A method for preparing cross-linked sodium hyaluronate gel and its composition.

Abstract

This invention discloses a method for preparing cross-linked sodium hyaluronate gel and its composition, comprising: S1: dissolving sodium hyaluronate in an alkaline solution and mixing thoroughly; S2: adding a cross-linking agent to the solution obtained in step S1 to obtain block-shaped cross-linked sodium hyaluronate gel, wherein the cross-linking agent is one or more of polyethylene glycol diglycidyl ether and polypropylene glycol diglycidyl ether with different molecular weights; S3: cutting the block-shaped cross-linked sodium hyaluronate gel into segments and adding them to a phosphate-sodium chloride buffer solution, allowing them to swell; S4: homogenizing and pulverizing the gel to obtain cross-linked sodium hyaluronate gel particles; S5: dissolving sodium hyaluronate in a buffer solution and then mixing it thoroughly with the cross-linked sodium hyaluronate gel particles obtained in step S4 to obtain the finished cross-linked sodium hyaluronate gel. The gel of this invention has the advantage of high cohesion, and the cross-linked sodium hyaluronate gel has good suspension and support capabilities for different polyester microspheres and inorganic microspheres.

Description

[0001] This invention relates to the field of cosmetic fillers, specifically to a method for preparing a cross-linked sodium hyaluronate gel and its composition. Background Technology

[0002] With the rise of the medical cosmetic surgery industry, hyaluronic acid (HA) dermal fillers have been widely used in minimally invasive maxillofacial surgery. Among them, cross-linked sodium hyaluronate, due to the three-dimensional network structure formed between HA molecules through cross-linking agents, exhibits enhanced stability and mechanical properties, leading to its wider application in the medical and cosmetic fields. Different HA filler products employ different cross-linking methods and manufacturing processes, resulting in variations in the physical properties of cross-linked HA gels, such as concentration, gel structure, and degree of cross-linking. Commercially available and under-developed products typically consist of a mixture of cross-linked sodium hyaluronate and free sodium hyaluronate in the gel portion, mixed in a specific ratio with polymer microspheres (polymethyl methacrylate, polycaprolactone, etc.) or inorganic microspheres (such as hydroxyapatite) to create injectable fillers for use in the medical cosmetic filling field.

[0003] Existing gel carriers have insufficient suspending capacity, and microspheres may settle before storage or injection, leading to uneven injection effects. This can result in localized excessive concentrations forming nodules, or in some areas with insufficient microspheres, leading to poor results. Furthermore, uneven distribution of microspheres can significantly reduce their effectiveness in stimulating collagen regeneration. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing cross-linked sodium hyaluronate gel and its composition, thereby solving the above-mentioned problems.

[0005] In order to achieve these objectives and other advantages according to the present invention: In a first aspect, a method for preparing cross-linked sodium hyaluronate gel includes the following steps: S1: Take sodium hyaluronate with a molecular weight of 500,000 to 3,000,000 Da, dissolve it in an alkaline solution, mix well, and the mass ratio of sodium hyaluronate to alkaline solution is 0.1 to 0.2:1; S2: Add a crosslinking agent to the solution obtained in step S1, and crosslink at 15~40℃ for 8~24h to obtain block crosslinked sodium hyaluronate gel. The crosslinking agent is one or more of polyethylene glycol diglycidyl ether and polypropylene glycol diglycidyl ether with different molecular weights. S3: Cut the block cross-linked sodium hyaluronate gel obtained in step S2 into segments and put them into phosphate-sodium chloride buffer. After swelling at 50~70℃ for 28~32 min, discard the buffer. Add new buffer and continue to swell at 50~70℃ for 28~32 min. Repeat 4~10 times until the weight of the gel no longer increases. S4: The gel that has been swollen and cleaned in step S3 is homogenized and pulverized to obtain cross-linked sodium hyaluronate gel particles. S5: Dissolve sodium hyaluronate with a molecular weight of 500,000 to 3,000,000 Da in a buffer solution to obtain a sodium hyaluronate solution. Then mix the solution with the cross-linked sodium hyaluronate gel particles obtained in step S4 to obtain the finished cross-linked sodium hyaluronate gel.

