NMN and bisabolol supramolecule as well as preparation method and application thereof

Abstract

The invention discloses an NMN and bisabolol supramolecule as well as a preparation method and application of the NMN and bisabolol supramolecule. The method comprises the following steps: mixing NMN, choline chloride and glucosamine to form a transparent homogeneous liquid under the protection of nitrogen; and adding (-) alpha-bisabolol into the transparent homogeneous liquid, stirring and mixing, and carrying out vacuum degassing treatment to obtain the NMN and (-) alpha-bisabolol supramolecules. The obtained supramolecule solves the problem that the NMN and the (-) alpha-bisabolol are immiscible with each other, and meanwhile, the permeation resistance is reduced, and the transdermal absorption is promoted, so that the NMN and the (-) alpha-bisabolol cooperate with the skin to resist light aging. The invention further provides a method for preparing the supramolecules into the lipidosome through a lipidosome wrapping technology, the supramolecules achieve supramolecular smearing type skin care by means of the lipidosome wrapping technology, operation is easy and convenient, and the effect is fast. The method is suitable for preparing the skin anti-photoaging preparation.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomaterials and cosmetics, and more specifically, relates to a supramolecule of NMN and bisabolol, a preparation method thereof, and an application thereof. Background Art

[0002] β-Nicotinamide mononucleotide, abbreviated as NMN, is a precursor of nicotinamide adenine dinucleotide (NDA+). NDA+ is an important redox cofactor and is crucial for regulating cell growth, energy metabolism, stress resistance, inflammation, and neuronal function. NMN exerts its anti-aging effect by affecting the NAD+ level in the body and has become a popular anti-aging ingredient in the cosmetics field. Photoaging belongs to exogenous aging and is a common skin disease, mainly caused by skin aging due to sunlight exposure. Long-term exposure to sunlight can cause complex changes and damage to the skin. Therefore, inhibiting photoaging is one of the important means of skin anti-aging. Research shows that NMN can resist ultraviolet light-induced photo-damage, maintain the normal and intact structure of the skin, and achieve skin anti-aging effect. However, current research mainly focuses on injection or oral administration, which is inconvenient to operate or has a slow onset of action. Topical administration is limited by the molecular properties of NMN and is difficult to penetrate the skin barrier, resulting in little effect.

[0003] (-)-α-Bisabolol is a light yellow or colorless viscous liquid with a slightly sweet smell, almost insoluble in water, and easily soluble in organic solvents such as methanol and ethanol. (-)-α-Bisabolol has activities such as reducing skin inflammation, antibacterial and anti-irritant, promoting blood circulation to remove stasis and relieve pain. At the same time, (-)-α-bisabolol has high permeability in the skin cortex, and its effect is dozens of times that of common penetrants, and it can be used as the main agent or adjuvant for skin prevention and conditioning.

[0004] NMN has skin anti-aging effect, and (-)-α-bisabolol has anti-inflammatory, antibacterial, and permeability-enhancing effects. In theory, the combination of the two can achieve a coordinated effect, promote the penetration of active ingredients, improve bioavailability, and achieve topical skin anti-photoaging effect. However, due to the difference in polarity between the two, they are incompatible with each other, and there are certain difficulties in combining the two, and their application remains to be developed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art, provide a supramolecule of NMN and bisabolol, a preparation method thereof, and an application thereof, solve the problem of their incompatibility with each other, and achieve a synergistic skin anti-photoaging effect. At the same time, this supramolecule realizes supramolecular topical skin care by means of micro-liposome encapsulation technology, which is simple to operate and has a fast onset of action.

[0006] To achieve the above purpose, the present invention first provides a preparation method of a supramolecule of NMN and bisabolol, which includes the following steps:

[0007] 1) Mix NMN, choline chloride, and glucosamine in a molar ratio of 0.7 - 1.3:2:1.6 to form a transparent homogeneous liquid under nitrogen protection.

[0008] 2) Add (-)-α-bisabolol to the transparent homogeneous liquid in an amount of 2 - 6 wt% of the transparent homogeneous liquid, stir and mix, and perform vacuum degassing treatment to obtain the NMN and (-)-α-bisabolol supramolecule.

