Preparation method of conus anti-wrinkle element

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a preparation method of conotoxin. The invention provides a conus anti-wrinkle element preparation method, which comprises: mixing a naked peptide, a solvent, a buffer salt, a catalyst and an oxidizing agent to obtain a mixed solution, introducing oxygen into the mixed solution, and carrying out a reaction to obtain the conus anti-wrinkle element, the catalyst comprises at least one of cysteine and glutathione. The preparation method of the conus anti-wrinkle element is simple to operate, short in preparation period and high in oxidation efficiency, also improves the yield of the conus anti-wrinkle element, and can be applied to industrial production of the conus anti-wrinkle element.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to a method for preparing cono anti-wrinkle factor. Background Art

[0002] Cono anti-wrinkle is an active polypeptide that is highly specific to different ion channels and nerve receptors. It is secreted by the venom glands of the venom tube and the inner wall of the venom sac of the marine gastropod mollusk cone snail. It is a highly folded peptide with 22 amino acids in the main chain. The peptide chain is long and has three pairs of disulfide bonds to form a specific spatial structure. Cono anti-wrinkle has strong penetration ability and can relax muscles. Studies have shown that cono anti-wrinkle can inhibit the transmission of nerve signals and inhibit muscle contraction by inhibiting sodium ion channels, calcium ion channels and acetylcholine receptors, ultimately achieving the effect of reducing wrinkles. Therefore, cono anti-wrinkle has the advantages of high selectivity and small side effects, and is widely used in the field of cosmetics.

[0003] The structure of conotin is as follows:

[0004] H-Pyr 1 -Gly 2 -Cys 3 -Cys 4 -Asn 5 -Gly 6 -Pro 7 -Lys 8 -Gly 9 -Cys 10 -Ser 11 -Ser 12 -Lys 13 -Trp 14 -Cys 15 -Arg 16 -Asp 17 -His 18 -Ala 19 -Arg 20 -Cys 21 -Cys 22 -NH2

[0005] (disulfide bonds: C3-C15; C4-C21; C10-C22);

[0006] The content of cono-antiwrinkle factor in organisms is extremely low. If biological extraction is used, it has the disadvantages of low yield and high cost. At present, the common industrial production method of cono-antiwrinkle factor is solid phase synthesis, but in actual production, inappropriate oxidation methods are very likely to generate disulfide bond mismatched isomers, and ordinary oxidation methods are inefficient, the purity of crude peptides is poor, and it also increases the difficulty of subsequent product purification, resulting in low yield and high cost. The commonly used cono-antiwrinkle factor oxidation methods in the prior art include ammonia water natural oxidation method, DMSO (dimethyl sulfoxide) oxidation method, and fixed-point oxidation method. These methods either have high requirements for oxidation (relatively stringent reaction conditions) or have low yields. Therefore, it is necessary to provide a preparation method for cono-antiwrinkle factor with high yield and loose oxidation reaction conditions. Summary of the invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the oxidation method for preparing cono anti-wrinkle in the prior art, that is, high oxidation requirement and low yield of the prepared cono anti-wrinkle, thereby providing a method for preparing cono anti-wrinkle.

[0008] To this end, the present invention provides the following technical solutions.

[0009] The present invention provides a method for preparing cono anti-wrinkle agent, comprising: mixing naked peptide, solvent, buffer salt, catalyst and oxidant to obtain a mixed solution, introducing oxygen into the mixed solution, reacting, and obtaining cono anti-wrinkle agent;

[0010] The catalyst comprises at least one of cysteine ​​and glutathione;

[0011] The naked peptide structure is as follows:

[0012] H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2.

[0013] In an optional embodiment, the mass ratio of the catalyst, buffer salt, oxidant and naked peptide is (1-20):(1-20):(1-20):(50-200); preferably (2.5-3.5):(6-7):(7-8):(110-130).

[0014] In an optional embodiment, the volume ratio of the total mass of naked peptide, buffer salt, catalyst, and oxidant to the solvent is 500-2000 mg:500 mL.

[0015] In an optional embodiment, the reaction time is 10-48 h; preferably 22-26 h.

[0016] In an optional embodiment, the step of adjusting the pH value of the mixed solution to 7-10 is further included before the reaction;

[0017] In an optional embodiment, the flow rate of oxygen is 1-2 L / min;

[0018] In an optional embodiment, the reaction further includes a step of purifying the reaction solution after the reaction.

[0019] In an optional embodiment, the pH value is adjusted by an alkaline solution;

[0020] Preferably, the alkaline solution comprises aqueous ammonia.

