Hyaluronic acid

hyaluronic acid: A glycosaminoglycan(mucopolysaccharide) that ispart of the matrix of connective tissue.Hyaluronic acid binds cells togetherand helps to lubricate joints.It may play a role in the migration ofcells at wounds; this activity ceaseswhen hyaluronidase breaks downhyaluronic acid. ChEBI: A mucopolysaccharide composed of N-acetylglucosamine and glucuronic acid subunits. It is found in the connective tissues of vertebrates.

Method 1. Take the cockscomb as a raw material for dehydration, crush it, soak it in distilled water for 24h, fully swell it, filter, extract the filter residue with distilled water 3 times, combine the filtrate, add 100g/L (10%) sodium chloride, and dissolve Then, add an equal volume of chloroform, stir for 3 hours, separate the water phase, add 2 times the volume of 95% ethanol to precipitate hyaluronic acid, dehydrate, and dry to obtain a crude product. Cockscomb [acetone] → dehydrated broken cockscomb [distilled water] → [24h] filtrate [NaCl, chloroform] → water phase [95% ethanol] → hyaluronic acid precipitation → crude product dissolve the crude product in a 0.1mol/L sodium chloride solution, Adjust pH 4.5-5 with dilute hydrochloric acid, add equal volume of chloroform and stir for 2 times. Adjust the pH to 7.5 with NaOH solution for the aqueous phase, add pronase and keep at 37°C for 24 hours. Treat the hydrolysate with chloroform for 2-3 times. Add an equal volume of 10g/L (1%) cetylpyridinium chloride solution, place the precipitate, add 0.4mol/L sodium chloride solution to the precipitate, stir to dissociate, centrifuge, divide the supernatant, use 3 times the volume The 95% ethanol is repeatedly precipitated, dehydrated, and dried to obtain fine products. Crude product [0.1mol/L NaCl]→dissolving solution [dilute HCl, chloroform]→[pH4.5]water phase [NaOH, bond mold protease]→[pH7.5, 37℃, 24h] enzymatic hydrolysis solution[chloroform]→ Aqueous phase [hexadecylpyridinium chloride]→precipitation [NaCl]→supernatant [95% ethanol]→precipitation→refined product Foreign literature reports, with distilled water, 93% hyaluronic acid can be extracted from cockscomb, crude The recovery rate is above 90%, and the total yield is as high as 6%. Method 2. Human umbilical cord was taken as raw material. The acetone dehydrated umbilical cord fragments were soaked in distilled water for 24 hours, extracted, extracted repeatedly 4 times, filtered, and the filtrate was combined. Umbilical cord [acetone] → dehydrated umbilical cord fragments [distilled water] → [24h] extract → filtrate Add 100g/L (10%) of sodium chloride to the above filtrate and stir to dissolve, adjust pH 5 with dilute hydrochloric acid, and add equal volume Stir in chloroform, separate the water phase after stratification, and treat it twice with the same operation, then adjust the pH to 7.5 with dilute sodium hydroxide, add 4g/L (0.4%) pronase, and keep it in a 37℃ water bath for 24h, enzymatic hydrolysis solution Add an equal volume of chloroform and repeat the treatment, and then use 3 times the volume of 95% ethanol to precipitate hyaluronic acid. The fibrous precipitate floating on the upper part of the ethanol solution and the powdery precipitate at the bottom are collected, dehydrated, and dried to obtain hyaluronic acid component I and component II. Filtrate [NaCl, dilute HCl, chloroform]→[pH5] water layer [NaOH, pronase]→[pH7.5, 37℃, 24h] enzymatic hydrolysis solution [chloroform, ethanol]→hyaluronic acid to combine hyaluronic acid Part I was dissolved in physiological saline, filtered through a vertical melting funnel No. 6 to sterilize, precipitated hyaluronic acid with sterile acetone, filtered, dried, and dissolved in an appropriate amount of sterile buffer to make a 1% solution, sterile The injection is obtained in aliquots. The total yield is 2% of the acetone dehydrated umbilical cord block. Method three, skin-based preparation method Shimada and Matsumura have designed two different methods. The first preparation method is to grind the freeze-dried rabbit skin block, defatted with acetone, add 0.5mol/L sodium chloride solution to make a homogenate, precipitate with cetylpyridinium chloride, and continuously dissolve it in increasing concentration of chlorine Sodium chloride solution, and then dissolved in 0.5mol/L sodium chloride solution, the 0.5mol/L sodium chloride component is further purified by DEAE-Sephadex chromatography to obtain hyaluronic acid with a relative molecular weight of 1×104-7.2× 104. The second preparation method, rabbit skin block is directly defatted (not freeze-dried and mechanically crushed), suspended in water, heated for extraction at 100°C, and the extract is treated with pronase and Dnase, and further purified by Sepharose G-75 and Sepharose 4B. Got. The relative molecular weight is 1.6×105-1.3×106. The yields of the two methods are similar. The first method may degrade hyaluronic acid due to mechanical treatment. Method 4. Preparation of sheep eyeballs as raw materials. Take frozen sheep eyeballs by thawing, peel off the vitreous body, thaw and centrifuge, separate the supernatant, add acetone, place, centrifuge, and dissolve the precipitate in 1mol/L sodium chloride solution. Stir and centrifuge. Add trichloroacetic acid to the supernatant, centrifuge, separate the supernatant, adjust the pH to neutral with sodium oxychloride, add 3 times the amount of 95% ethanol for precipitation, then dehydrate with ethanol and acetone, and dry it in a P2O5 vacuum dryer. The yield of crude sodium hyaluronate is 2.8% of the dry weight of the vitreous. Dissolve the crude product in sodium hydroxide solution, add the treated bleaching earth for adsorption, centrifuge, collect the supernatant, add cetylpyridinium bromide (CPB) solution to obtain sodium hyaluronate-CPB complex precipitate. Take the precipitate and wash it, dissociate it with 0.4mol/L chlorination custom, filter with suction, collect the clear liquid, add ethanol for precipitation, then dehydrate with ethanol and acetone, and dry it in a P2O5 vacuum dryer to obtain high-quality sodium hyaluronate. Yield It accounts for 62% of the dry weight of the crude product, and the total yield is 1.8%.

Used as a moisturizer in creams, creams, milks, masks and essences.Used as high-end cosmetic additives, also used in medicine.Necessary drugs for ophthalmology "viscous surgery". Applied to cataract surgery, the sodium salt is easy to be stored in the anterior chamber to maintain a certain depth of the anterior chamber, maintain a clear surgical field of vision, reduce inflammation and complications after surgery, and improve the effect of surgical correction of vision. It is also used for complex retinal detachment surgery.