[0009] According to a preferred embodiment of the present invention, in step 1), after mixing, grind the mixture, stir in a water bath at 35 - 55 °C for 3 - 4 h at a rotation speed of 250 - 350 rpm to form a transparent homogeneous liquid. The water bath is further preferably 40 - 50 °C.

[0010] According to a preferred embodiment of the present invention, the molar ratio of NMN, choline chloride, and glucosamine is 0.8 - 1.2:2:1.6.

[0011] According to a preferred embodiment of the present invention, in step 2), the stirring and mixing time is 1 - 2 h, and the vacuum degree of the vacuum degassing treatment is ≤ -0.09 MPa. Further, the addition amount of (-)-α-bisabolol is 3 - 5 wt% of the transparent homogeneous liquid.

[0012] The present invention also provides an NMN and bisabolol supramolecule prepared by the above method.

[0013] The present invention further discloses a method for preparing an NMN and bisabolol supramolecule micro-liposome, which comprises the following steps:

[0014] Add water, butylene glycol, polysorbate 80, and an antioxidant to the NMN and bisabolol supramolecule; the mass ratio is supramolecule:polysorbate 80:antioxidant:butylene glycol:water = 10:1.5:(0.01 - 0.1):13.5:75; homogenize and shear the above mixture to obtain a primary emulsion; the primary emulsion is circulated 2 - 6 times in a high-pressure homogenizer at 60 °C to obtain the micro-liposome of the NMN and (-)-α-bisabolol supramolecule.

[0015] According to a preferred embodiment of the present invention, the antioxidant is one or more of vitamin C, vitamin C ethyl ether, and vitamin E.

[0016] According to a preferred embodiment of the present invention, the operating pressure of the high-pressure homogenizer is 600 - 900 bar.

[0017] The present invention also provides an NMN and bisabolol supramolecule micro-liposome obtained by the above method.

[0018] The NMN and bisabolol supramolecule described in the present invention and the NMN and bisabolol supramolecular micro-liposome solve the problem of immiscibility between NMN and bisabolol. NMN and (-)-α-bisabolol can synergistically act on skin anti-photoaging, so they can be used to prepare skin anti-photoaging preparations. Preferably, the skin anti-photoaging preparation is a topical skincare product or a drug.

[0019] Compared with the prior art, the present invention prepares the NMN and (-)-α-bisabolol supramolecule based on the deep eutectic solvent technology to solve the problem of immiscibility between the two. The deep eutectic solvent technology can improve the solubility of active molecules, improve the oil-water partition coefficient of active molecules, reduce the penetration resistance at the same time, and promote transdermal absorption, so as to achieve the synergistic skin anti-photoaging effect of NMN and (-)-α-bisabolol. Through the micro-liposome encapsulation technology, the topical skincare effect of the supramolecule is realized, which simplifies the operation, has a fast onset, and realizes the synergistic skin anti-photoaging effect of topical application.

[0020] Other features and advantages of the present invention will be described in detail in the following specific implementation part. Description of the Drawings

[0021] Figure 1 It is the particle size distribution diagram of the NMN and (-)-α-bisabolol supramolecular micro-liposome.

[0022] Figure 2 It is the transmission electron microscope image of the NMN and (-)-α-bisabolol supramolecular micro-liposome.

[0023] Figure 3 It is the result diagram of anti-inflammatory activity.

[0024] Figure 4 It is the result diagram of ROS content detection.

[0025] Figure 5 It is the result diagram of MMP-1 content detection.

