Dehydrate the cockscomb with acetone, pulverize, take in distilled water for 24 hours, completely swell, filter, as well as repeatedly remove the filter deposit with distilled water for 3 times, combine the filtrates, add 100g/L (10%) sodium chloride, liquify After that, add an equal quantity of chloroform, stir for 3 hours, different the water phase, include 2 times the volume of 95% ethanol to speed up Pure Hyaluronic Acid, dry out, as well as completely dry to get the unrefined item. Cockscomb [acetone] → dehydrated broken chickencomb [distilled water] → [24h] filtrate [NaCl, chloroform] → water stage [95% ethanol] → Pure Hyaluronic Acid precipitation → crude item Liquify unrefined product in 0.1 mol/L sodium chloride solution, Adjust the pH to 4.5-5 with thin down hydrochloric acid, add an equal volume of chloroform and also stir, and also deal with for 2 times. The aqueous stage is adjusted to pH 7.5 with NaOH option, and also pronase is included at 37 ° C for 1 day, and the enzymatic hydrolysis solution is treated with chloroform for 2-3 times., add an equal volume of 10g/L (1%) cetylpyridine chloride solution to the water phase, put the precipitate, include 0.4 mol/L sodium chloride solution to the precipitate, stir and dissociate, centrifuge, separate the supernatant, usage 3 Repeated rainfall with twice the quantity of 95% ethanol, dehydration, and also drying out to acquire fine items. Unrefined item [0.1 mol/LNaCl] → liquifying remedy [weakened HCl, chloroform] → [pH4.5] liquid stage [NaOH, key mold and mildew protease] → [pH7.5, 37 ℃, 24h] enzymatic hydrolysis remedy [chloroform] → water Phase [Cetylpyridinium chloride] → Precipitation [NaCl] → Supernatant [95% ethanol] → Precipitation → Improved item According to foreign literature records, 93% Pure Hyaluronic Acid can be drawn out from cockscomb with distilled water, and the recuperation of unrefined product The yield is above 90%, and also the complete return is as high as 6%.
Human umbilical cable is taken as resources, and acetone dehydrated umbilical cable pieces are included in distilled water for saturating for 1 day, drawn out, drawn out repetitively for 4 times, filtered, as well as the filtrate is incorporated. Umbilical cord [acetone] → dehydrated umbilical cable fragments [pure water] → [24h] extract → filtrate Include 100g/L (10%) sodium chloride to the above filtrate, mix to liquify, adjust pH to 5 with weaken hydrochloric acid, as well as include an equal quantity of chloroform Mix the treatment, different the water phase after stratification, as well as perform the exact same procedure two times, then change the pH to 7.5 with dilute salt hydroxide, include 4 g/L (0.4%) pronase, as well as location it in a water bathroom at 37 ° C for 24 hours. Add an equivalent quantity of chloroform repetitively, and then use 3 times the volume of 95% ethanol to precipitate Pure Hyaluronic Acid. The fibrous precipitate floating on the top part of the ethanol remedy and the grainy precipitate on the bottom are gathered, dehydrated and also dried specifically to get Pure Hyaluronic Acid element I and part II. Filtrate [NaCl, weaken HCl, chloroform] → [pH5] aqueous layer [NaOH, pronase] → [pH7.5, 37 ℃, 24h] enzymatic hydrolysis service [chloroform, ethanol] → hyaluronic acid to make Pure Hyaluronic Acid team Part 1 is liquified in physiological saline, filteringed system as well as sterilized via No. 6 upright melting channel, as well as Pure Hyaluronic Acid is precipitated with clean and sterile acetone, filtered, dried out, and then dissolved in an ideal amount of sterilized barrier to prepare a 1% remedy, sterilized Dispense for shot. The overall return was 2% of the acetone-dehydrated umbilical cord items.
Technique of making skin as basic material Shimada and Matsumura have developed two various methods. For the initial preparation method, freeze-dried rabbit skin is ground, degreased with acetone, homogenized by adding 0.5 mol/L sodium chloride solution, precipitated with cetylpyridinium chloride, and constantly liquified in boosting focus of chlorine. The 0.5 mol/L sodium chloride element was further detoxified by DEAE-Sephadex chromatography to acquire Pure Hyaluronic Acid, with a family member molecular weight of 1 × 104-7.2 × 104. In the 2nd technique, the bunny skin block is straight defatted (without freeze-drying and also mechanical grinding), put on hold in water, warmed at 100 ° C for removal, the extract is treated with pronase and Dnase, and also further purified by SephadexG-75 and also Sepharose4B. The loved one molecular weight is 1.6 × 105-1.3 × 106. The yields of both approaches are comparable, and the very first technique may degrade Pure Hyaluronic Acid due to mechanical therapy. Technique 4. Preparation method of sheep eyeball as resources: Freeze the icy sheep eyeball with water, peel off the glasslike body, thaw as well as centrifuge, different the supernatant, add acetone, location, centrifuge, as well as liquify the precipitate in 1 mol/L sodium chloride solution. Stir, centrifuge. Trichloroacetic acid was added to the supernatant, centrifuged, the supernatant was divided, the pH was gotten used to neutrality with sodium oxychloride, 3 times the quantity of 95% ethanol was included for rainfall, then dehydrated with ethanol and acetone, as well as dried in a P2O5 vacuum desiccator. The unrefined product of Pure Hyaluronic Acid salt, the yield is 2.8% of the completely dry weight of the vitreous body. Dissolve the crude product in salt hydroxide solution, add dealt with whitening earth for adsorption, centrifuge, accumulate the supernatant, include cetyl pyridine bromide (CPB) option, and acquire Pure Hyaluronic Acid sodium-CPB complicated rainfall. The precipitate was cleaned, dissociated with 0.4 mol/L chlorination, suction filtration, gathered the clear fluid, precipitated with ethanol, dried out with ethanol and acetone, as well as dried in a P2O5 vacuum cleaner clothes dryer to obtain Pure Hyaluronic Acid salt. The yield It accounts for 62% of the dry weight of the unrefined item, and also the overall yield is 1.8%.
