INTRODUCTION:

The interest of hydrolysed collagen as a useful source in the pharmaceutical, cosmetic industry, and as a food supplement has been growing within the last few years¹⁴. Hydrolysed collagen is a polypeptide composite¹⁹ that made by further hydrolysis of denatured collagen. The hydrolysis involves breaking down molecular bonds between collagen strands either using heat, acids or enzymes²². Hydrolysed collagen consists of small peptides with low molecular weight, enriched in specific amino acids such as glycine, proline, and hydroxyproline¹⁰. Due to its low molecular weight, hydrolysed collagen is highly digestible, absorbed and distributed in the different tissues of the human body. Several experiments have shown that hydrolysed collagen can be efficiently absorbed and distributed to the dermis, where they can stimulate the proliferation and motility of fibroblast cells³⁰.

The nature of hydrolysed collagen from the fish source is that it has a low denaturation temperature. It will result in more sensitive to heat denaturation rather than from the bovine source.

Thus, they concluded that the fish source is difficult to be used as biomaterials¹⁶. Lower stability is caused by the moderate hydroxyproline content in the fish collagen source when comparing with the bovine source. Hydroxyproline content will be affecting thermal stability because of the intramolecular hydrogen bonds between hydroxyl (OH^-) groups of hydroxyproline stabilized the triple helix of collagen¹⁰. To overcome this problem, the tilapia fish scale found to be very heat stable.. It has higher thermal stability with higher resistance of heat, and their better structural stability might be useful to replace the mammalian and bovine collagen⁷. Another hydrolysed collagen from the fish scale (Rohu and Catla fish) was found to have denaturation temperature, which is advantageous for biomedical application, food and cosmetic industries due to closeness in denaturation temperatureto mammalian and bovine collagen²⁰. Besides, the fish source becomes more valuable, due to the fact that bovine source leads to the spread of bovine spongiform encephalopathy (BSE) and is strictly not preferred by certain religious groups³².

The extraction process of proteins from the fish scales in the form of collagen and hydrolysed collagen involved a few parameters such as time, pH value, temperature, the enzyme being used and species of sources. For example, enzymatic hydrolysis has the benefit of salt-free and the peptides formed are more consistent²³. Although many reports and publications have been carried out by using acid-based and enzymatic extraction³³, extraction of hydrolysed collagen from fish scales using hydrothermal extraction is satisfied as it is less complicated and cost-efficient because it does not require the usage of enzymes or additional chemicals in the extraction process¹⁷.

Tilapia fish (Oreochromis mossambicus) is a high-value fish and also is one of many fish species that are cultured in Malaysia and other southeast Asia countries. During the filleting process, the scales are considered as waste and will be discarded²⁵. Therefore, this waste can be another potential source of raw material for collagen and hydrolysed collagen extraction⁶. Thus, the usage of this waste material was attempted in this study as a source of hydrolysed collagen. The properties of the extracted hydrolysed collagen were compared to that collagen from another study and publications. Besides, only a few reported publications on the toxicity of collagen showed that collagen is very beneficial for the growth of cells, especially to fibroblast cell³⁴, but the studies were very specific regarding the sources of collagen. To address the issue, this study was undertaken to establish a profile for various concentrations of hydrolysed collagen from Oreochromis mossambicus‘s scale towards the viability of normal skin fibroblast primary cells.

MATERIAL AND METHODS:

Materials:

The fish scale of Tilapia (Oreochromis mossambicus) was imported from Medan, Indonesia. The fish scale was manually removed and washed with water to remove dirt and impurities. The scales were then cut into small pieces to facilitate the extraction process. The process of hydrolysed collagen extractions was done by a group of researchers in the Chemistry Department, Faculty of Science and Mathematics, Sultan Idris of Education University. This hydrolysed collagen was extracted from fish scales by the hydrothermal heating method.

Chemical Reagents:

All the chemicals used for macronutrients evaluation were purchased from Pronadisa, Laboratories Conda, S.A., while buffers were purchased from Merck, Germany. All SDS-PAGE chemicals and buffers were purchased from Bio-Rad Laboratories, United State of America. All chemicals and buffers for cell viability test were purchased from Gibco Invitrogen by Life Technologies, California.

Measurement of pH:

The pH of extracted hydrolysed collagen was measured according to the British Standard Method BS 757 8. Ten percent (w/v) aqueous solution was prepared in distilled water and cooled to room temperature of about 25°C. Samples solution was centrifuged for 15 minutes at 2000 xg, and the pH was measured using a Sartorius PB-10 pH meter with Sartorius pH/ATC electrode after standardizing with pH 4.0, 7.0 and 10.0 buffers. The pH readings were carried out in triplicates.

