A synergistic, stable proanthocyanidin supramolecular complex and a preparation method and application thereof

Abstract

This invention relates to a supramolecular complex for enhancing and stabilizing proanthocyanidins, its preparation method, and its applications. The supramolecular complex is prepared by the non-covalent self-assembly of proanthocyanidins and high-molecular-weight polynucleotides (PN). This invention utilizes high-molecular-weight, fibrous polynucleotides and proanthocyanidins to self-assemble into a three-dimensional network supramolecular complex under mild aqueous conditions through multiple non-covalent interactions. This complex utilizes the grooves and interchain spaces of the long PN chains to trap and uniformly distribute proanthocyanidin molecules, forming a "molecular cage" network. This improves the stability of proanthocyanidins, solving the problem of their easy inactivation under heat, light, and alkaline environments. Furthermore, it allows for the slow and continuous release of proanthocyanidins from the "molecular cage" network, providing long-lasting antioxidant protection. In addition, the supramolecular complex of this invention achieves synergistic anti-wrinkle and whitening effects, making it suitable for facial cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology and relates to a method for preparing and applying a supramolecular structure of proanthocyanidins in camellia extract that enhances and stabilizes its efficacy. Background Technology

[0002] As the largest organ in direct contact with the external environment, the skin is constantly exposed to environmental stressors such as ultraviolet radiation, pollution, and blue light, leading to excessive generation of reactive oxygen species (ROS) and triggering "oxidative stress." This process is the core pathological mechanism driving photoaging, inflammatory pigmentation, impaired barrier function, and collagen degradation in the skin. Therefore, supplementing exogenous antioxidants to neutralize free radicals and repair oxidative damage has become a cornerstone strategy for many product categories, including whitening, anti-aging, and repair products, in functional skincare.

[0003] Among numerous natural antioxidants, proanthocyanidins (PCs) are widely recognized as top-tier antioxidants due to their exceptional free radical scavenging ability, inhibition of matrix metalloproteinase (MMP) activity, and anti-inflammatory properties. Theoretically, they show clear promise in improving photoaging wrinkles, brightening skin tone, strengthening capillaries, and reducing skin inflammation. However, their enormous application potential has long been hampered by insurmountable technical bottlenecks in cosmetic practice, resulting in actual efficacy falling far short of theoretical expectations. Specifically: First, extreme instability leads to formulation failure and shelf-life activity degradation. Proanthocyanidins are plant polyphenols, possessing the fatal weakness of instability in hot and alkaline environments. Therefore, proanthocyanidins face multiple threats of inactivation throughout the entire lifecycle of cosmetics, from production, filling, and storage to consumer use: 1) Thermal instability: Conventional emulsification, high-temperature sterilization (e.g., above 80°C), or long-term high-temperature storage environments can lead to oxidation and ring-opening of their phenolic hydroxyl structures, resulting in a sharp decline in activity; 2) pH sensitivity: To maintain stability or meet the gentle needs of the skin, most cosmetic formulations adjust the pH value to a slightly acidic to neutral range of 5.5-7.0, or even higher. Proanthocyanidins exhibit significantly reduced stability in environments with pH > 6.0, especially under alkaline conditions, where they rapidly degrade. This makes it difficult for them to coexist stably with many active ingredients (such as certain amino acid surfactants and B vitamin derivatives) under a wide range of pH conditions; 3) Photooxidation: Although proanthocyanidins are photoprotectants, they are prone to photo-induced self-oxidation under transparent packaging or light exposure, leading to product discoloration (browning) and inactivation. Existing technologies, such as using opaque packaging, adding antioxidants (e.g., sulfites), or simple microencapsulation, can only provide limited protection and may introduce new incompatibilities or safety concerns, failing to fundamentally solve the stability problem in complex formulations. Secondly, existing technologies struggle to balance stability and synergistic activity. To address the instability of proanthocyanidins, the industry has attempted to encapsulate them with cyclodextrin or lecithin. For example, Chinese invention patents CN101884617A and CN102793665A disclose a method for preparing proanthocyanidin liposomes, using soybean lecithin and a reverse-phase evaporation method to embed proanthocyanidins in an internal aqueous phase, reducing their sensitivity to the external environment and improving their stability and transdermal absorption. Chinese invention patent CN115918903B discloses a grape seed proanthocyanidin and α-tocopherol complex liposome and its preparation method. This patent describes an ultrasound-assisted preparation method for grape seed proanthocyanidins and α-tocopherol complex liposomes. It utilizes lipid bilayer materials (lecithin, hydrogenated lecithin, and cholesterol) and liposome protein modification to form stable complex liposomes, thereby achieving synergistic encapsulation and controlled release of the two active substances.The encapsulation carriers in this type of technology are basically inert delivery carriers: cyclodextrin, ordinary liposomes, and polysaccharide nanoparticles. The carrier's function is limited to "carrying" and "delivering," and the carrier itself does not participate in skincare efficacy. There is no "pharmacological synergy between carrier and active ingredient," and it cannot produce a "1+1>2" biological synergistic effect with proanthocyanidins. This results in a huge waste of technology, investing high costs in delivery but failing to maximize the comprehensive efficacy value of the final product.

