Extraction of hyaluronic acid (HA) from rooster comb and characterization using flow field‐flow fractionation (FlFFF) coupled with multiangle light scattering (MALS)

Abstract

Hyaluronic acid (HA) was extracted in a relatively large scale from rooster comb using a method similar to that reported previously. The extraction method was modified to simplify and to reduce time and cost in order to accommodate a large-scale extraction. Five hundred grams of frozen rooster combs yielded about 500 mg of dried HA. Extracted HA was characterized using asymmetrical flow field-flow fractionation (AsFlFFF) coupled online to a multiangle light scattering detector and a refractive index detector to determine the molecular size, molecular weight (MW) distribution, and molecular conformation of HA. For characterization of HA, AsFlFFF was operated by a simplified two-step procedure, instead of the conventional three-step procedure, where the first two steps (sample loading and focusing) were combined into one to avoid the adsorption of viscous HA onto the channel membrane. The simplified two-step AsFlFFF yielded reasonably good separations of HA molecules based on their MWs. The weight average MW (M(w) ) and the average root-mean-square (RMS) radius of HA extracted from rooster comb were 1.20×10(6) and 94.7 nm, respectively. When the sample solution was filtered through a 0.45 μm disposable syringe filter, they were reduced down to 3.8×10(5) and 50.1 nm, respectively.

Detailed characterization of hyaluronan using aqueous size exclusion chromatography with triple detection and multiangle light scattering detection

“…For the three lots of HA tested, the values of the conformation plot slope are 0.6, which is consistent with HA having a random coil conformation in the aqueous solution tested. This result is in good agreement with the general findings in the literature reports 15, 16, 27, 37…”

“…This will help ensure that a certain molecular weight of HA is maintained over the shelf life of the product, which will help enhance comfort. Size exclusion chromatography (SEC) and flow field–flow fractionation with various advanced detection methods have been proved to be effective tools to evaluate the solution properties of HA and other biomaterials to ensure the quality of the products for specific claims 15, 16…”

Hyaluronan – A Functional and Structural Sweet Spot in the Tissue Microenvironment

“…The commercially available kits for measuring HA concentration (ELISA and ELISA-like assays) also vary, with some unable to detect HA polymers accurately at the extremes of the MW spectrum ( 220 ). On the other hand, thanks to a number of dedicated groups, a variety of reliable methodologies for HA MW analysis do exist, using combinations of size-exclusion chromatography ( 221 ), flat-bed polyacrylamide electrophoresis ( 222 , 223 ), agarose gel electrophoresis ( 223 – 227 ), gas phase electrophoretic mobility molecular analysis [GEMMA ( 228 )], or asymmetrical flow field fractionation with multi-angle light scattering ( 229 , 230 ), for continuous HA size profiling. Consequently, we anticipate the increased use of these robust methodologies to monitor HA size in various pathological settings in vivo in future studies.…”

SEC Separation of Polysaccharides Using Macroporous Spherical Silica Gel as a Stationary Phase

“…For example, cellulose is obtained from wood biomass in the oxidative delignification process by acetic acid and hydrogen peroxide with a catalytic amount of sulfuric acid [ 7 ]. HA is obtained from rooster combs by extraction with sodium acetate and precipitation with an ethanol solution [ 8 ]. Dextran is now synthesized by lactic acid bacteria, such as Leuconostoc mesenteroides using sucrose [ 9 ].…”