INTRODUCTION:

One of the most health-threatening problems in the world is burn injury¹. Over 6.6 million people worldwide suffer from burns, and almost 256.000 of them die annually^2,3. About 1% of all deaths are related to burn injuries⁴. Burns are classified into three degrees, depending on severity⁵. The increasing of free radical-mediated damage and reactive oxygen species on burns can delay granulation tissue formation, reduce angiogenesis, and decrease collagen reorganization⁶.

Collagen is the major part of the extracellular dermal matrix, which is also involved in scar formation during the healing of connective tissues.

Wound repair, particularly of non-infected, chronic, indolent skin ulcers have been enhanced with collagen dressing⁷. Collagen has long been used in wound care and management in various forms such as pad, gel-impregnated dressing, gels/pastes, and powder⁸.

About 30% of animal protein is consist of collagen. A common source of collagen has been come from bovine and porcine. However, in the last decades, the outbreak of bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE), and foot and mouth disease (FMD) have limited their use. Fish, fish wastes, starfish, sponges, and jellyfish are marine organisms that have recently been explored as the alternative source⁹.

Pempek is a typical food of Palembang city, Indonesia, made from flour and fish, especially snakehead murrel (Channa striata). Fish wastes such as skin, head, tail, fins, and bones have not been used optimally. These are contains amount of collagen. Fish bones are contain 33% of collagen and organic compound such as calcium (39%), potassium (0,2%), sodium (0,7%), magnesium (0,5%), carbonate (9,8%), and phosphate (17%)¹⁰.

Collagen extract from snakehead murrel bones can be formulated in spray gel preparation. Spray gel can provide a cooling and moisturizing effect on the wound during the proliferation phase¹¹. Besides that, spray gel delivers the drugs to the wound without direct contact. This can minimize the occurrence of contamination or infection. This study aimed at extracting collagen from snakehead murrel (Channa striata) bones, formulating into spray gel preparation, and investigate their wound healing potential in burn injury.

MATERIALS AND METHOD:

Chemicals and instrumentations:

Fresh snakehead murrel bones were collect from 16 ilir market in Palembang city and transport in ice to the laboratory and stored at freezer (-20^oC). All reagent used were analytical grade (Sigma-Aldrich®). The instruments of this study were spectrophotometer UV-Vis (Shimadzu®), Brookfield viscometer (S-62, model LVDV-E), pH meter (ATC®), and glassware (Pyrex®).

Extraction of collagen:

Collagen in snakehead murrel bones were extracted with acid soluble collagen (ASC) method¹².

Measurement of total protein:

Measurement of total protein in collagen extract was determined used lowry method spectrophotometrically.

Preparation of spray gel formulation:

Spray gel formulation was prepared using HPMC-60SH and Carbopol 940 as gelling agent. Table 1 shows the details of formulation compositions. HPMC-60SH was dissolved slowly with stirring in aquadest then heated at 90^oC for 15 min. Carbopol 940 was also dissolved slowly with stirring in aquadest then heated at 90^oC until dispersed. Methylparaben and propylparaben are dissolved in propylene glycol. Triethanolamine, HPMC-60SH, and methylparaben and propylparaben solutions were added to Carbopol solution. NaCl was added gradually and crushed homogeneously. Then extracted collagen was added gradually and crushed homogeneously.

Quality control of spray gel formulation:

Adhesive strength:

Samples (200 mg) were placed between 2 horizontal plates, then pressed with 20 g load object for 5 min. Lift the weight of the plates, then it was mounted on the test equipment. The test equipment was given 20 g load object then record the release time of the gel from the plates.

Table 1. Spray gel collagen extract formulations

Materials	F
Collagen extract (% w/v)	0,5
Carbopol^®940 (% w/v)	1
HPMC-60SH (% w/v)	1
Propylene glycol (% w/v)	15
Triethanolamine (drops) NaCl (mL)	10 7
Methylparaben (% w/v)	0,18
Propylparaben (% w/v)	0,02
Aquadest (mL)	Ad 20

pH measurement:

pH measurement of the samples was carried out using a digital pH meter by dipping the glass electrode completely into the system of spray gel until cover the electrode. The measurement was carried out in triplicate and the average of the three readings was recorded¹³.

Appearance and homogeneity:

Physical appearance and homogeneity of the prepared spray gel were evaluated by visual perception¹⁴.

Viscosity measurement:

The viscosity of spray gel was determined using Brookfield viscometer (S-62, model LVDV-E) with a 12 rpm rotation of spindle speed of viscometer at 25^oC¹⁵.

Spraying pattern:

Samples was sprayed on a sheet of plastic that has been weighed at 5 cm of distance. Then, diameter of spray was determined. The measurement was carried out in triplicate.

