for tropical countries like the Philippines. One way to solve such problems is to look for new applications and uses for these waste materials.
The utilisation of waste materials from nature could be the answer to a variety of needs in skin treatment or cosmetic improvement. The main thrust of this study was the investigation of chitosan from squid pen in the formulation of a scar remover. The characterisation of prepared chitosan was carried out by determining the viscosity-average molecular weight (MV), degree of deacetylation (DD) and the scanning of prepared chitosan films’ nanostructure. Preparation of chitosan films demonstrated significantly different viscosity and average molecular weight, which is one of the important parameters that could influence the performance of chitosan as a scar remover. The effectiveness of the formulated product was evaluated by comparing it with a commercial product. Evaluation of the product was based on the personal perception of the respondents and not on the assessment of panelists. The results obtained led to the conclusion that using 10% lactic acid for decalcification and 1M NaOH for deproteination is effective as an active ingredient in the formulation of a scar remover cream. The quick removal of the scar, confirmed by the respondents/ test subjects, shows that chitosans can replace commercial product formulations.
The Philippines, because of its strategic location in the tropics, is abundant in natural resources, both in land and in water. Marine resources such as crabs, shrimps and squid are tapped for food production, which results in coastal waste from canning industries. Management of coastal waste is one of the major problems in tropical countries. One way to solve such problem is to recycle these waste materials for productive application. A wide variety of medical applications for chitin and chitin derivatives have been reported over the past three decades.1-3 It has been suggested that chitosan may be used to inhibit fibroplasia in wound healing and to promote tissue growth and differentiation in tissue culture.4 Much attention has been paid to chitosan as a potential polysaccharide resource. Recently, a squid-derived woundhealing gel was invented by University of Otago scientists and is attracting attention from international medical companies, due to its unique blend of properties (http://www.scoop.co.nz/stories/SC0711/ S00054.htm). The “Chitodex” medical gel, which uses a polymer derived from squid, has been patented by researchers from the university’s department of chemistry (http://news.softpedia.com/news/Better- Surgery-Gel-Made-from-Squid-72459.html). Research team leader Professor Brian Robinson says Australian medical trials show the gel possesses both anti-bleeding and anti-scarring properties. In this study, the ?-chitin in squid pen was extracted and deacetylated to form ?-chitosan, characterised, and examined for scar removal reactions. The traditional extraction method using strong acid and alkali at elevated temperatures was considered as the normal procedure in the characterisation of chitosan. Different methods utilising less harsh processes to obtain a high molecular weight product was one of the objectives of this study (http://neon-otago.ac.nz/chemistry/ research/paa/defauylt.html). The best method, producing high viscosity and high molecular weight of chitosan, was used as an active component in the formulation of scar remover cream. Hyperthrophic scars should be aggressively treated with topical steroids along with the use of a moisturiser containing lanolin and 7% lactic acid for cell renewal and restoration (Scar Prevention and Treatment, Skin Health Reference Library, April 26, 2005) and the use of lactic acid for decalcification of squid pen was investigated in this study.
Materials and methods
Collection and preparation of the sample
The squid pens collected from the wet market were washed with water and airdried. The dried squid pens were cut into small pieces (1 cm x 1 cm) before they were ground in Glen Mills MHM4 grinder using a 2 mm sieve.
Decalcification of squid pen
Decalcification was carried out by soaking 10 g of ground squid pen in 150 mL of 10% lactic acid or 1N HCl for two hours at room temperature with constant stirring using a magnetic stirring motor with stirrer. The residue was filtered and washed with pH 7 buffer to about pH 6 to remove any excess acid and to prevent an acid–alkali reaction occuring during deproteination. The sample was dried in a vacuum oven at 60°C-70°C.
Deproteination of squid pen
Deproteination was done using 1M NaOH solution (150 mL/10 g of demineralised squid pen) or 2000 ppm papain at room temperature for two hours with constant stirring. Then, several washes were carried out up to pH 8 to the filtered chitin and dried in the vacuum oven at 60°C-70°C.
Deacetylation of chitin
The N-deacetylation of chitin was done following the procedure of Hirano5 with little modification. Seven grams of prepared chitin was treated with 210 mL 40% NaOH aqueous solution at 120°C for one hour using soxhlet apparatus, and the precipitate was washed with water. The precipitate was further washed with phosphate buffer 8. The chitosan was dried in a vacuum oven at 60°C-70°C. The dried chitosan was kept in a dessicating cabinet with silica gel.
Characterisation of prepared chitosan
Determination of viscosity-average molecular weight (Mv)
One per cent (w/v) of the chitosan was prepared by dissolving 0.5 g of purified chitosan in 50 mL of 1% acetic acid with stirring using a magnetic stirring bar and motor for about one hour. The viscosity of the solution was determined using Brookfield viscometer with RV number 6 spindle. The viscosity-average molecular weight was calculated using Equation 1: [?]=KmMva Equation 1 shows the relationship of viscosity and average molecular weight where [?] is the intrinsic viscosity, Km=1.81 x 10-3 and a=0.93 are the empirical Mark-Houwink viscometric constants that are specific for a given polymer.6
Analysis of the degree of deacetylation (DD)
The chitosan films were prepared according to the method of Khan et al.7 with slight modifications. Chitosan films were prepared by casting 1.0% w/v of purified chitosan in 1% acetic acid, followed by drying in a vacuum oven at 60°C for 12 hours. The chitosan films were deprotonated by washing 3-4 times with 1:1 methanol:ammonium hydroxide followed by distilled water and methanol. Chitosan films were heated in a vacuum oven at 80°C for 3-16 hours and kept in a desiccating cabinet with silica gel prior to scanning. The spectra of chitosan films were obtained using Impact 400 Nicolet FTIR with a frequency range of 4000-400 cm-1. The degree of deacetylation (DD) of the chitosan films was calculated using baseline (a), which was proposed by Domszy and Roberts8 as internal standard to correct for film thickness or for differences in chitosan
Materials and methods
Collection and preparation of the sample
The squid pens collected from the wet market were washed with water and airdried. The dried squid pens were cut into small pieces (1 cm x 1 cm) before they were ground in Glen Mills MHM4 grinder using a 2 mm sieve.