Macronutrients Evaluation:

The macronutrient evaluation includes moisture content, ash content, fat content and protein content. The moisture, ash, fat and protein content of hydrolysed collagen were determined according to Association Official Analytical Chemist (AOAC 2000) standard procedure⁸. Each evaluation was carried out in triplicates.

Molecular Weight Determination:

The molecular weight determination used was SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) analysis¹⁸. 10mg of hydrolysed collagen samples were diluted in distilled water. Hydrolysed collagen sample was withdrawn and mixed with sample buffer (containing 0.5M Tris-HCl, pH 6.8, containing 4% SDS, 20% glycerol) at 1:2 (v/v) ratio, before heating at 95°C for 4 minutes.

Two layers of gel were prepared, which were separating and stacking gel. 12.5% separating gel with 5% stacking gel of recipe was used with 0.75mm thickness. A gel cassette sandwich was placed into the Mini PROTEAN Tetra Cell. The Mini PROTEAN Tetra Cell was filled with electrode buffer. The samples and protein marker were loaded slowly into the wells, and a constant current of 200 volts (120 mA) was passed through for 45 minutes. Gels were then stained with 0.05% (w/v) Coomassie Blue R-250 and destained overnight. Precision Plus ProteinTM Standards (MW range of 10 kDa to 250 kDa) were used to estimate the molecular weights of peptides.

Viability of Cell towards Hydrolysed Collagen:

The viability of cell includes MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay²⁹ and trypan blue assay³⁵.

For MTT assay, cell suspension (2 X 10³ cells/well) was mixed with F12: Dulbecco’s Modified Eagle Medium and were added to each of a 96-well plate. The concentrations of hydrolysed collagen that were used to treat fibroblast cell are 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10mg/mL. Control was the cells with medium only (without hydrolysed collagen). Cells were then incubated at 37°C with 5% CO₂for 24 hours, 48 hours, 72 hours and 7 days. At the end of incubation, the entire medium was removed from the wells and washed with phosphate buffer saline. MTT dye solution was added to each well, and the incubation continued for 4 hours. After the incubation, solubilisation solution/stop solution was added to each well. Cell optical density was measured using a microplate reader at 570nm wavelength (Recorders and Medicare System, India).

For trypan blue assay, cell suspension (1 X 10⁵ cells/well) was mixed with F12: Dulbecco’s Modified Eagle Medium and were added to each of a 6-well plate with flat bottomed. The concentrations of hydrolysed collagen that were used to treat fibroblast cell are 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10mg/mL. Control was the cells with medium only (without hydrolysed collagen). Cells were then incubated at 37°C with 5% CO₂, for 24 hours, 48 hours, 72 hours and 7 days. At the end of incubation, the entire medium was removed from the wells and washed with phosphate buffer saline. Trypsin solution was added into each well to detach the cells. 10 µL of the cells were resuspended with 90µL of Trypan Blue dye to determine cell viability. The 40µL of the mixture was pipette onto both sides of haemocytometer, and the number of cells was counted.

Statistical Analysis:

The data were presented as means ± standard deviation of three determinations. Statistical analyses were done using one way ANOVA and the T-test comparisons of means were done using LSD test, and p < 0.05 was considered significant. All statistical analyses were made using IBM SPSS Statistics 20 software.

RESULTS AND DISCUSSIONS:

Measurement of pH and Macronutrients Evaluation:

The pH, moisture content, ash content, fat content and protein content of the hydrolysed collagen shown in Table 1. The pH of hydrolysed collagen from O. Mossambicus’s scale was in the range of weak acidic, which is at pH 5.13 and quite similar to the pH of collagen from chicken feet sources (pH 5.57)¹³. This weak acidic range of pH is advantageous because it will provide higher gel strength as close to pH 5.0 that approaching their isoelectric point¹³.

