Preparation method of sodium hyaluronate oligosaccharide composition, prepared composition and application

Abstract

The invention belongs to the field of biochemistry, particularly relates to a preparation method of a sodium hyaluronate oligosaccharide composition, and further discloses the prepared composition and application. According to the preparation method of the sodium hyaluronate oligosaccharide, macromolecular sodium hyaluronate is used as a raw material and is prepared into an acidic aqueous solution, then ethanol is added for a precipitation reaction, supernate is separated and subjected to impurity removal through an ultrafiltration membrane and secondary ethanol precipitation reaction, and separated precipitate is purified through a reverse-phase ODS column, so that the sodium hyaluronate oligosaccharide with required performance can be obtained. The method has the advantages of simple and feasible preparation process, appropriate reaction conditions and simple and smooth production process, the production period and cost are greatly reduced, and the product yield can reach 96% or above. The sodium hyaluronate oligosaccharide composition prepared by the method disclosed by the invention comprises disaccharide, tetrasaccharide, hexasaccharide, octasaccharide, decasaccharide and dodecasaccharide, and has the advantages of better product stability, excellent water solubility, good transdermal property, moisture retention, good oral absorbability and the like.

Description

Preparation method of sodium hyaluronate oligosaccharide composition and the composition obtained and its application Technical Field

[0001] The present invention belongs to the field of biochemistry, and specifically relates to a method for preparing a sodium hyaluronate oligosaccharide composition, and further discloses the prepared composition and its application. Background Art

[0002] Sodium hyaluronate, a sodium glucuronate with the chemical formula (C14H20NO11Na)n, is an endogenous component of the human body. It is non-species specific and is widely present in tissues such as the placenta, amniotic fluid, lens, articular cartilage, and the dermis of the skin, as well as in the cytoplasm and intercellular matrix of organs, where it lubricates and nourishes the cells and organelles contained therein. Furthermore, sodium hyaluronate is a major component of the extracellular matrix and a key constituent of skin, vitreous humor, synovial fluid, and cartilage. It possesses unique physical and chemical properties and biological functions, playing a vital role in maintaining tissue integrity and in tissue formation and remodeling during infection, injury, and embryonic development.

[0003] Sodium hyaluronate is classified based on its molecular weight, typically including: large-molecule sodium hyaluronate (molecular weight range ≥ 1.6 million Da); medium-molecule sodium hyaluronate (molecular weight range 200,000-1.6 million Da); small-molecule sodium hyaluronate (molecular weight range 10,000-200,000 Da); and low-molecule sodium hyaluronate (molecular weight range 3,000-10,000 Da). Although sodium hyaluronate has been widely used in medical and pharmaceutical applications, daily chemical products, nutritional foods, and pet food, the currently used sodium hyaluronate generally suffers from high molecular weight and excessive viscosity, resulting in poor transdermal absorption, poor oral absorption, and poor bioavailability, resulting in many limitations in its application scenarios. Therefore, the research and development of low-molecular sodium hyaluronate and even sodium hyaluronate oligosaccharides has long been a concern for technicians in this field.

[0004] In the prior art, the preparation of low-molecular-weight sodium hyaluronate products is mostly based on methods for degrading macromolecular sodium hyaluronate, mainly including physical methods, chemical methods, and biological enzymatic hydrolysis methods. However, these methods generally suffer from problems such as the large molecular weight of the degradation products, a wide molecular weight range of the products, unstable degradation effects, and high costs. For example, the preparation method of low-molecular-weight sodium hyaluronate disclosed in Chinese patent CN101020724A can control the average molecular weight of the sodium hyaluronate produced to be between 8000 and 20000 Da, with a minimum of approximately 5000 Da. However, the molecular weight range of the sodium hyaluronate produced by this method is relatively wide and cannot meet market demand. The method of producing low-molecular-weight hyaluronic acid using hydrogen peroxide and ascorbic acid oxidative degradation disclosed in Chinese patent CN101293934A suffers from problems such as residual organic solvents, complex post-processing processes, and environmental damage. For example, Chinese patents CN103484513A and CN101507733A disclose enzymatic methods for preparing small-molecule oligomeric hyaluronic acid. However, these methods utilize genetic recombination technology, making the methods and operations very complex and costly. In summary, existing sodium hyaluronate products often suffer from unstable quality and significant product variability, such as excessive molecular weight distribution, uneven product color, low solubility, and prolonged dissolution time. Furthermore, sodium hyaluronate preparation methods generally suffer from low efficiency (approximately 50%, rarely exceeding 90%), high costs, and residual organic solvents. Technical issues

[0005] Therefore, the technical problem to be solved by the present invention is to provide a method for preparing a sodium hyaluronate oligosaccharide composition, which has the advantages of simple process operation, low production cost and high production efficiency;

[0006] The second technical problem to be solved by the present invention is to provide a sodium hyaluronate oligosaccharide composition product prepared by the above-mentioned preparation method, which has stable product quality, high solubility, and rapid dissolution; the sodium hyaluronate oligosaccharide composition product can not only effectively improve the transdermal absorption effect, but also effectively increase the skin's moisture content and have good moisturizing properties;

[0007] The third technical problem to be solved by the present invention is to provide the use of the sodium hyaluronate oligosaccharide composition. Technical Solutions

[0008] In order to solve the above technical problems, the present invention provides a method for preparing the sodium hyaluronate oligosaccharide composition, comprising the following steps:

[0009] (1) Taking macromolecular sodium hyaluronate as a raw material to prepare an acidic aqueous solution and reacting;

[0010] (2) adjusting the pH of the reaction solution to neutral, adding ethanol to a concentration of 50-70%, and collecting the first precipitate and the supernatant for later use;

[0011] (3) The supernatant is subjected to ultrafiltration to remove impurities, ethanol is added to a concentration of 70-90%, and the precipitated second precipitate is collected;

[0012] (4) Dissolve the second precipitate in water and purify it using a reversed-phase ODS column to obtain the product.

[0013] Specifically, the ODS column preferably has a filler particle size of 20-50 μm.