[0021] In an optional embodiment, the buffer salt includes at least one of ammonium sulfate, ammonium carbonate and ammonium acetate;

[0022] In an optional embodiment, the oxidant includes at least one of ethylenediaminetetraacetic acid, hydrogen peroxide and DMSO.

[0023] In an optional embodiment, the catalyst is cysteine, the buffer salt is ammonium sulfate, and the oxidant is ethylenediaminetetraacetic acid.

[0024] In an optional embodiment, the preparation method further comprises the step of adding an acidic solution to terminate the reaction.

[0025] When the target peak in HPLC analysis is the only main peak, the reaction is completed and an acidic solution is added to terminate the reaction;

[0026] In an optional embodiment, the acidic solution adjusts the pH value of the reaction solution to 5-6;

[0027] In an optional embodiment, the acidic solution comprises acetic acid.

[0028] In an optional embodiment, the solvent is at least one of acetonitrile and water. Optionally, the solvent is an acetonitrile aqueous solution with a volume percentage concentration of 1-10%.

[0029] In an optional embodiment, the preparation method comprises the following steps: mixing naked peptide, solvent, buffer salt, catalyst and oxidant to obtain a mixed solution, adding alkaline solution to adjust the pH value to 7-10; introducing oxygen into the mixed solution to react, and adding acidic solution to terminate the reaction after the reaction is completed; purifying the reaction solution to obtain cono anti-wrinkle factor.

[0030] The technical solution of the present invention has the following advantages:

[0031] 1. A method for preparing conoantiwrinkle provided by the present invention comprises mixing naked peptide, solvent, buffer salt, catalyst and oxidant to obtain a mixed solution, introducing oxygen into the mixed solution, reacting, and preparing conoantiwrinkle; the catalyst comprises at least one of cysteine ​​and glutathione. The preparation method can form three pairs of disulfide bonds on the naked peptide of conoantiwrinkle, and no isomers with mismatched disulfide bonds will appear; wherein, the buffer salt can stabilize the pH value and provide a stable chemical environment to reduce the risk of naked peptide degradation; cysteine ​​or glutathione can avoid the defect that the naked peptide breaks due to oxidation of its amino acid residues, thereby reducing the activity of conoantiwrinkle, and can also maintain the chemical stability of the prepared conoantiwrinkle; the oxidant helps the formation of disulfide bonds and stabilizes the spatial structure of the prepared conoantiwrinkle. The preparation method of the present invention is simple to operate, short in preparation cycle, high in oxidation efficiency, and also improves the yield of conoantiwrinkle, and can be applied to the industrial production of conoantiwrinkle.

[0032] 2. A method for preparing cono anti-wrinkle agent provided by the present invention, wherein the present invention preferably uses cysteine ​​and ammonium sulfate as buffer salts and ethylenediaminetetraacetic acid as oxidants to prepare cono anti-wrinkle agent, and the oxidation efficiency is high, the preparation cycle is short, and the yield of the prepared cono anti-wrinkle agent is high; the present invention preferably uses a mass ratio of cysteine, buffer salts, oxidants, and naked peptides of (2.5-3.5): (6-7): (7-8): (110-130), which improves the yield of cono anti-wrinkle agent; the present invention preferably uses a reaction time of 22-26h, which can effectively improve the yield of cono anti-wrinkle agent. The present invention controls the reaction rate by controlling the oxygen flow rate to 1-2L / min, and the yield of the prepared cono anti-wrinkle agent is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The HPLC analysis of the target peak as the only main peak in Example 1 of the present invention after 24 hours of reaction is performed;

[0035] Figure 2 It is the high performance liquid chromatogram of the cono anti-wrinkle factor prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0037] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0038] Example 1

[0039] This embodiment provides a method for preparing cono anti-wrinkle factor, comprising the following steps:

[0040] (1) Dissolve 30.29 mg of cysteine, 66.07 mg of ammonium sulfate and 73.06 mg of ethylenediaminetetraacetic acid in 500 mL of water, add 1.2 g of naked peptide, stir, and after the solution is clarified, add 1 mol / L of ammonia water to adjust the pH value of the solution to 8.0. Pass oxygen into the solution and stir the reaction at an oxygen flow rate of 1 L / min; react for 24 hours (the pH value of the solution is tested after every 2 hours of reaction, and the pH value of the solution is always maintained at 8.0±0.2). Add 1 mol / L of acetic acid to the solution to adjust the pH value to 5-6 and terminate the reaction. The target peak in the reaction solution was analyzed by HPLC and was the only main peak. The results are shown in Figure 1 ;

[0041] (2) The solution obtained by the above reaction was purified for the first time. The conditions of the first purification are shown in Table 1, and the eluate with a purity of more than 90% of the target peak was collected; the obtained eluate was purified for the second time. The conditions of the second purification are shown in Table 2. The eluate with a purity of more than 98% of the target peak was collected and freeze-dried to obtain 0.688 g of cono anti-wrinkle factor. The HPLC chromatogram of the cono anti-wrinkle factor obtained in Example 1 is shown in Figure 2 .