Table 1 : The pH, moisture, ash, fat, and protein content. Values were mean±standard deviation of three replicates

Properties	Percentage (%) /pH
Moisture content	3.65±0.07
Ash content	0.79±0.03
Fat content	0.22±0.09
Protein content	63.1±0.30
pH at 25°C	5.13±0.01

The macronutrient evaluation was performed as a parameter in ensuring the removal of fat, mineral and determining whether the hydrolysis processes are carried out efficiently¹⁵. These content values are also useful in determining the quality and purity of samples⁹. The moisture content of hydrolysed collagen from O. Mossambicus’s scale (3.65%) was nearly similar to collagen from rainbow trout skin, which is 3.48%²⁸. The low moisture content in our study will be an advantage in the maintenance and improvement of the shelf life of sample⁴. In addition, low moisture will also give benefits to the rheological properties such as elasticity and viscosity of products¹³. At the contrary, high moisture content contributes to the growth and proliferation of microbes²⁶, fungi¹¹ and also made it to be sticky that will lead to structural damage¹³.

The quantity of ash in any sample assumes importance because it determines the nutritionally essential minerals⁴. High ash content is found on the scale due to the presence of hydroxyapetite. Therefore, the demineralisation process is done to remove approximately 98% of the ash content in the sample. The complete demineralisation might cause the looser matrix of the scale, which could be easier for hydrolysed collagen extraction¹². Ash content of hydrolysed collagen from O. Mossambicus’s scale (0.79%) was nearly similar to collagen from barramundi skin, which is 0.93%⁸.

The fat content of hydrolysed collagen from O. Mossambicus’s scale (0.22%) was similar to collagen from the silver carp scale (0.35%)³⁶. Low fat content in our study is essential for determining the quality of product⁷. In addition, the fat content in collagen-based material should be less than 0.5%¹⁵. Other than that, the low percentage of fat showed that the hydrolysed collagen extraction process was done successfully¹³.

Hydrolysed collagen’s protein content was high, which is 63.1% (Table 1). This could be attributed to the removal of most lipids and other impurities after hydrolysis process⁵. The high protein content of hydrolysed collagen is essential because of high protein content in a sample emphasizes their value as a vital source of nutrients⁴. The protein content of hydrolysed collagen from O. Mossambicus’s scale (63.1%) was nearly similar to collagen from barramundi skin (68.72%)⁸.

Molecular Weight Determination:

Soluble protein profile analysis was detected by SDS PAGE, which separates protein components according to their molecular weight. Figure 1 showed Coomassie blue-stained SDS-PAGE gel captured by the densitometer machine. SDS PAGE separated approximately bands between 17.75 to 23.50 kDa. These bands reading were nearly similar to the molecular weight of hydrolysed collagen from Spanish Mackerel Skin which their bands were between 5 to 25 kDa⁵.

The molecular weight of hydrolysed collagen was usually less than 50 kDa, and this less molecular weight could accelerate normal human skin fibroblast proliferation³⁴. Besides, the band distributions were wide because of their preparations were done under severe condition, which above the denaturation temperature. It means that the triple helices of gelatin and hydrolysed collagen had been destroyed, and parts of their peptide bonds were also broken out. Thus, it will lead to wide distributions of the band at lower molecular weight regions³⁸.

Fig. 1: Coomassie blue stained SDS PAGE gel.

Lane 1; molecular weight marker. Lane 2; molecular weight of hydrolysed collagen from O. Mossambicus’s scale

Viability of Cell towards Hydrolysed Collagen:

The effect of hydrolysed collagen on skin fibroblast primary cell was carried out by using cell viability MTT assay and cell viability Trypan blue assay. Cellular viability was assessed by the formation of formazon, which MTT as substrate²⁴. The formazon is a cleavage product formed by the release of mitochondrial dehydrogenase from the viable cells²⁹. Cell viability also represents the active mitochondrial enzymes in a cell that capable of reducing the MTT¹. The ability of mitochondrial enzymes of viable cells to reduce MTT substrate into blue formazon crystal was studied at 24 hours interval.

Skin fibroblast primary cell was chosen in this study because fibroblast cells are the most common types of connective tissues. It also actively engaged in the synthesis of collagenous extracellular matrix²⁷. Thus, biological evaluation with fibroblast cell cultures is a general bioassay that provides information regarding cell responses². The hydrolysed collagen concentration range chosen was 0.1-10mg/mL (0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10), while 0 mg/mL was control (cells with medium only, without hydrolysed collagen). The cells were exposed to varying concentrations of hydrolysed collagen for 24 hours, 48 hours, 72 hours, and 7 days as different sets. The cell viability is expressed as the absorbance at 570nm.