[0014] Specifically, in step (1), the molecular weight of the macromolecular sodium hyaluronate is 50,000-2,000,000 Da;

[0015] The acid used to prepare the acidic aqueous solution is sulfuric acid, hydrochloric acid, hydrofluoric acid or nitric acid, preferably sulfuric acid and hydrochloric acid, particularly preferably sulfuric acid;

[0016] The concentration of the acid is 0.5-4.0M; more preferably 0.5M-1.5M, particularly preferably 0.8-1.2M;

[0017] The mass ratio of the macromolecular sodium hyaluronate to the acid is 1:5-15; the temperature of the reaction step is 50-150°C, and the reaction time is 3-15 hours, more preferably 60-120°C, and particularly preferably 70-80°C; and the reaction time is more preferably 6-8 hours.

[0018] Specifically, in step (2), the base for adjusting the pH value of the reaction solution includes sodium hydroxide, potassium hydroxide, sodium carbonate, etc., preferably sodium hydroxide.

[0019] Specifically, in step (3), the molecular weight cut-off of the ultrafiltration membrane is 1500-3000da.

[0020] Specifically, in step (4), the purification step includes the steps of eluting with a 0.5-3 v / v% ethanol aqueous solution and eluting with a 40-60 v / v% ethanol aqueous solution, collecting the 40-60 v / v% ethanol elution fraction, recovering the ethanol and drying to obtain the product.

[0021] Preferably, in step (4), the purification step includes the steps of eluting with 1 v / v% ethanol aqueous solution for 1-4 column volumes, and eluting with 50 v / v% ethanol aqueous solution for 2-4 column volumes, collecting the elution fraction with 50 v / v% ethanol, recovering ethanol and drying to obtain the product.

[0022] Specifically, the method further includes the steps of dissolving the first precipitate in water, purifying it with an activated carbon column, collecting the purified product and repeating the operations of steps (1)-(2); the number of times steps (1)-(2) are repeated is 1-3 times.

[0023] Specifically, the step of purifying with an activated carbon column includes a step of eluting with water and a step of eluting with a 20-40 v / v% ethanol aqueous solution, collecting the elution fraction of the 20-40 v / v% ethanol aqueous solution, recovering ethanol and drying to obtain the desired purified product.

[0024] Preferably, the step of purifying with an activated carbon column includes the step of eluting with water for 4-6 times the column volume, and the step of eluting with a 30v / v% ethanol aqueous solution, collecting the elution fraction of the 30v / v% ethanol aqueous solution, recovering ethanol and drying to obtain the desired purified product.

[0025] Specifically, the preparation method of the sodium hyaluronate oligosaccharide is:

[0026] The second precipitate is controlled to be dissolved in water to a concentration of 10-25 mg / mL.

[0027] The first precipitate is controlled to be dissolved in water to a concentration of 20-35 mg / mL.

[0028] The primary purpose of adjusting the pH in the preparation method of the sodium hyaluronate oligosaccharide composition of the present invention is to convert hyaluronic acid under acidic conditions into sodium hyaluronate salt. In an acidic solution, the raw sodium hyaluronate is acidified into hyaluronic acid. Adjusting the pH to neutral here neutralizes the acidity of the solution, allowing the hyaluronic acid to form sodium hyaluronate salt, which is more soluble in water and more easily precipitated when ethanol is added.

[0029] The present invention has found that the optimal final concentration of ethanol is 70-90%. When the concentration is lower than 70%, the precipitated sodium hyaluronate is viscous and doughy, which makes subsequent processing very troublesome. When the concentration is higher than 90%, the amount of impurities increases, and the cost also increases.

[0030] The present invention uses an activated carbon column to desalinate and decolorize the first precipitate. The activated carbon column can adsorb sodium hyaluronate of various molecular weights. The salt in the first precipitate is removed by washing with water, and the sodium hyaluronate is then eluted with an ethanol-water solution, while the pigment remains on the activated carbon column. Activated carbon is low-cost and not only effectively desalinates but also has a decolorizing effect, effectively purifying sodium hyaluronate.

[0031] ODS was originally a chromatographic filler for purifying highly polar substances, but the present invention creatively uses it for the purification and desalination of sodium hyaluronate oligosaccharides, and adopts specific purification and desalination conditions, including first eluting and desalting with a low-concentration ethanol aqueous solution, and then eluting the hyaluronate oligosaccharides with a high-concentration ethanol aqueous solution. The present invention has found that the use of ODS to purify sodium hyaluronate oligosaccharides not only has a good desalting effect and high purification efficiency, but also has a high yield of sodium hyaluronate oligosaccharides. After a single ODS treatment, the purity of sodium hyaluronate oligosaccharides can reach more than 96%, which has obvious advantages over other purification methods that require repeated purification treatments to achieve a more ideal purity.

[0032] The present invention also discloses a sodium hyaluronate oligosaccharide composition prepared by the above method, wherein the sodium hyaluronate oligosaccharide has a general molecular formula of (C14H20NO11Na)1-6 and comprises: sodium hyaluronate disaccharide, sodium hyaluronate tetrasaccharide, sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, sodium hyaluronate decasaccharide and sodium hyaluronate dodecasaccharide;

[0033] Wherein, based on the total amount of the oligosaccharide composition being 100%, the sum of the mass contents of the sodium hyaluronate hexasaccharide, the sodium hyaluronate octasaccharide and the sodium hyaluronate decasaccharide is 40-60wt%.

[0034] Preferably, the mass content of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide and sodium hyaluronate decasaccharide is 45%-60%, more preferably 45%-55%, and further preferably 50%-55%.

[0035] Specifically, based on 100% of the total amount of the oligosaccharide composition, the content of sodium hyaluronate disaccharide is 10-24%, the content of sodium hyaluronate tetrasaccharide is 20%-33%; and the content of sodium hyaluronate dodecasaccharide is 3%-8%.

