TWI864716B - Method of continuously obtaining hyaluronic acids with multiple molecular weights and hyaluronic acid composition - Google Patents

Abstract

A method of continuously obtaining hyaluronic acids with multiple molecular weights is provided in some embodiments of the present disclosure, including: providing hy-aluronic acid powder; and placing the hyaluronic acid powder in an environment not lower than 80℃ and not higher than 100℃ and obtaining a portion of hyaluronic acid powder multiple times within 48 hr to 864 hr after placing the hyaluronic acid powder. A hyaluronic acid composition is further provided in some embodiments of the present disclosure.

Description

Method for continuously obtaining hyaluronic acid of various molecular weights and hyaluronic acid composition

This disclosure relates to a method for continuously obtaining hyaluronic acid of various molecular weights and a hyaluronic acid composition.

Hyaluronic acid has excellent biocompatibility, excellent bioabsorbability, and low carcinogenic risk, and is the main functional ingredient in current skin care.

In order to obtain hyaluronic acid of a specific molecular weight, hyaluronic acid needs to be degraded. The current degradation methods include chemical degradation (such as acid hydrolysis, free radical-assisted degradation, etc.), bioenzyme degradation, and physical degradation (such as ultrasound treatment, gamma ray irradiation, electron beam irradiation, microwave, liquid phase heating, etc.).

Chemical degradation is usually difficult to control, resulting in a wide molecular weight distribution. This method can add other chemicals to terminate the reaction, but the added chemicals often require additional cleaning processes, and if the cleaning is not complete, it may cause the finished product to continue to degrade. Depending on the added chemicals, the hyaluronic acid product after degradation may contain chemical byproducts, which in turn affects the safety and effectiveness of the final product.

The bio-enzyme degradation method is relatively expensive, sensitive to environmental conditions (such as temperature and pH), and requires strict maintenance of precise conditions for each reaction. In addition, the enzymes after degradation cannot be completely removed, which may contaminate the finished product and affect its stability, stability and effectiveness.

Ultrasonic degradation has high requirements for equipment. The viscosity of high-molecular hyaluronic acid is high and it is not easy to dissolve. The amount of liquid that can be treated at one time is limited. When the volume of the solution treated by ultrasound increases, the ultrasound will act unevenly in the system, resulting in too wide a molecular weight distribution of the finished product, making it difficult to obtain a product with consistent properties. Therefore, this method is not suitable for large-scale industrial use. High-power ultrasound may generate high heat locally, causing unexpected changes in the molecular structure of hyaluronic acid, affecting the performance of the finished product.

Both gamma ray and electron beam irradiation require the use of expensive equipment. Gamma ray irradiation of hyaluronic acid liquid will cause unexpected degradation, causing the liquid to turn yellow. The process of irradiating hyaluronic acid powder with electron beams is not conducive to scale-up to industrial processes. Gamma ray or electron beam irradiation of hyaluronic acid powder will produce free radicals, which have unpredictable side effects.

Microwave treatment requires expensive special equipment to maintain a stable temperature. The heat generated when microwaving hyaluronic acid powder is uneven, which can easily lead to extremely high temperatures in some areas and damage the molecular structure. When microwaving hyaluronic acid solution, acid needs to be added to assist, and there are volume restrictions, which is not conducive to mass production.

Liquid phase heating treatment requires dissolving hyaluronic acid back into the solvent to allow the liquid to be heated evenly. A high temperature and high pressure sterilization autoclave can be used, but its degradation efficiency is very limited and the molecular weight distribution of the finished product is too wide. Acid will be added to increase the degradation efficiency.

The method of utilizing free radicals to degrade hyaluronic acid includes using strong oxidants to add free radicals (such as hydrogen peroxide). Although the molecular weight distribution of hyaluronic acid after degradation by this method is narrow, the types of molecular weights of hyaluronic acid produced are quite limited.

Another method is to use copper ions as a catalyst (such as copper chloride) to activate free radicals in the system to cleave glycosidic bonds. This method requires precise control of the reaction process and may cause copper ions to remain in the finished product. In addition, this method will result in a wide molecular weight distribution of hyaluronic acid after degradation, and the waste solution containing metal ions needs to be properly handled to prevent damage to the environment.

The above existing degradation methods have limitations such as difficulty in removing impurities, high difficulty in purification, cumbersome operation, high cost and too wide molecular weight distribution. In addition, the hyaluronic acid liquid needs to be dried into powder particles before it can become a finished product.

Therefore, how to provide a method for obtaining hyaluronic acid with a variety of molecular weights and narrow molecular weight distribution in a continuous manner with simple steps, low cost and low residual impurities is a problem to be solved.

Some embodiments of the present disclosure provide a method for continuously obtaining hyaluronic acid of multiple molecular weights, comprising: providing hyaluronic acid powder; and placing the hyaluronic acid powder in an environment of not less than 80°C and not more than 100°C, and taking out part of the hyaluronic acid powder from the environment multiple times between 48 hours and 864 hours after placement.

In some embodiments, the initial weight average molecular weight of hyaluronic acid in the hyaluronic acid powder is 1.5x10 ⁶ Daltons to 3x10 ⁶ Daltons.

In some embodiments, the step of placing the hyaluronic acid powder at 80°C to 100°C is placing it at 90°C.

