Cyclodextrin and flavone inclusion compound as well as preparation method and application thereof

Abstract

The invention discloses a cyclodextrin and flavone inclusion compound and a preparation method and application thereof, and relates to the technical field of skin care product development, the inclusion compound comprises an inclusion subject and an inclusion object in a molar ratio of 1: 2-2: 1, the inclusion subject is cyclodextrin, and the inclusion object is a low-water-solubility natural flavone compound. After the clathrate provided by the invention is redissolved, the clathrate is smeared on an exposed blank skin area of a 2cm * 2cm mouse, ultraviolet damage caused by UVB can be improved to different degrees, and the clathration method provided by the invention increases the water solubility and bioavailability of flavone. After the inclusion system is directly applied to the skin or mixed with other matrixes, ultraviolet absorption and skin collagen metabolism stabilization can be achieved by applying the inclusion system to the skin, a better effect can be achieved compared with a pure ultraviolet blocking sunscreen method, and a new thought is provided for preparation of sunscreen skin care products.

Description

Technical Field

[0001] This invention relates to the field of skincare product development technology, specifically to a cyclodextrin and flavonoid inclusion complex, its preparation method, and its application. Background Technology

[0002] As a barrier organ directly exposed to the external environment, the skin maintains a dynamic balance between collagen synthesis and degradation under normal physiological conditions. However, ultraviolet radiation disrupts this balance, accelerating collagen degradation on one hand and inhibiting collagen synthesis on the other, ultimately leading to a reduction in total collagen volume and structural damage. Prolonged exposure to UVB (medium-wave ultraviolet radiation) can trigger characteristic degenerative damage, known as photoaging, characterized by epidermal thickening and dermal collagen fiber loss, resulting in deeper wrinkles, decreased elasticity, and a rough, dull complexion.

[0003] UVB induces oxidative stress by increasing the production of reactive oxygen species (ROS), and malondialdehyde (MDA), a hallmark product of oxidative stress, directly reflects the degree of UVB-induced skin oxidative damage. As oxidative damage intensifies, the mitogen-activated protein kinase (MAPK) pathway is activated, with phosphorylated p38 (p-p38) as a core node. Increased activity of p38 further regulates downstream target genes, promoting the expression of matrix metalloproteinase-1 (MMP-1). MMP-1, a key enzyme in collagen degradation, specifically targets dermal α-type I collagen (COL1A1), leading to a significantly accelerated degradation rate and continuous loss of collagen fibers. Simultaneously, UVB further exacerbates collagen metabolic imbalance by inhibiting collagen synthesis pathways. The TGF-β / Smad pathway is a core pathway regulating collagen synthesis, and Smad2 / 3 are key transcription factors in this pathway that directly regulate the expression of COL1A1 synthesis genes. Under normal conditions, Smad2 / 3, once activated, enters the cell nucleus, promoting COL1A1 synthesis and maintaining a stable total collagen level. However, UVB-induced oxidative damage inhibits the activity of this pathway, reducing the regulatory capacity of Smad2 / 3 on COL1A1 synthesis and leading to a decrease in new collagen synthesis. Thus, UVB, through its dual effects of "accelerating degradation + inhibiting synthesis," disrupts the collagen metabolic balance, ultimately causing photoaging of the skin.

[0004] Natural flavonoids, due to their excellent antioxidant and anti-inflammatory activities, hold promise for application in skincare product development to combat photoaging. For example, quercetin (Que) and naringenin (Nar) (chemical formula as shown in Formula 1), widely found in plants, while structurally different, have both been shown in pharmacological studies to inhibit collagen degradation through the P38 protein of the MAPK pathway and promote collagen synthesis through the TGF-β / Smad pathway. However, both Que and Nar share the drawback of low water solubility, making them unsuitable for dissolving in topical formulations. This low water solubility results in their inability to penetrate the stratum corneum and reach the dermis after application, leading to extremely low bioavailability and limiting their practical application in photoaging protection.

[0005]

[0006] Formula 1. Summary of the Invention

[0007] This invention provides a cyclodextrin and flavonoid inclusion complex, its preparation method, and its application, to solve the technical problem that existing natural flavonoids such as Que and Nar have low water solubility, which makes it difficult to meet the dissolution requirements of topical preparations and thus limits their practical application in photoaging protection.

