CN114920805B - Novel scorpion venom having wrinkle-improving activity and composition comprising the same - Google Patents
Novel scorpion venom having wrinkle-improving activity and composition comprising the same Download PDFInfo
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- CN114920805B CN114920805B CN202210483328.8A CN202210483328A CN114920805B CN 114920805 B CN114920805 B CN 114920805B CN 202210483328 A CN202210483328 A CN 202210483328A CN 114920805 B CN114920805 B CN 114920805B
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Abstract
The invention discloses a novel scorpion toxin with wrinkle improving activity and a composition containing the same; belongs to the field of polypeptide preparation; the method comprises the following steps: placing the conotoxin, the 3-beta-hydroxy oleanolic acid butyrate and the catalyst in a solvent to be completely dissolved, placing the mixture in a reactor to react, washing, drying and purifying to obtain the novel red scorpion toxin. The invention also relates to compositions comprising the novel scorpion toxin. The novel scorpion toxin prepared by the invention can improve the wrinkle activity; the composition is prepared by compounding the components with other components, has good anti-aging and anti-oxidation properties, can better promote the elasticity of skin and achieves the effects of moisturizing, locking water and preserving moisture.
Description
Technical Field
The invention belongs to the field of polypeptide preparation, and particularly relates to novel scorpion toxin with wrinkle improving activity and a composition containing the same.
Background
Wrinkles are the result of skin aging. Skin aging not only directly affects the normal structure and physiological functions of skin tissues, but also indirectly affects the stability of the internal and external environments of the organism, causes imbalance of metabolism in different degrees, and may promote and accelerate aging of the organism, even death. The normal course of muscle contraction is: nerve impulse-acetylcholine (Ach) -end plate potential-muscle action potential-muscle contraction. Wrinkles are formed due to the long-term repeated contraction of expression muscles in successive months; it is apparent that blocking signal transmission is one of the methods for improving wrinkle generation.
The red scorpion toxin is also called conotoxin, conotoxin wrinkle-resistant agent, conotoxin and the like, is a safe and efficient polypeptide which is derived from red scorpion toxin and simulates artificial synthesis of conotoxin and has the efficacy comparable to BOTOX, and is a highly folded peptide. It is commonly used as a molecular tool for neuroscience research and drug development by selectively targeting different types of neurotransmitter receptors or voltage-gated ion channels.
The prior art, for example publication No. CN 104334155A, discloses a cosmetic composition comprising a μ conopeptide; which comprises as active substance a cosmetically effective amount of at least one mu-conotoxin peptide. It also relates to compositions wherein the mu-conotoxin peptide is an arginine, lysine polypeptide, CAS number: 937286-43-6, molecular formula C 92 ,H 139 ,N 35 ,O 28 ,S 6 Acetate (molar mass 2376 g/mol); the use of the compositions involved therein to improve the mechanical properties of the skin, the tension and/or stiffness and/or elasticity of the skin.
Disclosure of Invention
The invention aims to provide a novel scorpion toxin capable of improving wrinkle activity; the composition is prepared by compounding the components with other components, and has good anti-aging effect and oxidation resistance, and can well promote the elasticity of skin and achieve the effects of moisturizing, locking water and preserving moisture.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a novel red scorpion toxin is characterized in that 3-beta-hydroxy oleanolic acid butyrate is grafted on the side chain of amino acid or the amino group at the N terminal of conotoxin;
the conotoxin is mu-conotoxin peptide; wherein the amino acid sequence of the mu-conotoxin peptide is as follows: pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-amide.
According to the invention, 3-beta-hydroxy oleanolic acid butyrate is grafted on the side chain of amino acid in the conotoxin or the amino group at the N terminal to prepare the novel red scorpion toxin, which can better improve the wrinkle activity so as to achieve the purpose of removing wrinkles.
The invention also discloses application of the novel scorpion toxin in preparing skin care products for improving wrinkle activity.
The invention also discloses a preparation method of the novel scorpion toxin, which comprises the following steps:
placing the conotoxin, the 3-beta-hydroxy oleanolic acid butyrate and the catalyst into a solvent to be completely dissolved, transferring the solvent into a reactor to react, washing, drying and purifying to obtain the novel red scorpion toxin.
