Study Of The Mechanism Of Formation Of Hyaluronan Putty In Lower PH Region

Hyaluronan(or named Hyaluronic acid, HA) with simple chemical structure and complexphysicochemical properties is an acidic mucopolysaccharide and an important member ofglycoaminoglycan family. It plays an important role in vertebrate life activities. With variousbioactivities, it has wide applications in medicine, cosmetics and health food industries.Previous Studies mainly focused on its biological activities, various physicochemicalproperties and applications. But the formation mechanism of its special properties discoveredby Dr. Balazs is still not clear until now. That is hyaluronan can form “viscoelastic gel” orputty in narrow lower pH region. When the pH is higher or lower than this region, thephenomenon will disappear. To resolve this question, this paper qualitatively andquantitatively explored the formation mechanism using the rheological method. Meantime,with molecular modeling and circular dichroism techniques, the formation mechanism of HAputty has been proved and supplemented. Furthermore, NMR, laser light scattering and SEMtechniques have been applied to explore it for more proof. Finally, possible application of HAputty has also been explored.First of all, with addition of sodium chloride and hydrogen-bond-breaking agent urea, itis believed that the main driving force of HA putty formation is hydrogen bonding rather thanionic force. Competition experiments between hyaluronic acid chains and small moleculeswhich have similar structure and same hydrogen bond donor/acceptor groups proved thatcarboxyl groups of glucuronic acid of HA chain and amido groups of N-acetylglucosamine ofneighbour chain formed hydrogen bonds which formed double helical structure and ultimatelyled to the formation of the rigid network covering the whole system.Secondly，A new rheological method was successfully developed to quantitative studythe entanglement networks of hydrocolloids. With the stress relaxation time spectrum ofhydrocolloids, the entanglement network numbers could be calculated:With this method, the effects of hyaluronic acid concentration, temperature, pH and ureaand other small molecules on HA network structure were successfully quantified. Furthermore,this method can be easily proformed on rotary rheometer.In addition, molecular modeling of ionized HA chains and protonated HA chains wereperformed. The results revealed that the double helical structure formed by protonated HAchains has better stability and rigidity than that form by ionized HA chains. It was alsodiscovered that HA putty formed when the ionization degree of HA chain was between11%-39%. In this state, the rigid networks formed by rigid double helix structure covered thewhole HA aqueous solution system, so the putty was formed. Furthermore, when theionization degree reached22%, the gel strength of HA putty would reach maximum. Researchof circular dichroism confirms the existance of the double helix structure and showed the effects of pH and temperature of the double helix structure.In the process of study of formation mechanism of HA putty, NOE analysis and T2determination with NMR technique, aggregation analysis with laser light scatteringtechniques and scanning electron microscope analysis technology have all been used. Theresults showed that this kind of technologies needed to be improved to solve this kind ofquestions. Finally，preliminary exploration for HA putty applied into food had been carriedon.At the end, the formation mechanism of HA putty is summarized:It is well known that HA is a weak polyelectrolyte and ionized HA chain and protonatedHA chain can exist in neutral or acidic aqueous solutions. In neutral aqueous solutions，mostcarboxyl groups are ionized which makes intermolecular electrostatic repulsion is large.However, higher HA concentration makes molecular chains close to each other,sointermolecular hydrogen bonds formed. The hydrogen bonds between amino groups andcarboxyl groups make HA chain form the double helix structure and network structure. Due tolower content of protonated HA chain, the double helix structure formed by ionized HAchains has poor rigidity low stability, so gel strength of solution is weak. With the decrease ofthe pH value, more carboxyl groups are protonated, double helix structure formed by ionizedHA chains are gradually place by that formed by protonated HA chains, network structuresbecome rigid and stable. When the ionization rate is less than39%, the rigid double helixstructure can cover the entire system, gel strength is significantly enhanced. When theionization rate reached22%, gel strength reaches the maximum. Lower pH form more rigiddouble helix and makes the network mesh smaller. When the ionization rate is less than11%and the rigid network can not cover the entire system, putty disappears. The rigid networks inthe system hinder the free movement of water molecules, which makes the whole system hasvery high viscosity. Because the hydrogen ion and amino groups makes HA networks havemore positive charges, aggregation of HA chains occur, but not precipitated.