What Are the Uses of Hyaluronic Acid Powder?

Hyaluronic acid, also known as hyaluronic acid, is an acidic mucopolysaccharide that forms a viscoelastic substance when combined with water molecules [1]. Hyaluronic acid is mainly found in the skin and connective tissues of the human body, where it acts as an extracellular matrix for cellular insertion. In addition to providing a certain volume of extracellular matrix for the cells in the body, hyaluronic acid also influences the stability, binding and viscoelasticity of the tissues. The molecular structure of all natural hyaluronic acids is the same, with virtually no species- or tissue-specific differences, so pure hyaluronic acid is not immunogenic [2].

Hyaluronic acid was first isolated from the vitreous body of the bovine eye by Meyer et al, Professor of Ophthalmology at Columbia University, USA, in 1934 [3]. Since Kendall et al. discovered that Streptococcus haemolyticus could produce hyaluronic acid in 1937, many scientists and scholars have been engaged in the study of hyaluronic acid production by microbial fermentation. In 1985, Shiseido of Japan first reported the production of hyaluronic acid by streptococcal fermentation. In 2003, the United States also approved a patent for the production of hyaluronic acid by fermentation [4].

Since then, there have been many studies on the selection and breeding of hyaluronic acid fermentation strains. For example, Fong et al. [5] obtained non-hemolytic and non-hyaluronidase-producing strains of Streptococcus zooepidemicus by mutagenicity with nitrosoguanidine. Up to now, the technology of hyaluronic acid production by microbial fermentation has been matured in foreign countries and has entered the industrialisation stage. In China, the production of hyaluronic acid by fermentation of Streptococcus faecalis has also become a research hotspot and has gradually entered into the industrialisation stage, and the development, production and application of hyaluronic acid has a very attractive prospect.

1 Physical and chemical properties and physiological functions of hyaluronic acid

1.1 Physical and chemical properties

Hyaluronic acid is soluble in water, and its solution is acidic with a certain negative charge. The molecule of hyaluronic acid is a high molecular polysaccharide formed by the polymerisation of the disaccharide units of β-D-glucuronic acid and β-D-N-acetylglucosaminoglucose.

Hyaluronic acid is a major component of some bacterial pods such as Streptococcus and Pseudomonas aeruginosa. Unlike other mucopolysaccharides, hyaluronic acid is an acidic mucopolysaccharide and does not contain sulphur in its molecular structure. The specific intermolecular configuration of hyaluronic acid gives it a high viscosity and moisturising effect. Hyaluronic acid molecules can carry about 500 times as much water as themselves, which is recognised as the best moisturising substance, and is therefore mass-produced and widely used in functional foods and cosmetics [6].

1.2 Physiological functions

1.2.1 Water retention function:

Hyaluronic acid has strong water absorption and water retention. In higher concentration solution, hyaluronic acid has strong hydrophilicity, and its long molecular chains are interwoven in a grid shape, which are combined with water molecules through hydrogen bonding.

1.2.2 Skin protection:

The effect of hyaluronic acid on the skin depends on its molecular weight. Hyaluronic acid with large molecular weight is mainly used for skin moisturising. Hyaluronic acid coated on the skin surface can quickly form a breathable hydration film wrapped around the skin surface, which softens the skin's stratum corneum and further enhances the absorption and utilisation of active substances by the stratum corneum of the skin, thus making the skin delicate and smooth [7].

1.2.3 Anti-aging function:

Hyaluronic acid has the function of scavenging free radicals, so it can slow down skin aging and protect the skin from damage.

1.2.4 Other functions:

Hyaluronic acid has the function of drug-carrying, which can be used as a carrier to embed drugs, not only slow release but also promote drug absorption. Hyaluronic acid also has antibacterial and anti-inflammatory effects [8].

2 Application of hyaluronic acid

2.1 Application of hyaluronic acid in health foods

Hyaluronic acid-based oral health products became popular in Japan in the 1980s [8]. As a functional food, hyaluronic acid does not directly exert health functions after consumption, but increases the precursors for the synthesis of hyaluronic acid in the body and promotes the synthesis of hyaluronic acid in skin and other tissues [9].

2.2 Application of hyaluronic acid in cosmetics

As mentioned earlier, hyaluronic acid has strong moisturising properties and is a naturally occurring substance found in a wide range of skin and other tissues. This property has led to its popularity in the cosmetic industry worldwide [9]. Currently, there are many cosmetic products containing hyaluronic acid in their formulations, such as toners, lotions, serum capsules, masks, body lotions, powders, lipsticks, shampoos, sunscreens, conditioners, mousses, and so on [10].

