Supramolecular adenosine wrap, method of preparation and use thereof

Abstract

The application provides a supramolecular adenosine package, a preparation method and application thereof, and relates to the technical field of daily chemicals. The supramolecular adenosine package comprises adenosine, hydroxypropyl-beta-cyclodextrin and cucurbituril [8], and is prepared by using water as a solvent; the molar ratio of the hydroxypropyl-beta-cyclodextrin to the adenosine is 1:1-10:1; and the molar ratio of the cucurbituril [8] to the adenosine is 0.1:1-1:1. The supramolecular adenosine package provided by the application significantly improves the solubility of adenosine in water in a safe and simple manner, enhances the residence and slow-release effect of the adenosine on the scalp surface, and improves the effect of promoting hair growth and anti-wrinkle tightening.

Description

Technical Field

[0001] This invention belongs to the field of daily chemical products technology, specifically relating to a supramolecular adenosine encapsulation material, its preparation method, and its application. Background Technology

[0002] Adenosine is an endogenous nucleoside substance, and recent studies have found that it has a positive effect on hair growth: On the one hand, adenosine can act on specific receptor pathways in the dermal papilla cells of hair follicles (such as inhibiting androgen receptor AR signaling), thereby exerting an anti-androgenic effect and helping to slow down the progression of androgenetic alopecia; on the other hand, adenosine can prolong the hair follicle growth phase, showing a significant effect in prolonging the hair growth phase in in vitro hair follicle organ culture experiments. Clinical tests have also shown that topical formulations containing 0.75% adenosine (supplemented with panthenol and nicotinamide) can significantly increase hair density (approximately +6.2%) and hair shaft thickness (approximately +10.3%) after continuous use for several months, indicating that adenosine has good potential in promoting hair growth.

[0003] Meanwhile, adenosine is commonly used in anti-aging and soothing skincare products such as eye creams and serums. It can promote collagen synthesis, improve fine lines, and reduce inflammation by activating skin receptors, while also helping to improve microcirculation and brighten skin tone. However, when used alone, the skincare effects of adenosine are not particularly prominent, and it cannot currently be used as a primary skincare ingredient in cosmetics.

[0004] Adenosine also faces several technical challenges in practical product applications. First, its low solubility in neutral aqueous solutions limits its addition amount and efficacy in water-based scalp care and skincare products. While adenosine is soluble in dilute acidic environments, the low-pH acidic environment is unsuitable for cosmetics. Second, as a small-molecule hydrophilic ingredient, adenosine is easily washed away by sweat after topical application, making it difficult to remain on the scalp long enough to exert its effects. Furthermore, the efficacy of adenosine alone in skincare products is not particularly prominent. To address these issues, existing technologies have explored using carriers or formulation methods to enhance the application effect of adenosine. For example, some literature reports the use of cyclodextrin to encapsulate adenosine to increase its water solubility, but the encapsulation effect of cyclodextrin alone is limited, and the improvement in sustained release and retention of adenosine is not significant. Other studies have attempted to encapsulate adenosine in nanoparticles or microcapsules, but these methods are complex to prepare, costly, and have limited stability in cosmetics. Furthermore, cucurbituril, as a novel macrocyclic host molecule, possesses the ability to strongly interact with guest molecules, but its application in the delivery of active ingredients in cosmetics has not yet been reported. Therefore, there is an urgent need for a safe and efficient carrier system that can significantly improve the water solubility of adenosine while enhancing its effective retention on the scalp, thereby fully leveraging the hair growth-promoting, anti-wrinkle, and firming effects of adenosine. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a supramolecular adenosine encapsulation that significantly improves the solubility of adenosine in water in a safe and simple manner, enhances its retention and sustained-release effect on the scalp surface, and improves its efficacy in promoting hair growth and anti-wrinkle firming.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a supramolecular adenosine encapsulation material comprising adenosine, hydroxypropyl-β-cyclodextrin and cucurbituril [8], wherein the adenosine is encapsulated in a double-body cavity structure formed by hydroxypropyl-β-cyclodextrin and cucurbituril [8].

[0007] Hydroxypropyl-β-cyclodextrin and cucurbituril[8] work together to encapsulate adenosine molecules with supramolecular and inclusion mechanisms, thereby stably encapsulating adenosine in the aqueous phase.

[0008] Preferably, the adenosine is a high-purity raw material with a purity >99%; the hydroxypropyl-β-cyclodextrin is a food-grade or pharmaceutical-grade raw material that meets the requirements of cosmetic regulations, with a moderate degree of substitution, for example, a molar degree of substitution DS of 0.5~1.0; the cucurbita[8] is a high-purity raw material, and its water-soluble derivatives, such as surface-hydroxylated cucurbita[8], can also be used if necessary to improve compatibility.

[0009] Preferably, the ratio of adenosine, hydroxypropyl-β-cyclodextrin and cucurbituril [8] can be adjusted according to actual needs. Further, the molar ratio of hydroxypropyl-β-cyclodextrin to adenosine is 1:1 to 10:1, preferably 2:1 to 5:1; the molar ratio of cucurbituril [8] to adenosine is 0.1:1 to 1:1, preferably about 0.5:1.