It was seen that in the initial stages of cell attachment, which is at 24 hours, the number of viable cells on hydrolysed collagen was similar to the control and not significantly different for all concentrations. After the initial attachment, cell viability on hydrolysed collagen remained increase until 48 hours, but there was no significant differences recorded between control and all the concentrations After 72 hours of seeding, it showed comparable cell viability starting from concentration 4 mg/mL (Figure 2). In addition, all concentrations did not induce significant cytotoxic effect because it exhibits higher cell viability than the control at 72 hours. However, 4mg/mL, 6mg/mL, 8mg/mL, 10mg/mL concentrations at 72 hours MTT resulted in significantly higher in cell viability as compared to the control.

Fig. 2 : Fibroblast cell viability by using MTT assays.

Data were expressed as the mean value and standard deviations. Data followed by * were statistically different when compare with 0 mg/mL.

The establishment of a biomaterial requires a longer term of observation. Therefore, the ability of hydrolysed collagen to support cell viability after 7 days of incubation was evaluated. The reading trend was the same like 72 hours of observation, which the comparable cell viability started from concentration 4mg/mL. In addition, cell viability difference between control and 4 mg/mL, 6mg/mL, 8mg/mL, 10mg/mL concentrations at 7 days MTT were statistically significant which p < 0.001. Therefore, our hydrolysed collagen samples do not give any mortality to the fibroblast cells; in fact, it supported the cell viability and allowed them to replicate.

The significant difference of cell viability starting from 4 mg/mL at the incubation of 72 hours and above may be because hydrolysed collagen provide higher surface area. Thus, the cell was able to proliferate and migrate well on the hydrolysed collagen itself²¹. Hydrolysed collagen was reported to promote cell viability due to the presence of the tripeptide, which is Arg(R) – Gly(G) – Asp (D) in their molecules¹. These RGD peptides were also reported in our hydrolysed collagen of O. mossambicus’s scale³.

These tripeptides are the recognition site for cell membrane receptors like integrin, and thus can effectively enhance cell attachment and migration. Sequence lacking this tripeptide will exhibit lower cell proliferation and attachment compared to the sequence where RGD peptides are attached. When the integrin activates, the intracellular will signalling cascades, result in transcription events that eventually regulate cell cycling and differentiation²¹. In addition, it has been reported that collagen fibres from the scales could effectively provide the space binding site because of the similarity of their structure to natural collagen arrangement in extracellular matrix³⁷.

The other effect of hydrolysed collagen on skin fibroblast primary cells was carried out by using cell viability Trypan blue assay. The cells were exposed to varying concentrations (0.1-10mg/mL) of hydrolysed collagen for 24 hours, 48 hours, 72 hours, and 7 days as different sets. The viability number of cells on hydrolysed collagen at 24 hours was nearly similar to the control and not significantly different for all concentrations (p>0.05)). Otherwise, it showed statistically different between control and the concentrations starting from 4mg/mL, 6mg/mL, 8 mg/mL, 10mg/mL at 48 hours, 72 hours and 7 days of incubation (Figure 3). The number of fibroblast cells increased rapidly for all concentrations from 24 hours to 48 hours. However, within 48 hours and above, the number of fibroblast cells only increased slightly. It might be due to the cells had reached confluence, and thus, it slowly increasing their number.

Fig. 3: Fibroblast cell viability by using Trypan blue assays.

Data were expressed as the mean value and standard deviations. Data followed by * were statistically different when compare with 0 mg/mL.

Based on the results of MTT assay and Trypan blue assay, it showed that hydrolysed collagen of O. mossambicus’s scale supported cell viability of skin fibroblast primary cell. This finding may be explained by the high affinity of cells towards hydrolysed collagen, thus resulting in in the migration of cells towards all areas of the hydrolysed collagen surface. In fact, a high number of cell attachment and proliferation on collagen scaffolds derived from freshwater and marine fish origin were also reported from previous studies^{31 ,21}.

CONCLUSION:

This study has proven that Oreochromis mossambicus’s scale can be an essential source of commercial hydrolysed collagen. The properties of hydrolysed collagen from the sample showed nearly similar to the properties of collagen from a few studies and publications. Based on MTT and Trypan blue assay, 4 mg/mL until 10 mg/mL concentrations of hydrolysed collagen increased the number of normal skin fibroblast cells. Thus, this range of concentration was assumed as beneficial to the viability of skin fibroblast primary cell. In addition, all concentrations of hydrolysed collagen did not induce a significant cytotoxic effect and had considerable cell viability.

ACKNOWLEDGEMENT:

We would like to thank to the Biology and Chemistry Department, Universiti Pendidikan Sultan Idris and Physiology Department, Universiti Kebangsaan Malaysia, for providing us with all necessary information, facilities and instruments.