[0036] Specifically, the mass ratio of the sodium hyaluronate hexasaccharide, the sodium hyaluronate octasaccharide and the sodium hyaluronate decasaccharide is 20-26:13-19:7-17;

[0037] Specifically, the average molecular weight of the sodium hyaluronate oligosaccharide is 1100-1600 Da, and the pH value of a 1 wt% aqueous solution is 6-7;

[0038] According to the Chinese Pharmacopoeia colorimeter method, the L* value of the sodium hyaluronate oligosaccharide composition product is 95-99.99, the a* value is -0.2-0.2, the b* value is -0.2-0.2, the ΔL value is 18-21, the Δa value is 36-39, the Δb value is 32-35, and the ΔE value is 93-96;

[0039] The sodium hyaluronate oligosaccharide composition product has a maximum absorption peak at 190 nm in the ultraviolet spectrum, a maximum solubility in water of 25 g / 100 mL, a clear and transparent solution, an optical rotation of -0.7 to 0.9 at a concentration of 1 wt%, an ash content of 12% to 15%, and the aqueous solution can pass through an ultrafiltration membrane with a cutoff value of 3000 Da, with a dry matter conversion rate of 100%.

[0040] The present invention also discloses the use of the sodium hyaluronate oligosaccharide composition or the sodium hyaluronate oligosaccharide composition prepared by the method in preparing food, medical supplies and / or daily chemical products.

[0041] Specifically, the food includes solid food, semi-solid food and liquid food.

[0042] The sodium hyaluronate oligosaccharide of the present invention is used in oral foods to improve beauty and bone and joint health. It can improve skin moisture, provide antioxidant properties, enhance skin elasticity, reduce osteoarthritis, and improve bone and joint health. Therefore, it can be widely used in powders, granules, soft capsules, capsules, tablets, solid beverages, liquid beverages, dairy products, and other products.

[0043] Specifically, the food may include pet food, specifically solid food, semi-solid food, and liquid food for pets. The sodium hyaluronate oligosaccharide of the present invention is used in pet food and, through oral administration, effectively supplements the sodium hyaluronate deficiency in dogs and cats, providing multiple benefits such as nourishing the skin, smoothing the hair, improving joint health, and protecting the stomach and intestines. Therefore, it can be used in the development and application of various pet foods, including staple foods for dogs and cats, and health supplements for dogs and cats.

[0044] Specifically, the medical supplies may include conventional medicines in this field; more specifically, they may include eye drops, eye moisturizing solutions, contact lens care solutions, eye wash solutions, cavity lubricants, oral pharmaceutical preparations, auxiliary medications for ophthalmic surgery, medications for improving knee arthritis, osteoarthritis, and frozen shoulder, dermal fillers, anti-adhesion preparations, etc.

[0045] Specifically, the daily chemical products include conventional daily necessities and cosmetics.

[0046] Specifically, the cosmetics can include products commonly used in the field, including facial masks, essences, gels, facial cleansers, toothpaste, mouthwashes, scalp care products, sunscreens, eye creams, cushion creams, sprays, freeze-dried powders, and the like. The sodium hyaluronate oligosaccharide of the present invention can be instantly absorbed through the skin and directly reach the dermis to moisturize, repair damaged cells, relieve skin sensitivity, reduce inflammation, scavenge free radicals, nourish epidermal cells, improve skin elasticity, reduce wrinkles, reshape a healthy skin barrier, and exert anti-aging effects. Therefore, it can be widely used in cosmetics for anti-aging, sun protection, moisturizing, soothing, anti-redness, repair, and acne treatment.

[0047] The sodium hyaluronate oligosaccharide of the present invention can be used for oral care, has multiple functions such as moisturizing, lubricating, anti-inflammatory, and repairing, and can be used in the development and application of various oral cleaning and care products such as mouthwash, spray, and gel.

[0048] The sodium hyaluronate oligosaccharide described in this invention is used in the field of family planning and has the advantages of good compatibility with natural latex, complete water solubility, easy cleaning, long-lasting lubrication, non-stickiness, and moisturizing and nourishing properties. The sodium hyaluronate oligosaccharide lubricant can be mixed with other water-soluble ingredients or directly soaked in condoms to reduce mucosal damage and nourish delicate skin. Beneficial effects

[0049] The method for preparing sodium hyaluronate oligosaccharides of the present invention comprises using macromolecular sodium hyaluronate as a raw material, preparing an acidic aqueous solution, adding ethanol of a selected concentration ratio to conduct a precipitation reaction, separating the supernatant, removing impurities through an ultrafiltration membrane, and conducting a second precipitation reaction with ethanol of a selected concentration ratio. The separated precipitate is purified through a reverse-phase ODS column to obtain sodium hyaluronate oligosaccharides with the desired properties. The method of the present invention has a simple and easy preparation process, suitable reaction conditions, and a simple and smooth production process, which greatly reduces the production cycle and cost, and the product yield can reach over 96%. It has the advantages of simple operation and low cost, effectively overcoming the defects of traditional bio-enzymatic hydrolysis methods, which are complex and costly, and traditional physical methods, which are relatively complex and require high equipment, and is suitable for large-scale industrial production of sodium hyaluronate oligosaccharide products.

[0050] The sodium hyaluronate oligosaccharide composition prepared by the method of the present invention comprises oligosaccharides with the general molecular formula (C₁₄H₂₀NO₁₁)₁ ...

[0051] In the sodium hyaluronate oligosaccharide composition product prepared by the method of the present invention, the sum of the mass contents of sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide and sodium hyaluronate decasaccharide is more than 40%, which not only has good transdermal absorption performance, but also because its molecular weight is larger than that of sodium hyaluronate disaccharide and sodium hyaluronate tetrasaccharide, its metabolism in the skin is relatively slow, and it can better retain moisture in the skin. Compared with other sodium hyaluronate oligosaccharide products, it can not only effectively improve the transdermal absorption effect, but also effectively increase the skin water content and have good moisturizing properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0053] FIG1 is a gel chromatogram of the sodium hyaluronate oligosaccharide sample described in Experimental Example 1;

[0054] Figure 2 is a comparison of the skin moisture test results of rats in Experimental Example 2;

[0055] FIG3 is a curve showing the absorbance change of the receptor solution at different transdermal times after treatment in Experimental Example 3;

[0056] FIG4 is a comparison chart of the moisturizing abilities of different moisturizers in Experimental Example 4. Modes for Carrying Out the Invention

[0057] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention.

[0058] In the following examples of the present invention, the purity of the sodium hyaluronate oligosaccharide is determined by ultraviolet spectrophotometry according to QB / T4416-201 "Cosmetic Raw Materials - Sodium Hyaluronate".