Pump delivery:

Samples was sprayed into the surface of weighed glass once. Then calculated the weight of the gel that comes out. The measurement was carried out in triplicate.

Animal study:

The rats were maintained under standard acclimatization condition of 12-h dark/light circle at approximately 25^oC and were provided with standard rodent food. All the experimental protocols to use animals were approved by Health Research Review Committee Mohammad Hoesin Central General Hospital and Faculty of Medicine Sriwijaya University, and the registration number is 213/kepkrsmhfkunsri/2019. Each rat was weighted and anesthetized by subcutaneuosly injection of 4 mg/Kg 2% lidocaine. Dorsum was shaved using depilatory cream and 70% alcohol was used to disinfect the dorsal area. Deep anesthetized rats were kept in a prone position. A deep second degree burn wound was induced by a hot metallic device (diameter: 5x2,5 cm²) warmed for 5 minutes within boiling water and put for 10 seconds on the dorsum of rat skin with an equal weight and pressure¹⁶. All animals were resuscitated with injection of 5 mL normal saline after burning. The burned rats were randomly divided into 4 groups of six rats. Group 1 (NS) was control and rats were only washed with normal saline during dressing without any topical treatment. Group 2 was treated with base spray gel (BSG) without any effective agent. Group 3 was treated with Bioplacenton® gel 1g/Kg (Kalbe Pharmaceutical Company, Indonesia). Group 4 was treated with spray gel collagen extract (SCE) 1g/Kg. The wound area (cm²) was evaluated using a ruler every 2 days for 14 days. Wound contraction was expressed as a reduction in percentage of original wound size. Percentage wound contraction on day X = [(area in day 0 – open area on day X)/area on day 0] x 100%¹⁷.

Skin irritation study:

Three young adults rats were maintained under standard acclimatization condition of 12-h dark/light circle at approximately 25^oC and were provided with standard rodent food. Hair on the back of each rat was shaved cleanly, exposing approximately 6 cm² area of skin, 24 h before dose application. The spray gel formulation was evenly applied to 4 cm² area. Skin reaction at the site of application was assessed and scored at 24, 48, and 72 h post-test observation period.

Statistical analysis:

Statistical analysis was carried out using the IBM SPSS (Version 25) software. The data were tested for normality. One-way analysis of variance (ANOVA) was used for comparing quantity variables in the groups, followed by a post hoc multiple comparing test. If the data has not normal, the analysis of data using a Kruskal-Wallis test. The difference data were considered significant at p<0,05

RESULTS AND DISCUSSION:

Extraction collagen from fish (Channa striata) wastes bones was done by acid soluble collagen method (ASC). Acid (0,5M acetic acid solution) helps increase hydrogen ions which causes water enter into collagen fibers more easely. The influx of water is caused by electrostatic force between polar groups on collagen fibers or the formation of hydrogen bonds between nonpolar groups in collagen fibers with hydrogen ions from acid. The hydrogen ions lead collagen fibers structure damage through disruption of the non-covalent bond so that it can facilitating extraction and solubility of collagen^{18, 19}. The total protein of collagen extract from snakehead murrel bones was 1.927,14 ppm.

Spray gel preparation with collagen extract (SCE) was chosen for treatment burn injury because the spray technique allows the drugs to be delivered directly to the wound without direct contact. It could be reducing the possibility of contamination or infection and less pain when applied to the wound. Spray gel formulation prepared using Carbopol^®940 and HPMC-60SH as a gelling agent was compatible with collagen extract (Table 1). Quality control of spray gel collagen extract (SCE) was evaluated for physical appearance, adhesive strength, pH, viscosity, pump delivery, and spraying pattern. Results of the study are recorded in Table 2.

Table 2. Spray gel collagen extract quality control

Parameters	Test value	Standard value
Physical appearance	Homogenous, white, smooth, translucent	Homogenous, translucent
Adhesive strength	1,58±0,004	>1
Ph	4,86	4,5-6,5
Viscosity	1740	800-3000
Pump delivery (mg)	90±0,01	-
Spraying pattern (mm)	5,12±0,02	-
Total protein in collagen extract (ppm)	1.927,14±4,76	-