[0004] Existing publicly available preparation methods for proanthocyanidins from red camellia flowers mostly focus on directly extracting and separating proanthocyanidin monomers or oligomers for use in cosmetic formulations. They maintain their activity by simply adding antioxidants or adjusting the pH of the formulation, which is a remedial measure at the formulation level rather than a fundamental solution to the stability problem at the material level. The few methods that use encapsulation technology mostly use traditional inert carrier systems such as cyclodextrin and ordinary liposomes, which cannot achieve synergistic activity and have not yet broken through the bottleneck of proanthocyanidin application. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a supramolecular complex that enhances and stabilizes proanthocyanidins, along with its preparation method and applications.

[0006] High molecular weight polynucleotides (INCI: SODIUM DNA, derived from salmon testes, with a weight-average molecular weight Mw ≥ 1500 kDa and A260 / A280 = 1.5-2.0) can activate the A2A receptor pathway to exert repair signals. Their fibrous long-chain structure provides superior physical support and hydration. However, in existing technologies, this substance is mostly used as an injection filler or high-viscosity matrix. Its large molecular weight makes it difficult to penetrate the skin, and its use alone lacks an effective carrying capacity and synergistic effect mechanism for active ingredients. This invention breaks away from the traditional "active ingredient + inert carrier" approach, combining two large molecular weight bioactive substances with clear efficacy but each having limitations in application. Through supramolecular engineering, a novel supramolecular complex is obtained that combines high stability, long-lasting sustained release, excellent skin feel, and multiple biological activities.

[0007] The objective of this invention can be achieved through the following methods: In a first aspect, the present invention provides a supramolecular complex for enhancing and stabilizing proanthocyanidins, which is prepared by non-covalent self-assembly of proanthocyanidins and high molecular weight polynucleotides (PN), wherein the weight-average molecular weight of the high molecular weight polynucleotides is 1500~6500 kDa.

[0008] In one embodiment of the invention, the source of the high molecular weight polynucleotide includes salmon testes. In some embodiments, the high molecular weight polynucleotide is derived from natural nucleic acid fragments of salmon testes and has a fibrous long-chain structure.

[0009] In one embodiment of the present invention, the proanthocyanidins are selected from one of the following: proanthocyanidins from red camellia flowers, proanthocyanidins from grape seeds, and proanthocyanidins from apple peels. The main characteristic component is proanthocyanidin dimer B2; preferably, it is a proanthocyanidin B2 enrichment from red camellia flowers.

[0010] As one embodiment of the present invention, the mass ratio of proanthocyanidins to high molecular weight polynucleotides is 1:1~5.

[0011] In this invention, the proanthocyanidin molecules are embedded in the double helix grooves of the polymer polynucleotide and / or entangled between its polymer chains through hydrogen bonds, π-π stacking and hydrophobic interactions, forming a supramolecular complex with a three-dimensional network structure.

[0012] The high-molecular-weight polynucleotide chains intertwine to form a three-dimensional fibrous network. The pores between network nodes hydrate and expand to a large scale under wet conditions. Proanthocyanidin molecules embed into the minor grooves of the double helix and interchain entanglement regions, forming a dual localization of "interchain entanglement + groove embedding." After spreading on the skin surface, this network macroscopically forms a breathable and skin-friendly extracellular matrix (ECM)-like hydration membrane, while molecular-level protection occurs at the groove scale. Proanthocyanidins are slowly and continuously released from the high-molecular-weight polynucleotide network, providing long-lasting antioxidant protection. Simultaneously, the high-molecular-weight polynucleotide chains provide immediate physical hydration as micro-support, improving fine lines and skin feel, brightening the complexion, and producing a synergistic effect.

[0013] Secondly, the present invention provides a method for preparing the supramolecular complex, comprising the following steps: S1. Dissolve proanthocyanidins in water to form a (homogeneous) aqueous solution; S2. Add the high molecular weight polynucleotide to the aqueous solution in step S1, stir until a (homogeneous) gel is formed, and continue stirring to form a supramolecular inclusion structure. S3. Disperse the supramolecular inclusion structure from step S2 in a polyol aqueous solution to obtain the supramolecular complex.