Preferably, according to the preparation method of the invention, 50 to 80 parts of conotoxin, 4.5 to 10 parts of 3-beta-hydroxy oleanolic acid butyrate, 1.75 to 5.5 parts of catalyst and 25 to 50 parts of solvent are calculated by weight.
Preferably, according to the preparation method of the present invention, the catalyst is at least one of DCC, HOBt, HOAt, EDCI, HBTU, DIPCDI, pyBOP.
Preferably, according to the preparation method of the present invention, the solvent is at least one of dimethylformamide, tetrahydrofuran and dichloromethane.
It is another object of the present invention to provide a composition comprising a novel scorpion toxin.
Preferably, the composition according to the present invention further comprises palmitoyl tripeptide-1, a humectant, a preservative, a pH regulator, an emulsifier, deionized water.
The composition is prepared by compounding the novel scorpion toxin, palmitoyl tripeptide-1, a humectant, a preservative, a pH regulator, an emulsifier and deionized water, and can improve the cell activity of HDF-a and NHEK, promote the proliferation of HDF-a and NHEK cells, further reduce the growth of wrinkles and achieve the anti-aging effect; meanwhile, the composition has good oxidation resistance, and can better promote the elasticity of skin and achieve the effects of moisturizing, locking water and preserving moisture.
Preferably, the composition according to the invention comprises the following components in parts by weight: 0.5 to 3 portions of novel scorpion toxin, 1 to 5 portions of palmitoyl tripeptide-1, 8 to 15 portions of humectant, 0.15 to 0.45 portion of preservative, 0.05 to 1.5 portions of pH regulator, 4 to 10 portions of emulsifier and 50 to 100 portions of deionized water.
The invention also discloses application of the composition in preparing a skin care product for improving wrinkle activity and/or firming skin.
The technical scheme of the invention has the following beneficial effects:
(1) 3-beta-hydroxy oleanolic acid butyrate is grafted on the side chain of amino acid or the amino group at the N terminal in the conotoxin to prepare the novel scorpion venom, which can better improve wrinkle activity so as to achieve the aim of removing wrinkles.
(2) The composition is prepared by compounding the novel scorpion toxin, palmitoyl tripeptide-1, a humectant, a preservative, a pH regulator, an emulsifier and deionized water, and can improve the cell activity of HDF-a and NHEK, promote the proliferation of HDF-a and NHEK cells, further reduce the growth of wrinkles and achieve the anti-aging effect; meanwhile, the composition has good oxidation resistance, and can better promote the elasticity of skin and achieve the effects of moisturizing, locking water and preserving moisture.
Therefore, the invention is a novel scorpion toxin capable of improving wrinkle activity; the composition is prepared by compounding the components with other components, and has good anti-aging effect and oxidation resistance, and can well promote the elasticity of skin and achieve the effects of moisturizing, locking water and preserving moisture.
Drawings
FIG. 1 is an infrared spectrum of the mu-conotoxin peptide and the novel red scorpion toxin in example 1;
FIG. 2 is an IR spectrum of protopectin and a pectin derivative in example 3.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
More preferably, in some embodiments of the present invention, a method for preparing a novel scorpion toxin comprises the steps of:
50 to 80 parts of conotoxin, 4.5 to 10 parts of 3-beta-hydroxy oleanolic acid butyrate and 1.75 to 5.5 parts of catalyst are placed in 25 to 50 parts of solvent by weight for complete dissolution, transferred into a reactor for reaction at room temperature for 3 to 6 hours, washed by citric acid aqueous solution and saturated sodium chloride solution with the concentration of 0.15 to 0.25mol/L for 2 to 3 times in sequence, dried by anhydrous sodium sulfate for 20 to 40min, and purified by HPLC, wherein the purification conditions are as follows: the chromatographic column is Agilent Zorbax SB-C 18 (4.6X 150mm,5 μm), a flow rate of 1 to 2mL/min, a detection wavelength of 220 to 280nm, and a mobile phase: solution A is an aqueous solution containing 0.1 to 0.2% TFA, solution B is an acetonitrile solution containing 0.1 to 0.2% TFA, the elution time is 20 to 40min, acetonitrile and TFA in the system are removed by rotary evaporation, and the mixture is freeze-dried to obtain the novel scorpion toxin.