[0042] Table 1 First purification conditions

[0043]

[0044]

[0045] Table 2 Second purification conditions

[0046]

[0047] Example 2

[0048] The present embodiment provides a method for preparing cono anti-wrinkle factor, which is basically the same as that in Example 1, the only difference is that in the present embodiment, 60.58 mg of cysteine, 132.4 mg of ammonium sulfate and 146.12 mg of ethylenediaminetetraacetic acid are dissolved in 500 mL of water instead of 30.29 mg of cysteine, 66.07 mg of ammonium sulfate and 73.06 mg of ethylenediaminetetraacetic acid dissolved in 500 mL of water in Example 1, and the remaining process conditions and operations are the same as those in Example 1, and finally 0.6739 g of cono anti-wrinkle factor is obtained.

[0049] Example 3

[0050] This embodiment provides a method for preparing cono anti-wrinkle factor, which is basically the same as that in Example 1, except that the reaction in this embodiment is 12 hours instead of 24 hours in Example 1, and the other process conditions and operations are the same as those in Example 1, and finally 0.546 g of cono anti-wrinkle factor is obtained.

[0051] Example 4

[0052] This embodiment provides a method for preparing cono anti-wrinkle factor, which is basically the same as that of Example 2, except that the reaction time in this embodiment is 48 hours instead of 24 hours in Example 1, and the other process conditions and operations are the same as those in Example 2, and finally 0.6612 g of cono anti-wrinkle factor is obtained.

[0053] Example 5

[0054] This embodiment provides a method for preparing cono anti-wrinkle factor, comprising the following steps:

[0055] (1) 181.74 mg of cysteine, 132.14 mg of ammonium carbonate and 146.12 mg of hydrogen peroxide were dissolved in 500 mL of water, 0.6 g of naked peptide was added, and the mixture was stirred. After the solution was clarified, 1 mol / L of ammonia was added to adjust the pH value of the reaction system to 8.0, oxygen was introduced into the solution and stirred for reaction, and the oxygen flow rate was 2 L / min; the reaction was continued for 24 h (the pH value of the solution was detected after every 2 h of reaction, and the pH value of the solution was always maintained at 8.0±0.2), 1 mol / L of acetic acid was added to the solution to adjust the pH value to 5-6, and the reaction was terminated;

[0056] (2) The preparation method is the same as step (2) of Example 1 to obtain 0.31746 g of cono-anti-wrinkle factor.

[0057] Example 6

[0058] This embodiment provides a method for preparing cono anti-wrinkle factor, which is basically the same as that of Example 1, except that the same mass of ammonium acetate is used instead of ammonium sulfate in this embodiment, and the other process conditions and operations are the same as those of Example 1, and finally 0.658 g of cono anti-wrinkle factor is obtained.

[0059] Example 7

[0060] This example provides a method for preparing cono anti-wrinkle agent, which is basically the same as that in Example 1, except that glutathione of the same mass is used to replace cysteine ​​in this example, and the other process conditions and operations are the same as those in Example 1, and finally 0.682 g of cono anti-wrinkle agent is obtained.

[0061] Example 8

[0062] This embodiment provides a method for preparing cono anti-wrinkle factor, which is basically the same as that in Example 1, the only difference is that in this embodiment, 1.2 g of naked peptide is dissolved in 500 mL of acetonitrile aqueous solution (the volume percentage concentration of the acetonitrile aqueous solution is 2% (v / v)), 30.29 mg of cysteine, 66.07 mg of ammonium sulfate and 73.06 mg of ethylenediaminetetraacetic acid are added, instead of 30.29 mg of cysteine, 66.07 mg of ammonium sulfate and 73.06 mg of ethylenediaminetetraacetic acid in Example 1 being dissolved in 500 mL of water, 1.2 g of naked peptide is added, and the other process conditions and operations are the same as those in Example 1, and finally 0.734 g of cono anti-wrinkle factor is obtained.