[0014] In one embodiment of the present invention, in step S2, the stirring temperature is 25~30℃, and the stirring time is 6~8 hours. The purpose is to allow the proanthocyanidins and polynucleotides to fully interact.

[0015] In one embodiment of the present invention, in step S2, the mass concentration of the high molecular weight polynucleotide in the supramolecular inclusion structure is 2-3%.

[0016] In one embodiment of the present invention, in step S3, the polyol is selected from one or more of propylene glycol, butylene glycol, and glycerol.

[0017] In one embodiment of the present invention, in step S3, the mass concentration of proanthocyanidins in the supramolecular complex is 0.01%~0.1%.

[0018] Thirdly, the present invention provides an application of the supramolecular complex in the preparation of facial cosmetics with skin-brightening, whitening, and anti-wrinkle effects.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes high molecular weight, fibrous polynucleotides (PN) and proanthocyanidins to self-assemble into a three-dimensional network supramolecular complex under mild aqueous conditions through multiple non-covalent bond interactions. This complex is not a simple physical mixture or short-chain inclusion, but rather utilizes the grooves and interchain spaces of the long PN chains to trap and uniformly distribute proanthocyanidin molecules, forming a "molecular cage." This improves the stability of proanthocyanidins, solving the problem of their easy inactivation under heat, light, and alkaline environments. Furthermore, it allows for the slow and continuous release of proanthocyanidins from the "molecular cage" network, providing long-lasting antioxidant protection.

[0020] 2. The proanthocyanidins of this invention, as potent antioxidants, primarily function to scavenge free radicals, inhibit tyrosinase activity, and block melanin production. Meanwhile, the high-molecular-weight polynucleotides (PN) not only provide physical protection as a carrier but also possess biological activities such as activating the A2A receptor pathway, promoting fibroblast proliferation, inhibiting melanin production, and accelerating DNA damage repair. The combination of these two components forms a stable supramolecular structure at the molecular level, achieving a synergistic effect of anti-wrinkle and whitening at the efficacy level.

[0021] 3. The dense polymer network of PN in this invention forms a "three-dimensional cage" protection for the proanthocyanidins trapped within it, which can more effectively shield against the attack of heat, light (especially ultraviolet rays) and alkaline environment. Its thermal stability and pH stability are significantly better than those of free proanthocyanidins.

[0022] 4. The PN structure of this invention has a large molecular pore size and a structural chamber width in the micrometer range, thus giving it a film-forming feel on the skin. This complex can form a breathable, skin-friendly, extracellular matrix (ECM)-like hydration film on the skin surface. Proanthocyanidins are slowly and continuously released from the PN network, providing long-lasting antioxidant protection; at the same time, the long-chain structure of PN itself provides immediate physical hydration and micro-support, improving fine lines and skin feel, and brightening the skin.

[0023] 5. The PN-proanthocyanidin complex aqueous dispersion system of the present invention has pseudoplastic rheological properties, which can provide excellent suspension stability and smooth application feel for the formulation without the need for or with a small amount of traditional polymer thickeners, thereby reducing the complexity of the formulation and the potential risk of irritation. Attached Figure Description

[0024] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The characteristic spectrum of proanthocyanidins; Figure 2 Characteristic spectra of high molecular weight polynucleotides; Figure 3 The characteristic spectrum of the sample in Example 1; Figure 4 The characteristic spectrum of the sample in Comparative Example 1; Figure 5 The images are electron microscope (EM) images of supramolecular proanthocyanidin complexes; where a is an image of the polymeric polynucleotide (PN) raw material, b is an EEM image of the supramolecular proanthocyanidin complex of the sample in Example 1, c is an EEM image of the supramolecular proanthocyanidin complex of Example 2, and d is an EEM image of the sample in Comparative Example 1. Figure 6 The images show electron micrographs of the PDRN-proanthocyanidin complex; where a is an image of the PDRN raw material and b is an electron micrograph of the sample from Comparative Example 2. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0026] The raw materials for the embodiments and comparative examples of this invention are as follows: Polynucleotide (PN): Purchased from Ruijiming (Shandong) Biotechnology Co., Ltd., traded under the name Polynucleotide (PN), derived from salmon sperm DNA, with a weight-average molecular weight of 5346 kDa.

[0027] PDRN: Purchased from Ruijiming (Shandong) Biotechnology Co., Ltd., trade name RJMPDRN-C02, derived from salmon sperm DNA, with a weight-average molecular weight of 198 kDa.

[0028] Pure proanthocyanidins: purchased from Shanghai Maclean Biochemical Technology Co., Ltd., Lot# C16517597, CAS 4852-22-6.