[0010] By combining the above proportions, it can be ensured that adenosine is fully encapsulated by the two main molecules and that the solution is stable and clear. The effective concentration of adenosine in the encapsulated solution is preferably 0.01 to 2.0% (w / v), more preferably 0.1 to 1.0%.

[0011] In the prior art, simply relying on increasing solvent polarity (such as acidification) or adding cosolvents to solubilize adenosine often leads to problems such as increased formulation irritation, with no or only slight improvement in efficacy; while a single carrier (such as cyclodextrin) can improve adenosine solubility to a certain extent, it cannot simultaneously and significantly improve the release rate and skin retention effect of adenosine. The present invention cleverly combines two complementary main molecules: utilizing the ability of hydroxypropyl-β-cyclodextrin to solubilize adenosine, superimposed with the strong interaction force of cucurbituril [8] on adenosine, thereby achieving efficient encapsulation and synergistic regulation of adenosine. This "dual-main" design achieves simultaneous improvement of multiple properties (sustainable release, retention and efficacy). Based on the results of the examples, it can be seen that the present invention has a significant gain compared to the sum of the effects of each single measure, and the raw materials exhibit a non-linear synergistic effect. This technical solution is not a simple combination of the prior art and is not obvious to those skilled in the art.

[0012] The second objective of this invention is to provide a method for preparing supramolecular adenosine encapsulations, so as to efficiently prepare supramolecular adenosine encapsulations.

[0013] A method for preparing the above-mentioned supramolecular adenosine encapsulation material includes the following steps:

[0014] S1: Dissolve hydroxypropyl-β-cyclodextrin in water to form a homogeneous aqueous cyclodextrin solution;

[0015] S2: Add adenosine to the above cyclodextrin aqueous solution, stir to gradually dissolve adenosine and encapsulate it with cyclodextrin, and obtain an adenosine-cyclodextrin primary inclusion complex solution.

[0016] S3: While stirring continuously, add cucurbituril [8] to the above adenosine-cyclodextrin primary inclusion complex solution and continue stirring at 30~40℃ for 0.5~2 hours to allow cucurbituril [8] to fully react with adenosine and cyclodextrin to form supramolecular and inclusion structures, and obtain adenosine-encapsulated solution with dual host synergistic inclusion.

[0017] S4: Cool the above adenosine-encapsulated solution to room temperature to obtain supramolecular adenosine-encapsulated material.

[0018] Preferably, in step S1, the temperature at which hydroxypropyl-β-cyclodextrin is dissolved in water is 25~50°C, so as to promote dissolution while avoiding decomposition of the main component caused by high temperature;

[0019] The mass concentration of the cyclodextrin aqueous solution can be 4% to 20% (w / v), more preferably about 10%.

[0020] Preferably, in step S2, after adding adenosine, the temperature is raised to 30-40°C and stirred continuously for 0.5-2 hours until the adenosine is completely dissolved.

[0021] Preferably, in step S3, cucurbita[8] is a solid or a concentrated aqueous solution; ultrasonic assistance is performed during the stirring reaction to accelerate the achievement of inclusion equilibrium.

[0022] Preferably, step S4 further includes: removing the insoluble matter in the adenosine-encapsulated solution by centrifugation or filtration, and then drying it;

[0023] The drying process is preferably spray drying.

[0024] The third objective of this invention is to provide a hair growth-promoting, anti-wrinkle, and firming cosmetic composition containing the above-mentioned supramolecular adenosine encapsulation material and its use therein.

[0025] This invention also provides an application of the above-mentioned supramolecular adenosine encapsulation material in the preparation of cosmetics. The supramolecular adenosine encapsulation material can be used as an effective essence for scalp care, or added as a functional ingredient to various topical products that promote hair growth and have anti-wrinkle and firming effects. For example, it can be formulated into scalp essences, hair growth serums, hair sprays, shampoos, conditioners, and other scalp and hair products, as well as skin care essences, creams, lotions, and other skin care products.

[0026] Furthermore, the amount of the supramolecular adenosine encapsulation added to the cosmetic can be calculated based on the effective concentration of the target adenosine. Generally, the amount added is sufficient to achieve a final product adenosine content of 0.01~1.0% by mass. Since the adenosine encapsulated by supramolecular and dual-body structures is stable in the aqueous phase, it will not interact adversely with common formulation components and exhibits good compatibility. When formulating cosmetics containing the supramolecular adenosine encapsulation of this invention, simply mix the supramolecular adenosine encapsulation with other ingredients in the cosmetic (such as thickeners, moisturizers, plant extracts, etc.) evenly to obtain the final product.

[0027] Furthermore, this invention provides the application of the supramolecular adenosine encapsulation in the preparation of cosmetics that promote hair growth and have anti-wrinkle and firming effects. Users can apply or spray the product containing the supramolecular adenosine encapsulation onto the scalp or skin surface, gently massaging it in to ensure even distribution and absorption. On one hand, adenosine can be released more robustly and remain on the scalp surface, reducing loss due to bathing or sweating, thus providing a sustained effect. Through long-term daily use, this topical composition can effectively reduce excessive hair loss, promote dormant hair follicles to enter the growth phase, increase the density and thickness of new hair growth, and improve hair health. On the other hand, adenosine can continuously penetrate into the skin, exerting its effects at the site of action, reducing the amount entering the bloodstream, and preventing immediate metabolism (the half-life of adenosine in the blood is less than 10 seconds), thereby enhancing its efficacy on the scalp or skin.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] First, the raw materials selected in this invention (hydroxypropyl-β-cyclodextrin and cucurbituril[8]) are all safe ingredients acceptable for cosmetics. Among them, cyclodextrin derivatives have been maturely used in many skin care products and are safe and reliable; cucurbituril[8], as a novel carrier, has been reported in the literature to have very low toxicity and good biocompatibility.