In some embodiments, in the step of removing a portion of the hyaluronic acid powder from the environment, the number average molecular weight of the hyaluronic acid in the hyaluronic acid powder is 5x10 ⁴ Daltons to 6x10 ⁵ Daltons.

In some embodiments, in the step of removing a portion of the hyaluronic acid powder from the environment, the polymer dispersibility index of the hyaluronic acid in the hyaluronic acid powder is 1 to 1.2.

In some embodiments of the present disclosure, a hyaluronic acid composition is provided, comprising a first hyaluronic acid, a second hyaluronic acid, and a third hyaluronic acid. The first hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a first time. The second hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a second time. The third hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a third time, wherein the first time, the second time, and the third time are all different.

In some embodiments, the first time is 48 hours to 60 hours, the second time is 84 hours to 108 hours, and the third time is 132 hours to 864 hours.

In some embodiments, the number average molecular weight of the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid is less than 6x10 ⁵ Daltons.

In some embodiments, the number average molecular weight of the first hyaluronic acid is 4x10 ⁵ Daltons to 6x10 ⁵ Daltons, the number average molecular weight of the second hyaluronic acid is 3x10 ⁵ Daltons to 3.95x10 ⁵ Daltons, and the number average molecular weight of the third hyaluronic acid is 2x10 ⁵ Daltons to 2.95x10 ⁵ Daltons.

In some embodiments, the polymer dispersibility index of the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid are all 1 to 1.2.

In some embodiments, the weight ratio of the first hyaluronic acid to the second hyaluronic acid is 5:1 to 1:5, and the weight ratio of the second hyaluronic acid to the third hyaluronic acid is 1:1 to 1:10.

In some embodiments, the hyaluronic acid composition further includes water, wherein the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid are dissolved in the water to form a hyaluronic acid aqueous solution, the viscosity of the hyaluronic acid aqueous solution is 100 to 200 mPa.s at a shear rate of 0.1/s to 22/s, and the viscosity of the hyaluronic acid aqueous solution is 30 to 150 mPa.s at a shear rate of 22/s to 2000/s.

In some embodiments, the hyaluronic acid composition further includes water, wherein the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid are dissolved in the water to form a hyaluronic acid aqueous solution, and when the weight percentage of the hyaluronic acid aqueous solution is 100%, the total weight percentage of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid in the hyaluronic acid aqueous solution is 0.1% to 1%.

In order to make the description of the present invention more detailed and complete, the following describes in detail the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The various embodiments disclosed below can be combined or replaced with each other in beneficial situations, and other embodiments can be added to one embodiment without further recording or explanation. In the following description, many specific details will be described in detail so that the reader can fully understand the following embodiments. However, the embodiments of the present invention can be practiced without these specific details.

In this document, unless the context specifically limits the article, "a", "an" and "the" may refer to one or more. It will be further understood that the words "include", "including", "have" and similar words used in this document specify the features, regions, integers, steps, operations, elements and/or components recorded therein, but do not exclude other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the term "about" means a value of a given amount that varies within 5% of the value (e.g., ±1%, ±2%, ±3%, ±4%, 5%). These values are examples only and are not intended to be limiting. It should be understood that the term "about" may mean a percentage of the value of a given amount as interpreted by a person skilled in the relevant art in light of the teachings herein.

Herein, the molecular weight may include the number average molecular weight Mn and the weight average molecular weight Mw.

Although a series of operations or steps are used below to illustrate the methods disclosed herein, the order in which these operations or steps are shown should not be construed as a limitation of the present invention. For example, certain operations or steps may be performed in a different order and/or simultaneously with other steps. In addition, not all operations, steps, and/or features must be performed to implement the present invention. Furthermore, each operation or step described herein may include a number of sub-steps or actions.

Referring to FIG. 1 , some embodiments of the present disclosure provide a method 100 for continuously obtaining hyaluronic acid of various molecular weights, comprising: step S110, providing hyaluronic acid powder; step S120, placing the hyaluronic acid powder in an environment of not less than 80°C and not more than 100°C; and step S130, taking out a portion of the hyaluronic acid powder from the environment multiple times between 48 hours and 36 days after placement. By heating the hyaluronic acid powder and taking it out at different times, hyaluronic acid of different molecular weights can be continuously obtained from the same source, and the molecular weight distribution of each hyaluronic acid is highly concentrated (i.e., the polymer dispersity index (PDI; PDI=Mw/Mn) is between 1.0 and 1.2).

Compared to the known chemical degradation method, the thermal degradation powder method disclosed herein is a temperature-controlled powder physical degradation method. Compared to the traditional decomposition method that requires the use of chemical reagents, this method avoids the need for added substances, thereby avoiding the impact on the properties of hyaluronic acid, and improving the purity, achieving the goal of low-allergy ingredients. In addition, compared to the bio-enzyme degradation method, the thermal degradation powder method disclosed herein does not require the use of hyaluronic acid degrading enzymes, and can omit the step of removing biological impurities such as proteins or endotoxins, and has the advantages of lower cost and simpler operation. On the other hand, compared with the liquid phase heating method which is also a physical degradation method, the liquid phase heating method has the problem of too wide distribution of hyaluronic acid molecular weight after degradation, and the concentration of hyaluronic acid in the solution is limited due to the large molecular weight of hyaluronic acid, so the liquid phase heating method also has the problem of poor efficiency. The thermal degradation powder method disclosed in the present disclosure directly uses powder for thermal degradation, so there is no problem of concentration limitation after dissolution, and the molecular weight of hyaluronic acid can be maintained at a high concentration after degradation. Therefore, the properties of hyaluronic acid after degradation can be more accurately controlled.