[0029] Camellia proanthocyanidins: Sourced primarily from red camellia flowers; the specific preparation process is as follows: (1) Pretreatment: Take fresh red camellia flowers, dry them to obtain dried flowers, and pulverize the dried flowers; (2) Degreasing and impurity removal: Degrease the dried flower powder by supercritical CO2 extraction to obtain camellia supercritical extract residue; (3) Enzyme-acid-alcohol synergistic extraction: Add the extract (70% ethanol (v / v), containing 0.3% citric acid, pH 3.0-4.0) at a mass ratio of 1:10 to the camellia supercritical extract residue, add 0.5% cellulase (to the weight of the camellia supercritical extract residue), and enzymatically hydrolyze at 50℃ for 1h; raise the temperature to 65℃ and keep warm for 2 extractions, 1h each time, and combine the filtrates; (4) Coarse filtration and flocculation to remove impurities: While the extract is hot, filter it through a 200-mesh nylon cloth or a plate and frame filter press to remove impurities. Large fiber residue), cooled to 40°C, added chitosan-pectin flocculant (chitosan: pectin = 3:1, total 0.5%), stirred slowly for 30 min, stood for 30 min, centrifuged and removed the supernatant; (5) Ultrafiltration pre-purification: the supernatant was passed through a 5 kDa ultrafiltration membrane to retain large molecular proteins and polysaccharides, and the permeate was collected; (6) Macroporous resin adsorption and elution: the permeate from step (5) was taken and passed through the resin column at a rate of 2 BV / h (BV bed volume) (sample volume: resin volume = 2:1) until the proanthocyanidin concentration in the effluent reached 10% of the sample volume; then 2 BV of water was passed through the resin column at a rate of 1 BV / h and discarded; then 3 BV of water was used. Elute monosaccharides, amino acids, and some flavonoid glycosides with 30% ethanol; elute with 60% ethanol at 2 BV / h and collect the effluent; (7) Vacuum concentration: concentrate the effluent from step (6) to 1 / 10 of its original mass to obtain camellia flower concentrate; (8) Drying: add 3-5 times the volume of 90% ethanol to the concentrate from step (7) to precipitate, and let it stand at 4℃ for 12h to crystallize; after filtration, wash the precipitate with 90% ethanol and freeze-dry to obtain camellia flower proanthocyanidin powder.

[0030] Example 1 This embodiment provides a method for preparing a supramolecular complex that enhances and stabilizes proanthocyanidins, comprising the following steps: (1) Weigh 0.5g of pure proanthocyanidins and dissolve them in 15g of water to form a homogeneous aqueous solution; (2) Weigh 0.5g of high molecular weight polynucleotide PN and add it to the aqueous solution in step (1), stir slowly until a homogeneous gel is formed, and continue stirring for 6-8 hours to allow proanthocyanidins and polynucleotides to fully interact and form a supramolecular inclusion complex; (3) Prepare a polyol aqueous solution: Weigh 90g of propylene glycol, 40g of butanediol, and 10g of glycerol and dissolve them in 344g of water; (4) Disperse the supramolecular inclusion complex in step (2) in the polyol aqueous solution in step (4) to obtain a supramolecular complex.

[0031] Example 2 This embodiment provides a method for preparing a supramolecular complex that enhances and stabilizes proanthocyanidins. The difference from Example 1 is that pure proanthocyanidins are replaced with camellia proanthocyanidins.

[0032] Comparative Example 1 The preparation method of this comparative example is basically the same as that of Example 2, except that camellia proanthocyanidins and high molecular weight polynucleotides PN are directly dispersed in a polyol aqueous solution. The specific steps are as follows: (1) Preparation of raw materials: Weigh 0.5g of camellia proanthocyanidins and 0.5g of high molecular weight polynucleotide PN respectively, and set aside; (2) Preparation of polyol aqueous solution: Weigh 90g of propylene glycol, 40g of butylene glycol, and 10g of glycerol and dissolve them in 359.25g of water; (3) Add the camellia proanthocyanidins from step (1) to step (2) and stir until completely dissolved; (4) Add the high molecular weight polynucleotide PN to step (3) and continue stirring for 6~8h to obtain the complex.

[0033] Comparative Example 2 The only difference from Example 2 is that the high molecular weight polynucleotide PN is replaced with low molecular weight PDRN.

[0034] Comparative Example 3 This comparative example provides an aqueous solution of proanthocyanidins. The specific preparation method is as follows: weigh 0.5g of proanthocyanidins, dissolve them in 359.5g of pure water, and then add 90g of propylene glycol, 40g of butylene glycol, and 10g of glycerol to obtain an aqueous solution of proanthocyanidins.