[0026] Figure 6 It is the result diagram of Collagen I content detection. Specific Embodiments

[0027] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0028] Example 1

[0029] Mix NMN, choline chloride, and glucosamine in a molar ratio of 1:2:1.6. Under nitrogen protection, grind the mixture and stir it in a 45 °C water bath for 4 h at a rotation speed of 350 rpm to form a transparent homogeneous liquid. Add 4 wt% of (-)-α-bisabolol and continue stirring for 2 h. Perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa) to obtain the NMN and (-)-α-bisabolol supramolecule. Let it stand for 14 days without stratification, indicating that the supramolecule is relatively stable. Mix the supramolecule, polysorbate 80, vitamin C, butanediol, and water in a mass ratio of 10:1.5:0.05:13.5:75 and homogenize and shear to obtain the primary emulsion. Then, circulate the primary emulsion through a high-pressure homogenizer (800 bar) three times at 60 °C to obtain the micro-liposomes of the NMN and (-)-α-bisabolol supramolecule. The prepared micro-liposomes are round-like particles. After measurement, the average particle size is 195.2 nm and the PDI is 0.15. Figure 1 is the particle size distribution diagram, Figure 2 and this is the transmission electron microscope image.

[0030] Example 2

[0031] Mix NMN, choline chloride, and glucosamine in a molar ratio of 0.7:2:1.6. Under nitrogen protection, grind the mixture and stir it in a 35 °C water bath for 3 h at a rotation speed of 250 rpm to form a transparent homogeneous liquid. Add 2 wt% of (-)-α-bisabolol and continue stirring for 1 h. Perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa) to obtain the NMN and (-)-α-bisabolol supramolecule. Let it stand for 14 days without stratification. Mix the supramolecule, polysorbate 80, vitamin C ethyl ether, butanediol, and water in a mass ratio of 10:1.5:0.01:13.5:75 and homogenize and shear to obtain the primary emulsion. Then, circulate the primary emulsion through a high-pressure homogenizer (600 bar) two times at 60 °C to obtain the micro-liposomes of the NMN and (-)-α-bisabolol supramolecule. After measurement, the average particle size is 205.1 nm and the PDI is 0.18.

[0032] Example 3

[0033] Mix NMN, choline chloride, and glucosamine in a molar ratio of 1.3:2:1.6. Under nitrogen protection, grind them, and stir in a water bath at 55 °C for 4 h at a rotation speed of 350 rpm to form a transparent homogeneous liquid. Add 6 wt% of (-)-α-bisabolol, continue stirring for 2 h, and perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa) to obtain the supramolecule of NMN and (-)-α-bisabolol. After standing for 14 days, no stratification occurs. Mix the supramolecule: polysorbate 80: vitamin E: butanediol: water in a mass ratio of = 10:1.5: 0.1:13.5:75, and homogenize and shear to obtain the primary emulsion. Then, the primary emulsion is circulated 6 times in a high-pressure homogenizer (900 bar) at 60 °C to obtain the micro-liposome of the supramolecule of NMN and (-)-α-bisabolol. The average particle size is measured to be: 185.2 nm, and the PDI is 0.11.

[0034] Comparative Example 1

[0035] Mix NMN and choline chloride in a molar ratio of 1:2, cancel glucosamine, but keep the total mass the same as in Example 1. Under nitrogen protection, grind them, and stir in a water bath at 45 °C for 4 h at a rotation speed of 350 rpm to form a transparent homogeneous liquid. Add 4 wt% of (-)-α-bisabolol, continue stirring for 2 h, perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa), and stratification occurs after standing for 1 h. The supramolecule of NMN and (-)-α-bisabolol is not formed stably.

[0036] According to the process of Comparative Example 1, but only add glucosamine to NMN (molar ratio of NMN and glucosamine is 1: 1.6), cancel choline chloride, keep the total mass the same as in Example 1, and other treatments are the same as in Example 1 and Comparative Example 1. Then, stratification also occurs and the supramolecule cannot be formed. Obviously, the addition of choline chloride and glucosamine is important for the formation of the supramolecule.

[0037] Comparative Example 2

[0038] Mix NMN, choline chloride, and glucosamine in a molar ratio of 1.5:2:1.6. Under nitrogen protection, grind them, and stir in a water bath at 45 °C for 4 h at a rotation speed of 350 rpm to form a transparent homogeneous liquid. Add 4 wt% of (-)-α-bisabolol, continue stirring for 2 h, perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa), and the obtained product can only maintain stability for a short time. Stratification will occur after standing for 1 h, and the supramolecular state cannot be formed.