Decalcification of squid pen
Decalcification was carried out by soaking 10 g of ground squid pen in 150 mL of 10% lactic acid or 1N HCl for two hours at room temperature with constant stirring using a magnetic stirring motor with stirrer. The residue was filtered and washed with pH 7 buffer to about pH 6 to remove any excess acid and to prevent an acid–alkali reaction occuring during deproteination. The sample was dried in a vacuum oven at 60°C-70°C.
Deproteination of squid pen
Deproteination was done using 1M NaOH solution (150 mL/10 g of demineralised squid pen) or 2000 ppm papain at room temperature for two hours with constant stirring. Then, several washes were carried out up to pH 8 to the filtered chitin and dried in the vacuum oven at 60°C-70°C.
Deacetylation of chitin
The N-deacetylation of chitin was done following the procedure of Hirano5 with little modification. Seven grams of prepared chitin was treated with 210 mL 40% NaOH aqueous solution at 120°C for one hour using soxhlet apparatus, and the precipitate was washed with water. The precipitate was further washed with phosphate buffer 8. The chitosan was dried in a vacuum oven at 60°C-70°C. The dried chitosan was kept in a dessicating cabinet with silica gel.
Characterisation of prepared chitosan
Determination of viscosity-average molecular weight (Mv)
One per cent (w/v) of the chitosan was prepared by dissolving 0.5 g of purified chitosan in 50 mL of 1% acetic acid with stirring using a magnetic stirring bar and motor for about one hour. The viscosity of the solution was determined using Brookfield viscometer with RV number 6 spindle. The viscosity-average molecular weight was calculated using Equation 1: [?]=KmMva Equation 1 shows the relationship of viscosity and average molecular weight where [?] is the intrinsic viscosity, Km=1.81 x 10-3 and a=0.93 are the empirical Mark-Houwink viscometric constants that are specific for a given polymer.6
Analysis of the degree of deacetylation (DD)
The chitosan films were prepared according to the method of Khan et al.7 with slight modifications. Chitosan films were prepared by casting 1.0% w/v of purified chitosan in 1% acetic acid, followed by drying in a vacuum oven at 60°C for 12 hours. The chitosan films were deprotonated by washing 3-4 times with 1:1 methanol:ammonium hydroxide followed by distilled water and methanol. Chitosan films were heated in a vacuum oven at 80°C for 3-16 hours and kept in a desiccating cabinet with silica gel prior to scanning. The spectra of chitosan films were obtained using Impact 400 Nicolet FTIR with a frequency range of 4000-400 cm-1. The degree of deacetylation (DD) of the chitosan films was calculated using baseline (a), which was proposed by Domszy and Roberts8 as internal standard to correct for film thickness or for differences in chitosan (Fig. 4) showed a smooth surface and the globular clusters are larger compared to the film of chitosan prepared using 1M HCl and 1M NaOH (Fig. 3). The film of chitosan prepared using HCl and 2,000 ppm papain (Fig. 5) appeared similar to the film prepared using lactic acid and 1M NaOH but the globular clusters are smaller. These photographs support the results of the molecular weights of the chitosan samples.
Evaluation of prepared scar remover cream
The formulated scar remover as well as one commercial product were evaluated by 11 respondents whose ages ranged from 17 to 50 years old. The formulated scar remover was applied on identified prominent scars twice a day every morning after bathing and before bedtime for a period of one month. The skin condition of volunteers who used the formulated product was monitored for a week to assess the safety of the formulated cream. No one among the volunteers had complained of irritation or experienced an allergic reaction while using the prepared product. A remarkable thinning of the keloid scar after 10 days of using the formulated product was observed from one of the volunteers. The same procedure was followed using the commercial product. An evaluation form was given to each respondent after the use of both products to collect data for analysis. Table 3 showed that in terms of quick removal of the scar, the formulated product had a very significant difference compared to the known commercial product. This means that the prepared scar remover is more effective than the commercial product. The non-irritating effect, spreadability and moisturising effect showed no significant differences, but the odour showed significant difference. This means that the odour of the prepared scar remover cream is preferred to the commercial product.
Conclusion
This study has successfully found productive applications for the chitosan produced from squid pen. These are the production of polymer films with high average molecular weight and degree of deacetylation; and the application of the polymer produced as an active ingredient in the formulation of a scar remover. Using HCl for decalcification and papain for deproteination suggests that this combination of purifying processes produces chitosan with a high molecular weight and high degree of deacetylation.
Acknowledgements
The author acknowledges the financial support extended by Rev. Fr. Honorato C. Castigador, O.P., Rector and President, Colegio de San Juan de Letran-Calamba, Philippines, in the attendance to the International Conferences. She is also thankful to Centro Escolar University, Manila, Philippines, where the study was conducted.
References
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