Preferably, according to the preparation method, the weight parts of the conotoxin, the 3-beta-hydroxy oleanolic acid butyrate and the solvent are respectively 50 to 80 parts, 4.5 to 10 parts, 1.75 to 5.5 parts and 25 to 50 parts.
Preferably, according to the preparation method of the present invention, the catalyst is at least one of DCC, HOBt, HOAt, EDCI, HBTU, DIPCDI, pyBOP.
Preferably, according to the preparation method of the present invention, the solvent is at least one of dimethylformamide, tetrahydrofuran and dichloromethane.
In order to optimize the anti-wrinkle and moisturizing properties of the composition, preferred measures taken also include: adding 0.5 to 2.5 parts by weight of pectin derivative into the composition; the anti-wrinkle water-replenishing emulsion has interaction with each component of the composition, and further improves the anti-wrinkle and water-replenishing performances of the composition.
According to the composition of the present invention, the pectin derivative is prepared from aspartame-modified pectin.
Preferably, the pectin derivative is prepared according to the composition of the invention by:
dissolving pectin in a solvent, adding aspartame, carrying out heating reflux reaction, cooling to room temperature after the reaction is finished, washing with absolute ethyl alcohol, and freeze-drying to obtain the pectin derivative.
More preferably, according to the preparation method of the pectin derivative, the pectin is 4 to 8 parts by weight, the solvent is 150 to 450 parts by weight, and the aspartame is 10 to 15 parts by weight.
More preferably, the heating temperature is 75 to 90 ℃ and the heating time is 10 to 15h according to the method for producing the pectin derivative.
In some embodiments of the invention, a composition is prepared by: dissolving a humectant, a preservative and an emulsifier in deionized water, stirring and mixing uniformly at room temperature, then adding the novel scorpion toxin and palmitoyl tripeptide-1, mixing uniformly, and adding a pH regulator to regulate the pH of a system to be 5.0 to 6.5 to obtain the composition.
Preferably, according to the composition of the present invention, the humectant is at least one of glycerin, xylitol, sorbitol, sodium hyaluronate, sodium lactate, collagen.
Preferably, according to the composition of the present invention, the preservative is at least one of benzyl alcohol, benzoic acid, ethylhexyl glycerol, salicylic acid, sorbic acid.
Preferably, according to the composition of the present invention, the emulsifier is at least one of sodium stearate, sodium lauryl sulfate, tween-20, cetostearyl alcohol.
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
example 1:
a method for preparing novel scorpion toxin comprises the following steps:
according to the weight portion, 60 portions of mu-conotoxin peptide, 7.5 portions of 3-beta-hydroxy oleanolic acid butyrate and 3.5 portions of DCC are placed in 40 portions of dimethylformamide to be completely dissolved,transferring the mixture into a reactor to react for 4h at room temperature, washing the mixture for 3 times by using citric acid aqueous solution with the concentration of 0.2mol/L and saturated sodium chloride solution in sequence, then drying the mixture for 30min by using anhydrous sodium sulfate, and purifying the mixture by adopting HPLC, wherein the purification conditions are as follows: the chromatographic column is Agilent Zorbax SB-C 18 (4.6X 150mm,5 μm), flow rate of 1.5mL/min, detection wavelength of 245nm, mobile phase: solution A is an aqueous solution containing 0.15% TFA, solution B is an acetonitrile solution containing 0.1% TFA, the elution time is 30min, acetonitrile and TFA in the system are removed by rotary evaporation, and the solution is freeze-dried to obtain the novel red scorpion toxin.
In the implementation, the mu-conotoxin peptide is prepared according to a conventional polypeptide synthesis method; the amino acid sequence of the mu-conotoxin peptide is as follows: pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-amide.