[0006] Furthermore, the alkaline solution mentioned in step S1 is a 1% NaOH solution, and the mass ratio of sodium hyaluronate to the alkaline solution is 0.15:1.

[0007] Furthermore, the crosslinking agent mentioned in step S2 is polyethylene glycol diglycidyl ether with a molecular weight of 500~6000 Da.

[0008] Furthermore, the buffer solution in step S3 contains 6 g / L sodium chloride, 4 g / L disodium hydrogen phosphate, and 2 g / L sodium dihydrogen phosphate.

[0009] Furthermore, in step S4, the particle size D of the cross-linked sodium hyaluronate gel particles is... 50 Within 100~250 μm.

[0010] Furthermore, in the sodium hyaluronate solution described in step S5, the mass ratio of sodium hyaluronate to buffer solution is 0.01~0.03:1.

[0011] Furthermore, in step S5, the mass ratio of sodium hyaluronate solution to cross-linked sodium hyaluronate gel particles is 0.1~0.5:1.

[0012] Secondly, a method for preparing a cross-linked sodium hyaluronate gel composition includes the following steps: R1: Take the cross-linked sodium hyaluronate gel product prepared in the first aspect and sterilize it by moist heat at 120~125℃ for 10~15min; R2: The cross-linked sodium hyaluronate gel obtained in step R1 is mixed with microspheres to obtain a filler containing cross-linked sodium hyaluronate. The microspheres are one or more of hydroxyapatite, PEGylated polycaprolactone, and PEGylated poly-L-lactic acid. The mass ratio of cross-linked sodium hyaluronate gel to microspheres is 2~4:1.

[0013] The beneficial effects of this invention are: 1. The gel prepared by the process has the advantage of high cohesion, and the cross-linked sodium hyaluronate gel has good suspension and support capabilities for different polyester microspheres and inorganic microspheres.

[0014] 2. The crosslinking agent used in this process contains polyethylene glycol segments. According to the principle of like dissolves like, the same PEG segments will form good compatibility, reduce the repulsive force between the interfaces of the two substances, and make them more likely to approach each other in the mixed system rather than separate, thereby improving the macroscopic dispersion uniformity and binding tightness.

[0015] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0016] Figure 1 The results of the elastic modulus test are as follows: Figure 2 The results of the cohesive strength test in Example 1 of this invention; Figure 3 The cohesive strength test results are from Example 3 of this invention. Figure 4 This is the cohesive strength test result of Comparative Example 1 in this invention; Figure 5 The results of the bonding test in Embodiment 4 of the invention; Figure 6 The results of the bonding test in Comparative Example 2 of the invention are shown. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0018] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this specification. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this specification as detailed in the appended claims.

[0019] The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of this specification. The singular forms “a,” “the,” and “the” as used in this specification and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0020] It should be understood that although the terms first, second, third, etc., may be used in this specification to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, without departing from the scope of this specification, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0021] The problem-solving approach of this invention: In a first aspect, a method for preparing cross-linked sodium hyaluronate gel includes the following steps: S1: Take sodium hyaluronate with a molecular weight of 500,000 to 3,000,000 Da, dissolve it in an alkaline solution, mix well, and the mass ratio of sodium hyaluronate to alkaline solution is 0.1 to 0.2:1; the alkaline solution is 1% NaOH solution, and the optimal mass ratio of sodium hyaluronate to alkaline solution is 0.15:1.

[0022] S2: Add a crosslinking agent to the solution obtained in step S1, and perform crosslinking at 15~40℃ for 8~24h to obtain block crosslinked sodium hyaluronate gel. The crosslinking agent can be one or more of polyethylene glycol diglycidyl ether and polypropylene glycol diglycidyl ether with different molecular weights. The crosslinking agent can be a single polyethylene glycol diglycidyl ether or polypropylene glycol diglycidyl ether, or a combination of polyethylene glycol diglycidyl ether with multiple molecular weights, or a combination of polypropylene glycol diglycidyl ether with one or more molecular weights, or a combination of polyethylene glycol diglycidyl ether with one or more molecular weights.