[0008] The technical solution adopted in this invention is as follows:

[0009] In a first aspect, the present invention provides an inclusion complex of cyclodextrin and flavonoids, comprising an inclusion host and an inclusion guest in a molar ratio of 1:2 to 2:1, wherein the inclusion host is cyclodextrin and the inclusion guest is a low-water-soluble natural flavonoid compound.

[0010] Furthermore, the cyclodextrin is at least one of β-cyclodextrin, hydroxyethyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin, and the low water-soluble natural flavonoid compound is quercetin and / or naringin.

[0011] Furthermore, the cyclodextrin is hydroxypropyl-β-cyclodextrin, and the low water-soluble natural flavonoid compound is quercetin.

[0012] Furthermore, the cyclodextrin is hydroxypropyl-β-cyclodextrin, and the low water-soluble natural flavonoid compound is naringenin.

[0013] In a second aspect, the present invention provides a method for preparing the inclusion complex of cyclodextrin and flavonoids described in the first aspect, the preparation method comprising the following steps:

[0014] (1) Dissolve low water-soluble natural flavonoids in ethanol to prepare saturated or near-saturated ethanol solutions (the ratio of anhydrous ethanol to quercetin is 1500-2000 mL / mol, and the ratio of anhydrous ethanol to naringenin is 1500-2000 mL / mol, ensuring that the prepared flavonol solutions are close to saturation), and dissolve cyclodextrin in water to prepare cyclodextrin aqueous solutions of 10-30 g / L.

[0015] (2) Add the cyclodextrin aqueous solution dropwise into the ethanol solution of low water-soluble natural flavonoids under high-speed stirring, and stir until the solution is in a colloidal state;

[0016] (3) The colloidal system is evaporated under reduced pressure, deionized water is added, and after stirring and dissolving, it is filtered through a microporous membrane. The clear filtrate is retained, and the filtrate is evaporated to obtain the finished powder of the inclusion compound.

[0017] Furthermore, the concentration of the cyclodextrin aqueous solution prepared in step (1) is 20 g / L.

[0018] Furthermore, the microporous filter membrane used in step (3) has a pore size of 0.45 μm.

[0019] Thirdly, the present invention further discloses the application of the cyclodextrin and flavonoid inclusion complexes described in the first aspect in the preparation of sunscreen skin care products and skin care products that promote collagen regeneration.

[0020] As a preferred embodiment: the inclusion complex used to prepare skin care products with both anti-collagen degradation and sun protection functions has hydroxypropyl-β-cyclodextrin as its cyclodextrin and quercetin as its low water-soluble natural flavonoid compound.

[0021] As a preferred embodiment: the inclusion complex used to prepare skin care products that have both collagen regeneration and sun protection functions has hydroxypropyl-β-cyclodextrin as its cyclodextrin and naringin as its low water-soluble natural flavonoid compound.

[0022] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] 1. When the encapsulated co-inhibitor provided by the present invention was reconstituted and applied to the bare skin area of ​​a 2cm×2cm mouse, it could improve the ultraviolet damage caused by UVB to varying degrees. It can be seen that the encapsulated co-inhibitor provided by the present invention can achieve sun protection by being applied directly to the skin and has good bioavailability. Compared with the simple ultraviolet blocking sun protection method, it can achieve better results and provides a new idea for the preparation of sun protection skin care products.

[0024] 2. The raw materials for the preparation of the encapsulation co-linked compounds provided by the present invention have good biosafety, and the preparation steps are simple, the conditions are mild, and they are easy to produce on a large scale.

[0025] 3. The encapsulated conjugates provided by this invention can be stored by drying and are easily reconstituted with water to form part of other compound preparations, that is, they have good reprocessing potential. Attached Figure Description

[0026] Figure 1 The infrared spectra of the six inclusion complexes, two flavonoids, and three blank cyclodextrins prepared in Example 1 are shown.

[0027] Figure 2 X-ray diffraction patterns of the six inclusion complexes prepared in Example 1, two flavonoids, and three blank cyclodextrins;

[0028] Figure 3 The above are the 1H NMR spectra of the six inclusion compounds and two flavonoids prepared in Example 1.

[0029] Figure 4 The UV spectra of the six inclusion complexes, two flavonoids, and three blank cyclodextrins prepared in Example 1 are shown.

[0030] Figure 5 The chart shows the drug loading and inclusion rate data of the six inclusion compounds prepared in Example 1, along with two flavonoids and three blank cyclodextrins.