Example 2:
a method for preparing novel scorpion toxin, which is different from the embodiment 1 in that:
according to the weight portion, 60 portions of mu-conotoxin peptide, 10 portions of 3-beta-hydroxy oleanolic acid butyrate and 4.5 portions of EDCI are placed in 50 portions of dimethylformamide to be completely dissolved, transferred into a reactor to react for 6 hours at room temperature, washed for 3 times by citric acid aqueous solution with the concentration of 0.25mol/L and saturated sodium chloride solution in sequence, then dried for 30min by anhydrous sodium sulfate, and purified by HPLC, wherein the purification conditions are as follows: the chromatographic column is Agilent Zorbax SB-C 18 (4.6X 150mm,5 μm), flow rate of 1.5mL/min, detection wavelength of 245nm, mobile phase: solution A is an aqueous solution containing 0.2% TFA, solution B is an acetonitrile solution containing 0.2% TFA, the elution time is 30min, acetonitrile and TFA in the system are removed by rotary evaporation, and the solution is freeze-dried to obtain the novel red scorpion toxin.
The amino acid sequence of the mu-conotoxin peptide in this example was the same as in example 1.
Example 3:
a method for preparing pectin derivatives comprises: dissolving 7.5 parts by weight of protopectin (purchased from Shandong Lusen Biotechnology Co., ltd.) in 250 parts by weight of deionized water, adding 12 parts by weight of aspartame, stirring and heating to 85 ℃, carrying out reflux reaction for 12 hours, cooling to room temperature after the reaction is finished, washing 3 times with absolute ethyl alcohol, and carrying out freeze drying to obtain the pectin derivative.
Example 4:
the preparation method of the composition comprises the following steps: dissolving 10 parts by weight of sorbitol, 0.25 part by weight of ethylhexyl glycerol and 6 parts by weight of sodium dodecyl sulfate in 80 parts by weight of deionized water, stirring and mixing uniformly at room temperature, then adding 1.5 parts by weight of mu-conotoxin peptide and 2.5 parts by weight of palmitoyl tripeptide-1, mixing uniformly, and adding triethanolamine to adjust the pH of the system to 5.5, thereby obtaining the composition.
The amino acid sequence of the mu-conotoxin peptide in this example was the same as in example 1.
Example 5:
the preparation method of the composition comprises the following steps: 10 parts by weight of sorbitol, 0.25 part by weight of ethylhexyl glycerin and 6 parts by weight of sodium lauryl sulfate were dissolved in 80 parts by weight of deionized water, and the mixture was stirred and mixed at room temperature, 1.5 parts by weight of the novel red scorpion toxin of example 1 and 2.5 parts by weight of palmitoyl tripeptide-1 were added and mixed, and the mixture was mixed uniformly, and triethanolamine was added to adjust the pH of the system to 5.5, thereby obtaining a composition.
Example 6:
a method of preparing a composition, different from example 5: the novel red scorpion toxin of example 1 was replaced with the novel red scorpion toxin of example 2.
Example 7:
a method of preparing a composition, different from example 4: dissolving 10 parts by weight of sorbitol, 0.25 part by weight of ethylhexyl glycerol, 6 parts by weight of sodium lauryl sulfate and 0.5 part by weight of protopectin (purchased from Shandong Lusen Biotech Co., ltd.) in 80 parts by weight of deionized water, stirring and mixing uniformly at room temperature, adding 1.5 parts by weight of mu-conotoxin peptide and 2.5 parts by weight of palmitoyl tripeptide-1, mixing uniformly, and adding triethanolamine to adjust the pH of the system to 5.5, thereby obtaining the composition.
Example 8:
a method of preparing a composition, different from example 7: mu-conotoxin peptides were replaced with the novel red scorpion toxin of example 1.
Example 9:
a method of preparing a composition, different from example 7: mu-conotoxin peptides were replaced with the novel red scorpion toxin of example 2.
Example 10:
a method of preparing a composition, different from example 7: protopectin was replaced with the pectin derivative in example 3.
Example 11:
a method of preparing a composition, different from example 8: the protopectin was replaced with the pectin derivative in example 3.
Example 12:
a method of preparing a composition, different from example 9: protopectin was replaced with the pectin derivative in example 3.
