A supramolecule of NMN and bisabolol, and its preparation method and application

Abstract

The present invention discloses a NMN and bisabolol supramolecule, its preparation method, and application. The method comprises: mixing NMN, choline chloride, and glucosamine under nitrogen protection to form a transparent homogeneous liquid; adding (-)α-bisabolol to the transparent homogeneous liquid, stirring and mixing, and vacuum degassing to produce the NMN and (-)α-bisabolol supramolecule. The resulting supramolecule solves the problem of miscibility between the two, while simultaneously reducing permeation resistance and promoting transdermal absorption, thereby achieving a synergistic anti-photoaging effect of NMN and (-)α-bisabolol on the skin. The present invention further proposes a method for preparing the supramolecule into liposomes using liposome encapsulation technology. Utilizing this liposome encapsulation technology, the supramolecule achieves supramolecular smearable skin care, which is simple to operate and has a rapid onset of action. The present invention is suitable for preparing skin anti-photoaging preparations.

Description

Technical Field

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

[0002] β-Nicotinamide mononucleotide, or NMN, is a precursor to nicotinamide adenine dinucleotide (NDA+). NAD+ is an important redox cofactor crucial for regulating cell growth, energy metabolism, stress resistance, inflammation, and neuronal function. NMN achieves anti-aging effects by affecting NAD+ levels in the body, making it a popular anti-aging ingredient in cosmetics. Photoaging, a common form of extrinsic aging, is primarily caused by sunlight exposure. Long-term exposure to sunlight causes complex changes and damage to the skin. Therefore, inhibiting photoaging is a key anti-aging approach. Research has shown that NMN can protect against UV-induced photodamage, maintain the skin's normal and intact structure, and achieve anti-aging effects. However, current research has focused on injections or oral administration, which can be inconvenient or slow to take effect. Topical administration is limited by the molecular properties of NMN, making it difficult to cross the skin barrier, resulting in minimal effectiveness.

[0003] (-)α-Bisabolol is a pale yellow or colorless viscous liquid with a slightly sweet odor. It is nearly insoluble in water but readily soluble in organic solvents such as methanol and ethanol. (-)α-Bisabolol has been shown to reduce skin inflammation, inhibit bacteria and irritation, and promote blood circulation and relieve pain. (-)α-Bisabolol also has high permeability in the skin, several dozen times that of commonly used penetrants. It can be used as a primary agent or adjuvant in skin treatment and conditioning.

[0004] NMN has anti-aging properties, while (-)α-bisabolol has anti-inflammatory, antibacterial, and penetration-enhancing effects. Theoretically, their combined use could achieve a synergistic effect, promoting the penetration of active ingredients, increasing bioavailability, and achieving a topical anti-photoaging effect. However, their polarity differences and incompatibility make their combined use difficult, and their application remains underdeveloped. Summary of the Invention

[0005] The present invention aims to overcome the deficiencies of the prior art by providing a supramolecule of NMN and bisabolol, its preparation method, and its application, to address the problem of their mutual incompatibility and achieve a synergistic anti-photoaging effect on the skin. Furthermore, this supramolecule utilizes liposome encapsulation technology to achieve supramolecular smearable skin care, which is easy to operate and has a rapid onset of action.

[0006] In order to achieve the above objectives, the present invention first provides a method for preparing NMN and bisabolol supramolecules, which comprises the following steps:

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

[0008] 2) adding (-)α-bisabolol to the transparent homogeneous liquid in an amount of 2 to 6 wt % of the transparent homogeneous liquid, stirring and mixing, and vacuum degassing to prepare NMN and (-)α-bisabolol supramolecules.

[0009] According to a preferred embodiment of the present invention, in step 1), after mixing, the mixture is ground and stirred in a water bath at 35-55°C for 3-4 hours at a rotation speed of 250-350 rpm to form a transparent homogeneous liquid. The water bath temperature is more 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 hours, and the vacuum degree of the vacuum degassing treatment is ≤ -0.09 MPa. Furthermore, (-)α-bisabolol is added to a transparent homogeneous liquid at 3-5 wt%.