[0023] This invention primarily uses polyethylene glycol diglycidyl ether with a degree of polymerization of 1-50 and a molecular weight of 500-6000 Da (preferably 500-2000 Da). Polypropylene glycol diglycidyl ether with a molecular weight of 380 is also preferred.

[0024] S3: Cut the block-shaped cross-linked sodium hyaluronate gel obtained in step S2 into segments and put them into sodium phosphate-chloride buffer solution. After swelling at 50~70℃ for 28~32 min, discard the buffer solution; add new buffer solution and continue to swell at 50~70℃ for 28~32 min. Repeat 4~10 times until the gel weight no longer increases; the buffer solution contains 6 g / L sodium chloride, 4 g / L disodium hydrogen phosphate and 2 g / L sodium dihydrogen phosphate.

[0025] S4: The gel that has undergone swelling and cleaning in step S3 is homogenized and pulverized to obtain cross-linked sodium hyaluronate gel particles; the particle size D of the cross-linked sodium hyaluronate gel particles is... 50Within 100~250 μm.

[0026] S5: Dissolve sodium hyaluronate with a molecular weight of 500,000 to 3,000,000 Da in a buffer solution (the mass ratio of sodium hyaluronate to buffer solution in the sodium hyaluronate solution is 0.01 to 0.03:1, preferably 0.02:1) to obtain a sodium hyaluronate solution, and then mix it evenly with the cross-linked sodium hyaluronate gel particles obtained in step S4 (the mass ratio of sodium hyaluronate solution to cross-linked sodium hyaluronate gel particles is 0.1 to 0.5:1, preferably 0.25:1) to obtain a cross-linked sodium hyaluronate gel.

[0027] Secondly, a method for preparing a cross-linked sodium hyaluronate gel composition includes the following steps: R1: Take the cross-linked sodium hyaluronate gel product prepared in the first aspect and sterilize it by moist heat at 120~125℃ for 10~15min; R2: The cross-linked sodium hyaluronate gel obtained in step R1 is mixed with microspheres to obtain a filler containing cross-linked sodium hyaluronate. The microspheres are one or more of hydroxyapatite, PEGylated polycaprolactone, and PEGylated poly-L-lactic acid. The mass ratio of cross-linked sodium hyaluronate gel to microspheres is 2~4:1, preferably 2.3:1.

[0028] The specific implementation method is as follows:

[0029] Example 1

[0030] 3g of sodium hyaluronate with a molecular weight of 1.4 million Da was dissolved in 20 ml of 1% NaOH solution and mixed thoroughly. PEGDE500 (polyethylene glycol diglycidyl ether) was added to the resulting solution and mixed thoroughly. The mixture was cross-linked at 37°C for 24 h to prepare block-shaped cross-linked sodium hyaluronate gel. A phosphate-sodium chloride buffer solution was prepared. The cross-linked sodium hyaluronate gel was cut into rectangular blocks and placed in the buffer solution. It was allowed to swell at 70°C for 30 min, the buffer solution was discarded, new buffer solution was added, and the process was repeated for another 30 min. This process was repeated 5 times. The swollen and washed gel was homogenized and pulverized to prepare cross-linked sodium hyaluronate gel particles. 2g of sodium hyaluronate with a molecular weight of 1.4 million Da was dissolved in 100 ml of buffer solution to obtain a sodium hyaluronate solution. 20g of the sodium hyaluronate solution was mixed thoroughly with 80g of the cross-linked sodium hyaluronate gel particles to obtain the finished cross-linked sodium hyaluronate gel. The obtained cross-linked sodium hyaluronate gel was subjected to moist heat sterilization at 121°C for 12 minutes. The obtained cross-linked sodium hyaluronate gel was then mixed with hydroxyapatite microspheres at a mass ratio of 7:3 to obtain a filler containing cross-linked sodium hyaluronate.