[0031] Figure 6 HE staining, Masson staining images and statistical graphs of the six inclusion complexes prepared in Example 1, two flavonoids and three blank cyclodextrins on mouse skin;

[0032] Figure 7 The six inclusion complexes prepared in Example 1, along with two flavonoids and three blank cyclodextrins, were analyzed using MDA fluorescence images and statistical graphs in mouse skin.

[0033] Figure 8 Immunofluorescence double staining images and statistical graphs of six inclusion complexes prepared in Example 1, two flavonoids, and three blank cyclodextrins on mouse skin p-p38 / MMP-1;

[0034] Figure 9 Immunofluorescence double staining images and statistical graphs of the six inclusion complexes prepared in Example 1, along with two flavonoids and three blank cyclodextrins, on mouse skin using Smad2 / 3-COL1A1. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to various embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0036] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in these embodiments, unless otherwise specified, employs conventional testing methods in the art. The terminology used in this invention is merely for describing particular implementations and is not intended to limit the scope of the disclosure.

[0037] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; other raw materials, reagents, test methods and techniques not specifically mentioned herein refer to raw materials and reagents commonly used by one of ordinary skill in the art, as well as commonly employed test methods and techniques.

[0038] Example 1

[0039] In this embodiment, β-cyclodextrin (β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD) were used as inclusion bodies, and quercetin (Que) and naringenin (Nar) were used as inclusion guests, respectively, to prepare six inclusion complexes. The unincluded raw materials were used as controls for the studies. Specific experiments are as follows:

[0040] 1. Preparation of the six inclusion compounds: The preparation process is as follows:

[0041] (1) Quercetin (Que) and naringenin (Nar) were dissolved in a certain amount of anhydrous ethanol at 50°C to obtain quercetin ethanol solution and naringenin ethanol solution respectively. Specifically, the amount of ethanol used was 15 mL, and the amounts of quercetin (Que) and naringenin (Nar) were Que (0.1511 g) and Nar (0.1351 g) respectively.

[0042] (2) Based on the solubility of β-cyclodextrin (β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD) at 50℃, 20 g / L aqueous solutions of the three cyclodextrins were prepared by stirring. The structural formulas of β-cyclodextrin (β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD) are shown in Formula 2.

[0043]

[0044] Formula 2.

[0045] (3) Add equimolar amounts of aqueous solutions of quercetin (Que) and β-cyclodextrin (β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD) to a saturated alcoholic solution of quercetin, respectively. Stir at 1000-1500 rpm for 24 hours, cool to room temperature, and then evaporate to dryness. Crude products of three quercetin cyclodextrin inclusion complexes, Que / β-CD, Que / HE-β-CD, and Que / HP-β-CD, are obtained. Add equimolar amounts of aqueous solutions of β-cyclodextrin (β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD) to an alcoholic solution of naringenin (Nar), respectively. Stir at 1000-1500 rpm for 24 hours, cool to room temperature, and then evaporate to dryness. Crude products of three cyclodextrin inclusion complexes of naringenin, namely Que / β-CD inclusion complex, Que / HE-β-CD inclusion complex, and Que / HP-β-CD inclusion complex, were obtained.

[0046] (4) Add a certain amount of deionized water to the crude powder obtained in step (3), sonicate for 20 minutes, filter with a 0.45 μm microporous membrane, collect the supernatant, evaporate by rotary evaporation, dry by rotary evaporation, and grind to obtain Que / β-CD, Nar / β-CD, Que / HE-β-CD, Nar / HE-β-CD, Que / HP-β-CD, and Nar / HP-β-CD inclusion complexes.

[0047] To understand the structures of Que / β-CD, Nar / β-CD, Que / HE-β-CD, Nar / HE-β-CD, Que / HP-β-CD, and Nar / HP-β-CD inclusion complexes, the following structural characterization tests were performed using unincluded raw materials as a control group:

[0048] 1. Fourier Transform Infrared Spectroscopy (FTIR)