[0035] Comparative Example 4 This comparative example provides an aqueous solution of proanthocyanidins, which differs from Comparative Example 3 in that the mass of the proanthocyanidins is 1g.

[0036] Comparative Example 5 This comparative example provides an aqueous solution of a high molecular weight polynucleotide PN, which differs from Comparative Example 4 in that proanthocyanidins are replaced with high molecular weight polynucleotide PN.

[0037] Test Example 1: Fourier transform infrared spectroscopy (FT-IR) was used to test proanthocyanidins, high molecular weight polynucleotides (PN), the sample of Example 1, and the sample of Comparative Example 1, with wavenumber ranges from 4000 to 400 cm⁻¹. -1 The characteristic absorption peak results for each sample are as follows: Figure 1-4 As shown.

[0038] Figure 1-4 The characteristic spectra are those of the raw material proanthocyanidins, high molecular weight polynucleotides (PN), sample from Example 1, and sample from Comparative Example 1, respectively. Figure 1 It can be seen that the raw material for proanthocyanidins is at 3120cm. -1 There is a broad OH stretching vibration peak nearby; at 1601 cm⁻¹ -1 and 1518cm -1At 1279, there is a C=C skeletal stretching vibration peak of the aromatic ring and a skeletal vibration peak of the benzene ring; at 1279, there is a CO vibration peak of phenol, and at 1097 and 1056, there are vibration peaks of the sugar ring. Figure 2 It can be seen that high molecular weight polynucleotides (PN) at 3335 cm⁻¹ -1 There is a strong OH / NH contraction vibration peak nearby; at 1646 cm⁻¹ -1 1479cm -1 and 1368cm -1 There are amide bond or polysaccharide skeleton vibration peaks at 1219 cm⁻¹. -1 There is a strong peak at 1054 cm⁻¹, indicating a P=O asymmetric stretching of the phosphodiester bond. -1 There is a peak indicating the symmetric stretching vibration of the COC glycocyclic phosphate diester bond. Figure 3 The results showed that the sample was at 3322.98 cm. -1 A broad absorption peak for the OH / NH stretching vibration appears at [value missing], compared to 3335.40 cm⁻¹ in the PN support material. -1 A significant redshift of 12.42 cm was observed. -1 Furthermore, the peak width increased; at 1637.75 cm⁻¹ -1 The characteristic absorption peak of amide I band appears, compared to 1646.40 cm⁻¹ of the PN support raw material. -1 The redshift is approximately 8.65 cm. -1 The peak shape showed significant passivation and broadening; the proanthocyanidin raw material was at 1518.33 cm⁻¹. -1 The characteristic absorption peak of the aromatic ring C=C skeleton shifted to 1524 cm⁻¹ in the sample of Example 1. -1 Blue shift approximately 6cm -1 The peak intensity decreased significantly; proanthocyanidins 1279.15 cm⁻¹ -1 The characteristic Ar-CO absorption peak of the phenolic hydroxyl group disappeared in the sample of Example 1, and only appeared at 1601.76 cm⁻¹. -1 A weaker aromatic ring conjugation absorption signal is retained. The broadening of the peak position and shape in the hydroxyl region and amide I band indicates that the PN support molecule has undergone significant structural perturbation, and intermolecular hydrogen bonding interactions have been induced. FT-IR analysis was performed on the sample of Comparative Example 1 under the same conditions, and the results showed ( Figure 4 The sample was at 3329.58 cm. -1 A hydroxyl absorption peak appears at 3335.40 cm⁻¹, which is similar to the peak at 3335.40 cm⁻¹ of the PN carrier raw material. -1 Compared to only a small redshift of 5.81 cm -1 ; at 1645.68cm -1 The characteristic absorption peak of amide I appears at 1646.40 cm⁻¹, which is similar to that of the PN support raw material. -1 The offset is only about 0.72cm. -1The peaks are sharp and do not show obvious broadening; the proanthocyanidin raw material is at 1518.33 cm⁻¹. -1 The characteristic absorption peak of the aromatic ring C=C at that location shifted to 1524.66 cm⁻¹. -1 Blue shift approximately 6.33cm -1 The peak intensity also decreased significantly; the characteristic absorption peaks corresponding to the PN carrier remained basically intact.