[0031] Example 2

[0032] 3g of 1,000,000 Da sodium hyaluronate was dissolved in 20 ml of 1% NaOH solution and mixed thoroughly. PEGDE2000 was added to the resulting solution and mixed thoroughly. The mixture was cross-linked at 37°C for 24 h to prepare block-shaped cross-linked sodium hyaluronate gel. A phosphate-sodium chloride buffer solution was prepared. The cross-linked sodium hyaluronate gel was cut into cuboids and placed in the buffer solution. It was allowed to swell at 70°C for 30 min, the buffer solution was discarded, new buffer solution was added, and the swelling was repeated for another 30 min, for a total of 6 times. The swollen and washed gel was homogenized and pulverized to prepare cross-linked sodium hyaluronate gel particles. 2g of 1,400,000 Da sodium hyaluronate was dissolved in 100 ml of buffer solution to obtain a sodium hyaluronate solution. 20g of the sodium hyaluronate solution was mixed thoroughly with 80g of the cross-linked sodium hyaluronate gel particles to obtain the finished cross-linked sodium hyaluronate gel. The finished cross-linked sodium hyaluronate gel was then subjected to moist heat sterilization at 121°C for 12 min. The cross-linked sodium hyaluronate gel obtained above was mixed with hydroxyapatite microspheres at a mass ratio of 7:3 to obtain a filler containing cross-linked sodium hyaluronate.

[0033] Example 3

[0034] 3g of sodium hyaluronate with a molecular weight of 1.4 million Da was dissolved in 20 ml of 1% NaOH solution and mixed thoroughly. PEGDE2000 was added to the resulting solution and mixed thoroughly. The mixture was cross-linked at 37℃ for 24 h to prepare block-shaped cross-linked sodium hyaluronate gel. A phosphate-sodium chloride buffer solution was prepared. The cross-linked sodium hyaluronate gel was cut into cuboids and placed in the buffer solution. It was allowed to swell at 70℃ for 30 min, the buffer solution was discarded, new buffer solution was added, and the swelling was repeated for another 30 min, for a total of 7 times. The swollen and washed gel was homogenized and pulverized to prepare cross-linked sodium hyaluronate gel particles. 2g of sodium hyaluronate with a molecular weight of 1.4 million Da was dissolved in 100 ml of buffer solution to obtain a sodium hyaluronate solution. 20g of the sodium hyaluronate solution was mixed thoroughly with 80g of the cross-linked sodium hyaluronate gel particles to obtain the finished cross-linked sodium hyaluronate gel. The finished cross-linked sodium hyaluronate gel was then subjected to moist heat sterilization at 121℃ for 12 min. The cross-linked sodium hyaluronate gel obtained above was mixed with hydroxyapatite microspheres at a mass ratio of 7:3 to obtain a filler containing cross-linked sodium hyaluronate.

[0035] Example 4

[0036] 3g of sodium hyaluronate with a molecular weight of 1.4 million Da was dissolved in 20 ml of 1% NaOH solution and mixed thoroughly. PEGDE2000 was added to the resulting solution and mixed thoroughly. The mixture was cross-linked at 37℃ for 24 h to prepare block-shaped cross-linked sodium hyaluronate gel. A phosphate-sodium chloride buffer solution was prepared. The cross-linked sodium hyaluronate gel was cut into cuboids and placed in the buffer solution. It was allowed to swell at 70℃ for 30 min, the buffer solution was discarded, new buffer solution was added, and the swelling was repeated for another 30 min, for a total of 7 times. The swollen and washed gel was homogenized and pulverized to prepare cross-linked sodium hyaluronate gel particles. 2g of sodium hyaluronate with a molecular weight of 1.4 million Da was dissolved in 100 ml of buffer solution to obtain a sodium hyaluronate solution. 20g of the sodium hyaluronate solution was mixed thoroughly with 80g of the cross-linked sodium hyaluronate gel particles to obtain the finished cross-linked sodium hyaluronate gel. The finished cross-linked sodium hyaluronate gel was then subjected to moist heat sterilization at 121℃ for 12 min. The cross-linked sodium hyaluronate gel obtained above was mixed with PEG-PCL (polyethylene glycol-polycaprolactone) microspheres at a mass ratio of 7:3 to obtain a filler containing cross-linked sodium hyaluronate.

