A neuroleptic composition containing cyclic hexapeptide and its application

Abstract

This invention discloses a neuro-soothing composition containing cyclic hexapeptide and its application. The composition includes cyclic hexapeptide, as well as calvaline or capsaicin. This invention also discloses the application of the neuro-soothing composition containing cyclic hexapeptide in the preparation of cosmetics or skincare products. This invention features enhanced neuro-soothing effects and reduced material costs.

Description

Technical Field

[0001] This invention relates to a composition containing cyclic hexapeptide, and more particularly to a neuroleptic composition containing cyclic hexapeptide and its application. Background Technology

[0002] With the continuous improvement of living standards, how to alleviate wrinkles has always been a key research topic in the cosmetics industry. Skin is affected by changes in the external environment and internal factors, leading to aging and the appearance of wrinkles, sagging, and changes in skin tone, with wrinkles being one of the most obvious signs. The prolonged and frequent contraction of facial muscles gradually forms skin lines, eventually resulting in dynamic wrinkles. The contraction of facial muscles mainly relies on the transmission of nerve signals, a complex electrochemical reaction process.

[0003] Based on current research into the mechanisms of dynamic wrinkles, existing technologies primarily work by blocking neurotransmitter transmission, thereby relaxing muscles and achieving a soothing and anti-wrinkle effect. Currently, commonly used anti-wrinkle substances on the market include neurotransmitter peptides, plant-based active ingredients, and enzymes. However, peptide raw materials suffer from high production costs and difficulty in mass production. Furthermore, high concentrations make them difficult to combine with other ingredients, increasing R&D costs for companies. In addition, synaptic signal transmission is a complex dynamic process; a single ingredient has limited target sites and pathways. Using a small amount of a single ingredient yields poor results, while using a large amount increases costs. Summary of the Invention

[0004] The purpose of this invention is to provide a neuro-relieving composition containing cyclic hexapeptide and its application. This invention features enhanced neuro-relieving effects and reduced material costs.

[0005] The technical solution of the present invention is a neuro-soothing composition containing cyclic hexapeptide, comprising cyclic hexapeptide, and further comprising calvalactone or capsaicin.

[0006] In the aforementioned neuro-soothing composition containing cyclic hexapeptide, the amino acid sequence of the cyclic hexapeptide is Cyclo(His-Trp-Phe-Arg-Leu-Ala).

[0007] In the aforementioned neuro-soothing composition containing cyclic hexapeptide, the composition comprises cyclic hexapeptide and calvanolactone, wherein the mass ratio of cyclic hexapeptide to calvanolactone is (2.5-10):(3.125-6.25).

[0008] In the aforementioned neuro-soothing composition containing cyclic hexapeptide, the composition comprises cyclic hexapeptide and calvanolactone, wherein the concentration of cyclic hexapeptide is 2.5 ppm to 10 ppm and the concentration of calvanolactone is 3.125 ppm to 6.25 ppm.

[0009] In the aforementioned neuro-soothing composition containing cyclic hexapeptide, the composition comprises cyclic hexapeptide and calvanolactone, wherein the concentration of cyclic hexapeptide is 2.5 ppm and the concentration of calvanolactone is 6.25 ppm.

[0010] In the aforementioned neuro-soothing composition containing cyclic hexapeptide, the composition comprises cyclic hexapeptide and capsaicin, wherein the mass ratio of cyclic hexapeptide to capsaicin is (2.5-10):(2.5-5).

[0011] In the aforementioned neuro-soothing composition containing cyclic hexapeptide, the composition comprises cyclic hexapeptide and capsaicin, wherein the concentration of cyclic hexapeptide is 2.5 ppm to 10 ppm and the concentration of capsaicin is 2.5 ppm to 5 ppm.

[0012] In the aforementioned neuro-soothing composition containing cyclic hexapeptide, the composition comprises cyclic hexapeptide and capsaicin, wherein the concentration of cyclic hexapeptide is 5 ppm and the concentration of capsaicin is 2.5 ppm.

[0013] The present invention also provides the application of a neuro-soothing composition containing cyclic hexapeptide in the preparation of cosmetics or skin care products.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The cyclic hexapeptide in this invention is a neurotransmitter-inhibiting polypeptide that, compared to linear hexapeptides, has advantages such as strong receptor binding ability, high stability, and high bioavailability. Its amino acid sequence is Cyclo(His-Trp-Phe-Arg-Leu-Ala). The preparation method of the cyclic hexapeptide is disclosed in Chinese invention patent application CN114634554A. This invention combines the high-cost and difficult-to-obtain cyclic hexapeptide with the low-cost and more readily available calvanolide or the active monomer capsaicin. The cyclic hexapeptide acts on the presynaptic membrane, inhibiting the release of neurotransmitters to achieve nerve relaxation, while calvanolide or capsaicin acts on the postsynaptic membrane, blocking ion channels and inhibiting action potentials generated by neurons to achieve nerve relaxation. Both exert their neurosoothing effects through different mechanisms of action.