[0039] The results above show that the shift and attenuation behavior of proanthocyanidin characteristic peaks in the sample of Example 1 and the sample of Comparative Example 1 are almost identical. The core difference lies in the characteristic peak response of the PN carrier: in the sample of Example 1, the hydroxyl and amide I bands of the PN carrier show obvious red shift and peak broadening, indicating a significant change in the functional group environment of the PN carrier macromolecule itself; while in the sample of Comparative Example 1, the characteristic peaks of the PN carrier basically retain their original state, with only the intensity of the proanthocyanidin characteristic peak observed to decrease. This phenomenon indicates that: in the sample of Comparative Example 1, there is no obvious hydrogen bonding coupling at the molecular level between proanthocyanidins and the PN carrier, and the attenuation of the proanthocyanidin peak intensity is more likely due to physical dilution and particle scattering effects, rather than functional group interactions caused by molecular-level embedding; in the sample of Example 1, intermolecular interactions occur between proanthocyanidins and the PN carrier, changing the functional group microenvironment of the PN macromolecule, proving that the process of the present invention promotes the formation of molecular-level composites, rather than simple physical mixing; while in Comparative Example 1, although the proanthocyanidin peak intensity also decreases, it lacks the synergistic perturbation of the carrier functional groups, indicating a simple physical mixing system.

[0040] Test Example 2: This test example uses scanning electron microscopy (SEM) to test the morphology of PDRN raw material, PN raw material, sample of Example 1, sample of Example 2, sample of Comparative Example 1, and sample of Comparative Example 2, respectively. The test results are as follows: Figures 5-6 As shown.

[0041] Figure 5 The images shown are electron microscope (EM) images of supramolecular proanthocyanidin complexes. (a) is an image of the polymeric polynucleotide (PN) raw material, which is fibrous. (b) is an EEM image of the supramolecular proanthocyanidin complex of the sample from Example 1, showing that PN has a micron-sized macroporous structure and proanthocyanidins are trapped in the dense network of PN. (c) is an EEM image of the supramolecular proanthocyanidin complex of Example 2. (d) is an EEM image of the sample from Comparative Example 1. Figure 6 The images are electron microscope images of the PDRN-proanthocyanidin complex, where (a) is a picture of the PDRN raw material in powder form; and (b) is an electron microscope image of the sample of Comparative Example 2.

[0042] Depend on Figure 5 and Figure 6It can be seen that in the sample of Comparative Example 2, no obvious coating or network interweaving phenomenon was observed in the microstructure of the mixture of proanthocyanidins and PDRN, indicating that low molecular weight PDRN is difficult to form an effective spatial network structure to encapsulate proanthocyanidins. In contrast, Figure 5 (a) shows that the macroscopic morphology of the high molecular weight polynucleotide PN raw material is fibrous; Figure 5 (b) and (c) show that PN forms a loose, porous network of interwoven structures at the microscopic level. In the supramolecular complexes prepared in Examples 1 and 2, proanthocyanidins are uniformly encapsulated within the dense three-dimensional network structure formed by PN, resulting in a uniform and continuous microstructure. This directly confirms the formation of the supramolecular inclusion structure. Figure 5 The electron microscopy image of Comparative Example 1 in (d) shows that there is no obvious interaction between proanthocyanidins and PN fibers to form an integral structure, further confirming that it is only a physical mixture rather than a supramolecular composite. The above-mentioned differences in microstructure and the FT-IR test results corroborate each other, indicating that the present invention has successfully constructed a stable proanthocyanidin-PN supramolecular complex by utilizing the steric hindrance effect and hydrogen bonding of high molecular weight PN through a specific preparation process.

[0043] Test Example 3: Stability Test of Proanthocyanidins under High Temperature, Light, and Alkaline Conditions The samples from Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were placed under room temperature, 50°C, and light conditions, respectively, to investigate the effects of temperature and light on the content of proanthocyanidin dimers. The results are shown in Table 1. In addition, the pH of the above samples was adjusted to 6.0, 7.0, and 8.0, respectively, and placed in a room temperature environment to investigate the effect of alkaline pH environment on the content of proanthocyanidin dimers. The results are shown in Table 2.

[0044] Table 1

[0045] As shown in Table 1, after 8 weeks of storage at room temperature, the proanthocyanidin dimer content of the sample in Example 1 remained stable with only slight fluctuations (from 1021.47 ppm to 1020.34 ppm), indicating its excellent long-term storage stability. Under high temperature conditions of 50°C, the content decreased to 983.09 ppm after 8 weeks, with a retention rate of approximately 96.2%, which was significantly better than the comparative examples. Under light conditions, although the content decreased over time, it remained at 712.03 ppm after 8 weeks, with a retention rate of 69.7%, which was significantly higher than that of Comparative Example 1 (59.14 ppm, retention rate of approximately 5.8%), Comparative Example 2 (46.03 ppm, retention rate of approximately 4.5%), and Comparative Example 3 (51.93 ppm, retention rate of approximately 5.1%).