2.2.1 Moisturising:

Moisturising is the main function of hyaluronic acid in cosmetics. Compared with glycerol, propylene glycol and other commonly used humectants, hyaluronic acid has the strongest hygroscopicity at lower relative humidity (less than 33%), and the weakest hygroscopicity at higher relative humidity (more than 75%), which is suitable for the protection of the skin in different seasons, as well as for the different requirements for the moisturising effect of cosmetics in different humid environments (e.g., dry winter and humid summer). In other words, the moisturising properties of hyaluronic acid are not affected by the relative humidity of the surrounding environment [10].

Of course, the moisturising property of hyaluronic acid is also related to its relative molecular weight, the larger the molecular weight, the stronger the moisturising property; the smaller the molecular weight, the weaker the moisturising property [10]. Hyaluronic acid as a moisturising agent is seldom used alone, but is often used in conjunction with other moisturising agents, so as to complement each other's strengths and achieve better whitening and moisturising effects.

2.2.2 Supplementation of skin nutrition:

Exogenous supplementation of hyaluronic acid can be used as a supplement to the skin's endogenous hyaluronic acid. Regular use of relevant cosmetics is conducive to the transfer of skin nutrients and metabolic waste discharge, maintaining a certain level of water content in the skin, in order to achieve the effect of delaying skin aging, beauty and skin care.

2.2.3 Sun protection and repair of damaged skin:

Hyaluronic acid has a certain effect on sun protection, but its mechanism of action is different from that of ordinary sunscreen. Sunscreens often include ultraviolet absorbers to achieve the purpose of sun protection; hyaluronic acid can largely reduce the transmittance of ultraviolet rays and repair skin damage caused by the small amount of ultraviolet rays that pass through. When the skin is exposed to sunlight, resulting in redness, burning pain, peeling, etc., cosmetics containing hyaluronic acid can be used to promote the proliferation and differentiation of epidermal cells, and hyaluronic acid can be used to remove the effect of oxygen free radicals to help the injured part of the skin to recover [10].

2.2.4 Lubricating and film-forming properties:

Hyaluronic acid has certain lubricating and film-forming properties, which can be used in cosmetics to enhance lubrication, increase the feel of skin care, and make the face feel good, and its film can be formed on the surface of the skin, which can improve the cosmetic effect of the skin, and make the skin have a good feeling of smoothness and moistness. When using shampoo and hair care products containing hyaluronic acid, it is equivalent to coating the surface of the hair with a protective film that lubricates and removes static electricity, which has the effect of making the hair easy to take care of and preventing it from falling off.

2.2.5 Thickening:

Hyaluronic acid has a certain viscosity after absorbing water, and the 1% concentration of aqueous solution is in the form of a gel, which can be added to water, creams and other cosmetics to increase the viscosity and stability.

2.3 Application of hyaluronic acid in cosmetic injection

Whether in foreign countries or domestic big cities, injection cosmetic technology is a popular cosmetic technology today. Through subcutaneous injection of fillers, skin wrinkles can be eliminated quickly, such as lip augmentation, chin augmentation, rhinoplasty and other cosmetic surgeries once used collagen solution as a filler.

Although natural collagen, like hyaluronic acid, is the main component of the extracellular matrix of animal connective tissues, the commercial collagen used in the cosmetic market is generally a protein extracted from animal tissues, and its structure and amino acid composition are not exactly the same as that of collagen in human tissues, so it has a certain degree of immunogenicity, and once injected subcutaneously, it will most likely cause immune response, leading to serious consequences and physical harm and economic losses to consumers. This can lead to serious consequences, causing bodily harm and economic loss to the consumer. Therefore, when using natural collagen as a cosmetic filler, it is necessary to conduct a ‘skin test’ [10].

Hyaluronic acid soft-tissue fillers can be injected or used surgically to eliminate crow's feet, nasolabial folds, and other facial wrinkles to a certain extent. Lip augmentation fillers can reduce the local collapse caused by facial atrophy and some facial scars, such as acne and healing scars. Cross-linked hyaluronic acid fillers cannot be used for breast augmentation as they are not permanent and will be degraded and absorbed by the tissues after a few months.