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

[0013] The present invention further discloses a method for preparing NMN and bisabolol supramolecular liposomes, which comprises the following steps:

[0014] Water, butylene glycol, polysorbate 80 and antioxidant were added to the NMN and bisabolol supramolecules; the mass ratio was supramolecule:polysorbate 80:antioxidant:butylene glycol:water=10:1.5:(0.01~0.1):13.5:75; the above mixture was homogenized and sheared to obtain a primary emulsion; the primary emulsion was circulated 2~6 times in a high-pressure homogenizer at 60°C to obtain liposomes of NMN and (-)α-bisabolol supramolecules.

[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 NMN and bisabolol supramolecular liposomes obtained by the method.

[0018] The NMN and bisabolol supramolecular and NMN and bisabolol supramolecular liposomes described herein address the immiscibility of NMN and bisabolol. NMN and (-)α-bisabolol can synergistically enhance the anti-photoaging effect on the skin, and thus can be used to prepare an anti-photoaging preparation for the skin. Preferably, the anti-photoaging preparation for the skin is a spreadable skin care product or medication.

[0019] Compared to existing technologies, this invention utilizes deep eutectic solvent technology to prepare NMN and (-)α-bisabolol supramolecules, resolving their immiscibility. This deep eutectic solvent technology improves the solubility of the active molecules, improves their oil-water partition coefficient, and reduces permeation resistance, promoting transdermal absorption. This results in a synergistic anti-photoaging effect for NMN and (-)α-bisabolol. Liposome encapsulation technology achieves a smearable skincare effect for the supramolecule, simplifying operation and providing rapid onset of action, achieving synergistic anti-photoaging efficacy.

[0020] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 Transmission electron microscopy images of NMN and (-)α-bisabolol supramolecular liposomes.

[0023] Figure 3 The graph shows the anti-inflammatory activity results.

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

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

[0026] Figure 6 This is the result of Collagen I content detection. DETAILED DESCRIPTION

[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. Instead, 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] NMN, choline chloride, and glucosamine were mixed in a molar ratio of 1:2:1.6, ground under nitrogen, and stirred in a 45°C water bath at 350 rpm for 4 hours to form a transparent, homogeneous liquid. 4 wt% (-)α-bisabolol was added, and stirring continued for 2 hours. The mixture was vacuum degassed (vacuum ≤ -0.09 MPa) to obtain NMN and (-)α-bisabolol supramolecules. No demixing occurred after 14 days of stasis, indicating the stability of the supramolecules. The supramolecules: polysorbate 80: vitamin C: butylene glycol: water were mixed in a mass ratio of 10:1.5:0.05:13.5:75 and homogenized to obtain a primary emulsion. The primary emulsion was then subjected to three cycles of high-pressure homogenization (800 bar) at 60°C to obtain liposomes containing NMN and (-)α-bisabolol supramolecules. The prepared liposomes were quasi-spherical particles with an average particle size of 195.2 nm and a PDI of 0.15. Figure 1 is the particle size distribution diagram, Figure 2 Transmission electron microscope image.

[0030] Example 2

[0031] NMN, choline chloride, and glucosamine were mixed in a molar ratio of 0.7:2:1.6, ground under nitrogen, and stirred in a 35°C water bath at 250 rpm for 3 h to form a transparent, homogeneous liquid. 2 wt% (-)α-bisabolol was added, and stirring continued for 1 h. The mixture was vacuum degassed (vacuum ≤ -0.09 MPa) to obtain NMN and (-)α-bisabolol supramolecular products. No demixing occurred after 14 days of stasis. The supramolecular product: polysorbate 80: ascorbic acid ethyl ether: butylene glycol: water were mixed in a mass ratio of 10:1.5:0.01:13.5:75 and homogenized to obtain a primary emulsion. The primary emulsion was then subjected to two cycles of high-pressure homogenization (600 bar) at 60°C to obtain liposomes containing NMN and (-)α-bisabolol supramolecular products. The average particle size was measured to be 205.1 nm, with a PDI of 0.18.