In some embodiments, the initial weight average molecular weight (Mw) of hyaluronic acid in the hyaluronic acid powder is 1.5x10 ⁶ Daltons to 3x10 ⁶ Daltons, for example, 1.5x10 ⁶ Daltons, 1.75x10 ⁶ Daltons, 2x10 ⁶ Daltons, 2.25x10 ^{6 Daltons, 2.5x10 6 Daltons} , 2.75x10 ⁶ Daltons, 3x10 ⁶ Daltons or a combination thereof. In some embodiments, the initial number average molecular weight (Mn) of the hyaluronic acid powder is 1.25x10 ⁶ Dalton to 3x10 ⁶ Dalton, such as 1.25x10 ⁶ Dalton, 1.5x10 ⁶ Dalton, 1.75x10 ⁶ Dalton, 2x10 ⁶ Dalton, 2.25x10 ⁶ Dalton, 2.5x10 6 Dalton, 2.75x10 ⁶ Dalton, 3x10 ⁶ Dalton, or any value in the foregoing range. If the molecular weight of the hyaluronic ^acid powder is too large, it will take a long time to degrade to the desired molecular weight, and if the molecular weight is too small, fewer types of hyaluronic acid can be obtained by degradation. By selecting hyaluronic acid powder with an appropriate starting molecular weight, the process parameters can be controlled, thereby differentially producing hyaluronic acid with a wide range of molecular weights.

In some embodiments, the step of placing the hyaluronic acid powder at 80°C to 100°C includes placing it at 80°C, 85°C, 90°C, 95°C, 100°C or any value in the aforementioned range. If the temperature is too low, the thermal degradation effect is not good. If the temperature is too high, the hyaluronic acid powder will turn from white to yellow, indicating that an unexpected chemical reaction has occurred and impurities have increased. Therefore, it cannot support long-term heat treatment. In one embodiment, the step of placing the hyaluronic acid powder at 80°C to 100°C includes placing it at 90°C. By selecting 90°C, while maintaining good thermal degradation efficiency, the hyaluronic acid powder can be prevented from turning yellow prematurely, thereby increasing the thermal degradation duration of the hyaluronic acid powder and reducing the molecular weight of the extracted part of the hyaluronic acid to a lower limit.

In some embodiments, a portion of the hyaluronic acid powder (i.e., hyaluronic acid treated with thermal degradation) is removed from the environment between 48 hours and 864 hours after placement, including 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 72 hours, 84 hours, 88 hours, 92 hours, 96 hours, 100 hours, 104 hours, 108 hours, 132 hours, 144 hours, 192 hours, 216 hours, 240 hours, 288 hours, 336 hours, 408 hours, 480 hours, 552 hours, 600 hours, 672 hours, 744 hours, 816 hours, 864 hours or values in between after placement. If it is taken out too early, the molecular weight of the hyaluronic acid powder taken out will be too high and will not meet the actual needs; if it is taken out too late, the waiting time will be too long and will not meet the production efficiency.

In some embodiments, in the step of removing a portion of the hyaluronic acid powder from the environment, the number average molecular weight of the hyaluronic acid in the hyaluronic acid powder is 5x10 ⁴ Dalton to 6x10 ⁵ Dalton, such as 5x10 ⁴ Dalton, 6x10 ⁴ Dalton, 7x10 ⁴ Dalton, 8x10 ⁴ Dalton, 9x10 ⁴ Dalton, 1x10 ⁵ Dalton, 2x10 ⁵ Dalton, 3x10 ⁵ Dalton, 4x10 ⁵ Dalton, 5x10 ⁵ Dalton, 6x10 ⁵ Dalton or any value in the aforementioned range. The molecular weight of the degraded hyaluronic acid can be adjusted by selecting an initial hyaluronic acid powder with a suitable molecular weight and the placement time.

For example, when the initial number average molecular weight (Mn) of the hyaluronic acid in the hyaluronic acid powder is 1.5x10 ⁶ Daltons to 3x10 ⁶ Daltons, after being placed in an environment at 90°C for 52 hours (2 days and 4 hours), the number average molecular weight of the hyaluronic acid in the hyaluronic acid powder is 4x10 ⁵ to 6x10 ⁵ Daltons; when the hyaluronic acid powder is placed in an environment at 90°C for 96 hours (4 days), the number average molecular weight of the hyaluronic acid in the hyaluronic acid powder is 3x10 ⁵ to 3.95x10 ⁵ Daltons; when the hyaluronic acid powder is placed in an environment at 90°C for 144 hours (6 days), the number average molecular weight of the hyaluronic acid in the hyaluronic acid powder is 2x10 ⁵ to 2.95x10 ⁵ Daltons.