[0016] The compositions of this invention exert a neuro-soothing effect through different mechanisms of action. They not only overcome the shortcomings of single-function bioactive substances, but also achieve multi-target action and synergistic effects. Moreover, the combined effect is far greater than the neuro-soothing effect of a single component at the same concentration or with a similar total concentration. This fully utilizes the efficacy of peptides and improves the bioavailability of single components. Furthermore, it can reduce the amount of active ingredients while ensuring the same effect, thus greatly reducing material costs. Detailed Implementation

[0017] A neuroleptic composition containing cyclic hexapeptide, comprising cyclic hexapeptide, and further comprising calvalactone or capsaicin.

[0018] The amino acid sequence of the cyclic hexapeptide is Cyclo(His-Trp-Phe-Arg-Leu-Ala).

[0019] When the composition comprises cyclic hexapeptide and kavalactone, the mass ratio of the cyclic hexapeptide to kavalactone is (2.5–10):(3.125–6.25).

[0020] When the composition comprises cyclohexapeptide and capsaicin, the mass ratio of cyclohexapeptide to capsaicin is (2.5-10):(2.5-5).

[0021] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0022] Examples 1-5:

[0023] A neuroleptic composition containing cyclic hexapeptide, comprising cyclic hexapeptide and calvanolide. In Examples 1-5, the concentration of cyclic hexapeptide is 2.5-10 ppm, and the concentration of calvanolide is 3.125-6.25 ppm, respectively.

[0024] Examples 6-11:

[0025] A neuroleptic composition containing cyclic hexapeptide, comprising cyclic hexapeptide and capsaicin. In Examples 6-11, the concentration of cyclic hexapeptide is 2.5-10 ppm, and the concentration of capsaicin is 2.5-5 ppm, respectively.

[0026] Comparative Examples 1-1 to 1-3: The component with a neuroleptic effect is cyclic hexapeptide. In Comparative Examples 1-1 to 1-3, the concentrations of cyclic hexapeptide are 10 ppm, 5 ppm, and 2.5 ppm, respectively.

[0027] Comparative Examples 2-1 to 2-2: The component with a neuroleptic effect is calvaline. In Comparative Examples 2-1 to 2-2, the concentrations of calvaline are 6.25 ppm and 3.125 ppm, respectively.

[0028] Comparative Examples 3-1 to 3-2: The component with a nerve-soothing effect is capsaicin. In Comparative Examples 3-1 to 3-2, the concentrations of capsaicin were 5 ppm and 2.5 ppm, respectively.

[0029] The specific components of each embodiment and comparative example are shown in Table 1.

[0030] Table 1. Specific ingredient list for each embodiment and comparative example

[0031]

[0032]

[0033] Zebrafish embryo seizure relief test:

[0034] 1.1 Experimental Principle:

[0035] Zebrafish possess neuroregulatory mechanisms similar to those in humans and have been widely used in the research and testing of neuroactive drugs. Through zebrafish embryonic behavior, instruments were used to record and software to measure the activity levels of zebrafish embryos. Changes in embryonic activity levels in the test drug-treated group and the control group were compared in a PTZ-induced seizure model, and the zebrafish neurorelaxation rate was calculated.

[0036] 1.2 Experimental Instruments and Equipment:

[0037] Zebrafish farming equipment, pH meter, dissolved oxygen meter, salinity meter, conductivity meter, analytical balance, microscope (with photographic system, stereo microscope with maximum magnification greater than 80x), 96-well plate, petri dishes, incubator, zebrafish motion recorder, brine shrimp incubator, pipettes, centrifuge tubes, beakers, volumetric flasks, vortex mixer, ultrasonic water bath, centrifuge, low temperature freezer, adjustable pipettes and pipette tips.

[0038] 1.3 Experimental Reagents:

[0039] Water (compliant with GB / T6682), methanol, dimethyl sulfoxide, ethanol and other cosolvents (analytical grade), water for broodstock aquaculture, Artemia salina eggs, fish embryo culture medium (prepared by dissolving 2940 mg anhydrous calcium chloride, 1233 mg magnesium sulfate heptahydrate, 630 mg sodium bicarbonate, and 55 mg potassium chloride in 10 L of water, pH 6.5–8.5, all chemicals are analytical grade), pentylenetetrazole (PTZ) stock solution (362 mM PTZ stock solution prepared from fish embryo culture medium and frozen).