2.4 Orthopaedic applications of hyaluronic acid

2.4.1 Application in the treatment of joint diseases:

Hyaluronic acid is the main component of cartilage and synovial fluid, and its physiological properties play an irreplaceable role in the function of joints [10]. Abnormalities in the production and metabolism of hyaluronic acid in the joints can lead to the deterioration of osteoarthritis, rheumatoid arthritis, and other infectious and non-infectious joint diseases, as well as to the degradation and destruction of cartilage.

In the treatment of joint diseases, hyaluronic acid can be injected to replenish and replace synovial fluid, mainly through the supplementation of exogenous hyaluronic acid, to promote the repair of cartilage and restore the lubricating ability of synovial fluid, so as to improve the function of joints [11].

2.4.2 Application in the prevention of postoperative adhesions:

Hyaluronic acid has been widely used to prevent postoperative tissue adhesions. As early as 1980, hyaluronic acid was first successfully used in the treatment of tendon repair in order to reduce postoperative adhesions. Numerous animal studies and clinical trials have shown that hyaluronic acid is a safe and effective substance for the prevention and reduction of post-surgical tissue adhesions [12].

2.5 Hyaluronic Acid in Ophthalmic Treatment

2.5.1 Distribution and role of hyaluronic acid in the animal eye:

As an acidic mucopolysaccharide, hyaluronic acid is distributed in the intercellular matrix of various tissues in animals, and has the important physiological functions of maintaining a stable osmotic pressure in cells and bonding adjacent cells [13]. The content of hyaluronic acid in the vitreous humor of animal eyes is high, and the content of hyaluronic acid in the vitreous humor of adult animals is higher than that of juvenile animals. Of course, the distribution of hyaluronic acid in the vitreous body of the eye is not uniform, with a low level of hyaluronic acid in the middle of the vitreous body of the eye and a high level of hyaluronic acid in the vicinity of the ciliary body.

2.5.2 Sodium hyaluronate is used in ophthalmic surgery:

Hyaluronic acid has a wide range of applications in ophthalmic surgery, mainly due to its properties of viscosity, pseudoplasticity, elasticity, adhesion and coatability, which make it have important functions such as viscoelastic cushioning, intra-tissue detachment, viscous occlusion, viscous haemostasis, viscoelastic cushioning, elastic fixation and so on [14].

Currently, hyaluronic acid is used as a viscoelastic agent in many ophthalmic surgeries, such as retina-related surgeries, cataract-related surgeries, and artificial lens-related surgeries.

The sodium salt of hyaluronic acid plays a very important role in IOL implantation and cataract surgery. For example, hyaluronic acid-Na can be injected into the anterior chamber of the cornea in order to form a protective layer in the cornea's endothelium, which not only reduces the rate of endothelial cell loss, but also reduces the damaging effects on the endothelial cells due to mechanical shear and the implantation of IOLs; furthermore, in order to implant a lens into the cornea, hyaluronic acid can be injected into the anterior chamber. In addition, it can be used to deepen the anterior chamber and open the lens capsule so that the anterior chamber IOL can be easily slid into the anterior chamber for smooth lens implantation [15].

By injecting hyaluronic acid into the anterior chamber, the normal depth of the anterior chamber can be maintained, the complications of insufficient aqueous secretion and choroidal detachment after glaucoma surgery can be reduced, the occurrence of shallow anterior chamber can be prevented, and the injection of hyaluronic acid-Na in the anterior chamber and subconjunctival valve can reduce the rate of haemorrhage, post-surgical scarring and post-surgical adhesion, and increase the rate of functional follicle formation. Therefore, the use of hyaluronan-Na in ophthalmic surgery can better prevent the formation of postoperative scarring, reduce intraocular pressure and the incidence of shallow anterior chamber and choroidal detachment, and is a safe, reliable, and effective treatment for glaucoma [16].

Hyaluronic acid-Na also plays an important role in corneal surgery, mainly in its protective effects on tissues, such as avoiding damage to intraocular tissues by surgical instruments, reducing astigmatism in the postoperative period, reducing the sudden loss of aqueous humor, avoiding postoperative adhesions, and facilitating postoperative recovery of epithelial tissues. The use of hyaluronic acid-Na in corneal transplantation can effectively protect the corneal tissue and promote the recovery of the transparency of the corneal implant [16].