Spray gel collagen extract was found to be homogeneous, white colored, smooth, and translucent. Viscosity on spray gel preparation can affect the ease of gel to be sprayed from sprayer bottle. The viscosity value was expected to be low so that it can be sprayed easily. Spray gel collagen extract (SCE) showed low viscosity value that was 1740 cPs. Adhesive strength is one of the requirements for spray gel preparation so that the gel can be applied to the skin. There are no specific requirements regarding the adhesion of semisolid preparations, but it is better if the adhesive strength of semisolid preparation is more than 1 second.²⁰ Spray gel collagen extract (SCE) showed have adhesive strength more than 1 second (1,52 s). The pH values of spray gel collagen extract (SCE) was close of skin pH (4,5-6,5), so it caused no skin irritation, which is also supported by skin irritation study (Table 3). Pump delivery test is a test to determine the amount of gel that comes out of the sprayer bottle every single time. This test was expected to have uniformity of the weight of the gel that comes out every single time (CV<5%). The uniformity of the gel weight come out of the sprayer was expected to produce uniformity of the dose every time the gel was sprayed. Pump delivery test showed 90 mg gel come out every single time sprayed with CV = 4,53%. Spraying pattern evaluation was to determined a diameter of the spread of spray gel collagen extract. SCE showed had 5,12 mm diameter of spread. Based on the data from quality control from spray gel collagen extract formulation meets all standard value. So spray gel collagen extract was continued to wound burn healing in vivo test.

Table 3. Primary skin irritation test for spraygel collagen extract

	Rat Numbers			Rat		Combined index
	1	2	3	Control	Average	Combined index
24 h
Erythema score	0	0	0	0	0,00	0,00
Edema score	0	0	0	0	0,00	0,00
48 h
Erythema score	0	0	0	0	0,00	0,00
Edema score	0	0	0	0	0,00	0,00
72 h
Erythema score	0	0	0	0	0,00	0,00
Edema score	0	0	0	0	0,00	0,00

Collagen is the major protein in the extracellular matrix that provides proteins and a structure that is critical for wound healing²⁰. Collagen has a triple helical structure from abundance of three amino acids: glycine, alanine, proline, and glutamic acid. The most widely studied collagen components of the skin are type I, III, IV, V, and VII collagen^21,22. According to previous study, collagen extract from snakehead murrel (Channa striata) bones with acid soluble collagen (ASC) method contain 1,23%w/w glycine, 0,79%w/w alanine, and 0,92%w/w glutamic acid²³. Type I collagen, the most abundant and most readily available of collagen, is known to control many cellular functions of fibroblasts and keratinocytes, including cell shape, adhesion, differentiation, and migration²⁴. Type III collagen regulates fibril diameter and plays a critical role in maintaining cutaneous integrity, modulates scar formation via promotion of myofibroblast differentiation, and leads rapid wound closure^25,26. Type IV collagen is a major component extracellular matrix products used in wound healing. Type IV collagen also plays a crucial role in endothelial cell adhesion, migration, and angiogenesis²⁷. Type V collagen may have a role in angiogenesis and is essential for type I collagen fibril formation²⁸. Type VII collagen is the major component of anchoring fibrils that holds the epidermis and dermis together²⁹. Collagen is biocompatible and nontoxic to multiple tissue types. Based on that cited earlier suggest that collagen can be used as alternative matrix components or composite materials for wound healing. These studies have been examined fish wastes source, while porcine and bovine sources are most common for collagen, especially type I collagen, as being potentially more economical, environmentally sound and also fewer health problems with equivalent or improved properties.

CONCLUSION:

The developed formulation of spray gel collagen extract (SCE) consisting of 1% each of Carbopol® 940 and HPMC-60SH was found to be promising topical preparation for the treatment of burn wound. The SCE group has the best rate of burn wound healing among all control groups, and significantly better than NS and BSG groups. Further purification of collagen extract and histological studies can strengthen this study.

ACKNOWLEDGMENT:

The authors wholeheartedly thank Ministry of research, technology, and higher education, Indonesia, on the grant of student creativity program for providing fund to carry out this research.

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 12.07.2019 Modified on 14.08.2019

Res. J. Pharma. Dosage Forms and Tech.2019; 11(4):275-279.

DOI: 10.5958/0975-4377.2019.00046.6

Treatment Groups	Day 2 (%)	Day 4 (%)	Day 6 (%)	Day 8 (%)	Day 10 (%)	Day 12 (%)	Day 14 (%)
NS	0	0	1,2±0,7	7,9±1,5	16,2±2,5	26,8±2,5	38,7±2,4
BSG	0	2,6±1,6	6,1±2,0	14,6±1,8	25,6±3,2	39,3±2,6	58,1±3,5
Bioplacenton®	2,0±0,2	5,0±2,6	11,8±3,3	24,2±4,1	48,6±2,9	56,5±3,2	74,9±4,5
SCE	4,2±2,3	12,8±3,3	26,1±5,5	36,5±7,3	53,9±2,0	70,2±8,8	86,5±4,7
p value*	0,021	0,003	0,002	0,007	0,003	0,001	0,000

Treatment Groups	Period of Epithelialization in days (Mean±SE)
NS	15,5±0,8
BSG	13,5±1,4
Bioplacenton®	12,0±1,8^a
SCE	11,2±2,4^a,b