[0049] Fourier transform infrared (FT-IR) spectra of Que, Nar, β-CD, HE-β-CD, HP-β-CD, and six inclusion compounds were obtained using a Nicolet 6700 Thermo Fisher Scientific instrument. The sample (1 mg) and KBr (100 mg) were ground into a homogeneous powder in agate slurry, then prepared into thin slices for scanning. The infrared wavelength range during detection was 400-4000 cm⁻¹. -1 Between. For example Figure 1 As shown in (A, B), the vibrational peak of -OH in the FT-IR spectrum of β-CD is at 3406 cm⁻¹. −1 At this point, the vibrational peak of CH is at 2927 cm⁻¹. −1 Location: 1158cm −1The absorption peak at 1028 cm⁻¹ represents the CO group. −1 The position of the absorption band is due to the vibration of the COC bond. In the FT-IR spectra of the Que / β-CD and Nar / β-CD inclusion complexes, the characteristic peaks of Que and Nar clearly disappear, but are basically consistent with the characteristic peaks of β-CD, indicating that after inclusion, the small molecules Que and Nar enter the cavity of β-CD, and the characteristic peaks of Que and Nar are masked by β-CD. Figure 1 As shown in (C, D), in the FT-IR spectrum of HE-β-CD, at 3418 cm⁻¹ −1 The vibrational peak at 2930 cm⁻¹ is the stretching vibration of -OH. −1 The vibrational peak belongs to CH, and the bending vibration of C−H in hydroxyethyl is at 1460 cm⁻¹. −1 The vibrational peak of the CO group is at 1360 cm⁻¹. −1 Location, 1031cm −1 The vibrational peak at that location belongs to the stretching vibration of COC. In the FT-IR spectra of the Que / HE-β-CD and Nar / HE-β-CD inclusion complexes, the characteristic absorption peaks produced by Que and Nar completely disappeared, indicating that Que and Nar molecules entered the cavity of the HE-β-CD cyclodextrin molecule after the reaction, demonstrating the success of the inclusion reaction. Figure 1 As shown in (E, F), in the FT-IR spectrum of HP-β-CD, the stretching vibration of -OH is at 3408 cm⁻¹. −1 Location, 2929cm −1 The vibrational peak belongs to CH, and the bending vibration of the methyl group (CH3) in hydroxypropyl is at 1370 cm⁻¹. −1 Location, 1156cm −1 and 1033cm −1 The vibrational peak at 400 cm⁻¹ belongs to the stretching vibration of COC. The FT-IR spectra of Que / HP-β-CD and Nar / HP-β-CD inclusion complexes are quite similar to those of HP-β-CD, and compared to Que and Nar, the peak at 400 cm⁻¹ is more pronounced. -1 -1500cm -1 Significant changes occurred between them, with the characteristics of Que and Nar almost completely disappearing. This indicates that Que and Nar molecules entered the cavity of the cyclodextrin molecule, and the characteristic absorption peaks were masked by the cyclodextrin, indicating that the inclusion reaction was successful.

[0050] 2. X-ray diffraction (XRD)

[0051] Powder diffraction patterns of Que, Nar, β-CD, HE-β-CD, HP-β-CD, six inclusion compounds, and six physical mixtures were obtained using an X-ray diffractometer (D8 ADVANCE, Bruker, Germany). A Cu target Kα line was used as the radiation source, with a scan rate of 5° / min, and scans were performed within the range of 5° to 60° 2θ. Figure 2 As shown in (A, B), the X-ray diffraction pattern of β-CD also reveals a series of sharp, strong peaks, indicating the specific crystal structure of β-CD, with diffraction peaks appearing at 8°, 10°, 12°, and 19°. The crystal diffraction peaks of the physical mixtures of Que and Nar with β-CD are even denser, with peaks from both substances appearing. However, in the Que / β-CD and Nar / β-CD inclusion complexes, the diffraction peaks produced by Que and Nar do not appear, similar to β-CD, indicating that Que and Nar are successfully included with β-CD. Figure 2 As shown in (C, D), since HE-β-CD is an amorphous structure and therefore not crystalline, its X-ray diffraction pattern is relatively smooth. The diffraction peaks of the physical mixture of Que and Nar with HE-β-CD show that Que and Nar diffraction peaks appear on top of the HE-β-CD diffraction pattern, simply superimposed. However, in the diffraction patterns of the Que / HE-β-CD and Nar / HE-β-CD inclusion complexes, the crystalline diffraction peaks of Que and Nar completely disappear, similar to HE-β-CD. This indicates the success of the inclusion reaction and suggests that Que and Nar underwent a state change after inclusion, no longer being crystalline. Figure 2 As shown in (E, F), HP-β-CD is also an amorphous form, so it is similar to HE-β-CD. The diffraction patterns of the physical mixture of Que and Nar with HP-β-CD show that Que and Nar diffraction peaks appear on the basis of the HP-β-CD diffraction pattern. In the Que / HP-β-CD inclusion complex, all the diffraction peaks of Que disappear, and in the Nar / HP-β-CD inclusion complex, all the diffraction peaks of Nar disappear, similar to HP-β-CD. This indicates that Que and Nar successfully entered the cavity of HP-β-CD, thus losing their crystallinity.