[0032] Example 3

[0033] NMN, choline chloride, and glucosamine were mixed in a molar ratio of 1.3:2:1.6, ground under nitrogen, and stirred in a 55°C water bath at 350 rpm for 4 h to form a transparent, homogeneous liquid. 6 wt% (-)α-bisabolol was added, and stirring continued for 2 h. The mixture was vacuum degassed (vacuum ≤ -0.09 MPa) to obtain NMN and (-)α-bisabolol supramolecules, which remained stable for 14 days without demixing. The supramolecules: polysorbate 80: vitamin E: butylene glycol: water were mixed in a mass ratio of 10:1.5:0.1:13.5:75 and homogenized to obtain a primary emulsion. The primary emulsion was then subjected to six cycles of high-pressure homogenization (900 bar) at 60°C to obtain liposomes containing NMN and (-)α-bisabolol supramolecules. The average particle size was measured to be 185.2 nm, with a PDI of 0.11.

[0034] Comparative Example 1

[0035] NMN and choline chloride were mixed in a 1:2 molar ratio, omitting glucosamine. The total mass remained the same as in Example 1. Under nitrogen, the mixture was ground and stirred in a 45°C water bath at 350 rpm for 4 hours to form a transparent, homogeneous liquid. 4 wt% (-)α-bisabolol was added, and stirring was continued for 2 hours. The mixture was then vacuum-degassed (vacuum ≤ -0.09 MPa). After standing for 1 hour, stratification occurred, indicating that NMN and (-)α-bisabolol did not form a stable supramolecule.

[0036] Following the process of Comparative Example 1, but adding only glucosamine to NMN (NMN to glucosamine molar ratio of 1:1.6) and omitting choline chloride, the total mass remained the same as in Example 1. All other treatments were identical to those in Example and Comparative Example 1. Separation also occurred, and supramolecules failed to form. This clearly demonstrates the importance of the addition of choline chloride and glucosamine for supramolecule formation.

[0037] Comparative Example 2

[0038] NMN, choline chloride, and glucosamine were mixed in a molar ratio of 1.5:2:1.6, ground under nitrogen, and stirred in a 45°C water bath at 350 rpm for 4 hours to form a transparent, homogeneous liquid. 4 wt% (-)α-bisabolol was added, stirred for another 2 hours, and vacuum degassed (vacuum ≤ -0.09 MPa). The resulting product remained stable only for a short time; after standing for 1 hour, it began to separate into separate layers, failing to form a supramolecular phase.

[0039] Following the process of Comparative Example 2, NMN, choline chloride, and glucosamine were mixed in a molar ratio of 0.5:2:1.6 to form a homogeneous liquid, and then 4 wt% (-)α-bisabolol was added. All other conditions were the same as in Comparative Example 2. This condition also failed to maintain a stable supramolecular state. This suggests that the amount of NMN in the system is crucial for maintaining the supramolecular state.

[0040] Comparative Example 3

[0041] NMN, choline chloride, and glucosamine were mixed in a molar ratio of 1:2:1.6, ground under nitrogen, and stirred in a 45°C water bath at 350 rpm for 4 hours to form a transparent, homogeneous liquid. 1 wt% (-)α-bisabolol was added, stirred for another 2 hours, and vacuum degassed (vacuum ≤ -0.09 MPa). The resulting product remained stable only for a short period of time and separated after 24 hours of standing, indicating that it could no longer maintain a supramolecular state.

[0042] Following the process of Comparative Example 3, if the amount of (-)α-bisabolol added exceeds 6 wt%, the stability cannot be maintained for a long time and delamination is easy to occur, indicating that the amount of (-)α-bisabolol has a significant impact on the formation of stable supramolecules.

[0043] Comparative Example 4

[0044] NMN, choline chloride, and glucosamine were mixed in a molar ratio of 1:2:1.6, ground under nitrogen, and stirred in a 45°C water bath at 350 rpm for 4 hours to form a transparent, homogeneous liquid. 4 wt% (-)α-bisabolol was added, and stirring was continued for 2 hours. The mixture was vacuum degassed (vacuum ≤ -0.09 MPa) to obtain stable NMN and (-)α-bisabolol supramolecular vesicles. The supramolecular vesicles were mixed with polysorbate 80, butanediol, and water in a mass ratio of 10:1.5:13.5:75, and homogenized to obtain a primary emulsion. The primary emulsion was then subjected to three cycles of high-pressure homogenization (800 bar) at 60°C to obtain liposomes containing the NMN and (-)α-bisabolol supramolecular vesicles. The difference from Example 1 is that no antioxidant was added during the preparation of the liposomes. During the high-pressure homogenization process at 60°C, the sample gradually turned yellow, which may be caused by the gradual oxidation of NMN during this process.