[0037] Example 5

[0038] 3g of sodium hyaluronate with a molecular weight of 2 million Da was dissolved in 20 ml of 1% NaOH solution and mixed thoroughly. PEGDE2000 was added to the resulting solution and mixed thoroughly. The mixture was cross-linked at 37℃ for 24 h to prepare block-shaped cross-linked sodium hyaluronate gel. A phosphate-sodium chloride buffer solution was prepared. The cross-linked sodium hyaluronate gel was cut into cuboids and placed in the buffer solution. It was allowed to swell at 70℃ for 30 min, the buffer solution was discarded, new buffer solution was added, and the swelling was repeated for another 30 min, for a total of 8 times. The swollen and washed gel was homogenized and pulverized to prepare cross-linked sodium hyaluronate gel particles. 2g of sodium hyaluronate with a molecular weight of 1.4 million Da was dissolved in 100 ml of buffer solution to obtain a sodium hyaluronate solution. 20g of the sodium hyaluronate solution was mixed thoroughly with 80g of the cross-linked sodium hyaluronate gel particles to obtain the finished cross-linked sodium hyaluronate gel. The finished cross-linked sodium hyaluronate gel was then subjected to moist heat sterilization at 121℃ for 12 min. The cross-linked sodium hyaluronate gel obtained above was mixed with hydroxyapatite microspheres at a mass ratio of 7:3 to obtain a filler containing cross-linked sodium hyaluronate.

[0039] Compared with Example 1, all process data are basically the same as those in Example 3, except that the crosslinking agent is replaced with BDDE (butanediol diglycidyl ether).

[0040] Compared with Example 2, all process data are basically the same as those in Example 3, except that the crosslinking agent is replaced with BDDE (butanediol diglycidyl ether), and the microspheres are made of PEG-PCL. Performance evaluation methods:

[0041] 1) Evaluation of elastic modulus: The test method is as follows: Take a 2 g sample, use a rheometer, and perform frequency scanning at 25℃ and 0.5% shear strain. Read the elastic modulus at 1 Hz. The test results are as follows. Figure 1 As shown in the table below.

[0042] Examples 3 and Comparative Example 1, and Examples 4 and Comparative Example 2 differ only in the crosslinking agent used. The corresponding examples all use PEGDE crosslinking, resulting in higher elastic modulus. This demonstrates that the composition prepared using PEGDE as a crosslinking agent has a higher elastic modulus than the composition prepared using BDDE, indicating that it can provide better mechanical support.

[0043] 2) Evaluation of cohesion: The test method was as follows: 2 g of the moist heat sterilized cross-linked sodium hyaluronate gel (before mixing with microspheres) was stained with 2% toluidine blue solution. The stained sample was then injected into 1 L of slowly stirred purified water. The morphology was observed and recorded at 15s, 70s, and 95s after injection. Tests were performed on Examples 1, 3, and Comparative Example 1, and the results are as follows: Figure 2-4 As shown in the table below.

[0044] As can be seen, the comparative example disperses quickly when injected into water, while neither Example 1 nor Example 3 shows dispersion within the observation range. The order of the degree of dispersion of the gel in water is: Example 3, Example 1, and the comparative example. This indicates that the gel obtained by this technique has better cohesion and is more conducive to post-injection use.

[0045] 3) To evaluate the binding affinity between the gel and microspheres, the test method was as follows: the sample was filled into a 1 mL pre-filled syringe, centrifuged at 500 rpm for 10 min, and the layering effect was observed. Tests were performed on Example 4 and Comparative Example 2, and the results are as follows: Figure 5 and 6 As shown in the table, judging from whether or not the gel separates into layers, it is clear that in Example 4, no separation of the gel and microspheres occurs after centrifugation, while in Comparative Example 2, the separation of the gel and microspheres is more obvious.