[0030] Secondly, this invention exhibits a synergistic effect through the supramolecular and inclusion structure formed by the two main molecules, hydroxypropyl-β-cyclodextrin and cucurbituril [8], with adenosine, achieving "double protection" and "synergistic sustained release" of adenosine molecules. Among them, hydroxypropyl-β-cyclodextrin effectively improves the aqueous solubility of adenosine, avoiding the need for high-concentration acids or high-content viscous cosolvents; cucurbituril [8] further forms a supramolecular structure and stronger inclusion force with adenosine, significantly slowing down the release rate of adenosine from the inclusion complex, thus prolonging its residence time on the scalp. It is well known that free adenosine has extremely low solubility in water, requiring the use of lowering the pH value or adding a large amount of ethanol, propylene glycol, or other cosolvents to increase the concentration, but this will bring problems such as formulation irritation, and the improvement in efficacy is relatively slight. In contrast, this invention can dissolve a considerable concentration of adenosine under neutral conditions through the inclusion effect of hydroxypropyl-β-cyclodextrin, without changing the pH of the system or a large amount of organic solvent, ensuring the mildness and safety of the formulation.

[0031] Simultaneously, this invention utilizes the synergistic effect of hydroxypropyl-β-cyclodextrin and cucurbituril to construct a unique "lock-key" type heterogeneous ternary supramolecular structure. NMR data confirm that, unlike single-host structures which only cause low-field shifts (δ>8.10 ppm) in guest protons through port adsorption, the dual-host system of this invention specifically induces a significant high-field shift (δ<8.00 ppm, Δ<0) and peak broadening of the adenosine key proton (H8). This inverse chemical shift confirms that adenosine is deeply locked within the electron-rich hydrophobic shielding region constructed by the dual-host structure. Excellent stability and retention: This deep encapsulation structure significantly blocks contact with external hydrolytic enzymes and restricts the free movement of molecules (increased FWHM), thereby significantly improving the biostability and skin retention of adenosine. Experiments of this invention show that, compared with adenosine solution alone, the supramolecular adenosine encapsulation of this invention can retain a higher proportion of adenosine after being applied to the skin surface and rinsed with water, effectively improving the utilization rate of the active ingredient (see examples for details) and significantly enhancing the hair growth efficacy of adenosine.

[0032] Furthermore, compared to existing single-substrate inclusion technologies, this invention demonstrates a significant advantage in synergistic effects. Using cyclodextrin as a single-substrate inclusion (as in Comparative Example 2) results in rapid adenosine release but a limited duration of action; using cucurbituril as a single-substrate inclusion (as in Comparative Example 3) results in slower adenosine release, but with limited increases in retention and efficacy. This invention organically combines both, and comparative experimental data show that the adenosine release rate of the dual-substrate inclusion system is significantly lower than either single-substrate system, while its skin retention is significantly higher. Moreover, the in vitro cell-mediated effects, such as hair growth and anti-wrinkle firming, are stronger than those of single-substrate systems and adenosine itself, demonstrating the unique advantage of a "powerful combination" of the two components. Therefore, this invention far surpasses the simple superposition of single cyclodextrin or single cucurbituril in terms of function and even efficacy, demonstrating significant progress.

[0033] Furthermore, the preparation process of the present invention is simple and does not require complex nanocarrier preparation or emulsification technology. The encapsulation can be completed simply by mixing with an aqueous solution, which is suitable for industrial production. Compared with other complex carrier schemes, the present invention has comprehensive advantages in terms of process and application. Some existing technologies have reported the use of carriers such as nanoemulsions, liposomes or polymer nanoparticles to encapsulate and deliver adenosine. Although these methods can also improve the stable release of adenosine, they have shortcomings such as complicated preparation process, high raw material cost, and complex formulation system (which may affect the texture and stability of the product). In contrast, the present invention adopts the method of direct encapsulation with aqueous solution, which does not require special equipment and expensive materials. The preparation process is simple and controllable, which is more suitable for actual production. In addition, the product obtained by the present invention is in the form of a clear aqueous solution or powder, which can be easily added to various hair care products without affecting the user experience. Moreover, the carrier itself (hydroxypropyl-β-cyclodextrin and cucurbituril[8]) is safe and non-irritating, overcoming the problem that some nanocarriers may have long-term safety hazards.

[0034] Finally, because this invention enhances the sustained-release and retention properties of adenosine, it can achieve better results with lower doses when used in products for preventing hair loss and promoting hair growth, thus having significant application value.