[0040] 1.4 Experimental subjects:

[0041] Spawning tests were conducted using wild-type AB strain zebrafish (Daniorerio) from reliable sources (such as the China National Zebrafish Resource Center). The broodstock exhibited good reproductive capacity and were 6–12 months old. Lateral crosses were used to maintain genetic diversity, and purebred broodstock were replaced with a new batch after five generations. The broodstock showed no obvious signs of infection or disease and had not undergone drug treatment within the past two months. Prior to the spawning tests, the broodstock were acclimatized to the laboratory for at least 14 days after being introduced.

[0042] 1.5 Cultivation and treatment of experimental subjects:

[0043] The ideal water temperature for aquaculture is between 26℃ and 28.5℃, and the indoor temperature should be between 20℃ and 25℃. The stocking density should be controlled at 1-2 fish per liter of water. Provide a fixed 12-16 hours of light daily and maintain a good filtration system. Feed at least twice a day, including at least one feeding of Artemiasalina larvae, with feeding intervals of at least 3 hours to avoid overfeeding, which could negatively impact water quality and cleanliness.

[0044] Male and female zebrafish were placed in the spawning tank at a 2:1 ratio, 1-2 hours before the lights were turned off the day before spawning. Since zebrafish occasionally do not spawn, multiple spawning tanks were prepared as backups. To avoid genetic bias, eggs collected from at least three spawning tanks were mixed and then selected for later use. After the eggs were removed from the collection box, the embryos were cleaned with embryo culture medium. Healthy zebrafish embryos were selected and cultured at 28℃±1℃ at a density of no more than one embryo per 200μL of embryo culture medium until 5 days post-fertilization.

[0045] 1.6 Experimental Methods:

[0046] Experimental materials: Healthy zebrafish embryos, 5 days after fertilization, were selected.

[0047] Blank control group: 16 fish embryos were randomly selected and placed into a 30 mm culture dish, and 3.6 mL of fish embryo culture medium was added;

[0048] Model control group: 16 fish embryos were randomly selected and placed in a 30 mm culture dish, and 3.6 mL of fish embryo culture medium was added.

[0049] Test substance treatment group: 16 fish embryos were randomly selected and placed in a 30 mm culture dish, and 3.6 mL of test substance solution (i.e., the composition of the example or comparative example) was added.

[0050] The blank control group, model control group, and test substance treatment group were placed in a constant temperature incubator at 28℃±1℃ for 2 hours. Then, 76 μL of fish embryo culture medium was added to the blank control group, and 76 μL of LPTZ stock solution was added to the model control group and the test substance treatment group.

[0051] Embryo activity detection: Zebrafish embryos from the blank control group, model control group, and test substance treatment group were transferred into 96-well plates, with one embryo and 0.2 mL of the corresponding culture medium in each well. After sample addition, the 96 wells were transferred to a zebrafish motion recorder and allowed to acclimatize for 20 minutes. The activity of each zebrafish embryo within 20 minutes was then recorded using infrared tracking software.

[0052] Replace 180 μL of the solution in each well with fish embryo culture medium, and then place the wells in a constant temperature incubator at 28℃±1℃ for 24h±2h before counting the fish embryo survival rate.

[0053] 1.7 Data and Result Calculation:

[0054] Zebrafish nerve relaxation rate = shortening rate of swimming distance of zebrafish embryonic activity = (MS) / (MB)*100%; where S is the average value of embryonic activity in the test substance treatment group; B is the average value of embryonic activity in the blank control group; and M is the average value of embryonic activity in the model control group.

[0055] The nerve-soothing effect was evaluated by the reduction rate of swimming distance in zebrafish; the greater the reduction rate, the better the nerve-soothing effect.

[0056] If the swimming distance shortening rate in the calculation results is negative, it indicates that at the added concentration of the substance, there is no nerve-soothing effect, but rather a certain degree of nerve stimulation.

[0057] The neuro-soothing improvement rate of the composition relative to the single component is equal to Sab-Sa or Sab-Sb; where Sab is the neuro-soothing rate of the example; and Sa and Sb are the neuro-soothing rates of the single components of the same concentration in the comparative example.

[0058] In a PTZ-induced zebrafish embryonic seizure model, samples from various embodiments and comparative examples were added. The activity levels of the zebrafish embryos were recorded using detection instruments and measured using software. Changes in embryonic activity levels were compared between the model control group and the test substance treatment group. The activity-induced neuroleptic rate was calculated to evaluate the anticonvulsant and neuroleptic efficacy of the samples. The experimental results are shown in Table 2.

[0059] Table 2. Nerve Relaxation Rate of Various Embodiments and Comparative Examples

[0060]

[0061]

[0062] As shown in Comparative Examples 1-1 to 1-3, in the PTZ-induced zebrafish embryonic seizure model, when the concentration of cyclic hexapeptide was 2.5 to 10 ppm, it could reduce the activity of zebrafish embryos and had a good neurosoothing effect.