It can be understood that the degraded hyaluronic acid obtained by heat-treating the hyaluronic acid powder has a highly concentrated molecular weight distribution. In some embodiments, in the step of removing a portion of the hyaluronic acid powder from the environment, the PDI of the hyaluronic acid in the hyaluronic acid powder is 1 to 1.2, such as 1.0, 1.1, 1.2 or a value in the aforementioned range. If the PDI is too high, the number average molecular weight distribution of the hyaluronic acid is too dispersed, and the properties of the hyaluronic acid are too diverse.

Some embodiments of the present disclosure provide a hyaluronic acid composition, comprising a first hyaluronic acid, a second hyaluronic acid, and a third hyaluronic acid. The first hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a first time. The second hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a second time. The third hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a third time, wherein the first time, the second time, and the third time are all different.

The hyaluronic acid composition uses hyaluronic acid obtained by treating hyaluronic acid powder with different heat treatment times to obtain individual hyaluronic acids with highly concentrated molecular weights (PDI is 1.0 to 1.2), which can be used to accurately adjust the properties of the hyaluronic acid composition. In addition, through the different molecular weight characteristics of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid, the hyaluronic acid composition can simultaneously perform three different skin care mechanisms on the skin, thereby achieving a more comprehensive skin care effect.

In some embodiments, the first time is 48 hours to 60 hours (e.g., 48 hours, 52 hours, 56 hours, 60 hours, or a value therebetween), the second time is 84 hours to 108 hours (e.g., 84 hours, 88 hours, 92 hours, 96 hours, 100 hours, 104 hours, 108 hours, or a value therebetween), and the third time is 132 hours to 864 hours (e.g., 132 hours, 144 hours, 192 hours, 216 hours, 240 hours, 288 hours, 336 hours, 408 hours, 480 hours, 552 hours, 600 hours, 672 hours, 744 hours, 816 hours, 864 hours, or a value therebetween). By selecting hyaluronic acid taken out at different time points, the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid with different molecular weights are obtained.

In some embodiments, the number average molecular weight of the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid is less than 6x10 ⁵ Daltons, for example, the number average molecular weight of the first hyaluronic acid is 4x10 ⁵ Daltons to 6x10 ⁵ Daltons, the number average molecular weight of the second hyaluronic acid is 3x10 ⁵ Daltons to 3.95x10 ⁵ Daltons, and the number average molecular weight of the third hyaluronic acid is 2x10 ⁵ Daltons to 2.95x10 ⁵ Daltons.

It is understandable that when the hyaluronic acid composition is applied to the skin (for example, when dissolved in water to form a hyaluronic acid aqueous solution), the number average molecular weight of hyaluronic acid is less than 6x10 ⁵ Daltons, which can achieve better skin absorption efficiency. In addition, through the different molecular weights of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid, the design of having different retention properties on the skin surface can make the skin continuously absorb hyaluronic acid of different molecular weights in stages, thereby extending the duration of hyaluronic acid acting on the skin. In addition, hyaluronic acid of different molecular weights can act on different depth layers of the skin to enhance the skin's moisturizing effect.

For example, the first hyaluronic acid with a molecular weight between 4x10 ⁵ Daltons and 6x10 ⁵ Daltons can stay on the surface of the stratum corneum to provide a moisturizing effect; the second hyaluronic acid with a molecular weight of about 3x10 ⁵ Daltons can penetrate the stratum corneum and act between the stratum corneum and the epidermis to achieve a surface hydrating effect; the third hyaluronic acid with a molecular weight below 3x10 ⁵ Daltons can penetrate the epidermis and enter the dermis to achieve a deep hydrating effect.

In one embodiment, the number average molecular weight of the first hyaluronic acid is 4x10 ⁵ Daltons, 5x10 ⁵ Daltons, 6x10 ⁵ Daltons, or values in the aforementioned ranges. In one embodiment, the number average molecular weight of the second hyaluronic acid is 3x10 ⁵ Daltons, 3.15x10 ⁵ Daltons, 3.25x10 ⁵ Daltons, 3.35x10 ⁵ Daltons, 3.45x10 ⁵ Daltons, 3.55x10 ⁵ Daltons, 3.65x10 ⁵ Daltons, 3.75x10 ⁵ Daltons, 3.85x10 ⁵ Daltons, 3.95x10 ⁵ Daltons, or values in the aforementioned ranges. In one embodiment, the number average molecular weight of the third hyaluronic acid is 2x10 ⁵ Daltons, 2.15x10 ⁵ Daltons, 2.25x10 ⁵ Daltons, 2.35x10 ⁵ Daltons, 2.45x10 ⁵ Daltons, 2.55x10 ⁵ Daltons, 2.65x10 ⁵ Daltons, 2.75x10 ⁵ Daltons, 2.85x10 ⁵ Daltons, 2.95x10 ⁵ Daltons or a value in the foregoing range.

In some embodiments, the PDI of the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid are all 1 to 1.2. It can be understood that the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid obtained by heat treatment of the hyaluronic acid powder have a highly concentrated molecular weight distribution, thus avoiding the problem of the properties of the hyaluronic acid composition being difficult to control due to the excessively high PDI and the overly dispersed molecular weight distribution of the hyaluronic acid.