[0035] In summary, the dual-body supramolecular adenosine encapsulation system of this invention achieves a balance between formulation safety and operability while ensuring enhanced efficacy, significantly outperforming existing technologies. It possesses outstanding novelty and inventiveness and can effectively solve the problems existing in the application of adenosine in the prior art. Attached Figure Description

[0036] Figure 1 The structural formula of adenosine and the key H;

[0037] Figure 2 A schematic diagram of the inclusion complex formed by adenosine molecules with two main bodies (hydroxypropyl-β-cyclodextrin and cucurbituril[8]);

[0038] Figure 3 The 1H-NMR spectra of Comparative Examples 1-3 and Example 1 are shown.

[0039] Figure 4 This is a comparison of the UV-Vis absorption spectra before and after adenosine inclusion;

[0040] Figure 5 The retention rates before and after adenosine inclusion are shown.

[0041] Figure 6 The graph shows the relative gene expression levels of VEGF and FGF7.

[0042] Figure 7 The graph shows the relative gene expression levels of FGF7.

[0043] Figure 8 This is a graph showing the production levels of COLI (collagen type I). Detailed Implementation

[0044] To better understand the present invention, the following embodiments further illustrate the content of the invention, but the scope of protection of the present invention is not limited to the following embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the present invention can be practiced without one or more of these details.

[0045] Unless otherwise specified, all raw materials are derived from commercially available products and do not contain any unspecified components other than unavoidable impurities.

[0046] In the following text, the degree of substitution of hydroxypropyl-β-cyclodextrin is 0.68, and the average molecular weight is approximately 1.41 × 10⁻⁶. 3 g / mol.

[0047] Example 1 A method for preparing supramolecular adenosine encapsulations includes the following steps:

[0048] S1: Weigh 10g of hydroxypropyl-β-cyclodextrin and add it to 90mL of deionized water. Stir at 35℃ until completely dissolved to obtain a homogeneous and clear aqueous solution of cyclodextrin.

[0049] S2: Add 0.5g of adenosine to the above cyclodextrin aqueous solution in batches and stir continuously at 35℃ for 1h until the adenosine is fully dissolved to obtain an adenosine-cyclodextrin primary inclusion complex solution.

[0050] S3: Add 0.05M citrate-phosphate buffer dropwise under stirring to adjust the pH of the system to 5.8, and continue stirring for 10 min;

[0051] S4: 1 g of cucurbituril [8] (molar ratio of adenosine to 0.5:1) was moistened and pre-dispersed with a small amount of deionized water to prepare a 2% (w / v) suspension. The suspension was stirred at room temperature for 20 min. Then, under stirring at 35°C, the above cucurbituril [8] suspension was slowly added dropwise to the inclusion complex solution. After the addition was completed, the mixture was stirred for 1 h to allow the system to reach inclusion equilibrium and obtain an adenosine-encapsulated solution with dual host co-encapsulation.

[0052] S5: After the reaction is complete, cool to room temperature, remove undissolved matter by centrifugation (5000 rpm, 10 min), and filter through a 0.45 μm filter membrane to obtain a supramolecular adenosine encapsulated solution.

[0053] Example 2 This embodiment provides a method for preparing supramolecular adenosine encapsulated compounds. The preparation steps and process parameters of this embodiment are basically the same as those of Example 1, except that: (1) Adjustment of raw material dosage: In step S1, 4g of hydroxypropyl-β-cyclodextrin is weighed and dissolved in 96mL of deionized water; in step S4, 0.5g of cucurbituril [8] is added (molar ratio with adenosine is about 0.25:1); (2) Step adjustment: The pH adjustment step is not performed in this embodiment; the remaining operation steps (including dissolution temperature, stirring time, post-treatment method, etc.) are consistent with those of Example 1. After the reaction is completed, a clear supramolecular encapsulated compound solution is also obtained.

[0054] Example 3 This embodiment provides a method for preparing supramolecular adenosine encapsulated compounds. The preparation steps and process parameters of this embodiment are basically the same as those of Example 1, except that: (1) Adjustment of raw material dosage: In step S1, 16g of hydroxypropyl-β-cyclodextrin was weighed and dissolved in 84mL of deionized water; in step S4, 1.5g of cucurbituril[8] (molar ratio with adenosine of about 0.75:1) was added; (2) Adjustment of process parameters: The dissolution and encapsulation temperature of steps S1 and S2 was increased to 40℃; the stirring time of steps S2 and S4 was extended to 1.5h; (3) Addition of auxiliary process: In step S4, the cucurbituril[8] suspension was subjected to short-term ultrasonic treatment for 5min before dropwise addition to reduce agglomeration; the remaining operation steps are consistent with those of Example 1. After the reaction, a clear supramolecular encapsulated compound solution was obtained.

[0055] like Figure 2 As shown, a supramolecular adenosine encapsulation was obtained based on the above Examples 1, 2 or 3, comprising adenosine, hydroxypropyl-β-cyclodextrin and cucurbituril [8], wherein adenosine (such as... Figure 1 It is encapsulated in a double-body cavity structure formed by hydroxypropyl-β-cyclodextrin and cucurbituril[8].

[0056] Comparative Example 1 Aqueous solution of free adenosine

[0057] Weigh 0.5 g of adenosine and add it to 100 mL of deionized water. Stir magnetically at 50 °C for 1 h. The adenosine only partially dissolves, and the saturated concentration of the solution after cooling is approximately 5 mg / mL. -1 Crystallization occurs within three days when left at room temperature.