[0039] According to the procedure of Comparative Example 2, but mixing NMN, choline chloride, and glucosamine in a molar ratio of 0.5:2:1.6 to form a homogeneous liquid, and then adding 4 wt% of (-)-α-bisabolol. Other conditions are the same as in Comparative Example 2. Under this condition, a stable supramolecular state cannot be maintained either. This indicates that the amount of NMN in the system is crucial for maintaining the supramolecular state.

[0040] Comparative Example 3

[0041] Mix NMN, choline chloride, and glucosamine in a molar ratio of 1:2:1.6, grind under nitrogen protection, stir in a 45 °C water bath for 4 h at a rotation speed of 350 rpm to form a transparent homogeneous liquid. Add 1 wt% of (-)-α-bisabolol, continue stirring for 2 h, and perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa). The obtained product can only maintain stability for a short time and will layer after standing for 24 h, that is, the supramolecular state cannot be maintained.

[0042] According to the procedure of Comparative Example 3, if the amount of (-)-α-bisabolol added exceeds 6 wt%, it also cannot maintain stability for a long time and is prone to layering, indicating that the amount of (-)-α-bisabolol has a significant impact on the formation of stable supramolecules.

[0043] Comparative Example 4

[0044] Mix NMN, choline chloride, and glucosamine in a molar ratio of 1:2:1.6, grind under nitrogen protection, stir in a 45 °C water bath for 4 h at a rotation speed of 350 rpm to form a transparent homogeneous liquid. Add 4 wt% of (-)-α-bisabolol, continue stirring for 2 h, and perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa) to obtain stable NMN and (-)-α-bisabolol supramolecules. The supramolecules: polysorbate 80: butanediol: water are mixed in a mass ratio of 10:1.5:13.5:75, and homogenized and sheared to obtain a primary emulsion. Then the primary emulsion is circulated 3 times in a high-pressure homogenizer (800 bar) at 60 °C to obtain microcapsules of NMN and (-)-α-bisabolol supramolecules. The difference from Example 1 is that no antioxidant is added during the preparation of the microcapsules. During the high-pressure homogenization process at 60 °C, the sample will gradually turn yellow, which may be caused by the gradual oxidation of NMN during this process and the resulting color change.

[0045] Comparative Example 5

[0046] Mix NMN, choline chloride, and glucosamine in a molar ratio of 1:2:1.6. Under nitrogen protection, grind the mixture and stir it in a 45°C water bath for 4 h at a rotation speed of 350 rpm to form a transparent homogeneous liquid. Add 4 wt% of (-)-α-bisabolol and continue stirring for 2 h. Then, perform vacuum degassing treatment (vacuum degree ≤ -0.09 MPa) to obtain a stable NMN and (-)-α-bisabolol supramolecule. Mix the supramolecule, polysorbate 80, vitamin C, butanediol, and water in a mass ratio of 10:1.5:0.05:13.5:75 and homogenize and shear to obtain a primary emulsion. Then, circulate the primary emulsion through a high-pressure homogenizer (1000 bar) at 60°C for 3 times, but a stable micro-liposome cannot be obtained. The possible reason is that the excessive high-pressure homogenization pressure intensifies the effects of laminar shear, turbulent collision, and cavitation effect, etc., and destroys the stable interactions formed among the various components. If the number of cycles of the high-pressure homogenizer in Comparative Example 5 is greater than 6 times, a stable micro-liposome cannot be formed either. When the pressure of the high-pressure homogenizer in Comparative Example 5 is lower than 600 bar, or the number of cycles is less than 2 times, the sizes of the micro-liposomes produced are inconsistent. The possible reason is insufficient mechanical force, resulting in uneven particle refinement.