[0046] In contrast, Comparative Examples 1, 2, and 3 all exhibited a rapid degradation trend under both high temperature and light exposure conditions. Particularly under light exposure, the proanthocyanidin dimer content in Comparative Examples 1 to 3 decreased significantly to less than 10% of the initial value within four weeks, indicating that systems without an effective supramolecular inclusion structure are ill-suited to resist photo-induced degradation. These results demonstrate that the preparation process in Example 1, which involved pre-complexing proanthocyanidins with polymeric PN in an aqueous phase and then dispersing them in a polyol system, effectively promoted the intermolecular interaction between proanthocyanidins and PN, forming a stable supramolecular inclusion structure, thereby significantly improving their chemical stability under high temperature and light stress.

[0047] Table 2

[0048] As shown in Table 2, after 8 weeks of storage at pH 6.0 and 7.0, the proanthocyanidin dimer content of the sample in Example 1 remained at 1017.03 ppm and 1013.24 ppm, respectively, with retention rates exceeding 99%, demonstrating strong tolerance. Even in a strongly alkaline environment at pH 8.03, the content still reached 915.36 ppm after 8 weeks, with a retention rate of approximately 89.6%, significantly better than all comparative examples. In contrast, comparative examples 1, 2, and 3 all rapidly degraded at pH 8.00, with contents decreasing to 23.70 ppm, 23.96 ppm, and 23.89 ppm, respectively, after 8 weeks, with retention rates all below 3.5%, indicating extreme instability in alkaline environments.

[0049] It is noteworthy that within the pH range of 7.00-8.00, the degradation rates of all comparative examples accelerated significantly, especially showing a sharp decline between weeks 2 and 4, while the degradation trend in Example 1 was much gentler. This result further confirms that the supramolecular structure formed through aqueous pre-complexation effectively shields the active sites of proanthocyanidin dimers under alkaline conditions, inhibiting their decomposition reactions caused by deprotonation or oxidative ring-opening. In summary, the supramolecular complex provided by this invention not only exhibits excellent stability under high temperature and light conditions but also demonstrates significant protective effects in alkaline environments, providing a reliable guarantee for its application in complex formulation systems such as cosmetics and pharmaceuticals.

[0050] Test Example 4: Transdermal Sustained-Release Performance Test The transdermal absorption efficiency of proanthocyanidins in samples of Example 1, Comparative Example 1, and Comparative Example 3 was determined using the Franz diffusion cell method. The retention amount of proanthocyanidin dimers in pig skin was also tested. The results are shown in Table 3 below. Table 3: Absorption effect of Franz diffusion cell

[0051] As shown in Table 3, the intradermal retention of proanthocyanidins in all groups increased over time, but the release behavior differed. Comparative Examples 1 and 3 exhibited faster penetration rates within the first 12 hours, with cumulative penetration amounts of 66.07 μg / cm³ and 75.64 μg / cm³, respectively, significantly higher than the 50.22 μg / cm³ in Example 1. This result indicates that proanthocyanidins in the comparative samples exist in a free or physically mixed state, readily and rapidly penetrating the skin barrier, exhibiting a "burst release" characteristic. In contrast, the Example 1 sample, due to its supramolecular complex structure, effectively encapsulates proanthocyanidins within the PN carrier network. The release process is regulated by intermolecular forces and diffusion resistance, resulting in a slower initial release rate but a stronger sustained release capacity, exhibiting an overall sustained-release trend. This also demonstrates that it effectively delays the initial burst release of the active ingredient, which is beneficial for prolonging the duration of action and reducing the risk of irritation, providing dual protection for its long-lasting and safe application in functional skincare products.

[0052] Test Example 5: Whitening Efficacy Test This test case provides verification of the whitening efficacy of the samples in Example 1, Example 2, Comparative Example 4, and Comparative Example 5, specifically by inhibiting the production of melanin in melanocytes.

[0053] Experimental Principle: Melanin is a high-molecular-weight biological pigment whose content and distribution directly affect the color of skin, hair, and eyes. When ultraviolet rays (UVA and UVB) irradiate the skin, they easily stimulate melanocytes to secrete melanin. When excessive melanin is produced, it moves through layers of cell metabolism and eventually accumulates in the epidermis, easily leading to abnormal pigment accumulation, resulting in freckles, sunspots, dark spots, and other blemishes, further causing aesthetic problems such as melasma and age spots. Currently, in the in vitro efficacy evaluation of whitening cosmetics, the B16 melanocyte cell line is a typical cell line for studying melanin production. This experiment evaluates the melanin inhibition ability of the test samples, i.e., the whitening efficacy, by measuring the relative content of melanin production. The experimental results are shown in Table 4.

[0054] Table 4

[0055] Note: Compared with the model control group: significance is expressed as... express, This indicates that p < 0.05; This indicates that p < 0.01; This indicates that p < 0.001.