REFERENCES:

1. Acharya C, Ghosh SK, and Kundu SC. Silk fibroin protein from mulberry and non-mulberry silkworms: Cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion. Journal of Materials Science: Materials in Medicine. 2008; 19(8): 2827–2836.

2. Amaral M, Gomes PS, Lopes MA, Santos, JD, Silva RF, and Fernandes MH. Cytotoxicity evaluation of nanocrystalline diamond coatings by fibroblast cell cultures. Acta Biomaterialia. 2009; 5(2): 755–763.

3. Arifin A. 2013. Effect of Electron Beam Irradiation on Molecular Weight of Hydrolysed Collagen. Universiti Pendidikan Sultan Idris.

4. Bhat R, and Sridhar KR. Nutritional quality evaluation of electron beam irradiated lotus (Nelumbo nucifera) seeds. Food Chemistry. 2008 ; 107(1): 174–184.

5. Chi CF, Cao ZH, Wang B, Hu FY, Li ZR, and Zhang B. Antioxidant and functional properties of collagen hydrolysates from Spanish mackerel skin as influenced by average molecular weight. Molecules. 2014; 19(8): 11211–11230.

6. Christynal OR. Production of ecofriendly silver nanoparticle and evaluation of its potential antimicrobial activity. Research J. Pharm. and Tech. 2015; 8(10): 1374-1378.

7. Huang CY, Kuo JM, Wu SJ, and Tsai HT. Isolation and characterization of fish scale collagen from tilapia (Oreochromis sp.) by a novel extrusion–hydro-extraction process. Food Chemistry. 2016; 190: 997–1006.

8. Jamilah B, Umi Hartina MR, Mat Hashim D, Sazili AQ. Properties of collagen from barramundi (Lates calcarifer) skin. International Food Research Journal. 2013; 20(2): 835–842.

9. Leena K. Pappachen KS. Sreelakshmi. Phytochemical screening and in vitro cytotoxicity studies of Mussaenda frondosa Linn leaves. Research J. Pharm. and Tech 2017; 10(12): 4227-4230.

10. Liu D, Liang L, Regenstein JM, and Zhou P. Extraction and characterisation of pepsin-solubilised collagen from fins, scales, skins, bones and swim bladders of bighead carp (Hypophthalmichthys nobilis). Food Chemistry. 2012; 133(4): 1441–1448.

11. Magan N and Lacey J. Ecological determinants of mould growth in stored grain. International Journal of Food Microbiology. 1998; 7(3): 245–256.

12. Matmaroh K, Benjakul S, Prodpran T, Encarnacion AB, and Kishimura H. Characteristics of acid soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus heptacanthus). Food Chemistry. 2011; 129(3): 1179–1186.

13. Mohd Nazri AR, Shariffah Azzainurfina SK, and Azlan J. Extractions, physicochemical characterizations and sensory quality of chicken feet gelatin. Borneo Science. 2012; (1): 1–13.

14. Mohd Yaqub K, Poonam G, Bipin B, Vineet KS, Irfaan A. A reveiw- miracle of nanotechnology in cosmetics. Asian Journal Pharm Research. 2014; 4(1): 16-23.

15. Muyonga JH, Cole CGB, and Duodu KG. Characterisation of acid soluble collagen from skins of young and adult Nile perch (Lates niloticus). Food Chemistry. 2004; 85(1): 81–89.

16. Nagai N, Yunoki S, Suzuki T, Sakata M, Tajima K, and Munekata M. Application of cross-linked salmon atelocollagen to the scaffold of human periodontal ligament cells. Journal of Bioscience and Bioengineering. 2004; 97(6): 389–394.

17. Olatunji O, and Denloye A. Temperature - dependent extraction kinetics of hydrolyzed collagen from scales of croaker fish using thermal extraction. Food Science Nutrition. 2017; (5): 1015–1020.

18. Pan BQ, Su WJ, Cao MJ, Cai QF, Weng WY, and Liu GM. IgE reactivity to type I collagen and its subunits from tilapia (Tilapia zillii). Food Chemistry. 2012; 130(1): 127–133.

19. Pandey Rishabh, Singh AV, Pandey Awanish, Tripathi Poonam, SK Majumdar, Nath LK. Protein and peptide drugs: A brief review. Research J. Pharm. and Tech. 2009; 2(2): 228-233.