[0059] Example 1

[0060] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment comprises the following steps:

[0061] (1) Place 500 g of macromolecular sodium hyaluronate (average molecular weight 1.2 million Da) in a 20 L reactor, add 1 M sulfuric acid, and the mass ratio of macromolecular sodium hyaluronate to sulfuric acid is 1:10. Turn on the circulating water heating pump, heat to 70 ° C, start the timing reaction, and keep the constant temperature hydrolysis reaction for 7 hours;

[0062] (2) adding a sodium hydroxide aqueous solution to the reaction solution under stirring to adjust the pH to 7, adding an appropriate amount of ethanol to a concentration of 50%, and reacting until no precipitate is formed, and collecting the first precipitate and the supernatant respectively;

[0063] The first precipitate was dissolved in distilled water to a concentration of 30 mg / mL as a sample, and purified using an activated carbon column. After elution with water for 5 column volumes, elution was performed with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C, and freeze-dried to obtain a purified product.

[0064] Repeat the above steps (1)-(2) twice using the above purified product as raw material;

[0065] (3) After the above supernatants are combined and filtered through an ultrafiltration membrane to remove impurities, ethanol is added to a concentration of 70%, and the reaction is continued until no precipitate is formed, and the precipitated second precipitate is collected;

[0066] (4) The second precipitate was added to distilled water and dissolved to a concentration of 20 mg / mL. The precipitate was purified using a reversed-phase ODS column (particle size 20 µm), eluted with 1 v / v% ethanol-water solution for 2 column volumes, and then eluted with 50 v / v% ethanol for 4 column volumes. The elution fraction with 50 v / v% ethanol was collected, and after recovering the ethanol under reduced pressure, the fraction was freeze-dried to obtain 495.5 g of the desired sodium hyaluronate oligosaccharide, with a calculated yield of 99.1% and a purity of 98.8%.

[0067] Example 2

[0068] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment comprises the following steps:

[0069] (1) Place 500 g of macromolecular sodium hyaluronate (average molecular weight 600,000 Da) in a 20 L reactor and add 1 M hydrochloric acid. The mass ratio of macromolecular sodium hyaluronate to hydrochloric acid is 1:10. Turn on the circulating water heating pump, heat to 80 ° C, start the timing reaction, and keep the hydrolysis reaction at constant temperature for 8 hours.

[0070] (2) adding a sodium hydroxide aqueous solution to the reaction solution under stirring to adjust the pH to 7, adding an appropriate amount of ethanol to make the concentration reach 70%, and reacting until no precipitate is formed, and collecting the first precipitate and the supernatant respectively;

[0071] The first precipitate was dissolved in distilled water to a concentration of 25 mg / mL as a sample, and purified using an activated carbon column. After elution with water for 5 column volumes, elution was performed with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C, and freeze-dried to obtain a purified product.

[0072] Repeat the above steps (1)-(2) twice using the above purified product as raw material;

[0073] (3) After the above supernatants are combined and filtered through an ultrafiltration membrane to remove impurities, ethanol is added to reach a concentration of 90%, and the reaction is continued until no precipitate is formed, and the precipitated second precipitate is collected;

[0074] (4) The second precipitate was dissolved in distilled water to a concentration of 20 mg / mL, and purified using a reversed-phase ODS column (particle size 20 µm). The elution was performed with a 1 v / v% ethanol-water solution for 2 column volumes, and then with 50 v / v% ethanol for 4 column volumes. The elution fraction with 50 v / v% ethanol was collected, and after recovering the ethanol under reduced pressure, the fraction was freeze-dried to obtain 485.5 g of the desired sodium hyaluronate oligosaccharide, with a calculated yield of 97.1% and a purity of 97.6%.

[0075] Example 3

[0076] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment comprises the following steps:

[0077] (1) Place 1 kg of macromolecular sodium hyaluronate (average molecular weight 1.2 million Da) in a 100 L enamel reactor, add 1 M sulfuric acid (the mass ratio of macromolecular sodium hyaluronate to sulfuric acid is 1:10), stir to dissolve, and heat to 70 ° C. Start the timing reaction and keep the hydrolysis reaction at constant temperature for 7 hours;

[0078] (2) adding a sodium hydroxide aqueous solution to the reaction solution while stirring to adjust the pH to 7, slowly adding ethanol while stirring to an ethanol concentration of 60%, and reacting until no precipitate is formed, and collecting the first precipitate and the supernatant respectively;

[0079] The first precipitate was dissolved in distilled water to a concentration of 35 mg / mL as a sample, and purified using an activated carbon column. After elution with water for 4 column volumes, elution was performed with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C, and freeze-dried to obtain a purified product.

[0080] Repeat the above steps (1)-(2) twice using the above purified product as raw material;

[0081] (3) After the above supernatants are combined and filtered through an ultrafiltration membrane to remove impurities, ethanol is slowly added under stirring to an ethanol concentration of 80%, and the reaction is carried out until no precipitate is formed, and the precipitated second precipitate is collected;

[0082] (4) The second precipitate was added to distilled water and dissolved to a concentration of 25 mg / mL. The product was purified using a reverse phase ODS column (particle size 50 µm), eluted with 1 v / v% ethanol-water solution for 2 column volumes, and then eluted with 50 v / v% ethanol for 2 column volumes. The elution fraction with 50 v / v% ethanol was collected, and after recovering the ethanol under reduced pressure, the product was freeze-dried to obtain 968.6 g of the desired sodium hyaluronate oligosaccharide, with a calculated yield of 96.9% and a purity of 98.2%.

[0083] Example 4

[0084] The preparation method of the sodium hyaluronate oligosaccharide composition described in this embodiment comprises the following steps:

[0085] (1) Take 20 kg of macromolecular sodium hyaluronate (average molecular weight 1.2 million) and place it in a 1-ton enameled reactor. Add 1M sulfuric acid (the mass ratio of macromolecular sodium hyaluronate to sulfuric acid is 1:10). Stir and dissolve. Heat to 70°C, start the reaction timer, and hydrolyze at constant temperature for 7 hours.