[0045] Comparative Example 5

[0046] NMN, choline chloride, and glucosamine were mixed in a molar ratio of 1:2:1.6, ground under nitrogen, and stirred in a 45°C water bath at 350 rpm for 4 hours to form a transparent, homogeneous liquid. 4 wt% (-)α-bisabolol was added, and stirring was continued for 2 hours. The mixture was then vacuum degassed (vacuum ≤ -0.09 MPa) to obtain a stable NMN and (-)α-bisabolol supramolecule. The supramolecule: polysorbate 80: vitamin C: butylene glycol: water were mixed in a mass ratio of 10:1.5:0.05:13.5:75 and homogenized to obtain a primary emulsion. The primary emulsion was then subjected to three cycles of high-pressure homogenization (1000 bar) at 60°C, but no stable liposomes were obtained. This may be due to the excessively high homogenization pressure, which exacerbated laminar shear, turbulent collisions, and cavitation, disrupting the stabilizing interactions between the components. If the high-pressure homogenizer cycle number in Comparative Example 5 exceeds 6, stable liposomes cannot be formed. When the high-pressure homogenizer pressure in Comparative Example 5 is less than 600 bar or the number of cycles is less than 2, the liposomes produced vary in size. This may be due to insufficient mechanical force, resulting in uneven particle refinement.

[0047] Performance Testing

[0048] Transdermal testing: Using a Franz diffusion cell, pig skin was subjected to 1-, 2-, 4-, and 8-hour transdermal testing using a pig skin permeation model. Because NMN has difficulty penetrating the skin barrier, it was used as a marker active ingredient. The intradermal retention of the active ingredient was measured using a high-performance liquid chromatography (HPLC) detector to characterize its transdermal efficiency. An aqueous NMN solution served as Control 1, while a mixture of NMN and (-)α-bisabolol stirred together served as Control 2 (the ratio of NMN to (-)α-bisabolol in the mixture remained consistent with that in the supramolecule in Example 1). The NMN and (-)α-bisabolol supramolecules prepared in Example 1 served as Experimental Group 1, and the NMN and (-)α-bisabolol supramolecule liposomes prepared in Example 1 served as Experimental Group 2. The total amount of NMN in each group was maintained consistent. Three replicates were performed for each group, and the average values were calculated. See Table 1 for details.

[0049] Table 1-NMN retention in the skin

[0050]

[0051] Control 1 showed little skin penetration, indicating a very low NMN content. Control 2, despite the addition of (-)α-bisabolol, was incompatible with NMN, resulting in a poor penetration-enhancing effect. However, when NMN and (-)α-bisabolol formed a supramolecule, transdermal penetration was significantly improved, demonstrating the role of (-)α-bisabolol in enhancing penetration. When the supramolecules were further formulated into liposomes, their submicron particle size resulted in even better penetration.

[0052] Anti-inflammatory activity test: Interleukin-1α was released from keratinocytes and treated with UV to reconstruct an epithelial model; the cells treated with UV alone were used as the control group, and the cells treated with (-)α-bisabolol, NMN prepared in Example 2, and (-)α-bisabolol molecules and liposomes (keeping the absolute amount of (-)α-bisabolol the same in the three groups) were used as experimental groups 1 to 3 respectively. The survival amount of interleukin-1α in the four groups was tested. The results are shown in Figure 2. Figure 3 The results showed that (-)α-bisabolol, NMN, and (-)α-bisabolol supramolecules and liposomes all inhibited the production and release of interleukin-1α by UV-stimulated keratinocytes, demonstrating significant anti-inflammatory activity. Furthermore, the anti-inflammatory activity of the supramolecules and liposomes was slightly higher than that of pure (-)α-bisabolol, likely due to the addition of NMN, which coordinated the anti-inflammatory activity to a certain extent.

[0053] Anti-skin photoaging activity test: The present invention studies the effects of NMN and (-) α-bisabolol supramolecules and liposomes in inhibiting the reactive oxygen species (ROS) scavenging ability of skin cells under ultraviolet irradiation, the production of matrix metalloproteinases (MMP-1) and promoting collagen (Collagen I) synthesis, in order to evaluate the anti-skin photoaging efficacy of the supramolecules and liposomes.