Example 13:
1. and (3) infrared structural characterization:
performing structural characterization on the prepared substance by using a 2000-104 type Fourier transform infrared spectrometer, wherein the scanning range is 4000-500cm -1 。
FIG. 1 is an infrared spectrum of mu-conotoxin peptide and novel red scorpion toxin in example 1; curves a and b are respectively mu-conotoxin peptide and novel red scorpion toxin; as can be seen from FIG. 1, the mu-conotoxin peptide is 2900cm -1 、2850cm -1 The characteristic absorption peak appearing nearby is the asymmetric and symmetric absorption peak of C-H; at 1680cm -1 、1630cm -1 Expansion and contraction vibration absorption peaks of C = O and N-H in amide groups appear nearby; at 700cm -1 、530cm -1 Characteristic absorption peaks of disulfide bonds appear nearby; relative to mu-conotoxin peptide, the novel scorpion toxin is 2910cm -1 、2850cm -1 The asymmetric and symmetric absorption peaks of C-H appearing nearby are enhanced; at 1730cm -1 A stretching vibration absorption peak of C = O appears nearby in the ester group; the method is characterized in that 3-beta-hydroxy oleanolic acid butyrate is grafted on the side chain or the N terminal amino group of amino acid in the mu-conotoxin peptide to prepare the novel red scorpion toxin.
FIG. 2 is red color of protopectin and pectin derivative in example 3An external spectrogram; curves c and d are protopectin and pectin derivative, respectively; as can be seen from FIG. 2, protopectin is found at 1725cm -1 The characteristic absorption peak appearing nearby is the stretching vibration of C = O in the ester group; the pectin derivative is 3040cm relative to protopectin -1 The characteristic absorption peak appearing nearby is the stretching vibration of a benzene ring; at 1605cm -1 The characteristic absorption peak appearing nearby is the stretching vibration of C = O in amide; at 1465cm -1 Bending vibration with a characteristic absorption peak of N-H appears nearby; thus, the pectin derivatives are prepared by modifying pectin with aspartame.
2. Novel red scorpion toxin anti-wrinkle performance test
Testing a sample: the mu-conotoxin peptide and the novel red scorpion toxin in example 1 and the novel red scorpion toxin in example 2 were designated as test group 1, test group 2, and test group 3, respectively.
The testing steps are as follows: placing normal myoblasts in a medium containing 2/MEM and 1/3M199, 1.25mmol/L L-glutamine, 45U/mL penicillin, 40. Mu.g/mL streptomycin, 5% fetal bovine serum, and making 5% CO at 37 ℃% 2 And gelatin coated plating medium to form a monolayer of myofibrils; then placing the spinal cord explant of a mouse embryo born with 8d dorsal root ganglia on the single-layer myofibril, culturing for 48h, observing the contact of neurites growing from the explant with muscle cells, and beginning to shrink after 90 h; after 3 weeks of culture, the nerves and muscles were connected to form a mature neuromuscular junction differentiated striated muscle fiber model. Before adding the sample, observing the muscle contraction frequency of the culture model for 25s by using a microscope, observing for 6 times, and taking an average value; adding the sample to be tested with the concentration of 0.45mmol/L into the culture model, setting a blank group, observing the muscle contraction frequency for 25s at 5min, 1h and 12h, and recording the result.
TABLE 1 results of the wrinkle resistance test of the novel scorpion toxin
As can be seen from Table 1, after 5min of the test, the mu-conotoxin peptide in example 1 induced the muscle contraction less than 50 times, while the new scorpion toxin in examples 1-2 induced the muscle contraction not more than 15 times; after the test is carried out for 1h, the muscle contraction times are 0 times, which are lower than that of the mu-conotoxin peptide and far lower than that of a blank control group; the results show that 3-beta-hydroxy oleanolic acid butyrate is grafted on the side chain of amino acid or the amino group at the N terminal in the mu-conotoxin peptide to prepare the novel red scorpion toxin, so that the muscle contraction times are reduced, and the aim of better wrinkle removal is fulfilled.
3. Physicochemical Properties of the composition
The test samples were: compositions of examples 4-11.