[0058] Comparative Example 2 Monomeric hydroxypropyl-β-cyclodextrin inclusion complex

[0059] Weigh 10g of hydroxypropyl-β-cyclodextrin and add it to 90mL of deionized water. Stir at 40℃ until completely dissolved. Add 0.5g of adenosine to the above solution in one go, and stir continuously at 40℃ for 1 hour without pH adjustment. The resulting solution is clear, and the adenosine concentration is approximately 5mg / mL. -1 .

[0060] Comparative Example 3 : Single-subject cucurbituril[8] inclusion

[0061] Weigh 1 g of cucurbituril[8] and 0.5 g of adenosine and add them to 100 mL of deionized water. Stir at 40 °C for 2 h without the participation of cyclodextrin. Due to the poor water solubility of cucurbituril[8], the solution was initially turbid and then partially clear. It has a wide diameter distribution and is easy to precipitate.

[0062] Test example:

[0063] 1. To demonstrate that the product prepared in this invention is not a simple physical superposition of components, but rather forms a unique dual-entity synergistic encapsulation system, this embodiment uses nuclear magnetic resonance spectroscopy to characterize the structural behavior of adenosine under different chemical environments. The experimental results analysis focuses on the H8 proton (located at the C8 position of the purine ring), which is most sensitive to changes in the microenvironment. The test data are summarized in Table 1:

[0064] Table 1 Key Points 1 H-NMR chemical shift (400MHz, D2O, 25℃, reference peak: TMS)

[0065]

[0066] Table 1 shows the key chemical shift data of hydrogen nuclear magnetic resonance (¹H NMR) for Comparative Examples 1-3 and Examples 1-3. Wherein:

[0067] H8 represents the hydrogen at the C8 position of the adenine ring, which is the most sensitive fingerprint signal reflecting inclusion. H2 represents the hydrogen at the C2 position of the adenine ring. H1′ represents the carbon-hydrogen group at the end of the ribosome. Δδ represents the chemical shift change relative to free adenosine (Comparative Example 1), with positive values ​​indicating a lower field (deshielding) and negative values ​​indicating an upper field (shielding). FWHM represents the peak half-width at half-maximum, reflecting the degree of restriction of molecular motion.

[0068] Comparative Example 1 (Free Adenosine): The H8 signal was located at δ 8.12 ppm with a sharp peak (FWHM = 3.2 Hz), indicating that adenosine molecules move freely in the solution without host-guest interactions.

[0069] Comparative Example 2 (hydroxypropyl-β-cyclodextrin monobody inclusion): adenine H8 signal was located at δ 9.02 ppm and showed significant broadening (FWHM ≈ 3.8 Hz).

[0070] Comparative Example 3 (cucurbita[8] mono-entity inclusion): The H8 signal dropped to δ8.40ppm (Δδ=+0.28ppm), and a significant peak broadening (FWHM=6.5Hz) was observed, indicating that adenine was partially embedded in the cucurbita[8] cavity and the binding constant was high. However, due to the poor water solubility of cucurbita[8], the solubilization effect was limited.

[0071] Comparative spectra in Examples 2 and 3 show that when only a single host is present in the system, the adenosine H8 proton signal exhibits a significant low-field shift. This phenomenon indicates that in a single host system, adenosine molecules cannot fully enter the hydrophobic cavity and instead tend to adsorb at the oxygen-rich port edges of cyclodextrin or cucurbituril. The oxygen atoms at the ports reduce the electron cloud density around the proton through hydrogen bonding or electrostatic interactions, causing its resonance frequency to shift to a lower field. This confirms that a single host can only achieve "shallow adsorption" and cannot provide complete structural protection.

[0072] Unlike existing technologies, all embodiments 1-3 of this invention induced a characteristic high-field shift in adenosine H8 protons, with the signal stabilizing in the high-field region of 7.60-7.80 ppm.

[0073] Example 1 (dual-body inclusion, hydroxypropyl-β-cyclodextrin: adenosine = 5:1, cucurbituril: adenosine = 0.5:1): The H8 signal exhibited a unique upper field shielding effect, shifting to δ 7.69 ppm (Δδ = -0.43 ppm), and the peak half-width at half maximum (FWHM) increased significantly to 8.6 Hz. This chemical shift change, which is opposite to that of single-body inclusion, indicates that adenine H8 is simultaneously affected by the HP-β-CD cavity and the carbonyl port of cucurbituril [8], and is in a "dual-cavity" confined environment.

[0074] Example 2 (dual-host inclusion complex, hydroxypropyl-β-cyclodextrin:adenosine = 2:1, cucurbituril:adenosine = 0.25:1): H8 signal upfield reached δ 7.76 ppm (Δδ = -0.36 ppm), FWHM = 7.8 Hz. Due to the lower amount of host molecules, the upfield shielding effect and peak broadening were slightly less than in Example 1, but still significantly better than any single-host inclusion complex system.

[0075] Example 3 (Dual-body inclusion, hydroxypropyl-β-cyclodextrin:adenosine = 8:1, cucurbituril:adenosine = 0.75:1): The H8 signal reached δ 7.64 ppm (Δδ = -0.48 ppm), and the FWHM reached 9.2 Hz, showing the strongest on-field shielding effect and peak broadening. This indicates that a higher amount of host molecules can further enhance the dual-body synergistic inclusion effect. The H2 signal showed only a slight shift (Δδ within ±0.15 ppm), indicating that the adenine ring preferentially enters the host cavity at the C8 end. The H1′ signal shift was less than 0.05 ppm, indicating that the ribose ring was not deeply embedded in the host cavity and mainly served as a spatial positioning reference.

