ISOLATION, PURIFICATION, AND CHARACTERIZATION OF BOVINE TENDON COLLAGEN AND ANALYSIS OF GLYCINE, PROLINE, AND HYDROXYPROLINE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-FLUORESCENCE

  • Dwi Yulianti Department of Pharmacy, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia.
  • Harmita . Department of Pharmacy, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia.
  • Taufiq Indra Rukmana Department of Pharmacy, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, Indonesia.

Abstract

Objective: In this study, collagen isolated from bovine tendon was purified and characterized, and the optimum conditions for analysis of glycine,
proline, and hydroxyproline were determined.
Methods: The collagen isolation process used 0.1 N NaOH as a pretreatment, 0.5 M acetic acid in the extraction, 0.9 M NaCl in the salting-out
step, centrifugation and dialysis for purification, and freeze-drying as the final step. The characterization of the collagen included analysis of the
organoleptic properties, pH, moisture content, viscosity, and ash content. A Fourier-transform infrared (FTIR) spectroscopy analysis and Casson’s
trichrome staining were also performed. The collagen was hydrolyzed in 6 N HCl for 24 h and derivatized using 9-fluorenylmethoxycarbonyl chloride.
The optimum condition was conducted from the optimal wavelength, selection of mobile phase composition, and flow rate.
Results: The average content was 11.867±0.20% for glycine, 33.247±0.20% for proline, and 10.51±0.23% for hydroxyproline. The optimum condition
analysis for collagen was achieved by high-performance liquid chromatography (HPLC) with a C18® column and a fluorescence detector (excitation:
265 nm and emission: 320 nm) with mobile phase acetate buffer (pH 4.2):acetonitrile (55:45), and the flow rate was 0.8 mL/min.
Conclusion: The collagen isolated from bovine tendon was obtained at a yield of 0.690%, and the identity was confirmed by FTIR functional group
analysis and Casson’s trichrome staining. The HPLC conditions using a fluorescence detector for analysis of glycine, proline, and hydroxyproline
concentrations in the bovine tendon collagen were optimized. The analysis of amino acids gave the average levels of 33.247±0.20% for glycine,
11.867±0.20% for proline, and 10.51±0.23% for hydroxyproline.

Keywords: Amino acid, Bovine tendon collagen, Content, Derivatization, Fluorescence, Glycine, High-performance liquid chromatographic, Hydroxyproline, Optimization, Proline.

References

1. Silvipriya K, Kumar K, Bhat A, Kumar B, John A, Lakshmanan P.
Collagen: Animal sources and biomedical application. J Appl Pharm
Sci 2015;5:123-7.
2. Schmidt MM, Dornelles RC, Mello RO, Kubota EH, Mazutti MA,
Kempka AP, et al. Collagen extraction process. Int Food Res J
2016;23:913-22.
3. Tridhar, NA. Collagen Production Comparation from Scales and Bone
Fish Gurami (Osphronemus gourami) by Chemical and Enzyme.
Bandung: Universitas Pasundan; 2016.
4. Lestari, T. Isolation and Charaterization of Collagen from Tuna Bone
Fish (Thunnus albacares) as Raw Material for Pharmacy Industry.
Depok: Thesis of Faculty of Pharmacy, Universitas Indonesia; 2007.
5. Association of Official Analytical Chemists. Official Methods of
Analysis of AOAC International. 18th ed. Maryland: Association of
Official Analytical Chemists Int.; 2005.
6. Kong J, Yu S. Fourier transform infrared spectroscopic analysis of
protein secondary structures. Acta Biochim Biophys Sin (Shanghai)
2007;39:549-59.
7. National Standardization Agency. Crude Collagen from Fish Scales-
Quality and Preparation Requirement. Jakarta: Badan Standardisasi
Nasional; 2014.
8. Fakhri S, Mohammadi B, Jalili R, Hajialyani M, Bahrami G. Screening
and confirmation of different synthetic adulterants in slimming
products. Asian J Pharm Clin Res 2018;11:260-4.
9. Schrieber R, Gareis H. Gelatine Handbook: Theory and Industrial
Practice. Weinheim: Wiley-VCH Verlag GmbH and Co., KgaA.; 2007.
10. Khalaf FH, Salih SI. Clinical and histopathological evaluation of
using platelet-rich plasma and platelet-rich fibrin matrix in treatment
of induced chronic open wounds in bucks. Asian J Pharm Clin Res
2018;11:337-41.
Statistics
51 Views | 137 Downloads
Citatons
How to Cite
Yulianti, D., ., H., & Rukmana, T. I. (2018). ISOLATION, PURIFICATION, AND CHARACTERIZATION OF BOVINE TENDON COLLAGEN AND ANALYSIS OF GLYCINE, PROLINE, AND HYDROXYPROLINE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-FLUORESCENCE. International Journal of Applied Pharmaceutics, 10(1), 311-315. https://doi.org/10.22159/ijap.2018.v10s1.69
Section
Original Article(s)