2.5.3 The role of sodium hyaluronate in eye lubrication:

Dry eye disease is a common comprehensive ophthalmic disease, which is mainly caused by the dysfunction of certain ocular gland cells, including various types of conjunctivitis. Hyaluronic acid-Na has a prolonged residence time in the eye due to its non-Newtonian fluid properties. Hyaluronic acid-Na also has more hydrophilic groups, which can combine with water molecules to achieve hydrophilicity and lubrication, so it can alleviate the symptoms of dry eyes to a certain extent. This is because when blinking, hyaluronic acid-Na has a certain degree of viscoelasticity like tear mucin, so it can replace the role of tear mucin and alleviate the discomfort of dry eyes [17].

2.5.4 The role of hyaluronic acid in ophthalmic preparations:

The properties of dilute solution of hyaluronic acid are close to those of tear fluid, which belongs to non-Newtonian fluid, and its viscosity and elasticity are the same as those of animal tear fluid, with good biotolerance. Therefore, hyaluronic acid solution can be used as a pharmaceutical medium to thicken ophthalmic preparations, and its effect is better than the general chemical thickeners.

Currently, hyaluronic acid-Na is also widely used as an ophthalmic agent to treat intraocular inflammation by intraocular injection of highly concentrated hyaluronic acid mixed with anti-inflammatory drugs in a gel. This method is different from systemic medications such as oral medications or intravenous injections, and it has the characteristics of quick effect and less frequent administration, which can reduce the pain of patients [17].

2.5.5 Other applications of hyaluronic acid in ophthalmology:

In addition to the above applications, hyaluronic acid also plays a role in many other clinical applications in ophthalmology, such as ocular trauma, anterior chamber haemorrhage removal, extraocular muscle surgery and oculoplastic surgery.

2.5.6 Adverse effects:

Increased intraocular pressure is a common complication following ophthalmic surgery due to blockage of ocular outflow pathways, e.g., tissue debris, tissue oedema, and tissue residue [17]. This complication occurs mainly a few hours after injection and peaks when the postoperative intraocular pressure normalises, and the peak intraocular pressure is related to the concentration of hyaluronan-Na. In order to reduce the postoperative increase in intraocular pressure and in patients with glaucoma, it is generally recommended that hyaluronan be aspirated or flushed out of the anterior chamber after surgery.

When hyaluronic acid is used in ocular surgery, inflammatory reactions and aqueous clouding may occasionally occur within 24 hours, and some inflammatory reactions may persist for several weeks, mainly due to impurities such as protein or nucleic acid residues in the hyaluronic acid formulation, as well as damage to surgical instruments, residual tissue debris, and lack of timely removal of blood [17].

2.6 Other Applications of Hyaluronic Acid

2.6.1 Application in urology:

Hyaluronic acid can be injected directly into the bladder as a temporary replacement for the lack of a protective layer of bladder epithelial glycosaminoglycans, thus achieving the purpose of treating interstitial cystitis [18]. Currently, hyaluronic acid is being used for a wider range of indications than just interstitial cystitis, which provides a broader market opportunity for hyaluronic acid products [19].

2.6.2 Application in disease diagnosis:

Given that the level of hyaluronic acid in the body shows a significant increase in the onset of many diseases, it is possible to reflect the changes of various diseases through the measurement of hyaluronic acid in the blood serum.

3 Production methods of hyaluronic acid

Hyaluronic acid can be produced by two methods: animal tissue extraction and microbial fermentation. Among them, microbial fermentation is the main method of hyaluronic acid production at present.

3.1 Animal Tissue Extraction

Hyaluronic acid exists in almost all animal tissues and is widely distributed. Animal materials that can be used for hyaluronic acid extraction mainly include the vitreous body of chicken crown and cow's eye, and human umbilical cord and so on. The main operating procedures are:

Raw materials → acetone or ethanol to raw materials degreasing, dehydration, air drying → soaked in distilled water, filtration → aqueous sodium chloride and chloroform solution treatment → add trypsin insulation → ion exchange agent treatment, purification → refined hyaluronic acid.

Animal tissue extraction method has higher requirements for raw materials, which must be fresh and safe. The raw materials of animal tissues are expensive, and it is difficult to obtain some of them, which is greatly affected by the growth cycle of animals and the seasons, so the purity of hyaluronic acid in the raw materials of animal tissues is not high, and the extraction yield is low.

The extraction of hyaluronic acid is complicated by the consumption of large amounts of organic solvents and hydrolytic enzymes, and the number of operating units increases the cost of hyaluronic acid extraction. In addition, due to the low purity of hyaluronic acid extracted from animal tissues, further refinement and purification of the product is more complicated, which limits the application of the product. The extraction method can no longer meet the current market needs, so fermentation has basically replaced the extraction method for the production of hyaluronic acid [20].