In some embodiments, the weight ratio of the first hyaluronic acid to the second hyaluronic acid is 5:1 to 1:5 (e.g., 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5 or any value in the aforementioned range), and the weight ratio of the second hyaluronic acid to the third hyaluronic acid is 1:1 to 1:10 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any value in the aforementioned range). The contents of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid can be adjusted according to actual application requirements. For example, when the weight of the first hyaluronic acid with a higher molecular weight is relatively high, the first hyaluronic acid with a higher molecular weight has better retention in the skin and can therefore provide long-lasting moisturizing. When the weight of the third hyaluronic acid with a lower molecular weight is relatively high, the skin's absorption of hyaluronic acid can be accelerated.

In some embodiments, the hyaluronic acid composition further comprises water, and the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid are dissolved in the water to form a hyaluronic acid aqueous solution.

It can be understood that by adjusting the molecular weight and weight ratio of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid, the rheological properties of the hyaluronic acid aqueous solution can be adjusted, thereby formulating hyaluronic acid aqueous solutions with different application feels.

For example, when the shear rate of the hyaluronic acid aqueous solution is 0/s to 0.1/s, the viscosity is 150 mPa·s to 350 mPa·s (e.g., 150 mPa·s, 200 mPa·s, 250 mPa·s, 300 mPa·s, 350 mPa·s, or values in the aforementioned range). When the shear rate of the hyaluronic acid aqueous solution is 0.1/s to 22/s, the fluid properties are close to Newtonian fluids, and the hyaluronic acid aqueous solution has a certain shear resistance, and the viscosity is 100 mPa·s to 200 mPa·s (e.g., 100 mPa·s, 125 mPa·s, 150 mPa·s, 175 mPa·s, 200 mPa·s, or values in the aforementioned range). When the shear rate of the hyaluronic acid aqueous solution is between 22/s and 2000/s, it turns back into the commonly known pseudoplastic fluid, and the viscosity decreases with the increase of the shear rate. The viscosity is between 30 mPa.s and 150 mPa.s (for example, 30 mPa.s, 50 mPa.s, 75 mPa.s, 100 mPa.s, 125 mPa.s, 150 mPa.s, or values in the aforementioned range).

In other words, the hyaluronic acid aqueous solution has shear resistance at low shear rates, and as the shear rate increases, the viscosity gradually decreases. The above characteristics can give the hyaluronic acid aqueous solution a better application feel when applied to the skin.

In some embodiments, when the weight percentage of the hyaluronic acid aqueous solution is 100%, the weight percentage of the sum of the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid in the hyaluronic acid aqueous solution is 0.1% to 1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, or a value in the aforementioned range). By controlling the weight percentage of the sum of the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid within a suitable range, when applied to the skin, the effect of the hyaluronic acid aqueous solution on skin care and the skin feel can be adjusted.

Some embodiments of the present disclosure also provide a use of a hyaluronic acid aqueous solution for skin care, including applying the hyaluronic acid aqueous solution to the skin of an individual. Due to the different number average molecular weights of the first hyaluronic acid, the second hyaluronic acid, and the third hyaluronic acid, the hyaluronic acid has different retention properties on the skin surface, and can act on different depth layers of the skin, while achieving the effects of skin moisturizing and hydrating. For example, the molecular weight of hyaluronic acid is between 4x10 ⁵ Daltons and 6x10 ⁵ Daltons, so it can stay on the surface of the stratum corneum to provide a moisturizing effect. The molecular weight of hyaluronic acid is about 3x10 ⁵ Daltons, so it can act between the stratum corneum and the epidermis. The molecular weight of hyaluronic acid is less than 3x10 ⁵ Daltons, so it can penetrate the epidermis and enter the dermis to achieve a deep hydrating effect.

In order to further illustrate the methods of continuously obtaining hyaluronic acid of various molecular weights and hyaluronic acid aqueous solutions provided by various embodiments of the present disclosure, the following embodiments are performed. It should be noted that the following embodiments are provided for exemplary purposes only and are not intended to limit the present invention.

1. Comparison of different thermal degradation temperatures

To test the effect of different thermal degradation temperatures on the degradation of hyaluronic acid, hyaluronic acid powder (water content less than 15%) was placed in a room temperature (e.g. 25˚C, as a control group), 90˚C and 120˚C environment (placed in a hot air circulation oven (brand: DENG YNG; model: DO60), humidity range 8% to 12%) for 24 hours of thermal degradation. The results are shown in Figure 2.

Figure 2 shows that HA powder turns yellow rapidly in a 120°C environment. This phenomenon may be caused by the maillard reaction or caramelization caused by impurities in the HA powder. In contrast, HA powder can remain white and does not turn yellow in a 90°C environment. Therefore, 90°C was selected as the subsequent long-term thermal degradation condition.

2. Production methods and properties of hyaluronic acid with different molecular weights

2.1. Manufacturing method

First, hyaluronic acid powder with a weight average molecular weight of ^2.15x106 Daltons (water content less than 15%) was divided into 8 centrifuge tubes (brand: labcon), each centrifuge tube was filled with 10 grams of hyaluronic acid powder, and then the centrifuge tubes were placed in a hot air circulation oven at 90˚C (humidity range of 8% to 12%), and one tube of hyaluronic acid powder was taken out at each time point of 52 hours, 96 hours, 144 hours, 10 days, 14 days, 20 days, 25 days, and 31 days.