1) And (3) testing the anti-aging performance:
the test cells were: fibroblasts (HDF-a), human keratinocytes (NHEK);
placing HDF-a cells in FM complete medium containing 10% Fetal Bovine Serum (FBS), 1% pleiotrophin (FGS), 1% penicillin-streptomycin (PS) at 37 deg.C, 5% 2 The cells were cultured in the incubator of (1) until the exponential growth phase was reached, digested with 0.25% trypsin for 4min, and then collected in FM complete medium at 8X 10 3 Inoculating each cell/well in a 96-well plate, replacing the culture medium with FM basic culture medium without FBS and FGS after culturing for 24h, adding 0.25wt% composition diluent (diluted by the FM basic culture medium), culturing at 37 deg.C for 48h, and observing by microscopic examination and photographing. Setting an FM complete culture medium treatment group as a positive control group, setting an FM basic culture medium treatment group as a negative control group, setting a treatment group added with a composition sample as an experimental group, and setting 5 more wells per well; after the culture is finished, the culture medium is discarded, 120 mu L of MTT solution with the final concentration of 0.45mg/mL is added, the treatment is carried out for 5h at 37 ℃, the MTT solution is discarded, 80 mu L of DMSO is used for elution for 15min, the absorbance is detected at 492nm of an enzyme labeling instrument and is marked as A, and the cell activity is calculated according to the following calculation formula:
cell activity = a Experimental group /A Negative control group
In DMEM complete medium containing 10% FBS and 1% PS, at 37 ℃ and 5% CO 2 Is cultured in an incubator until the exponential growth is prolonged, and the culture is performed at 8 multiplied by 10 3 Inoculating one cell/well into a 96-well plate, culturing for 24h, and performing the post-treatment in the same manner as above,the final concentration of the test sample of the composition was 0.25wt%, and the cell activity was calculated in the same manner as above.
TABLE 2 Effect of compositions on HDF-a, NHEK cells
As can be seen from Table 2, the cell activities of the compositions in examples 5-6 on HDF-a are higher than 120.5%, the cell activities of NHEK are higher than 170%, and the cell activities of the compositions in comparative examples 4-6 and the FBS-free treatment group are higher than those of the compositions in examples 5-6 on HDF-a and NHEK than those of the compositions in example 4 and the FBS-free treatment group (negative control group), which shows that the amino group of the side chain or N terminal of the amino acid in the mu-conotoxin peptide is grafted with 3-beta-hydroxy oleanolic acid butyrate to prepare the novel scorpion toxin, and the novel scorpion toxin is compounded with other components of the compositions for use, so that the cell activities of HDF-a and NHEK are improved, the proliferation of HDF-a and NHEK cells is promoted, and the growth of the HDF-a and NHEK cells is further reduced, and the anti-aging effect is achieved. The compositions of examples 11-12 have a cellular activity on HDF-a of more than 133.5% and NHEK of more than 200%, close to the FBS-treated group (positive control group), comparing example 4, example 7 and example 10, example 5, example 8 and example 11, example 6, example 9 and example 12, the composition of example 10 has a cellular activity on HDF-a and NHEK of more than example 4 and example 7, the composition of example 11 has a cellular activity on HDF-a and NHEK of more than example 5 and example 8, and the composition of example 12 has a cellular activity on HDF-a and NHEK of more than example 6 and example 9; the pectin derivative prepared by adopting the aspartame modified pectin and the novel red scorpion toxin are used as components of the composition and interact with other components, so that the wrinkle activity can be better improved, and the wrinkle resistance and the anti-aging performance of the composition are further improved.