[0052] 3. Proton NMR analysis

[0053] For Que, Nar, and the six inclusion compounds, 15 mg was weighed and dissolved in DMSO-d6, and then ¹H NMR spectra were recorded on a nuclear magnetic resonance spectrometer (400MR, Agilent Technologies, USA). Chemical shifts are expressed in ppm.

[0054] like Figure 3As shown in (A), the ¹H NMR spectrum of Que exhibits a sharp characteristic peak for aromatic hydrogens in the δ 6.0–8.0 ppm region, while the δ 9.0–13.0 ppm region corresponds to the signal of phenolic hydroxyl hydrogens. Compared to free Que, the aromatic proton signals (δ 6.0–8.0 ppm) in its three inclusion compounds show a slight upper field shift. Notably, all phenolic hydroxyl proton signals (δ 9.0–13.0 ppm) are significantly attenuated, while the aromatic proton signals remain. This spectral change may be due to the participation of these hydroxyl groups in the inclusion of cyclodextrins. This indicates that Que has successfully entered the cavities of the three cyclodextrins, forming inclusion compounds. Figure 3 As shown in (B), the ¹H NMR spectrum of Nar exhibits a sharp aromatic proton signal in the δ 5.5–7.5 ppm region and a distinct phenolic hydroxyl proton signal in the δ 9.5–12.5 ppm region. Consistent with the behavior of the Que complex, a slight up-field shift was observed in the aromatic proton signals (δ 5.5–7.5 ppm) of the β-CD, HE-β-CD, and HP-β-CD inclusion complexes of Nar. Furthermore, all phenolic hydroxyl hydrogen signals in the δ 9.5–12.5 ppm region were significantly attenuated. This indicates that Nar has successfully entered the cavities of the three cyclodextrins and formed inclusion complexes. The proton signals of the two flavonoids in all inclusion complexes were right-biased. The intensity of the free hydroxyl signals of all flavonoids was significantly reduced relative to the benzene ring hydroxyl signals. Thus, the inclusion complex was ultimately confirmed as successful.

[0055] To understand the various properties of the target product, we also conducted the following tests using the unencapsulated raw material as a control group:

[0056] 1. Ultraviolet (UV) Spectrum

[0057] Que, Nar, β-CD, HE-β-CD, HP-β-CD, and six inclusion complexes were weighed and dissolved in anhydrous ethanol to obtain a 10 μg / mL test solution. After complete dissolution, their UV absorption spectra in the 200-500 nm range were measured using a UV spectrophotometer (Agilent 8453, Agilent Technologies, USA). Figure 4 As shown: Que has a characteristic UV absorption peak at 375 nm, and Nar has a characteristic UV absorption peak at 290 nm. It is known that β-CD, HE-β-CD, and HP-β-CD lack unsaturated bonds and have very weak UV absorption intensity, exhibiting no characteristic peaks. However, the prepared quercetin inclusion complex has characteristic absorption in the 220-270 nm wavelength range, and the prepared naringenin inclusion complex has characteristic absorption in the 330-410 nm wavelength range. This indicates that they can maintain their original UV absorption characteristics in the inclusion state, absorbing UV light of specific wavelengths.

[0058] 2. Drug loading and inclusion rate of the inclusion complex

[0059] Accurately weigh 2 mg of the inclusion complex and dissolve it in 5 mL of ethanol. Sonicate the solution for 10 min at room temperature (25°C). Filter the solution through a 0.45 μm microporous membrane to remove undissolved substances. Then, dilute the solution according to a specific concentration ratio. Measure the absorbance at the wavelengths of the maximum absorption peaks of Que and Nar using a UV spectrophotometer. Calculate the concentrations of Que and Nar based on the standard curve.

