ISOLATION, PURIFICATION, AND CHARACTERIZATION OF PORCINE SKIN COLLAGEN: ANALYSIS OF THE GLYCINE, PROLINE, AND HYDROXYPROLINE COMPONENTS USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The aim of this study is to produce collagen through the extraction and isolation of porcine skin.Methods: Collagen from porcine skin (Sus scrofa domesticus) was isolated, purified, and characterized. Major amino acid content of collagen (glycine,proline, and hydroxyproline) was determined. Samples were extracted with 0.5 N acetic acid and precipitated with 0.9 M NaCl. Characterization testsincluded those to determine the organoleptic content, pH, Fourier-transform infrared analysis, moisture content, ash content, viscosity, and Masson’strichrome staining on collagen tissue. The collagen was further analyzed using high-performance liquid chromatography using C-18® column anda fluorescence detector at 265 nm and 320 nm, acetic buffer (pH 4.2)–acetonitrile (55:45) as mobile phase, and optimum flow rate of 0.8 mL/min.Results: Our findings indicated that the best method for isolating collagen was with 0.1 M NaOH expressed by average contents of glycine, proline, andhydroxyproline in collagen which were 33.663±0.215%, 12.333±0.128%, and 11.303±0.354%, respectively.Conclusion: Porcine collagen has been successfully obtained with this method.

Download Full-text

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 ◽

10.22159/ijap.2018.v10s1.69 ◽

2018 ◽

Vol 10 (1) ◽

pp. 311 ◽

Cited By ~ 1

Author(s):

Dwi Yulianti ◽

Harmita . ◽

Taufiq Indra Rukmana

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Mobile Phase ◽

High Performance ◽

Wavelength Selection ◽

Spectroscopy Analysis ◽

Fluorescence Detector ◽

Selection Of ◽

Tendon Collagen

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-outstep, centrifugation and dialysis for purification, and freeze-drying as the final step. The characterization of the collagen included analysis of theorganoleptic properties, pH, moisture content, viscosity, and ash content. A Fourier-transform infrared (FTIR) spectroscopy analysis and Casson’strichrome 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 conditionanalysis 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 groupanalysis and Casson’s trichrome staining. The HPLC conditions using a fluorescence detector for analysis of glycine, proline, and hydroxyprolineconcentrations 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.

Download Full-text