[0086] (2) adding a sodium hydroxide aqueous solution to the reaction solution while stirring to adjust the pH to 7, slowly adding ethanol while stirring to an ethanol concentration of 60%, and reacting until no precipitate is formed, and collecting the first precipitate and the supernatant respectively;

[0087] The first precipitate was dissolved in distilled water to a concentration of 35 mg / mL as a sample, and purified using an activated carbon column. After elution with water for 4 column volumes, elution was performed with 30 v / v% ethanol. The fraction eluted with 30 v / v% ethanol was collected, and the ethanol was recovered under reduced pressure at a temperature not exceeding 40°C, and freeze-dried to obtain a purified product.

[0088] Repeat the above steps (1)-(2) twice using the above purified product as raw material;

[0089] (3) After the above supernatants are combined and filtered through an ultrafiltration membrane to remove impurities, ethanol is slowly added under stirring to an ethanol concentration of 80%, and the reaction is carried out until no precipitate is formed, and the precipitated second precipitate is collected;

[0090] (4) The second precipitate was dissolved in distilled water to a concentration of 25 mg / mL, and purified using a reversed-phase ODS column (particle size 50 µm), eluted with 1 v / v% ethanol-water solution for 1.5 column volumes, and then eluted with 50 v / v% ethanol for 2 column volumes. The elution fraction with 50 v / v% ethanol was collected, and after recovering the ethanol under reduced pressure, freeze-dried to obtain 19.3 kg of the desired sodium hyaluronate oligosaccharide, with a calculated yield of 96.5% and a purity of 96.3%.

[0091] Comparative Example 1

[0092] The preparation method of the sodium hyaluronate oligosaccharide composition described in this comparative example is the same as that in Example 4, the only difference being that in step (1), the hydrolysis reaction time is extended to 24 hours.

[0093] Comparative Example 2

[0094] The preparation method of the sodium hyaluronate oligosaccharide composition in this comparative example is the same as that in Example 4, the only difference being that in step (3), the molecular weight cutoff of the ultrafiltration membrane is 1000 Da.

[0095] Experimental example

[0096] Experimental Example 1: Performance Testing of Sodium Hyaluronate Oligosaccharide Composition Products

[0097] The sodium hyaluronate oligosaccharide composition prepared in Example 4 was used as a sample to perform the following tests.

[0098] Molecular weight and molecular weight distribution detection

[0099] The size exclusion chromatography method in accordance with the General Chapter 0514 of Part IV of the Pharmacopoeia of the People's Republic of China (2020 edition) was used for detection. The gel chromatogram of the sodium hyaluronate oligosaccharide sample (Example 4) is shown in FIG1 , and the molecular weight and molecular weight distribution of the sodium hyaluronate oligosaccharide are shown in the following table.

[0100] Molecular weight and molecular weight distribution results of sodium hyaluronate oligosaccharide Project test results Mw1.275×10 3 g / molMn1.162×10 3 g / molMw / Mn1.098

[0101] Filter membrane testing

[0102] The sodium hyaluronate oligosaccharide sample of the present invention (Example 4) was tested using a 3000 molecular weight filter membrane. The dry matter yield was 100%, indicating that the entire sample could pass 3000 Da, i.e., the maximum molecular weight of the obtained sodium hyaluronate oligosaccharide did not exceed 3000 Da.

[0103] Solubility testing

[0104] The solubility of the sodium hyaluronate oligosaccharide sample (Example 4) of the present invention is 2.5 g per 10 mL of water. When the amount exceeds 2.5 g, precipitation is generated, indicating that the solubility of the obtained sodium hyaluronate oligosaccharide is 25 g / 100 mL.

[0105] HPLC analysis was performed using the sodium hyaluronate oligosaccharide compositions prepared in Examples 1-4 and Comparative Examples 1-2 as samples. The HPLC analysis was conducted in accordance with the High Performance Liquid Chromatography method (Chinese Pharmacopoeia 2015 Edition, Part IV, General Rules 0512) using 20% ​​acetonitrile in water as the mobile phase, a flow rate of 0.8 mL / min, UV detection at a wavelength of 191 nm, and a column temperature of 30°C. It should be noted that HPLC analysis can only determine the content of the individual hyaluronate oligosaccharide components in the sample; salt impurities such as Na2SO4 and NaCl cannot be detected.

[0106] According to the HPLC test results, the sodium hyaluronate oligosaccharide composition prepared by the present invention includes sodium hyaluronate disaccharide, sodium hyaluronate tetrasaccharide, sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, sodium hyaluronate decasaccharide and sodium hyaluronate dodecasaccharide. The general molecular formula of sodium hyaluronate oligosaccharide is: (C 14 H 20 NO 11 Na) 1-6 , its systematic name is: [(1→4)-O-β-D-glucuronic acid-(1→3)-2-acetylamino-2-deoxy-β-D-glucose] 1-6 Sodium salt.

[0107] The mass content of each component in the sodium hyaluronate oligosaccharide composition sample is shown in Table 1 below, where Rt refers to retention time.

[0108] Table 1 Sodium hyaluronate oligosaccharide sample content

[0109]

[0110] Experimental Example 2: Animal Experiment

[0111] This experimental example is to observe the changes after feeding rats with the sodium hyaluronate oligosaccharide feed.

[0112] Experimental Materials

[0113] Sodium hyaluronate oligosaccharide sample (Example 4), sodium hyaluronate oligosaccharide control group (Comparative Example 1); experimental mouse diets were produced by an animal food factory. Based on the mouse diet, specially prepared mouse diets containing varying amounts of sodium hyaluronate oligosaccharide were prepared (the original mouse diet was crushed, and 0.02%, 0.06%, and 0.09% (by weight) of sodium hyaluronate oligosaccharide were added to the mouse diet, respectively). These experimental mouse diet granules were then prepared to contain varying amounts of sodium hyaluronate oligosaccharide.

[0114] Experimental testing instruments

[0115] The skin moisture tester Corneometer CM 825 is produced by Courage+Khazaka of Germany.

[0116] experimental animals

[0117] All experimental rats were SD clean-grade aged female rats, weighing 300-400 g, and aged 15 months. They were provided by the Laboratory Animal Center of the Chinese Academy of Military Medical Sciences. Their qualification certificate number was 035, and their laboratory animal environment and facility qualification certificate number was 027. The animals were randomly divided into seven groups, with 20 rats in each group.