[0063] Comparative Example 1

[0064] This comparative example provides a method for preparing cono anti-wrinkle factor, which is basically the same as that in Example 1, except that cysteine ​​is not added in this comparative example, and the other process conditions and operations are the same as those in Example 1, and finally 0.461 g of cono anti-wrinkle factor is obtained.

[0065] Test Example 1

[0066] The properties of the cono anti-wrinkle agent obtained in the above-mentioned Examples 1-8 and Comparative Example 1 are as follows:

[0067] (1) The yield of the conoanti-wrinkle peptide prepared in each of Examples 1-8 and Comparative Example 1 was calculated. The results are shown in Table 4. Yield = molar amount of the prepared conoanti-wrinkle peptide / molar amount of the naked peptide

[0068] (2) The purity of the cono anti-wrinkle extracts prepared in Examples 1-8 and Comparative Example 1 was detected. The chromatographic detection conditions were shown in Table 3, and the results were shown in Table 4.

[0069] Table 3 Chromatographic detection conditions

[0070]

[0071]

[0072] Table 4 Test results

[0073] Purity Yield Example 1 98.1％ 57.48％ Example 2 98.0％ 56.3％ Example 3 97.7％ 45.61％ Example 4 96.5％ 55.24％ Example 5 97.8％ 53.04％ Example 6 97.5％ 54.97％ Example 7 97.6％ 56.98％ Example 8 98.3％ 61.32％ Comparative Example 1 96.3％ 38.51％

[0074] As shown in Table 4, the yield of the cono anti-wrinkle prepared in Examples 1-8 is relatively high, among which Example 8 has better purity and higher yield; compared with Example 8, Example 1 uses water as the solvent, and its purity and yield are lower than Example 8 using acetonitrile aqueous solution; Example 2 is not within the preferred mass ratio of cysteine, buffer salt, oxidant and naked peptide of the present invention (2.5-3.5): (6-7): (7-8): (110-130), and its purity and yield are worse than Example 8; Examples 3-4 prove that the reaction time of 22-26h is the preferred reaction time, which can improve the yield of cono anti-wrinkle; Examples 5-7 prove that when ethylenediaminetetraacetic acid, ammonium sulfate and cysteine ​​are used to prepare cono anti-wrinkle, the yield of cono anti-wrinkle will be improved. Comparison of Example 1 with Comparative Example 1 shows that when cysteine ​​is not added, the purity and yield of the prepared cono anti-wrinkle are greatly reduced.

[0075] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the scope of protection of the invention.

Claims

1. A method for preparing conus anti-wrinkle factor, It is characterized in that The method comprises mixing naked peptide, solvent, buffer salt, catalyst and oxidant to obtain a mixed solution, introducing oxygen into the mixed solution to react and obtain cono anti-wrinkle factor; The catalyst comprises at least one of cysteine ​​and glutathione; The structure of the naked peptide is as follows: H-Pyr-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH 2 。 2. The preparation method according to claim 1, It is characterized in that The mass ratio of the catalyst, buffer salt, oxidant and naked peptide is (1-20):(1-20):(1-20):(50-200); preferably (2.5-3.5):(6-7):(7-8):(110-130).

3. The preparation method according to claim 1, It is characterized in that The reaction time is 10-48h; preferably 22-26h.

4. The preparation method according to claim 1, It is characterized in that Before the reaction, the step of adjusting the pH value of the mixed solution to 7-10 is also included; and / or, The oxygen flow rate is 1-2 L / min; and / or, After the reaction, the step of purifying the reaction solution is also included.

5. The preparation method according to claim 4, It is characterized in that The pH value is adjusted by an alkaline solution; Preferably, the alkaline solution comprises aqueous ammonia.

6. The preparation method according to claim 1, It is characterized in that The buffer salt comprises at least one of ammonium sulfate, ammonium carbonate and ammonium acetate; and / or, The oxidant includes at least one of ethylenediaminetetraacetic acid, hydrogen peroxide and DMSO.

7. The preparation method according to claim 1, It is characterized in that The catalyst is cysteine, the buffer salt is ammonium sulfate, and the oxidant is ethylenediaminetetraacetic acid.

8. The preparation method according to claim 1, It is characterized in that The preparation method further comprises the step of adding an acidic solution to terminate the reaction.

9. The preparation method according to claim 8, It is characterized in that The acidic solution adjusts the pH value of the reaction solution to 5-6; and / or, The acidic solution includes acetic acid.

10. The preparation method according to claim 1, It is characterized in that The solvent includes at least one of acetonitrile and water. Optionally, the solvent is an acetonitrile aqueous solution with a volume percentage concentration of 1-10%.

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