2) Oxidation resistance test
Preparing a test sample of the composition into a solution with the concentration of 0.5 wt%; placing 5mL of 0.05mol/L Tris-HCl buffer solution (pH = 8.2) in a water bath kettle at 23 ℃ for preheating for 25min, then adding 1.5mL of sample solution and 0.45mL of 25mmol/L pyrogallol solution, after uniform mixing, placing in the water bath kettle at 23 ℃ for reacting for 5min, and adding 1mL of 6mol/L hydrochloric acid solution to terminate the reaction; measuring absorbance value at 299nm by taking Tris-HCl buffer solution as reference; blank control was replaced with 1.5mL of water; the free radical clearance rate is calculated according to the following formula:
radical clearance (%) = [1- (sample absorbance value/blank absorbance value) ] × 100%
TABLE 3 radical scavenging Rate of the compositions
As can be seen from Table 3, the compositions of examples 5-6 have radical scavenging rates higher than 90%, and the compositions of comparative examples 4-6 and examples 5-6 have radical scavenging rates higher than example 4, which indicates that the amino group of the side chain or N-terminal of the amino acid in the mu-conotoxin peptide is grafted with 3-beta-hydroxy oleanolic acid butyrate to prepare the novel scorpion toxin, and the novel scorpion toxin is compounded with other components of the composition to improve the antioxidant performance of the composition. The compositions of examples 11-12 have a radical scavenging rate of greater than 92%, the compositions of examples 4, 7 and 10, examples 5, 8 and 11, examples 6, 9 and 12 are compared, the compositions of example 10 have a radical scavenging rate of greater than that of examples 4 and 7, the compositions of example 11 have a radical scavenging rate of greater than that of examples 5 and 8, and the compositions of example 12 have a radical scavenging rate of greater than that of examples 6 and 9; the pectin derivative prepared by adopting the aspartame modified pectin and the novel red scorpion toxin are used as the components of the composition and interact with other components, so that the antioxidant property of the composition is improved.
3) Skin elasticity test
Randomly selecting 45 volunteers, wherein the volunteers are 33 to 50 years old and randomly divided into 9 groups, each group comprises 5 persons, respectively using the composition to test samples, before testing, the test subjects need to be clean in face, before testing, the test subjects need to be stable for 30min under test conditions, and other products of the same type cannot be used during testing; each group was smeared with the test sample 2 times per day for 4 weeks at 1.5g each time, and the skin changes after 0, 2 and 4 weeks using the test sample were measured using a skin elasticity probe (model PVM 600) and the test structure was expressed by an index in units of 1, with larger numbers indicating better skin elasticity, and the average value of each group was taken.
TABLE 4 Effect of the compositions on skin elasticity
As can be seen from Table 4, the skin elasticity index of the compositions of examples 5 to 6 was higher than 0.61, the skin elasticity index of the compositions of comparative examples 4 to 6, and the compositions of examples 5 to 6 was higher than that of example 4 after the use of the test sample for 4 weeks, which shows that the amino group of the amino acid in the mu-conotoxin peptide was grafted with 3-beta-hydroxy oleanolic acid butyrate to prepare a novel class of androctoxin, which was used in combination with other ingredients of the composition, to improve the skin elasticity index and to be able to better promote the elasticity of the skin. After 4 weeks of use of the test sample, the skin elasticity index of the compositions of examples 11-12 was higher than 0.68, the skin elasticity index of the compositions of comparative examples 4, 7 and 10, examples 5, 8 and 11, examples 6, 9 and 12, the skin elasticity index of the compositions of example 10 was higher than that of examples 4 and 7, the skin elasticity index of the compositions of example 11 was higher than that of examples 5 and 8, and the skin elasticity index of the compositions of example 12 was higher than that of examples 6 and 9; the pectin derivative prepared by adopting the aspartame modified pectin and the novel red scorpion toxin as the components of the composition interact with other components, so that the elasticity effect of the composition on skin is further improved, and the anti-wrinkle effect is further achieved.
4) Skin moisture test
Selecting 45 subjects with normal and healthy skin and no cosmetic allergy history, wherein the subjects are 25 to 35 years old and randomly divided into 9 groups, 5 subjects in each group use the composition test sample respectively, the subjects need to clean the face before the test, the subjects need to be stable for 30min under the test condition before the test, and other products with the same type cannot be used during the test; each group of the smeared samples is 1.5g, the skin moisture content after 0h, 1h, 3h and 5h of the used samples is measured by a skin moisture test probe (CM 825), the relative humidity is measured to be 50%, and the average value of each group is taken.