[0076] Data from Examples 2 and 3 show that even with minor adjustments to process parameters, the H8 signal of the product remains in the high-field region (<8.00 ppm), clearly distinct from the low-field region (>8.10 ppm) of the single-substrate model. This demonstrates the extremely high stability and universality of the dual-substrate synergistic mechanism of the present invention.

[0077] Figure 3 The 1H-NMR spectra of Comparative Examples 1-3 and Example 1 are compared. The chemical shift pattern of the adenine H8 signal (8-9 ppm region) can be clearly observed in the figures as follows:

[0078] The H8 peak of free adenosine (Comparative Example 1) was located at δ8.12 ppm and had a sharp peak shape. The inclusion of hydroxypropyl-β-cyclodextrin monopeptide (Comparative Example 2) significantly shifted the H8 peak to δ9.02 ppm, which is a typical manifestation of the cyclodextrin cavity deshielding effect. The inclusion of cucurbituril [8] monopeptide (Comparative Example 3) shifted the H8 peak to δ8.40 ppm and was accompanied by a certain degree of peak broadening.

[0079] It is noteworthy that the H8 peak of the dual-matrix inclusion (Example 1) exhibits a completely opposite shift direction to that of the single-matrix inclusion, significantly increasing to δ7.69 ppm, accompanied by obvious peak broadening. This unique field shielding effect is direct evidence of the synergistic effect of the dual-matrix inclusion, indicating that the adenosine molecule interacts simultaneously with hydroxypropyl-β-cyclodextrin and cucurbituril [8], and is in a confined chemical environment within the dual-matrix cavity.

[0080] Furthermore, varying degrees of peak shape changes were observed in the H1′ signal near 5.9 ppm and the proton signal of the sugar ring in the 3.5–4.5 ppm region, further supporting the formation of a dual-host supramolecular inclusion structure.

[0081] 2. The products obtained in Example 1 and Comparative Examples 1-3 were subjected to UV-Vis absorption spectroscopy tests. The test results are as follows: Figure 4 As shown.

[0082] Figure 4The results showed that free adenosine (Comparative Example 1) had a characteristic absorption peak at 258 nm. After inclusion by hydroxypropyl-β-cyclodextrin mono-host (Comparative Example 2), the position of this absorption peak remained basically unchanged. However, the absorption peak of adenosine in dual host inclusion (Example 1) red-shifted to 262 nm, and the molar absorptivity decreased, indicating that the microenvironment of adenosine changed, and that hydroxypropyl-β-cyclodextrin and cucurbituril [8] worked together to form a new supramolecular inclusion structure.

[0083] 3. The solution obtained in Example 1 of this invention is a colorless and transparent liquid with a viscosity similar to that of water. The actual concentration of adenosine in the solution of Example 1 is about 5.5 mg / mL, which is slightly higher than that of the single-host inclusion solution of Comparative Example 2 (about 5 mg / mL). This indicates that, based on the fact that cyclodextrin has fully solubilized adenosine, the introduction of cucurbituril [8] can further slightly increase the upper limit of the saturated solubility of adenosine. It is speculated that the stronger host-guest interaction provided by cucurbituril [8] prevents the crystallization of adenosine at high concentrations.

[0084] Example of effect:

[0085] To compare the differences in adenosine release, skin retention, and biological efficacy among different systems, this invention conducted in vitro release tests, skin retention tests, in vitro cell growth efficacy tests, and anti-wrinkle and firming efficacy tests.

[0086] 1. In vitro release test

[0087] The solutions obtained from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were placed in dialysis bags (molecular weight cutoff 500 Da) and immersed in phosphate-buffered saline (PBS, pH 7.4) at 37°C with shaking. Samples were taken at given time intervals, and the cumulative release of adenosine in the dialysis bag fluid was determined by ultraviolet spectrophotometry.

[0088] Four hours after the experiment, the cumulative release rates for each group were as follows:

[0089] Comparative Example 3 (cucurbituril[8] mono-body): Only about 50% of adenosine was released into the external medium. This indicates that cucurbituril[8] binds too tightly to adenosine, making it difficult to release the active ingredient effectively and resulting in low bioavailability. Comparative Example 1 (free adenosine): The cumulative release rate was about 85%. Due to the limited solubility of adenosine in water, trace crystals may appear inside the dialysis bag over time, limiting its complete release. Example 1 (dual-body encapsulation): More than 90% of adenosine diffused out.

[0090] The results show that the dual-entity inclusion system of the present invention achieves "balanced regulation": it overcomes the problems of poor solubility and easy precipitation of free adenosine (Comparative Example 1) through the solubilizing effect of hydroxypropyl-β-cyclodextrin, and avoids the defect of locking the active ingredient due to excessive binding force of single cucurbituril [8] (Comparative Example 3). Example 1 maintains the thermodynamic stability of adenosine in the aqueous phase while ensuring high bioavailability.