20. Pati F, Adhikari B, and Dhara S. Isolation and characterization of fish scale collagen of higher thermal stability. Bioresource Technology. 2010; 101(10): 3737–3742.

21. Pati F, Datta P, Adhikari B, Dhara S, and Ghosh K. Collagen scaffolds derived from fresh water fish origin and their biocompatibility. Journal of Biomedical Materials Research. 2012; 100(4): 1068–1079.

22. Pei Y, Yang J, Liu P, Xu M, Zhang X, and Zhang L. Fabrication, properties and bioapplications of cellulose/collagen hydrolysate composite films. Carbohydrate Polymers. 2013; 92(2): 1752–1760.

23. Puja K, Shatabdi S, Suneetha V. Production of glue from tannery effluent by physical, chemical and biological methods. Research J. Pharm. and Tech. 2016; 9(8):1031-1035.

24. Rahamat U, Devaragattu BV, Naga SK, Chekalta S, Baddanapally SR. In vitro cytotoxicity of L-glutaminase against Hep-G2 cell lines. Research J. Pharm. and Tech. 2018; 11(6): 2213-2216.

25. Rajesh AS, Vijay LG. A biosorption of heavy metal ions from effluent using waste fish scale. Asian J. Research Chem. 2018; 11(5): 775-777.

26. Ravi Kumar, Rajarajeshwari N, Narayana Swmay VB. Isolation and characterization of Borassus flabellifer mucilage. Research J. Pharm. and Tech. 2012; 5(8): 1093-1101.

27. Sajid Ali, Maryam Shabbir, Nabeel Shahid. The structure of skin and transdermal drug delivery system- a review. Research J. Pharm. and Tech. 2015; 8(2): 103-109.

28. Shahiri Tabarestani H, Maghsoudlou Y, Motamedzadegan A, Sadeghi Mahoonak AR, Rostamzad H. Study on some properties of acid-soluble collagens isolated from fish skin and bones of rainbow trout (Onchorhynchus mykiss). International Food Research Journal. 2012; 19(1): 251–257.

29. Shrivastava HY, Ravikumar T, Shanmugasundaram N, Babu M, and Nair BU. Cytotoxicity studies of chromium ( III ) complexes on human dermal fibroblasts. Free Radical Biology and Medicine. 2005; 38: 58–69.

30. Sibilla S, Godfrey M, Brewer S, Budh-Raja A, and Genovese L. (2015). An Overview of the Beneficial Effects of Hydrolysed Collagen as a Nutraceutical on Skin Properties: Scientific Background and Clinical Studies. The Open Nutraceuticals Journal. 2015; 8: 29–42.

31. Song E, Yeon Kim S, Chun T, Byun HJ, and Lee YM. Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials. 2006; 27(15): 2951–2961.

32. Soumya Verma, Vamshi Krishna T. A Review on the polymers for vegetarian soft gel capsule films. Research J. Pharm. and Tech. 2017; 10(9): 3217-3222.

33. Wang B, Wang YM, Chi CF, Luo HY, Deng SG, Ma JY. Isolation and characterization of collagen and antioxidant collagen peptides from scales of croceine croacker Pseudosciaenia croceia. Marine Drugs. 2013; 11: 4641–4661.

34. Wang Y, Zhang C, Zhang Q, and Li P. Composite electrospun nanomembranes of fish scale collagen peptides/chito-oligosaccharides : antibacterial properties and potential for wound dressing, 2011; 667–676.

35. Younis AM. 2011. The Effect of Centella Asiatica Extract on Human Periodontal Ligament Fibroblast Cell Line. University of Technology Malaysia.

36. Zhang J, Duan R, Ye C, and Konno K. Isolation And Characterization Of Collagens From Scale Of Silver Carp (Hypophthalmichthys Molitrix). Journal of Food Biochemistry. 2010; 34(6): 1343–1354.

37. Zhang Y, Su B, Venugopal J, Ramakrishna S, and Lim C. Biomimetic and bioactive nano fibrous scaffolds from electrospun composite nano fibers. International Journal of Nanomedicine. 2007; 1(9): 623–638.

38. Zhang Z, Li G, and Shi B. Physicochemical properties of collagen, gelatin and collagen hydrolysate derived from bovine limed split wastes. Journal of the Society of Leather Technologists and Chemists. 2006; 90(1): 23–28.

Received on 13.12.2018 Modified on 15.05.2019

Research J. Pharm. and Tech. 2020; 13(12):5855-5860.

DOI: 10.5958/0974-360X.2020.01020.3