2.2. Properties of hyaluronic acid powder taken out at different time points

The hyaluronic acid powders taken out at different time points were analyzed by size-exclusion chromatography-multi-angle laser light-scattering-refractive index-ultraviolet (SEC-MALS-RI-UV) detection (miniDAWN-Optilab ^® ; brand: Wyatt; dn/dc=0.167) to obtain the Mn and PDI of the hyaluronic acid, which are summarized in Table 1 below. At the same time, based on the values in Table 1, the relationship between the Mn and PDI of hyaluronic acid sample 1 and the degradation time is plotted in Figure 3. In addition, Figures 4A and 4B illustrate the detection results of the molecular weight and PDI of hyaluronic acid powder with degradation time of 52 hours and 31 days using SEC-MALS-RI-UV, respectively, wherein the results of Figures 4A and 4B can correspond to Table 1.

Table 1. Correspondence between thermal degradation time, Mn, and PDI of hyaluronic acid Time point 1 2 3 Mn (x10 ⁵ Daltons) PDI Mn (x10 ⁵ Daltons) PDI Mn (x10 ⁵ Daltons) PDI 52 hours 5.162 1.034 5.677 1.038 4.869 1.037 96 hours 3.588 1.038 3.499 1.033 N/A N/A 144 hours 2.750 1.042 3.280 1.041 3.014 1.053 10 days 1.956 1.051 N/A N/A N/A N/A 14 days 1.543 1.055 N/A N/A N/A N/A 20 days 1.175 1.110 N/A N/A N/A N/A 25 days 0.995 1.119 N/A N/A N/A N/A 31 days 0.786 1.174 N/A N/A N/A N/A

Table 1 (See Figure 3) The results show that as the heat treatment time increases, the Mn of hyaluronic acid gradually decreases, while the PDI can be maintained between 1.0 and 1.2, and the color can be observed to remain white and not change color during the process. That is, by directly heating the hyaluronic acid powder in a hot air circulation oven at 90°C, the hyaluronic acid can be degraded while maintaining a highly concentrated distribution of the molecular weight. And in a 90°C environment, even if the hyaluronic acid powder is heated for a long time, the color of the hyaluronic acid powder can still remain white and will not turn yellow.

In contrast, please refer to Table 2, which illustrates the degradation conditions when hyaluronic acid is degraded by other methods, and the Mn and PDI of the degraded hyaluronic acid.

Table 2. Comparison of degradation conditions, Mn and PDI of hyaluronic acid degraded by other methods method result Description (A good PDI here means a PDI of 1 to 1.2) Name Parameters Mn (starting at 1000 kDa) PDI Free Radicals 10 _{mM H2O2} to 60 _{mM H2O2} 560 kilodaltons to 414 kilodaltons 1.106 to 1.063 Good PDI, but unable to obtain various molecular weight hyaluronic acid Copper ions 0.1 μM CuCl ₂ to 6.0 μM CuCl ₂ 413,000 Daltons to 83.5,000 Daltons 1.087 to 1.351 Hyaluronic acid is available in a variety of molecular weights, but good PDI is not achieved pH 3 to 10 82 kilodaltons to 220 kilodaltons 1.309 to 1.527 Unable to obtain various molecular weights of hyaluronic acid and good PDI Liquid phase heating 20°C to 60°C 85.5 thousand daltons to 45.35 thousand daltons 1.407 to 1.539 Unable to obtain various molecular weights of hyaluronic acid and good PDI Ultrasound 135W to 200W 91.11 thousand daltons to 51.67 thousand daltons 1.340 to 1.400 Unable to obtain various molecular weights of hyaluronic acid and good PDI

Table 2 shows that, although good PDI can be obtained by using the free radical degradation method, hyaluronic acid with multiple molecular weights cannot be continuously obtained in the same process. Although hyaluronic acid with multiple molecular weights can be obtained by using the copper ion degradation method, good PDI cannot be obtained (that is, the distribution of molecular weights is too dispersed). As for the pH value method, liquid phase heating method, and ultrasonic method, multiple molecular weights cannot be continuously obtained in the same process, and PDI cannot be maintained in a good range, and the distribution of molecular weights is too dispersed. Therefore, compared with the known method in Table 2, the method of thermal degradation of hyaluronic acid powder in the present application can not only obtain degraded hyaluronic acid with good PDI, but also obtain degraded hyaluronic acid with multiple molecular weights by adjusting the degradation time or the starting molecular weight.

In addition, it should be added that hyaluronic acid from different sources or with different molecular weights is applicable to the thermal degradation method; and, after thermal degradation, the PDI can be maintained at 1.0 to 1.2, and the molecular weight distribution is highly concentrated, for example, please refer to Table 3 below.