[0056] As shown in Table 4, the inhibition rates (f) of melanin production in B16 cells by the samples in Examples 1 and 2 at a concentration of 0.5% were 19.24% and 18.83%, respectively, comparable to the inhibition rate (20.37%) of the positive control group (hexylresorcinol, 10 μg / mL), and significantly better than those of Comparative Example 4 (12.59%) and Comparative Example 5 (8.29%). This result indicates that the supramolecular complex prepared in this invention possesses whitening activity. Although Comparative Examples 4 and 5 also showed whitening activity, their melanin inhibition effects were significantly weaker than those of Examples 1 and 2, indicating that the combination of proanthocyanidins and PN has a synergistic effect.

[0057] Test Example 6: Anti-wrinkle efficacy test This test case provides the whitening efficacy verification of the samples in Example 1, Example 2, Comparative Example 4, and Comparative Example 5, specifically by inhibiting the expression of MMP-1.

[0058] Experimental Principle: MMP-1 is the most important enzyme for degrading type I and type III collagen. When skin cells overexpress MMP-1, the structure of the extracellular matrix in the dermis is severely damaged, especially the normal structure of collagen and elastin fibers. Therefore, MMP-1 is the most important enzyme leading to aging symptoms such as wrinkles and fine lines. The detection of MMP-1 release is a key indicator for evaluating photoaging. The Earth's surface is predominantly exposed to UVA and UVB radiation. UVA exposure modeling leads to a reduction in matrix metalloproteinases in the extracellular matrix of fibroblasts in normal human skin. The anti-wrinkle and firming effects of the test substances were evaluated through pretreatment and post-irradiation treatment. The experimental results are shown in Table 5.

[0059] Table 5

[0060] Note: Compared with the model control group: significance is expressed as... express, This indicates that p < 0.05.

[0061] As shown in Table 5, the samples in Examples 1 and 2, at a concentration of 0.5%, exhibited inhibition rates of 67.58% and 64.45% against UVA-induced MMP-1 expression, respectively, significantly superior to Comparative Example 4 (57.38%) and Comparative Example 5 (23.53%). This result indicates that the supramolecular proanthocyanidin complex prepared in this invention demonstrates good bioactivity in anti-wrinkle effects, effectively inhibiting the excessive secretion of matrix metalloproteinase MMP-1, thereby protecting the dermal collagen structure from degradation. While Comparative Examples 4 and 5 also showed good anti-wrinkle effects, their MMP-1 inhibition effects were significantly weaker than those in Examples 1 and 2, suggesting that the composite system of proanthocyanidins and PN constructed using a specific process has a synergistic effect in anti-wrinkle efficacy.

[0062] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A supramolecular complex for enhancing and stabilizing proanthocyanidins, characterized in that, The supramolecular complex is prepared by non-covalent self-assembly of proanthocyanidins and high molecular weight polynucleotides; the weight-average molecular weight of the high molecular weight polynucleotides is 1500~6500 kDa.

2. The supramolecular complex according to claim 1, characterized in that, The sources of the high molecular weight polynucleotides include salmon testes.

3. The supramolecular complex according to claim 1, characterized in that, The proanthocyanidins are selected from one of the following: proanthocyanidins from red camellia flowers, proanthocyanidins from grape seeds, and proanthocyanidins from apple peels.

4. The supramolecular complex according to claim 1, characterized in that, The mass ratio of proanthocyanidins to high molecular weight polynucleotides is 1:1~5.

5. A method for preparing a supramolecular complex according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Dissolve proanthocyanidins in water to form an aqueous solution; S2. Add the high molecular weight polynucleotide to the aqueous solution in step S1, stir until a gel is formed, and continue stirring to form a supramolecular inclusion structure. S3. Disperse the supramolecular inclusion structure from step S2 in a polyol aqueous solution to obtain the supramolecular complex.

6. The preparation method according to claim 5, characterized in that, In step S2, the stirring temperature is 25~30℃, and the stirring time is 6~8h.

7. The preparation method according to claim 5, characterized in that, In step S2, the mass concentration of the high molecular weight polynucleotide in the supramolecular inclusion structure is 2-3%.

8. The preparation method according to claim 5, characterized in that, In step S3, the polyol is selected from one or more of propylene glycol, butylene glycol, and glycerol.

9. The preparation method according to claim 5, characterized in that, In step S3, the mass concentration of proanthocyanidins in the supramolecular complex is 0.01%~0.1%.

10. The use of a supramolecular complex as described in any one of claims 1-4 in the preparation of facial cosmetics with skin-brightening, whitening, and anti-wrinkle effects.

Images