[0047] Performance Test

[0048] Transdermal experiment: Use a Franz diffusion cell and conduct transdermal experiments on porcine skin for 1 h, 2 h, 4 h, and 8 h according to the porcine skin penetration model. Since NMN is difficult to penetrate the skin barrier, it is used as the marker active ingredient, and the retention amount in the skin is measured using a high-performance liquid chromatography detector to characterize the transdermal efficiency of the active substance. The NMN aqueous solution is used as Control Group 1, the mixture of NMN and (-)-α-bisabolol after stirring is used as Control Group 2 (the ratio of NMN and (-)-α-bisabolol in the mixture is the same as the ratio of the two in the supramolecule in Example 1), the NMN and (-)-α-bisabolol supramolecule prepared in Example 1 is used as Experimental Group 1, and the NMN and (-)-α-bisabolol supramolecule micro-liposome prepared in Example 1 is used as Experimental Group 2. Ensure that the total amount of NMN in each group is the same. Each group is measured in parallel 3 times and the average value is taken. See Table 1 for details.

[0049] Table 1 - Retention Amount of NMN in Skin

[0050]

[0051] In control group 1, it was rarely able to penetrate the skin and the NMN content was very low. In control group 2, although (-)-α-bisabolol was added, NMN and (-)-α-bisabolol were immiscible, and the penetration-enhancing effect of (-)-α-bisabolol was not good. When NMN and (-)-α-bisabolol formed a supramolecule, the transdermal effect was significantly improved, obviously (-)-α-bisabolol played a role in promoting penetration. When the supramolecule was further prepared into micro-liposomes, its particle size was in the sub-micron level, endowing better penetration.

[0052] Anti-inflammatory activity test: Release interleukin-1α from keratinocytes in an epithelial reconstruction model treated with UV; Use only the UV-treated as the control group, and use the cells treated with (-)-α-bisabolol, the NMN and (-)-α-bisabolol supramolecule and micro-liposomes prepared in Example 2 (keeping the absolute amount of (-)-α-bisabolol in the three groups the same) as experimental groups 1 to 3 respectively, and test the survival amount of interleukin-1α in the 4 groups. The results are shown in Figure 3 . The results showed that (-)-α-bisabolol, NMN and (-)-α-bisabolol supramolecule and micro-liposomes could all inhibit the production and release of interleukin-1α by UV-stimulated keratinocytes, showing good anti-inflammatory activity. At the same time, the anti-inflammatory activity of the supramolecule and micro-liposomes was slightly higher than that of pure (-)-α-bisabolol, probably because the addition of NMN played a certain role in coordinating the anti-inflammatory activity.

[0053] Anti-skin photoaging activity test: The present invention evaluates the anti-skin photoaging efficacy of the supramolecule and micro-liposomes by studying the effects of NMN and (-)-α-bisabolol supramolecule and micro-liposomes on the ability to scavenge reactive oxygen species (ROS), the production of matrix metalloproteinase (MMP-1) and the promotion of collagen (Collagen I) synthesis in skin cells under ultraviolet irradiation.

[0054] Inoculate fibroblasts into 6-well plates at a density of 1.85×10 5 cells / well and incubate overnight in an incubator at 37 °C and 5% CO2. When the cell confluence rate reaches 40% - 60%, administer the drugs. Add 2 mL of cell culture medium to each well of the blank control group and the negative control group; Dissolve the NMN and (-)-α-bisabolol supramolecule and micro-liposomes in Example 1 in 2 mL of the corresponding culture medium (ensuring the same mass of NMN and (-)-α-bisabolol for both), and record them as experimental group 1 and experimental group 2 respectively. The blank control group is not irradiated with ultraviolet light, and the negative control group, experimental group 1 and experimental group 2 are irradiated with ultraviolet light (UVA) with a total dose of 15 J / cm 2 .

[0055] Reactive oxygen species (ROS) detection: After the UVA irradiation was completed, the culture medium was replaced. The 6-well plate was placed in an incubator and incubated for 30 min. The supernatant was discarded, and the cells were digested with trypsin and collected. The cells were washed with PBS solution and resuspended, and then detected by flow cytometry. The results are shown in Figure 4 . Compared with the negative control group and the blank control group, the ROS was significantly increased, indicating that the ROS detection model was successfully constructed. The contents of the experimental group 1 and the experimental group 2 were equivalent, and compared with the negative control group, the ROS contents were decreased by 26.25% and 25% respectively, indicating that both the supramolecule and the micro-liposome of NMN and (-)-α-bisabolol could not only inhibit the increase of ROS content caused by UVA irradiation, but also scavenge the oxygen free radicals generated under UVA irradiation.