[0118] Experimental methods

[0119] A total of 140 SD clean-grade rats aged 15 months, weighing (360±21) g, were randomly divided into 7 groups, with 20 rats in each group.

[0120] There were three groups of normal feeding control group, three groups of sodium hyaluronate oligosaccharide feed, and three groups of sodium hyaluronate oligosaccharide feed containing comparative example 1, which were fed with low, medium and high doses of sodium hyaluronate oligosaccharide feed respectively for a total of 60 days; during this period, the experimental animals in each group were housed in a single cage and had free access to food and water.

[0121] During the feeding period, the rats in each group were observed daily for food intake, mental state, activity, nausea, vomiting, and the odor and characteristics of urine and feces. Body weight was measured regularly. No rats died or were near death during the experiment.

[0122] Detection of skin moisture content in experimental rats

[0123] Testing was conducted in a spacious, well-ventilated room with a relatively stable temperature (22°C ± 2°C) and humidity (60%). All rats were shaved on both sides of the dorsal spine, covering an area of ​​approximately 1.5 cm x 1.5 cm. The moisture content of the shaved area was measured using a skin moisture meter. All rats were tested using the same instrument and by the same staff in the same room. The contact force and vertical angle between the test head and the rat's skin were maintained consistent.

[0124] Experimental results

[0125] The skin moisture values ​​obtained in this experimental example are summarized in Table 2 below, and a comparison chart of rat skin moisture detection is shown in Figure 2.

[0126] Table 2 Summary and analysis of experimental rat skin moisture detection data Experimental groups Animal count Average water content (%) Statistical value Blank control group 20 25.4±5.3 0.02% Sodium hyaluronate oligosaccharide group 20 26.3±5.6 0.41 (P>0.05) 0.06% Sodium hyaluronate oligosaccharide group 20 32.1±4.9 0.021 (P<0.05) 0.09% Sodium hyaluronate oligosaccharide group 20 35.1±6.1 0.015 (P<0.05) 0.02% Sodium hyaluronate oligosaccharide control group 120 25.9±4.8 0.38 (P>0.05) 0.06% Sodium hyaluronate oligosaccharide control group 120 27.2±5.1 0.33 (P>0.05) 0.09% Sodium hyaluronate oligosaccharide control group 120 28.5±5.5 0.12 (P>0.05)

[0127] It can be seen from the data in the above table that as the amount of sodium hyaluronate oligosaccharide added to the feed increases, the water content of the skin of the experimental animals increases simultaneously.

[0128] The general condition of the rats was as follows: During the experiment, the rats in each group grew well, and no obvious abnormalities were found in their general conditions such as behavior, activity, mental state, drinking water and feces.

[0129] Skin moisture test data showed no significant difference between the low-dose sodium hyaluronate oligosaccharide group and the blank control group; however, significant differences were observed between the medium- and high-dose sodium hyaluronate oligosaccharide groups and the blank control group, indicating that medium- and high-dose sodium hyaluronate oligosaccharides can significantly increase skin moisture content in rats. While skin moisture content increased slightly in the three doses of the sodium hyaluronate oligosaccharide control group compared to the blank control group, no significant difference was observed. In the sodium hyaluronate oligosaccharide composition of the present invention, the content of sodium hyaluronate hexasaccharide to sodium hyaluronate decasaccharide ranged from 40-60%, while the sodium hyaluronate oligosaccharide control group (Comparative Example 1) contained approximately 75% sodium hyaluronate disaccharide and less than 15% sodium hyaluronate hexasaccharide to sodium hyaluronate decasaccharide. This is because sodium hyaluronate disaccharides have a low molecular weight and are rapidly metabolized after absorption, making them easily lost. In contrast, the sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, and sodium hyaluronate decasaccharide have high molecular weights and are slowly metabolized after absorption, making them less likely to be lost, thereby better maintaining skin moisture.

[0130] It can be seen that the sodium hyaluronate oligosaccharide composition product of the present invention has a good effect on improving the water content of rat skin, and high skin water content indicates high skin health. Since animal skin also contains sodium hyaluronate, the maturation and aging process of rodent skin also changes with the content and metabolism of sodium hyaluronate. When the sodium hyaluronate content is high, it can improve skin nutritional metabolism, make the skin soft, smooth, and promote health; similarly, sodium hyaluronate oligosaccharides are used in pet food, and through oral administration, they effectively supplement the lack of sodium hyaluronate in dogs and cats, play multiple roles such as nourishing the skin, smoothing hair, improving joints, and protecting the stomach. Therefore, it can be used for the development and application of various pet foods such as dog and cat staple food and dog and cat health products.

[0131] Experimental Example 3 Transdermal Effect

[0132] In this experimental example, ordinary sodium hyaluronate solution (200,000-400,000 Da), sodium hyaluronate oligosaccharide solution (sample in Example 4), and sodium hyaluronate oligosaccharide solutions of Comparative Examples 1-2 (respectively as control groups 1-2) with the same content were used for comparative study to investigate their transdermal effects on snake sloughs and provide a certain experimental basis for their application.

[0133] Experimental instruments and reagents

[0134] Drug transdermal diffusion tester (Shanghai Youpu Scientific Instrument Co., Ltd.); UV-visible spectrometer (Thermo Electron Corporation, USA); PBS solution is phosphate buffer solution, pH 7.5, self-prepared.

[0135] Experimental methods

[0136] In vitro transdermal diffusion test: Self-purchased snake skin was soaked in physiological saline for 30 min, and then stored in PBS solution at 4°C for 12 h before use.

[0137] Cut a piece of snake skin of appropriate size and fix it in the drug transdermal diffusion tester (diffusion area is 1.0 cm 2 The receiving pool volume is 8.5 cm 3 ) with the outer surface of the snake slough facing the donor chamber and the receiving chamber containing PBS buffer. The temperature was maintained at 37°C with stirring. 0.5 mL of sample was taken at fixed intervals and an equal amount of receiving solution was added simultaneously. After the experiment, the sample solution was appropriately diluted and passed through a 0.50 μm microporous filter membrane. Hydrolysis was performed and the absorbance at 550 nm was measured by UV spectrophotometry.