[0060] like Figure 5 As shown, the inclusion capacity of the three cyclodextrins with Que and Nar is in the order: HP-β-CD > HE-β-CD > β-CD. This indicates that HP-β-CD is the optimal carrier for Que and Nar among the three cyclodextrins. It can also be observed that in the inclusion complexes formed by β-CD with Que and Nar, the drug loading and inclusion rate of Que / β-CD are both lower than those of Nar / β-CD. However, in the inclusion complexes formed by HE-β-CD and HP-β-CD with Que and Nar, the drug loading and inclusion rate differ, indicating that different cyclodextrins have different inclusion effects on different flavonoid drugs.

[0061] 3. Mouse skin photoaging repair experiment

[0062] Healthy female Kunming mice were randomly divided into 10 groups: normal group, model group, Que group, Nar group, Que / β-CD group, Que / HE-β-CD group, Que / HP-β-CD group, Nar / β-CD group, Nar / HE-β-CD group, and Nar / HP-β-CD group. Before the experiment, the hair on the backs of the mice was removed with depilatory cream, exposing a skin area of ​​approximately 2cm × 2cm. Water was applied to the normal group and the UV model group. Since Que and Nar are insoluble in water, they were dissolved in 50% ethanol solution and then applied to the backs of the mice. The other six inclusion complex groups were applied to the backs of the mice at a concentration of 500 μg / mL. After 15 minutes for absorption, the mice were placed under a UV lamp at a distance of 15cm for 30 minutes each time, with a UVB irradiance of 0.38 mw / cm². 2 A skin photoaging model was established by continuous irradiation for 7 days, with a total UVB irradiation dose of 4.788 J / cm². 2 .

[0063] Subsequently, skin morphology in different groups was observed using HE staining and Masson staining. Figure 6As shown, skin tissue damage was improved to varying degrees in all six cyclodextrin inclusion complex groups (Que / β-CD, Que / HE-β-CD, Que / HP-β-CD, Nar / β-CD, Nar / HE-β-CD, and Nar / HP-β-CD). In the normal group, the skin structure of mice was intact, with a uniform epidermal layer thickness (10.54±2.13µm), tightly and orderly arranged cells, and deep blue, evenly distributed, and tightly interwoven dermal collagen fibers. In the model group, the epidermis of mice was significantly thickened (29.72±2.69µm), with disordered epidermal cell arrangement, abnormal keratinization in some areas, and lighter staining and severe breakage of dermal collagen fibers, exhibiting a sparse, reticular distribution. These differences were significant compared to the normal group (P<0.001), indicating that UVB irradiation successfully established a skin photoaging model. Compared with the model group, the skin tissue damage in the Que group, Nar group, and six cyclodextrin inclusion complex groups (Que / β-CD, Que / HE-β-CD, Que / HP-β-CD, Nar / β-CD, Nar / HE-β-CD, Nar / HP-β-CD) all showed varying degrees of improvement: epidermal thickness was significantly reduced, cell arrangement became more regular, and collagen fiber breakage was alleviated. Among them, the inclusion complex group with HP-β-CD as the carrier showed the most significant improvement, with epidermal thickness closest to the normal control group. It was also found that the Que / HP-β-CD group was more effective than the Nar / HP-β-CD group in improving epidermal thickening.

[0064] 4. Mouse skin oxidative repair experiment

[0065] In a mouse skin photoaging repair experiment, MDA expression levels were observed by staining mouse skin with MDA fluorescence. Images were acquired using a specific wavelength microscope; positive MDA signals appeared in red, and observation was performed via the CY3 channel. The fluorescence area ratio was measured using ImageJ software (five fields of view were randomly selected from each group, and the average value was taken). Figure 7 As shown, only weak MDA-positive fluorescence signals were observed in the normal group skin tissue, indicating that the level of skin oxidative damage was extremely low under normal physiological conditions; however, the MDA-positive fluorescence signal was significantly enhanced in the model group skin tissue, with both the fluorescence area ratio and fluorescence intensity reaching peak values, indicating that UVB irradiation can induce skin cells to produce a large number of oxidative stress products, leading to severe oxidative damage.