TABLE 5 Effect of the composition on skin moisture content
As can be seen from Table 5, after 1 hour of application, the skin moisture content of the compositions of examples 5-6 was higher than 60%, and the skin moisture content of the compositions of comparative examples 4-6 and examples 5-6 was not significantly different from example 4, while after 5 hours of application, examples 5-6 still had a higher moisture content, and the water loss was lower than example 4, indicating that the amino group of the amino acid side chain or N-terminal of the mu-conotoxin peptide was grafted with 3-beta-hydroxy oleanolic acid butyrate to prepare a novel class of androctoxin, which was used in combination with other components of the composition, had no significant effect on the skin moisture content, but had better water-locking and moisturizing effects. The skin moisture content of the compositions of examples 11-12 was greater than 70%, comparing example 4, example 7 and example 10, example 5, example 8 and example 11, example 6, example 9 and example 12, the skin moisture content of the compositions of example 10 was greater than that of examples 4 and example 7, the skin moisture content of the compositions of example 11 was greater than that of examples 5 and 8, and the skin moisture content of the compositions of example 12 was greater than that of examples 6 and 9; and still has higher water content after being smeared for 5 hours; the pectin derivative prepared by adopting the aspartame modified pectin and the novel red scorpion toxin are used as components of the composition and interact with other components, so that the water replenishing and moisturizing effects of the composition are improved, and the effect of improving the wrinkle activity is further achieved.
Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be described herein.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A novel red scorpion toxin is characterized in that 3-beta-hydroxy oleanolic acid butyrate is grafted on the side chain of amino acid or the amino group at the N terminal of conotoxin;
the conotoxin is mu-conotoxin peptide; the amino acid sequence of the mu-conotoxin peptide is as follows: pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-amide;
the preparation method of the novel scorpion toxin comprises the following steps:
50 to 80 parts of conotoxin, 4.5 to 10 parts of 3-beta-hydroxy oleanolic acid butyrate and 1.75 to 5.5 parts of catalyst are placed in 25 to 50 parts of solvent by weight for complete dissolution, transferred into a reactor for reaction at room temperature for 3 to 6 hours, washed by citric acid aqueous solution and saturated sodium chloride solution with the concentration of 0.15 to 0.25mol/L for 2 to 3 times in sequence, dried by anhydrous sodium sulfate for 20 to 40min, and purified by HPLC, wherein the purification conditions are as follows: the chromatographic column is Agilent Zorbax SB-C 18 Wherein the length of the column is 150mm, the inner diameter is 4.6mm, the diameter of the chromatographic column filler particles is 5 μm, the flow rate is 1-2mL/min, the detection wavelength is 220-280nm, and the mobile phase: solution A is an aqueous solution containing 0.1 to 0.2% TFA, solution B is an acetonitrile solution containing 0.1 to 0.2% TFA, the elution time is 20 to 40min, acetonitrile and TFA in the system are removed by rotary evaporation, and the mixture is freeze-dried to obtain the novel scorpion toxin.
2. The novel scorpion toxin according to claim 1, wherein: the catalyst is at least one of DCC, HOBt, HOAt, EDCI, HBTU, DIPCDI and PyBOP.
3. The novel scorpion toxin according to claim 1, characterized in that: the solvent is at least one of dimethylformamide, tetrahydrofuran and dichloromethane.
4. Use of a novel scorpion toxin according to claim 1 for the preparation of a skin care product with wrinkle-improving activity.
5. A composition comprising a novel scorpion toxin according to claim 1.
6. A composition according to claim 5, characterized in that: the composition also comprises palmitoyl tripeptide-1, a humectant, a preservative, a pH regulator, an emulsifier and deionized water.
7. A composition according to claim 6, characterized in that: the composition comprises the following components in parts by weight: 0.5 to 3 portions of novel scorpion toxin, 1 to 5 portions of palmitoyl tripeptide-1, 0.15 to 0.45 portion of preservative, 0.05 to 1.5 portions of pH regulator, 4 to 10 portions of emulsifier and 50 to 100 portions of deionized water.
8. A composition according to claim 6, wherein: 0.5 to 2.5 parts by weight of a pectin derivative is added to the composition.
9. Use of a composition according to claim 5 for the preparation of a skin care product for improving wrinkle activity and/or tightening skin.
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