[0046] The core reason for the improved binding properties of the gel prepared using this technology when mixed with PEGylated microspheres is the synergistic effect brought about by the same PEG fragments. After PEGDE crosslinks HA, PEG groups remain or are incorporated into its molecular chain; and the surface of the PEGylated polyester microspheres is also modified with PEG fragments. According to the principle of "like dissolves like," the same PEG fragments will form good compatibility, reducing the repulsive force between the two substances at the interface, making them easier to approach each other in the mixed system rather than separate, thereby improving the macroscopic dispersion uniformity and binding tightness.

[0047] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing cross-linked sodium hyaluronate gel, characterized in that, Includes the following steps: S1: Take sodium hyaluronate with a molecular weight of 500,000 to 3,000,000 Da, dissolve it in an alkaline solution, mix well, and the mass ratio of sodium hyaluronate to alkaline solution is 0.1 to 0.2:1; S2: Add a crosslinking agent to the solution obtained in step S1, and crosslink at 15~40℃ for 8~24h to obtain block crosslinked sodium hyaluronate gel. The crosslinking agent is one or more of polyethylene glycol diglycidyl ether and polypropylene glycol diglycidyl ether with different molecular weights. S3: Cut the block cross-linked sodium hyaluronate gel obtained in step S2 into segments and put them into phosphate-sodium chloride buffer. After swelling at 50~70℃ for 28~32 min, discard the buffer. Add new buffer and continue to swell at 50~70℃ for 28~32 min. Repeat 4~10 times until the weight of the gel no longer increases. S4: The gel that has been swollen and cleaned in step S3 is homogenized and pulverized to obtain cross-linked sodium hyaluronate gel particles. S5: Dissolve sodium hyaluronate with a molecular weight of 500,000 to 3,000,000 Da in a buffer solution to obtain a sodium hyaluronate solution. Then mix the solution with the cross-linked sodium hyaluronate gel particles obtained in step S4 to obtain the finished cross-linked sodium hyaluronate gel.

2. The method for preparing a cross-linked sodium hyaluronate gel as described in claim 1, characterized in that, The alkaline solution mentioned in step S1 is a 1% NaOH solution, and the mass ratio of sodium hyaluronate to the alkaline solution is 0.15:

1.

3. The method for preparing a cross-linked sodium hyaluronate gel as described in claim 1, characterized in that, The crosslinking agent mentioned in step S2 is polyethylene glycol diglycidyl ether with a molecular weight of 500~6000 Da.

4. The method for preparing a cross-linked sodium hyaluronate gel as described in claim 1, characterized in that, In step S3, the buffer solution contains 6 g / L sodium chloride, 4 g / L disodium hydrogen phosphate, and 2 g / L sodium dihydrogen phosphate.

5. The method for preparing a cross-linked sodium hyaluronate gel as described in claim 1, characterized in that, In step S4, the particle size D of the cross-linked sodium hyaluronate gel particles is... 50 Within 100~250 μm.

6. The method for preparing a cross-linked sodium hyaluronate gel as described in claim 1, characterized in that, In step S5, the mass ratio of sodium hyaluronate to buffer solution in the sodium hyaluronate solution is 0.01~0.03:

1.

7. A method for preparing a cross-linked sodium hyaluronate gel as described in claim 1 or 6, characterized in that, In step S5, the mass ratio of sodium hyaluronate solution to cross-linked sodium hyaluronate gel particles is 0.1~0.5:

1.

8. A method for preparing a cross-linked sodium hyaluronate gel composition, characterized in that, Includes the following steps: R1: Take any of the cross-linked sodium hyaluronate gel products prepared according to claims 1-7 and perform moist heat sterilization at 120~125℃ for 10~15min; R2: The cross-linked sodium hyaluronate gel obtained in step R1 is mixed with microspheres to obtain a filler containing cross-linked sodium hyaluronate. The microspheres are one or more of hydroxyapatite, PEGylated polycaprolactone, and PEGylated poly-L-lactic acid. The mass ratio of cross-linked sodium hyaluronate gel to microspheres is 2~4:1.

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