[0066] Compared with the model group, the MDA-positive fluorescence signals of the Que group, Nar group, and six cyclodextrin inclusion complex groups (Que / β-CD, Que / HE-β-CD, Que / HP-β-CD, Nar / β-CD, Nar / HE-β-CD, Nar / HP-β-CD) were all weakened to varying degrees. The fluorescence signal weakening of the inclusion complex groups was generally greater than that of the corresponding Que and Nar groups, indicating that cyclodextrin inclusion can enhance the antioxidant capacity of the two flavonoid compounds. Comparing the three types of cyclodextrin inclusion complexes, the inclusion complex group with HP-β-CD as the carrier (Que / HP-β-CD, Nar / HP-β-CD) had the weakest MDA fluorescence signal and the closest fluorescence area ratio to the normal control group; while the improvement effect of the β-CD inclusion complex group was relatively weak, and the effect of the HE-β-CD inclusion complex group was between the two. Meanwhile, it was found that the MDA fluorescence signal of the Que group and its inclusion complex was weaker than that of the Nar group and its inclusion complex, indicating that Que was more effective than Nar in inhibiting UVB-induced skin oxidative damage.

[0067] 5. Study on the regulation of the p-p38 / MMP-1 skin collagen degradation pathway by inclusion complexes

[0068] In a mouse skin photoaging repair experiment, the expression levels of the p-p38 / MMP-1 pathway were observed after fluorescence double staining. Positive signals for p-p38 were indicated by red, and positive signals for MMP-1 by green. The fluorescence area ratio was measured using ImageJ software (five fields of view were randomly selected from each group for measurement, and the average value was taken). Figure 8 The normal control group showed extremely weak positive fluorescence signals for p-p38 and MMP-1 in their skin tissue, indicating that under physiological conditions, the MAPK pathway that regulates collagen degradation maintains low activity, and the expression of p-p38 and MMP-1 is at a basal level. The collagen metabolism in the dermis is in stable balance, with no significant collagen loss.

[0069] The positive fluorescence signals of p-p38 and MMP-1 in the skin tissue of the model group were significantly enhanced, and the fluorescence area ratio reached its peak, indicating that UVB irradiation can effectively activate the MAPK pathway, promote the phosphorylation of p38, enter the cell nucleus, regulate the transcription and expression of downstream MMP-1, resulting in a large accumulation of MMP-1, which ultimately accelerates the degradation of COL1A1.

[0070] Compared with the model group, the p-p38 and MMP-1 fluorescence area ratios of the Que group, Nar group, and six cyclodextrin inclusion complex groups (Que / β-CD, Que / HE-β-CD, Que / HP-β-CD, Nar / β-CD, Nar / HE-β-CD, Nar / HP-β-CD) were all reduced to varying degrees. The signal reduction of all inclusion complex groups was greater than that of the corresponding Que group and Nar group. It was found that the inclusion complex group with HP-β-CD as the carrier (Que / HP-β-CD, Nar / HP-β-CD) had the weakest p-p38 and MMP-1 fluorescence signals and the fluorescence area ratio closest to the normal control group. This indicates that the HP-β-CD group has the best inhibitory effect on the MAPK pathway. In the comparison of the two flavonoids, the p-p38 and MMP-1 fluorescence signals of the Nar group and the inclusion complex group were stronger than those of the Que group and the inclusion complex group, indicating that Que and its inclusion complex were more effective than Nar and its inclusion complex in downregulating the MMP-1 pathway through p-p38.

[0071] 6. Study on the regulation of the skin collagen synthesis pathway Smad2 / 3-COL1A1 by inclusion complexes

[0072] In a mouse skin photoaging repair experiment, the expression levels of the Smad2 / 3-COL1A1 pathway were observed after fluorescence double staining of mouse skin. Smad2 / 3 positive signals appeared in red, and COL1A1 positive signals appeared in green. The fluorescence area ratio was measured using ImageJ software (five fields of view were randomly selected from each group for measurement, and the average value was taken).

[0073] like Figure 9 As shown, the highest fluorescence area ratio of Smad2 / 3 and COL1A1 was observed in the skin tissue of the normal control group. This indicates that under normal conditions, the TGF-β / Smad pathway, which regulates collagen synthesis, remains active, continuously activating and regulating the synthesis of downstream COL1A1, thus maintaining a dynamic balance between collagen regeneration and degradation in the dermis, and ensuring the integrity of collagen structure and the stability of total collagen amount.

[0074] In the model group, the fluorescence signals of Smad2 / 3 and COL1A1 were significantly weakened, and the fluorescence area ratio dropped to the lowest level. This indicates that UVB irradiation can inhibit the TGF-β / Smad pathway, reduce Smad2 / 3 activity, and ultimately lead to reduced COL1A1 synthesis, insufficient collagen regeneration, and disruption of collagen metabolism balance.