Table 3. Correspondence between thermal degradation time, Mn, and PDI of hyaluronic acid powders from different sources and different molecular weights Hyaluronic acid 1 Hyaluronic acid 2 Hyaluronic acid 3 Hyaluronic acid 4 Mn (x10 ⁴ ) PDI Mn (x10 ⁴ ) PDI Mn (x10 ⁴ ) PDI Mn (x10 ⁴ ) PDI Day 0 180 -- 200 -- 220 -- 50 -- 52 hours 52.3 1.02 -- -- 46.6 1.02 20.9 1.03 96 hours 35.2 1.02 -- -- 38.7 1.02 18.9 1.04 144 hours 29.3 1.02 29.4 1.04 32.2 1.03 15.5 1.03 10 days 22.7 1.02 22.1 1.03 22.1 1.02 11.5 1.07 14 days 13.5 1.11 15.7 1.04 18.4 1.03 9.7 1.11 20 days 11.8 1.08 11.2 1.08 13.8 1.07 -- -- 25 days 7.9 1.12 9.7 1.08 9.7 1.09 -- -- 31 days 6.5 1.20 6.3 1.20 8.2 1.12 -- --

Table 3 shows that hyaluronic acid powders from different sources and different initial molecular weights were placed at 90°C for degradation, and some hyaluronic acid powders were taken out at each time point in point 2.1. Table 3 shows the number average molecular weight of the hyaluronic acid powders taken out at each time point. It can be seen that no matter what the source or molecular weight of hyaluronic acid is, it can be degraded at 90°C, and the PDI can be maintained at 1.0 to 1.2. In other words, no matter what kind of hyaluronic acid powder, after thermal degradation, the molecular weight distribution is highly concentrated.

3. Analysis of rheological properties of hyaluronic acid aqueous solution

Since hyaluronic acid of different molecular weights can act on different depths of the skin and provide different usage experiences, in order to test the appropriate formula for application to human skin, hyaluronic acid powder that has not been thermally degraded (HA 0 hours), hyaluronic acid powder that has been thermally degraded for 52 hours (HA 52 hours), hyaluronic acid powder that has been thermally degraded for 96 hours (HA 96 hours), and hyaluronic acid powder that has been thermally degraded for 144 hours (HA 144 hours) were selected from the hyaluronic acid powder in point 2. According to Table 4, each group of hyaluronic acid powder was mixed with water according to different weight ratios to form experimental group 1, experimental group 2, and experimental group 3. Then, using a rheometer, the shear rate and viscosity change trends of experimental group 1, experimental group 2, and experimental group 3 were compared with those of commercial products, and the results are presented in Figure 5.

Table 4. Formula of hyaluronic acid aqueous solution Hyaluronic acid Experimental Group 1 Experimental Group 2 Experimental group 3 Commercially available products HA 0 hours (wt %) 0.1 0.1 HA 52 hours (wt %) 0.1 0.1 HA 96 hours (wt %) 0.1 0.1 HA 144 hours (wt %) 0.4 0.4 0.4 Total content (wt %) 0.6 0.6 0.6 0.6

The results in Figure 5 show that when the shear rate is between 0.131/s and 11.7/s, the viscosity of Experimental Group 3 changes from 168.0 to 123.6 (change rate 26.4%), the viscosity of Experimental Group 2 changes from 378.2 to 199.0 (change rate 47.4%), the viscosity of Experimental Group 1 changes from 470.8 to 221.4 (change rate 53.0%), and the viscosity of the commercial product changes from 73.6 to 21.3 (change rate 71.0%); when the shear rate is between 22.3/s and 2000/s, the viscosity of Experimental Group 3 changes from 118.5 to 30.3 (change rate 74.4%), and the viscosity of Experimental Group 2 changes from 166.1 to 29.4 The viscosity of experimental group 1 varied from 182.7 to 31.0 (variation rate 83.1%), and the viscosity of the commercial product varied from 21.0 to 11.7 (variation rate 44.1%). The overall viscosity ranking from high to low is experimental group 1> experimental group 2> experimental group 3> commercial product.

When the hyaluronic acid aqueous solution is at a low shear rate (0.131/s to 22.3/s), experimental groups 1, 2, and the commercial product show a trend of decreasing viscosity as the shear rate increases. In contrast, experimental group 3 has a certain shear resistance (the smallest rate of change), has unique fluid mechanics, and the fluid properties are close to Newtonian fluid. When the hyaluronic acid aqueous solution is at a high shear rate (22.3/s to 2000/s), each group shows a commonly known pseudoplastic fluid, and the viscosity change is consistent, and the viscosity decreases as the shear rate increases.

Compared with Experimental Groups 1 and 2, Experimental Group 3, which used all thermally degraded hyaluronic acid, had a viscosity greater than 168 mPa·s when the shear rate was less than 0.131/s. However, if the shear rate continued to increase, for example, when the shear rate was from 0.131/s to 22.3/s, the viscosity was maintained at 118.5 mPa·s to 168 mPa·s. Therefore, using Experimental Group 3, the viscosity can be controlled within a specific range, thereby maintaining a consistent hyaluronic acid aqueous solution feel at different application forces.

Therefore, by mixing hyaluronic acid of different molecular weights, in addition to allowing hyaluronic acid of different molecular weights to act on different skin depths, the rheological properties of the hyaluronic acid aqueous solution can also be adjusted, thereby customizing the formula of the hyaluronic acid aqueous solution according to individual skin needs.

Some embodiments of the present disclosure provide a method for continuously obtaining hyaluronic acid of various molecular weights. By heating hyaluronic acid powder and taking it out at different times, various hyaluronic acids that have been degraded into different molecular weights can be continuously obtained while maintaining a highly concentrated molecular weight distribution.