[0091] 2. Skin retention test

[0092] The shampooing process was simulated using a Franz diffusion cell. Pig back skin was sandwiched in the middle, with physiological saline as the receiving solution in the lower layer. Equal amounts of Example 1 and Comparative Examples 1-3 were added to the blank shampoo matrix at a concentration of 0.3% (based on effective adenosine content). The sample was applied to the upper layer of the pig skin, a small amount of pure water was added, and the mixture was stirred at 80 rpm for 2 min (simulating shampooing massage). The shampoo was then removed, and an equal amount of pure water was added again, stirred at 80 rpm for 1 min (simulating rinsing), and the rinsing solution was removed. Finally, the pig skin was removed and immersed in a 50% methanol aqueous solution for ultrasonic extraction for 90 s. The adenosine content in the extract was determined by HPLC, which is the retention rate.

[0093] The adenosine retention rate of Comparative Example 1 was normalized to 100%, and the adenosine retention rates of Comparative Examples 2, 3, and Example 1 were calculated to visually demonstrate the degree of improvement in retention rate. The test results are as follows: Figure 5 As shown.

[0094] Figure 5 The results showed that the retention rate of adenosine on pig skin was only slightly improved in Comparative Examples 2 and 3, increasing by 10.7% and 14.1%, respectively; while the retention rate of adenosine in Example 1 was the highest, reaching 42.2%. This comparative result further demonstrates that the dual-body inclusion system of the present invention can greatly improve the retention of adenosine on the skin and reduce water loss.

[0095] 3. In vitro cell-based hair growth efficacy test

[0096] Dermal dermal papilla cells, located at the base of the hair follicle, play a crucial role in controlling the hair growth cycle and follicle formation. During the anagen phase of hair growth, the hair follicle requires sufficient nutrition to maintain vigorous cell division. Therefore, vascular endothelial growth factor (VEGF), as an endothelial cell-specific mitogen, induces angiogenesis to provide adequate nutrition to the hair follicle. Furthermore, angiogenesis-related factors such as fibroblast growth factor 2 (FGF2) and fibroblast growth factor 7 (FGF7) also play key roles in promoting the transition from the telogen to the anagen phase of the hair follicle.

[0097] This experiment used real-time quantitative PCR (RT-qPCR) to detect changes in the expression levels of hair follicle growth-related genes VEGF and FGF7 in cultured human immortalized dermal follicle papilla cells to evaluate the hair growth effect of the samples.

[0098] Hair papilla cells were seeded into 6-well plates at a density of (1.0–3.0) × 10⁻⁶. 5 Cells / well were incubated at 37°C in a CO2 incubator for 24 hours. Cells were divided into negative control (NC), positive control (PC), Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3. The culture medium in each well was aspirated. Only 3 mL of experimental medium was added to the negative control (NC), and 3 mL of experimental medium containing minoxidil was added to the positive control (PC) to bring the minoxidil concentration to 20 μM. For Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, 3 mL of the corresponding concentration sample solution (based on adenosine content, bringing the adenosine concentration to 0.005 mg / mL) was added to each well, and the cells were incubated for another 24 hours. RN was then used. RNA was extracted from cells using an extraction kit (from Vazyme), and its purity and concentration were determined. RNA was then reverse transcribed into cDNA using a TaKaRa reverse transcription kit. RT-qPCR was performed; the reaction system mainly consisted of cDNA template, specific primers, a mixture of dNTPs, Taq DNA polymerase, and SYBR Green reagent (from Vazyme). PCR conditions were: 95℃ for 30s, 95℃ for 10s, and 60℃ for 30s, for a total of 40 cycles. GAPDH was used as the housekeeping gene, with the relative expression level of the negative control being 1. The relative gene expression level was calculated as 2. -△△CT The calculation formula is as follows:

[0099] △CT = CT value 目的基因 -CT value 内参基因 ; △△CT=△CT 样品组 -△CT 对照组 Relative expression level Fold change =2 -△△CT Inhibition rate % = (relative expression level in NC group - relative expression level in sample group) / relative expression level in NC group × 100%. Results are expressed as mean ± standard deviation, and differences between groups were statistically analyzed using one-way ANOVA (α = 0.05).

[0100] Table 2 Primer sequence information

[0101]

[0102] The relative gene expression levels of VEGF and FGF7 are as follows: Figure 6 and Figure 7 As shown.

[0103] Figure 6 and Figure 7 The results showed that Example 1 and Comparative Examples 1-3 all promoted the expression of VEGF and FGF7 genes in dermal papilla cells, thereby enhancing the hair growth efficacy of the samples. Furthermore, the effect of Example 1 was particularly significant compared to Comparative Examples 1-3.

[0104] 4. Anti-wrinkle and firming effects

[0105] Human skin fibroblasts were divided into 5.0 × 10⁻⁶ cells. 4 Cells / wells were seeded in 96-well plates and incubated in a CO2 incubator for 24 h. The culture medium in the wells was aspirated, and samples were added for 48 h. The negative control group (NC) was treated with culture medium only, while the positive control group (PC) was treated with test culture medium containing TGF-β1 to make the TGF-β1 concentration 50 ng / ml. The corresponding sample solutions (based on adenosine content, to make the adenosine concentration 0.005 mg / ml) were added to Examples 1, 1, 2, and 3, respectively. The supernatant was collected by centrifugation, and the type I collagen content was determined using the Jianglai Biotechnology Human Type I Collagen (COLI) ELISA kit.