[0075] Compared with the model group, the fluorescence of Smad2 / 3 and COL1A1 in the Que group, Nar group, and six cyclodextrin inclusion complexes (Que / β-CD, Que / HE-β-CD, Que / HP-β-CD, Nar / β-CD, Nar / HE-β-CD, Nar / HP-β-CD) were enhanced to varying degrees. Furthermore, the signal enhancement amplitude of all inclusion complexes was greater than that of the corresponding Que and Nar groups, indicating that inclusion can enhance the activation ability of the two flavonoids on the TGF-β / Smad pathway, thereby more effectively upregulating the expression of Smad2 / 3 and COL1A1. The inclusion complexes using HP-β-CD as the carrier (Que / HP-β-CD, Nar / HP-β-CD) showed the strongest Smad2 / 3 and COL1A1 fluorescence signals, and the fluorescence area ratio was closest to the normal control group, indicating that the HP-β-CD group had the best activation effect on the TGF-β / Smad pathway. In the comparison of the two flavonoids, the fluorescence signals of Smad2 / 3 and COL1A1 in the Que group and the inclusion complex group were stronger than those in the Nar group and the inclusion complex group, indicating that Que and its inclusion complex are more effective than Nar and its inclusion complex in activating the TGF-β / Smad pathway.

[0076] In summary, this embodiment uses quercetin (Que) and naringenin (Nar) as inclusion guests, and β-cyclodextrin (β-CD), hydroxyethyl-β-cyclodextrin (HE-β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD) as inclusion hosts to prepare a series of inclusion compounds. These inclusion compounds possess a triple effect of ultraviolet absorption, prevention of collagen degradation, and promotion of collagen regeneration.

[0077] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A complex containing cyclodextrin and flavonoids, characterized in that, It includes an inclusion host and an inclusion guest with a molar ratio of 1:2 to 2:1, wherein the inclusion host is a cyclodextrin and the inclusion guest is a low-water-soluble natural flavonoid compound.

2. The cyclodextrin and flavonoid inclusion complex as described in claim 1, characterized in that, The cyclodextrin is at least one of β-cyclodextrin, hydroxyethyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin, and the low water-soluble natural flavonoid compound is quercetin and / or naringin.

3. The cyclodextrin and flavonoid inclusion complex as described in claim 1, characterized in that, The cyclodextrin is hydroxypropyl-β-cyclodextrin, and the low-water-soluble natural flavonoid compound is quercetin.

4. The cyclodextrin and flavonoid inclusion complex as described in claim 1, characterized in that, The cyclodextrin is hydroxypropyl-β-cyclodextrin, and the low-water-soluble natural flavonoid compound is naringenin.

5. The method for preparing the inclusion complex of cyclodextrin and flavonoids as described in any one of claims 1 to 4, characterized in that, The preparation method includes the following steps: (1) Dissolve low water-soluble natural flavonoids in ethanol to prepare ethanol saturated or near-saturated solutions of them, and dissolve cyclodextrin in water to prepare cyclodextrin aqueous solutions of 10~30g / L; (2) Add the cyclodextrin aqueous solution dropwise into the ethanol solution of low water-soluble natural flavonoids under high-speed stirring, and stir until the solution is in a colloidal state; (3) The colloidal system is evaporated under reduced pressure, deionized water is added, and after stirring and dissolving, it is filtered through a microporous membrane. The clear filtrate is retained, and the filtrate is evaporated to obtain the finished powder of the inclusion compound.

6. The preparation method according to claim 5, characterized in that, The concentration of the cyclodextrin aqueous solution prepared in step (1) is 20 g / L.

7. The preparation method according to claim 5, characterized in that, The microporous filter membrane used in step (3) has a pore size of 0.45 μm.

8. The application of the cyclodextrin and flavonoid inclusion complex as described in any one of claims 1 to 4 in the preparation of sunscreen skin care products and skin care products that promote collagen regeneration.

9. The application as described in claim 8, characterized in that, The inclusion complex used to prepare skin care products with functions of preventing collagen degradation, promoting collagen regeneration and ultraviolet absorption has hydroxypropyl-β-cyclodextrin as its cyclodextrin and quercetin as its low water-soluble natural flavonoid compound.

10. The application as described in claim 8, characterized in that, The inclusion complex used to prepare skin care products with functions of preventing collagen degradation, promoting collagen regeneration and ultraviolet absorption has hydroxypropyl-β-cyclodextrin as its cyclodextrin and naringenin as its low water-soluble natural flavonoid compound.

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