[0054] According to 1.85×10 5 Fibroblasts were seeded into 6-well plates at a density of 100 cells / well and incubated overnight in an incubator at 37°C and 5% CO2. When the cell plating rate reached 40%-60%, the drug was administered. 2 mL of cell culture medium was added to each well of the blank control group and the negative control group; the NMN and (-) α-bisabolol supramolecules and liposomes in Example 1 were dissolved in 2 mL of the corresponding culture medium (both ensured that the quality of NMN and (-) α-bisabolol was consistent), and were recorded as experimental group 1 and experimental group 2, respectively. The blank control group was not irradiated with ultraviolet light, and the negative control group, experimental group 1, and experimental group 2 received a total dose of 15 J / cm 2 of ultraviolet light (UVA) irradiation.

[0055] Reactive oxygen species (ROS) detection: After UVA irradiation, the culture medium was replaced and the 6-well plate was placed in an incubator for 30 min. The supernatant was discarded, the cells were trypsinized and collected, the cells were washed with PBS solution and resuspended, and the cells were detected by flow cytometry. Figure 4 Compared with the blank control group, the negative control group showed a significant increase in ROS, indicating that the ROS detection model was successfully established. Experimental Groups 1 and 2 showed comparable levels of ROS, and compared with the negative control group, ROS levels decreased by 26.25% and 25%, respectively. This suggests that NMN and (-)α-bisabolol supramolecular and liposomes can not only inhibit the increase in ROS levels caused by UVA irradiation, but also scavenge the oxygen free radicals generated under UVA irradiation.

[0056] MMP-1 and Collagen I ELISA detection: Collect the cell culture medium incubated for 24 h after UVA irradiation into an EP tube, and add 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 into a 6-well plate to detect and analyze the content of MMP-1 and Collagen. Figure 5 The graph shows the results of MMP-1 content testing. Compared to the blank control group, MMP-1 levels in the negative control group increased significantly, demonstrating the effectiveness of the experimental conditions. Compared to the negative control group, levels in experimental groups 1 and 2 decreased by 37.5% and 35.4%, respectively, indicating that both NMN and (-)α-bisabolol supramolecular and liposomes significantly inhibited increases in MMP-1 levels. Figure 6 The following figure shows the results of Collagen I content testing. Compared to the blank control group, the negative control group showed a significant decrease in Collagen I content. However, experimental groups 1 and 2 showed significant increases compared to the negative control group, indicating that NMN and (-)α-bisabolol supramolecules and liposomes can promote Collagen I secretion, maintaining skin elasticity under oxygen stress and restoring its barrier function.

[0057] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing NMN and bisabolol supramolecules, characterized in that: The steps include: 1) Mix NMN, choline chloride, and glucosamine in a molar ratio of 0.7-1.3:2:1.6 under nitrogen protection to form a transparent homogeneous liquid; 2) adding (-)α-bisabolol to the transparent homogeneous liquid in an amount of 2 to 6 wt% of the transparent homogeneous liquid, stirring and mixing, and vacuum degassing to prepare NMN and (-)α-bisabolol supramolecules.

2. The preparation method according to claim 1, characterized in that In step 1), after mixing, the mixture is ground and stirred in a water bath at 35-55° C. for 3-4 hours at 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 to 2 hours, and the vacuum degree of the vacuum degassing treatment is ≤ -0.09 MPa.

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

5. A method for preparing supramolecular liposomes of NMN and bisabolol, characterized in that: The steps include: Water, butylene glycol, polysorbate 80, and an antioxidant are added to the NMN and bisabolol supramolecule of claim 4; the mass ratio is supramolecule: polysorbate 80: antioxidant: butylene glycol: water = 10:1.5: (0.01-0.1):13.5:75; the above mixture is homogenized and sheared to obtain a primary emulsion; The primary emulsion was circulated 2 to 6 times in a high-pressure homogenizer at 60 °C to obtain liposomes containing NMN and (-)α-bisabolol supramolecular molecules.

6. The preparation method according to claim 5, characterized in that 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 600~900bar.

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

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

10. The use according to claim 9, characterized in that The skin anti-photoaging preparation is a smearable skin care product.

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