[0138] Experimental results

[0139] In vitro transdermal diffusion experiments were conducted using three groups: a sodium hyaluronate oligosaccharide group containing 2 mL of a homemade 30 wt% sodium hyaluronate oligosaccharide solution; a control group containing 2 mL of a 30 wt% conventional sodium hyaluronate solution; and a control group containing 30 wt% sodium hyaluronate oligosaccharide solution from Comparative Examples 1-2. The absorbance of the receptor fluid at different transdermal times is shown in Table 3 below. The absorbance curves of the receptor fluid at different transdermal times after treatment are shown in Figure 3.

[0140] Table 3 Absorbance of the receptor solution at different transdermal times after treatment

[0141]

[0142] As can be seen from the table above, the absorbance of the sodium hyaluronate oligosaccharide group was significantly higher than that of the ordinary sodium hyaluronate control group at the same transdermal time, indicating that the sodium hyaluronate oligosaccharide group had a significantly higher transmittance through snake skin than the ordinary sodium hyaluronate control group at the same transdermal time, indicating that the sodium hyaluronate oligosaccharide group with a molecular weight less than 3000 can effectively improve its ability to penetrate snake skin. Due to the higher content of sodium hyaluronate disaccharide and sodium hyaluronate tetrasaccharide in sodium hyaluronate oligosaccharide control groups 1 and 2, the transdermal absorption effect was also better than that of the ordinary sodium hyaluronate group, but there was almost no difference from the sodium hyaluronate oligosaccharide group.

[0143] Experimental Example 4 Moisturizing Performance

[0144] This experimental example compares the moisturizing properties of sodium hyaluronate with different molecular weights and commonly used moisturizing ingredients.

[0145] Experimental reagents and instruments

[0146] Sodium hyaluronate (200,000-400,000 Da), small molecule sodium hyaluronate (10,000-100,000 Da), cosmetic grade, Huaxi Freda Biotechnology Co., Ltd.

[0147] Sodium hyaluronate oligosaccharide, sample of Example 4; sodium hyaluronate oligosaccharide control group 1, sample of Comparative Example 1; sodium hyaluronate oligosaccharide control group 2, sample of Comparative Example 2;

[0148] Aloe vera gel extract, cosmetic grade, Beijing Sanyou Huizhi Biotechnology Co., Ltd.;

[0149] Tremella fuciformis extract, cosmetic grade, Huanya Research Institute;

[0150] Ceramide 3, cosmetic grade, Doosan Corporation, South Korea;

[0151] Excipients such as hydrogenated polydecene, cetearyl alcohol, ARLACEL 165 (a mixture of glyceryl stearate and PEG-100 stearate), dimethicone (5CST), carbomer, triethanolamine, xanthan gum, aminomethyl propanol, and disodium EDTA are all cosmetic grade and provided by cosmetic companies.

[0152] Corneometer CM825 Skin Moisture Meter, Courage+Khazaka, Germany.

[0153] Experimental methods

[0154] Oil phase: Hydrogenated Polydecene 5.0, Cetearyl Alcohol 1.0, ARLACEL 165 3.0, Dimethicone 5.0;

[0155] Aqueous phase: 0.02% disodium EDTA, 0.3% carbomer, 0.1% xanthan gum, 0.05% triethanolamine, and the balance water;

[0156] Of the 8 moisturizers, except ceramide which is soluble in the oil phase, the other 7 are soluble in the water phase. The dosages are shown in Table 4 below.

[0157] After adding the moisturizer to the oil phase or water phase, completely dissolve the oil and water phases in an 80°C water bath. Slowly pour the oil phase into the water phase with the agitator at 1000 rpm for 3 minutes of pre-emulsification. Increase the homogenizer speed to 9000 rpm and homogenize for 3 minutes. After cooling to 50°C, add aminomethyl propanol and preservatives. After adding water, homogenize for another 1 minute. After the paste cools to room temperature, vacuum discharge and obtain the paste.

[0158] According to relevant literature and experimental experience, two levels of different moisturizers were selected for experimentation. The specific addition amounts are shown in Table 4 below.

[0159] Table 4 Addition amount of different moisturizers

[0160] Forty-five healthy volunteers aged 20-50 years without allergic diseases were selected and divided into nine groups with five people in each group. Eight moisturizer experiments were conducted at room temperature (23±2)℃ and humidity 40%-60%. One group served as a blank control group.

[0161] Before the experiment, volunteers need to stay quiet in the environment for at least 30 minutes. After the environment reaches equilibrium, draw a 4cm×4cm experimental area on each forearm, with an interval of at least 1cm between the areas. The left arm of the subject is the blank control area, and the right arm is the area for smearing the sample to be tested.

[0162] Take homemade moisturizing cream and press (2.0±0.1) mg / cm 2 Apply the dose to the test area. After 30 minutes and 2 hours of using the cosmetics, technicians use a skin moisture test probe to test the moisture content of the test area. The average value of 5 measurements is taken as the test value. The moisture value change rate is calculated as follows:

[0163] Moisture content change rate = (moisture content after use - moisture content before use) / moisture content before use × 100%. The test results are shown in Table 5 below.

[0164]

[0165] Table 5 Changes in skin moisture 30 min and 2 h after applying homemade moisturizing cream

[0166] As can be seen from the data in the above table, compared with the blank control, the homemade moisturizing cream of the present invention has a certain moisturizing effect on the skin.

[0167] After 30 minutes, the change rate of skin moisture value reached more than 20%. The moisturizing agents contained in the moisturizing cream were sodium hyaluronate group, small molecule sodium hyaluronate group, sodium hyaluronate oligosaccharide group, sodium hyaluronate oligosaccharide control group 1, sodium hyaluronate oligosaccharide control group 2, 3.00% aloe vera gel extract, and ceramide group.

[0168] The moisturizing creams that achieved a skin moisture value change rate of more than 20% after 2 hours contained only the small molecule sodium hyaluronate group, sodium hyaluronate oligosaccharide, sodium hyaluronate oligosaccharide control group 1, sodium hyaluronate oligosaccharide control group 2, and ceramide group.