[0056] MMP-1 and Collagen I ELISA detection: The cell culture medium incubated for 24 h after UVA irradiation was collected in an EP tube, and 0.1 mL of freshly diluted enzyme-labeled antibody, 0.1 mL of TMB substrate solution, and 0.05 mL of 2 mol / L sulfuric acid solution were added to the 6-well plate to detect and analyze the contents of MMP-1 and Collagen. Figure 5 It shows the result graph of the MMP-1 content detection. Compared with the blank control group, the MMP-1 content in the negative control group was significantly increased, indicating that the experimental conditions were effective. Compared with the negative control group, the contents of the experimental group 1 and the experimental group 2 were decreased by 37.5% and 35.4% respectively, meaning that both the supramolecule and the micro-liposome of NMN and (-)-α-bisabolol could significantly inhibit the increase of MMP-1 content. Figure 6 It is the result graph of the Collagen I content detection. Compared with the blank control group, the Collagen I content in the negative control group was significantly decreased. Compared with the negative control group, the contents of the experimental group 1 and the experimental group 2 were significantly increased, indicating that both the supramolecule and the micro-liposome of NMN and (-)-α-bisabolol could promote the secretion of Collagen I, enabling the skin to maintain elasticity under oxygen stress and regain the barrier function.

[0057] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A preparation method of NMN and bisabolol supramolecule, characterized in that, It includes the following steps: 1) Mix NMN, choline chloride and glucosamine in a molar ratio of 0.7 - 1.3:2:1.6 to form a transparent homogeneous liquid under nitrogen protection; 2) Add (-)-α-bisabolol to the transparent homogeneous liquid, with the addition amount being 2 - 6 wt% of the transparent homogeneous liquid, stir and mix, and perform vacuum degassing treatment to obtain the NMN and (-)-α-bisabolol supramolecule.

2. The preparation method according to claim 1, characterized in that, In step 1), after mixing, grind the mixture, stir in a water bath at 35 - 55 °C for 3 - 4 h, with a rotation speed of 250 - 350 rpm to form a transparent homogeneous liquid.

3. The preparation method according to claim 1, characterized in that, In step 2), the stirring and mixing time is 1 - 2 h, and the vacuum degree of the vacuum degassing treatment is ≤ -0.09 MPa.

4. A supramolecule of NMN and bisabolol prepared by the method according to any one of claims 1 - 3.

5. A preparation method of NMN and bisabolol supramolecular micro-liposomes, characterized in that, It includes the following steps: In the NMN and bisabolol supramolecule of claim 4, add water, butylene glycol, polysorbate 80 and an antioxidant; the mass ratio is supramolecule:polysorbate 80:antioxidant:butylene glycol:water = 10:1.5:(0.01 - 0.1):13.5:75; homogenize and shear the above mixture to obtain a primary emulsion; The primary emulsion is circulated 2 - 6 times in a high-pressure homogenizer at 60 °C to obtain the micro-liposomes of the NMN and (-)-α-bisabolol supramolecule.

6. The preparation method according to claim 5, wherein The antioxidant is one or more of vitamin C, vitamin C ethyl ether and vitamin E.

7. The preparation method according to claim 5, characterized in that, The operating pressure of the high-pressure homogenizer is 600 - 900 bar.

8. A micro-liposome of the NMN and bisabolol supramolecule prepared by the preparation method according to claim 5.

9. Use of the NMN and bisabolol supramolecule according to claim 4 or the micro-liposome of the NMN and bisabolol supramolecule according to claim 8 in the preparation of a skin anti-photoaging preparation.

10. The application according to claim 9, wherein The skin anti-photoaging preparation is a smear-type skin care product.

Images