In addition, the hyaluronic acid aqueous solution provided by the present disclosure includes hyaluronic acid powder that has been thermally degraded for different periods of time to produce hyaluronic acid of various molecular weights. Users can adjust the thermal degradation time and the molecular weight of hyaluronic acid according to skin needs, thereby precisely adjusting the application feel of the hyaluronic acid aqueous solution and the degree of skin absorption of hyaluronic acid.

Although the contents of this disclosure have been disclosed as above in the form of implementation, it is not intended to limit the contents of this disclosure. Anyone skilled in the art can make various changes and modifications without departing from the spirit and scope of the contents of this disclosure. Therefore, the protection scope of the contents of this disclosure shall be subject to the scope defined by the attached patent application.

100: Method S110, S120, S130: Steps

In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understandable, the attached figures are described as follows: Figure 1 presents a flow chart of a method for continuously obtaining hyaluronic acid of various molecular weights in some embodiments of the present disclosure. Figure 2 presents a comparison of the appearance of hyaluronic acid powders in each group when the hyaluronic acid powders are placed in different temperature environments for 24 hours in Example 1 of the present disclosure, wherein the color of the hyaluronic acid powder placed at 120˚C is yellow, and the color of the control group and the hyaluronic acid powder placed at 90˚C remains white. Figure 3 presents a relationship diagram between the number average molecular weight and the polymer dispersibility index and the degradation time in Example 2.2 of the present disclosure. Figures 4A and 4B show the detection results of molecular weight and polymer dispersity index at degradation times of 52 hours and 31 days in Example 2.2 of the present disclosure. Figure 5 shows the shear rate and viscosity change trend diagram of Experimental Group 1, Experimental Group 2, and Experimental Group 3 of the present disclosure and the commercial product.

Domestic storage information (please note in the order of storage institution, date, and number) None Foreign storage information (please note in the order of storage country, institution, date, and number) None

100: Method S110, S120, S130: Steps

Claims (11)

A method for continuously obtaining hyaluronic acid of various molecular weights comprises: providing hyaluronic acid powder, wherein the initial weight average molecular weight of hyaluronic acid in the hyaluronic acid powder is ^1.5x106 Dalton to ^3x106 Dalton; and placing the hyaluronic acid powder in an environment of not less than 80°C and not more than 100°C, and taking out part of the hyaluronic acid powder from the environment multiple times between 48 hours and 864 hours after placement. As described in claim 1, the step of placing the hyaluronic acid powder in an environment not lower than 80°C and not higher than 100°C is placing it at 90°C. A method as described in claim 1, wherein in the step of removing a portion of the hyaluronic acid powder from the environment, the number average molecular weight of the hyaluronic acid in the hyaluronic acid powder is 5 x 10 ⁴ Daltons to 6 x 10 ⁵ Daltons. As described in claim 1, in the step of removing a portion of the hyaluronic acid powder from the environment, the polymer dispersibility index of the hyaluronic acid in the hyaluronic acid powder is 1 to 1.2. A hyaluronic acid composition comprises: a first hyaluronic acid, wherein the first hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a first time; a second hyaluronic acid, wherein the second hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a second time; a third hyaluronic acid, wherein the third hyaluronic acid is prepared by placing hyaluronic acid powder at 80°C to 100°C for a third time, wherein the first time, the second time and the third time are different, and wherein the number average molecular weight of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid is less than ^6x105 Dalton. The hyaluronic acid composition as described in claim 5, wherein the first time is 48 hours to 60 hours, the second time is 84 hours to 108 hours, and the third time is 132 hours to 864 hours. A hyaluronic acid composition as described in claim 5, wherein the number average molecular weight of the first hyaluronic acid is 4x10 ⁵ Daltons to 6x10 ⁵ Daltons, the number average molecular weight of the second hyaluronic acid is 3x10 ⁵ Daltons to 3.95x10 ⁵ Daltons, and the number average molecular weight of the third hyaluronic acid is 2x10 ⁵ Daltons to 2.95x10 ⁵ Daltons. The hyaluronic acid composition as described in claim 5, wherein the polymer dispersibility index of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid are all 1 to 1.2. The hyaluronic acid composition as described in claim 5, wherein the weight ratio of the first hyaluronic acid to the second hyaluronic acid is 5:1 to 1:5, and the weight ratio of the second hyaluronic acid to the third hyaluronic acid is 1:1 to 1:10. The hyaluronic acid composition as described in claim 5 further includes water, wherein the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid are dissolved in the water to form a hyaluronic acid aqueous solution, wherein the viscosity of the hyaluronic acid aqueous solution is 100 mPa.s to 200 mPa.s at a shear rate of 0.1/s to 22/s, and the viscosity of the hyaluronic acid aqueous solution is 30 mPa.s to 150 mPa.s at a shear rate of 22/s to 2000/s. The hyaluronic acid composition as described in claim 5 further comprises water, wherein the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid are dissolved in the water to form a hyaluronic acid aqueous solution, wherein when the weight percentage of the hyaluronic acid aqueous solution is 100%, the total weight percentage of the first hyaluronic acid, the second hyaluronic acid and the third hyaluronic acid in the hyaluronic acid aqueous solution is 0.1% to 1%.