3.2 Microbial fermentation

The production of hyaluronic acid by microbial fermentation has been studied since the 1930s. Since hyaluronic acid in the fermentation broth exists in a free state, it is easy to isolate and purify hyaluronic acid. Therefore, the production of hyaluronic acid by microbial fermentation method has more advantages than that by animal tissue extraction method, such as low production cost and unlimited scale of raw materials.

There are two types of streptococci that can produce hyaluronic acid: group A and group C. Group A mainly includes Streptococcus pyogenes, which is generally not used as a production strain because of its strong pathogenicity; group C includes Streptococcus zooepidemicus, Streptococcus equi, and Streptococcus equi, etc., which are all used in the production of hyaluronic acid, and are also used in the production of hyaluronic acid. Group C includes S. zooepidemicus, S. equi, S. equisimilis, etc., all of which are non-pathogenic bacteria, so they can be used as hyaluronic acid production strains [21]. Luo Ruiming et al. [22] isolated Streptococcus zooepidemicus strain NUF-035 from the lung fluid of sheep with pneumonia, and the yield of hyaluronic acid obtained by optimising the medium was 1.88 g/L. Feng Jiansheng et al.

Feng Jianjian et al. [23] used Streptococcus equi SH0 as the starting strain and selected a genetically stable, non-hemolytic and hyaluronidase-deficient strain SH0201 by physical and chemical mutagenesis, and obtained hyaluronic acid with a relative molecular weight of 2.06×106 Da by shake flask fermentation. Li Zigang et al[24] isolated a strain of Streptococcus faecalis from the nasal mucosa of dairy cows, and the yield of hyaluronic acid could reach 6.94 g/L after UV mutagenesis and nitrosoguanidine mutagenesis.

The quality and yield of hyaluronic acid produced by microbial fermentation are mainly affected by the following aspects: the selection of strains, the optimisation of medium and fermentation conditions, and the downstream technology of biotechnology, i.e., the extraction of hyaluronic acid [25-27]. So far, many scholars at home and abroad have conducted research on the production of hyaluronic acid by microbial fermentation.

Guo Xueping et al. [28] carried out research on the fermentation production process of hyaluronic acid, and carried out laboratory pilot and production workshop pilot studies; Chen Yonghao et al. [29] used γ-rays and ultraviolet rays in combination with irradiation for mutation breeding, and obtained non-haemolytic strains, so that the yield of hyaluronic acid and relative molecular mass has been further improved; Yang Li et al. Yang Li et al. [30] explored the factors affecting the molecular weight of hyaluronic acid, and obtained the relationship between the dissolved oxygen level and stirring speed and the molecular weight of hyaluronic acid; Fu Li et al. [31] screened a hyaluronic acid-producing bacterium from the natural world; Ye Hua et al. [32] studied the process of adding hyaluronic acid to the fermentation medium; Shi Peng [33] researched on the process of hyaluronic acid production and extraction by the fermentation method; Hao Ning et al. Hao Ning et al. [34] carried out genetic modification of hyaluronic acid producing bacteria, and the yield of hyaluronic acid produced by recombinant bacteria was greatly improved.

Holmstrm [35] and Johns [36] optimized the fermentation nutrient conditions of hyaluronic acid by shaking flasks and then fermenting in small fermentation tanks; Kim et al [37] selected Streptococcus zooepidemicus and optimized the cultivation conditions; Armstrong [38] and Chong [39] studied the fermentation conditions of hyaluronic acid. 38] and Chong [39] obtained the relationship between the culture conditions of hyaluronic acid-producing bacteria on the yield and molecular weight of hyaluronic acid.

The main process of hyaluronic acid fermentation production is as follows: slant seed → shake bottle seed → inoculation → fermentation culture → replenishment → tank placement → fermentation broth (crude extraction with ethanol) → crude extraction (add filter aid, activated carbon, adjust pH value) → filtration (remove impurities) → filtrate (ethanol precipitation) → precipitate (dehydration and drying) → product hyaluronic acid [40-41].

4 Outlook

The market demand and sales of hyaluronic acid are increasing every year. The research and development of hyaluronic acid mainly focuses on the molecular modification of hyaluronic acid and the application of its derivatives in various industries. The selection of strains, optimisation of fermentation conditions, improvement of the extraction process and analytical modifications can expand the range of applications of hyaluronic acid and increase its economic value.

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