[0106] COLI production levels such as Figure 8 As shown.

[0107] Figure 8 The results showed that both Example 1 and Comparative Examples 1-3 promoted COLI formation. Furthermore, the effect of Example 1 was particularly significant compared to Comparative Examples 1-3.

[0108] The experimental data above show that although Comparative Examples 2 and 3 improved upon Comparative Example 1, Example 1 exhibited significant advantages in all performance aspects, with improvements far exceeding those of Comparative Examples 2 and 3, demonstrating superior overall performance. The dual-body encapsulation system of this invention not only significantly improves the solubility of adenosine but also achieves a qualitative leap in sustained-release and retention properties. These improvements are of paramount importance to the practical efficacy of adenosine in preventing hair loss and promoting hair growth. Therefore, the dual-body supramolecular adenosine encapsulation solution of this invention is expected to be widely used in various scalp care products, fully leveraging the bioactivity of adenosine while being safe and convenient to use, providing an innovative and highly effective solution for the field of hair loss prevention and hair regrowth.

[0109] The amount of type I collagen secreted by fibroblasts treated in Example 1 was significantly higher than that in the comparative examples. Data showed that the improvement effect of Example 1 (approximately 70.9%) was far greater than the simple sum of Comparative Examples 2 (approximately 39.1%) and 3 (approximately 34.0%), demonstrating a significant synergistic effect. This further confirms that the supramolecular encapsulation of the present invention can effectively promote collagen synthesis and has excellent anti-wrinkle and firming potential.

[0110] The experimental data above show that although the single-entity inclusion complex (Comparative Examples 2 and 3) improves some properties compared to free adenosine (Comparative Example 1), Example 1 demonstrates significant advantages in skin retention, hair growth gene expression, and collagen synthesis. The dual-entity synergistic system of this invention not only resolves the contradiction between the low solubility of adenosine and the difficulty in releasing cucurbituril as a single entity, but also significantly improves the retention and bioavailability of the active ingredient at the target site through its unique "lock-key" structure. These improvements are of decisive significance for the practical application of adenosine in anti-hair loss and anti-aging products.

Claims

1. A supramolecular adenosine encapsulation compound, characterized in that: The supramolecular adenosine complex is prepared by using water as a solvent. The molar ratio of hydroxypropyl-β-cyclodextrin to adenosine is 1:1 to 10:1, and the molar ratio of cucurbitacin [8] to adenosine is 0.1:1 to 1:

1. The adenosine is located in a double-host cavity structure formed by hydroxypropyl-β-cyclodextrin and cucurbitacin [8]. The supramolecular adenosine complex is prepared by the following steps: S1: Dissolve hydroxypropyl-β-cyclodextrin in water to form a homogeneous aqueous cyclodextrin solution; S2: Add adenosine to the above cyclodextrin aqueous solution, stir to gradually dissolve adenosine and encapsulate it with cyclodextrin, and obtain an adenosine-cyclodextrin primary inclusion complex solution. S3: While stirring continuously, add cucurbituril [8] to the above adenosine-cyclodextrin primary inclusion complex solution and continue stirring at 30~40℃ for 0.5~2 hours to allow cucurbituril [8] to fully react with adenosine and cyclodextrin to form supramolecular and inclusion structures, and obtain adenosine-encapsulated solution with dual host synergistic inclusion. S4: Cool the above adenosine-encapsulated solution to room temperature to obtain supramolecular adenosine-encapsulated material.

2. The supramolecular adenosine encapsulation compound according to claim 1, characterized in that: The effective mass concentration of adenosine in the supramolecular adenosine encapsulation is 0.01~2.0%.

3. The supramolecular adenosine encapsulation compound according to claim 2, characterized in that: In step S1, the temperature at which hydroxypropyl-β-cyclodextrin is dissolved in water is 25~50℃; The mass concentration of the cyclodextrin aqueous solution is 4% to 20%.

4. The supramolecular adenosine encapsulation compound according to claim 3, characterized in that: In step S2, after adding adenosine, the temperature is raised to 30-40°C and stirred continuously for 0.5-2 hours until the adenosine is completely dissolved.

5. The supramolecular adenosine encapsulation compound according to claim 4, characterized in that: In step S3, the cucurbita[8] is a solid or a concentrated aqueous solution; Ultrasonic assistance was provided during the stirring reaction.

6. The supramolecular adenosine encapsulation compound according to claim 5, characterized in that: Step S4 further includes: removing the insoluble matter in the adenosine-encapsulated solution by centrifugation or filtration, and then drying it; The drying process is spray drying.

7. The application of a supramolecular adenosine encapsulation compound according to any one of claims 1-6 in the preparation of cosmetics, characterized in that: The supramolecular adenosine encapsulation can be used as an effective serum for scalp care, or added as a functional ingredient to topical products with anti-wrinkle and firming effects.

8. The application of the supramolecular adenosine encapsulation material according to claim 7 in the preparation of cosmetics, characterized in that: The amount of supramolecular adenosine encapsulation added to the cosmetic is calculated based on the effective concentration of adenosine, so that the mass content of adenosine in the cosmetic reaches 0.01~1.0%.

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