| Low molecular weight hyaluronic acid and oigosacchariddes of hyaluronic are of good penetration, excellent biological compatibility and easy to assemble. And they have the effects of angiogenesis, immunoregulation in the human body, therefore they are becoming the hotspot in pharmacologist and biomaterial. The methods of hyaluronic acid degradation are chemical, physical, enzymatic. Enzymatic can get narrow molecular weight distribution and low molecular weight hyaluronic acid, but it is expensive, and can not be recycled. So it can not be used in industrial production. In this study, the research of low molecular weight hyaluronic acid is established, the key factors of ultrasonic and chemical is analyzed, kinetic model is created, low molecular weight hyaluronic acid is produced by multiple ultrasonic-chemical method and analyzed by the chromatogram.①The measurement of viscometry to show changes of viscosity-average molecular weight is to evaluate the method of chemical degradation and ultrasonic degradation. According to experiment result, it was found that high temperature and pH value have great effect on the degradation. HA solutions is decomposed quickly in 8h when the temperature is over 25℃; when the temperature is at 4℃and 10℃, the rate is low. Mr of all HA is stay the same at some time whatever temperature is. Also It was found it has significant effect if pH is above 9.0 on the degradation. with high rate. The experiments show that oxidant content has great influence on the degradation. At 4h, group of 8mL oxidant has 2.60 times Mr than group of 1mL, and it is 3.48 times at 24h. When using ultrasonic, similar degradation curves and same molecular weight indicate that temperature has little effect. Degradation with different frequencies, it is found the higher frequency it is used, the faster rate and lower final Mr it has, in the range of 300kHz to 1.7MHz. All the above is proved that oxidant content and pH value are the key factors on chemical degradation, especially Oxidant is the most important influence when pH is above 9.0, and frequency is the key factors on ultrasonic degradation.②In order to predict and evaluation of ultrasounic and chemical degradation of hyaluronic acid, a kinetic model based on Rice-Herzfeld theory is built and tested. As in the chemical and ultrasonic degradation, free radical content plays most important role in the reaction, Rice-Herzfeld theory is applicable. Model assumes that no side effects, ignoring the thermal effect, and the radical reaction is an instant reaction. mathematical formula prove that if Mr-t is known, xl correlated with the reaction rate. To calculate the activation rate, HPLC is used to detected that salicylic acid decompose to 2,3-DHBA. Compared by theoretical and experimental values, it is found that deviation is less than 20%. It shows that the model can effectively predict and evaluate the chemical and ultrasonic degradation.③In order to prepare low molecular weight hyaluronic acid with narrow distribution, it is established a new chemical-multiple ultrasonic method to decompose. Ultrasonic degradation of hyaluronic acid (HA) salt solutions was investigated by a ultrasonic instrument, which is designed to make use of multiple ultrasonic oscillator simultaneously. The Mr-t implies that the ultrasonic degradation of are faster and more effective, with final Mr 10kDa.With the conditions: 4℃, pH range 9.0-10.0, dual ultrasound (1.7MHz and 800kHz), low molecular weight hyaluronic acid of 10kDa is made in 8 hours, and 5kDa in 24 hours. Infrared spectroscopy prove the structure of hyaluronic acid did not change. Using the model to evaluate, the average deviation is 6.4%.④In order to analyze the molecular weight distribution of LMWHA, it is established HPLC method to analyze molecular weight distribution and structure of low molecular weight hyaluronic acid. HPLC method: SHISEIDO CAPCELL PAK NH2: 5μm, 4.6×250mm; mobile phase A: 0.1N NaH2PO4; mobile phase B: H2O, with gradient: B%: 100(0)→0(60min), the flow rate was 1.0 mL/min, detection wavelength was set at 210 nm, the temperature of column was 35℃. Comparison of GPC and HPLC, HPLC has economic, high sensitivity characteristic. Analysis of spectra, there are further evidences that ultrasound-chemical degradation method can quickly and efficiently get LMWHA and o-HA. |
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