[0169] The moisturizers with skin moisture value change rates of more than 20% at 30 minutes and 2 hours included the small molecule sodium hyaluronate group, sodium hyaluronate oligosaccharide group, sodium hyaluronate oligosaccharide control group 1, sodium hyaluronate oligosaccharide control group 2, and ceramide group.

[0170] Taking 4.0% glycerol as the standard, the formula for calculating the moisturizing ability of the moisturizer is:

[0171] Moisturizing ability = the rate of change in moisture content after using a moisturizer / the rate of change in moisture content after using glycerin. The results of the comparison of moisturizing abilities of moisturizers are shown in Figure 4.

[0172] The experimental results show that different moisturizers have varying moisturizing abilities, primarily due to the different moisturizing mechanisms of moisturizers with different molecular weights. Sodium hyaluronate, aloe vera gel extract, and Tremella fuciformis extract have higher molecular weights, and their polysaccharide components can form a film on the skin's surface, quickly preventing moisture evaporation. Low-molecule sodium hyaluronate, the sodium hyaluronate oligosaccharide group, the sodium hyaluronate oligosaccharide control group 1, the sodium hyaluronate oligosaccharide control group 2, and ceramides have lower molecular weights and can penetrate deep into the skin's inner layers, binding to free water and preventing it from evaporating. This enhances their ability to lock in moisture and prevent evaporation. Furthermore, once absorbed into the skin, the sodium hyaluronate oligosaccharide group exhibits the highest moisturizing effect, due to its high content of hexasaccharides to decasaccharides and relatively high molecular weight, resulting in slower metabolism within the skin and greater water retention.

[0173] In summary, the sodium hyaluronate oligosaccharide composition product prepared by the present invention has the advantages of good moisturizing performance and good transdermal absorption, and is safe to use and has no side effects. It can be widely used in food, daily chemical products, medical supplies and other fields.

[0174] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas. At the same time, for those skilled in the art, according to the ideas of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing a sodium hyaluronate oligosaccharide composition, characterized in that: The steps include: (1) Taking macromolecular sodium hyaluronate as a raw material to prepare an acidic aqueous solution and reacting; (2) adjusting the pH of the reaction solution to neutral, adding ethanol to a concentration of 50-70%, and collecting the first precipitate and the supernatant for later use; (3) The supernatant is subjected to ultrafiltration to remove impurities, ethanol is added to a concentration of 70-90%, and the precipitated second precipitate is collected; (4) Dissolve the second precipitate in water and purify it using a reversed-phase ODS column to obtain the product.

2. The method for preparing the sodium hyaluronate oligosaccharide composition according to claim 1, characterized in that: In the step (1), the molecular weight of the macromolecular sodium hyaluronate is 50,000-2,000,000 Da; The acid used to prepare the acidic aqueous solution is sulfuric acid, hydrochloric acid, hydrofluoric acid or nitric acid; the concentration of the acid is 0.5-4.0M; The mass ratio of the macromolecular sodium hyaluronate to the acid is 1:5-15; The temperature of the reaction step is 50-150°C and the reaction time is 3-15 hours; In the step (3), the molecular weight cut-off of the ultrafiltration membrane is 1500-3000da.

3. The method for preparing the sodium hyaluronate oligosaccharide composition according to claim 1 or 2, characterized in that: In the step (4), the purification step includes the steps of eluting with 0.5-3 v / v% ethanol aqueous solution and eluting with 40-60 v / v% ethanol aqueous solution, collecting the 40-60 v / v% ethanol elution fraction, recovering ethanol and drying to obtain the product.

4. The method for preparing the sodium hyaluronate oligosaccharide composition according to claim 1 or 2, characterized in that: The method further comprises the steps of dissolving the first precipitate in water, purifying it with an activated carbon column, collecting the purified product and using the purified product as a raw material to repeat steps (1) to (2); The step of purifying with an activated carbon column includes a step of eluting with water and a step of eluting with a 20-40 v / v% ethanol aqueous solution, collecting the elution fraction of the 20-40 v / v% ethanol aqueous solution, recovering ethanol and drying to obtain the desired purified product.

5. The method for preparing the sodium hyaluronate oligosaccharide composition according to claim 1 or 2, characterized in that: Controlling the second precipitate to be dissolved in water to a concentration of 10-25 mg / mL; The first precipitate is controlled to be dissolved in water to a concentration of 20-35 mg / mL.

6. A sodium hyaluronate oligosaccharide composition prepared by the method according to any one of claims 1 to 5, characterized in that: The oligosaccharide composition comprises: sodium hyaluronate disaccharide, sodium hyaluronate tetrasaccharide, sodium hyaluronate hexasaccharide, sodium hyaluronate octasaccharide, sodium hyaluronate decasaccharide and sodium hyaluronate dodecasaccharide; Wherein, based on the total amount of the oligosaccharide composition as 100%, the sum of the mass contents of the sodium hyaluronate hexasaccharide, the sodium hyaluronate octasaccharide and the sodium hyaluronate decasaccharide is 40-60wt%.

7. The sodium hyaluronate oligosaccharide composition according to claim 6, characterized in that Based on the total amount of the oligosaccharide composition as 100%, the content of the sodium hyaluronate disaccharide is 10-24wt%, the content of the sodium hyaluronate tetrasaccharide is 20-33wt%, and the content of the sodium hyaluronate dodecasaccharide is 3-8wt%.

8. The sodium hyaluronate oligosaccharide composition according to claim 6 or 7, characterized in that The mass ratio of the sodium hyaluronate hexasaccharide, the sodium hyaluronate octasaccharide and the sodium hyaluronate decasaccharide is 20-26:13-19:7-17.

9. The sodium hyaluronate oligosaccharide composition according to claim 6 or 7, characterized in that The sodium hyaluronate oligosaccharide composition has an average molecular weight of 1100-1600 Da, and a pH value of 1 wt % aqueous solution is 6-7.

10. Use of the sodium hyaluronate oligosaccharide composition according to any one of claims 6 to 9 or the sodium hyaluronate oligosaccharide composition prepared by the method according to any one of claims 1 to 5 in preparing food, medical supplies and / or daily chemical products.

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