[0063] As shown in Comparative Examples 2-1 to 2-2, in the PTZ-induced zebrafish embryonic seizure model, when the concentration of calvalactone was 6.25 ppm and 3.125 ppm, the reduction in zebrafish embryonic activity was not significant, and the effect on the soothing of zebrafish nerves was poor.

[0064] As shown in Comparative Examples 3-1 to 3-2, in the PTZ-induced zebrafish embryonic convulsion model, when the concentration of capsaicin was 2.5 ppm to 5 ppm, the reduction in zebrafish embryonic activity was not significant, and the effect on soothing the zebrafish nerves was poor.

[0065] As shown in Examples 1-5, in the PTZ-induced zebrafish embryonic seizure model, the combinations of cyclic hexapeptide at concentrations of 2.5 ppm, 5 ppm, and 10 ppm with calvaline at concentrations of 3.125 ppm and 6.25 ppm, as obtained in Examples 1-5, can effectively reduce the activity of zebrafish embryos and increase the neurosoothing effect in zebrafish. This effect is far superior to the neurosoothing effect of the single component at the corresponding concentrations, and also superior to the neurosoothing effect of the single component at a similar total concentration. Therefore, it is shown that cyclic hexapeptide and calvaline can play a synergistic role in neurosoothing.

[0066] Examples 6-11 show that in the PTZ-induced zebrafish embryonic seizure model, the combinations of cyclic hexapeptide at concentrations of 2.5 ppm, 5 ppm, and 10 ppm with capsaicin at concentrations of 2.5 ppm and 5 ppm, as described in Examples 6-11, can effectively reduce the activity of zebrafish embryos and increase the neurosoothing rate of zebrafish. These effects are far superior to the neurosoothing effects of the corresponding concentrations of single components, and even superior to the neurosoothing effects of single components with similar total concentrations. This indicates that cyclic hexapeptide and capsaicin can have a synergistic effect in neurosoothing.

[0067] In summary, the three single components—cyclic hexapeptide, kavanolactone, and capsaicin—can reduce embryonic activity in zebrafish within the tested concentration range, exhibiting good neurosoothing effects and reducing PTZ irritation in a dose-dependent manner. Combining cyclic hexapeptide with kavanolactone or capsaicin at specific concentrations effectively reduces zebrafish embryonic activity and enhances the neurosoothing effect, significantly outperforming the neurosoothing effects of the corresponding single components or single components at similar total concentrations, demonstrating a synergistic effect. Furthermore, it achieves the same effect while saving on material usage, greatly reducing material costs.

Claims

1. A neuro-soothing composition containing a cyclic hexapeptide for use in the preparation of cosmetics, characterized in that: The composition includes cyclic hexapeptide, and also includes kavanolide or capsaicin; the amino acid sequence of the cyclic hexapeptide is Cyclo(His-Trp-Phe-Arg-Leu-Ala); the composition includes cyclic hexapeptide and kavanolide, wherein the mass ratio of the cyclic hexapeptide to kavanolide is (2.5~10):(3.125~6.25); the composition includes cyclic hexapeptide and capsaicin, wherein the mass ratio of the cyclic hexapeptide to capsaicin is (2.5~10):(2.5~5).

2. The neuro-soothing composition containing cyclic hexapeptide for preparing cosmetics according to claim 1, characterized in that: The composition comprises cyclic hexapeptide and calvalactone, wherein the concentration of cyclic hexapeptide is 2.5 ppm to 10 ppm and the concentration of calvalactone is 3.125 ppm to 6.25 ppm.

3. A neuro-soothing composition containing cyclic hexapeptide for preparing cosmetics according to claim 2, characterized in that: The composition comprises cyclic hexapeptide and calvanolactone, wherein the concentration of cyclic hexapeptide is 2.5 ppm and the concentration of calvanolactone is 6.25 ppm.

4. A neuro-soothing composition containing cyclic hexapeptide for preparing cosmetics according to claim 1, characterized in that: The composition comprises cyclohexapeptide and capsaicin, wherein the concentration of cyclohexapeptide is 2.5 ppm to 10 ppm and the concentration of capsaicin is 2.5 ppm to 5 ppm.

5. A neuro-soothing composition containing cyclic hexapeptide for preparing cosmetics according to claim 4, characterized in that: The composition comprises cyclohexapeptide and capsaicin, wherein the concentration of cyclohexapeptide is 5 ppm and the concentration of capsaicin is 2.5 ppm.

6. The use of the cyclic hexapeptide-containing neuro-soothing composition for preparing cosmetics according to any one of claims 1-5 in the preparation of cosmetics with neuro-soothing effects.

7. The application according to claim 6, characterized in that: The cosmetics mentioned are skincare products.

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