Ginsenoside CK-aescin supramolecule and preparation method thereof

Abstract

The invention relates to the technical field of supramolecules, in particular to a ginsenoside CK-aescin supramolecule, in the supramolecule, ginsenoside CK and aescin are formed through a non-covalent bond, the mass ratio of ginsenoside CK to aescin is 1: (1-25), and a preparation method comprises the following steps: dispersing aescin in an organic solvent aqueous solution to prepare a suspension A, dissolving ginsenoside CK in ethanol to prepare a solution D for later use; stirring the suspension A to obtain a suspension B; performing ultrasonic treatment on the suspension B to obtain emulsion C; slowly adding the emulsion C into the solution D while stirring, stirring to obtain a ginsenoside CK-aescin supramolecular solution crude product, and dialyzing. According to the ginsenoside CK-aescin supramolecule prepared by the invention, an IVPT test shows that the transdermal absorption capacity of the ginsenoside CK-aescin supramolecule is remarkably improved, so that the ginsenoside CK-aescin supramolecule can be better absorbed by human skin, and the efficacy of the ginsenoside CK-aescin supramolecule is favorably exerted.

Description

Technical Field

[0001] This invention relates to the field of supramolecular technology, and in particular to a ginsenoside CK-aescin supramolecular compound and its preparation method. Background Technology

[0002] Ginsenoside CK (Compound K), also known as rare ginsenoside CK, is the main metabolite and final absorption form of diol-type ginsenosides such as ginsenoside Rb1 under the action of human intestinal flora. It cannot be obtained through traditional acid-base hydrolysis and requires biotransformation technologies such as enzymatic conversion or microbial fermentation. Ginsenoside CK is a tetracyclic triterpenoid saponin with the chemical formula C2. 36 H 62 O8, with a molecular weight of approximately 622.87, belongs to the dammarane-type triterpenoids. In the skincare field, ginsenoside CK significantly promotes type IV collagen regeneration, with a measured activation rate of 397%, higher than that of pro-xylane and retinol. Simultaneously, it inhibits matrix metalloproteinase-1 (MMP-1) activity, reducing collagen degradation and thus enhancing skin elasticity and reducing wrinkles. Furthermore, it upregulates hyaluronic acid synthase 2 gene expression, increasing skin hydration, and promotes microvascular angiogenesis by regulating the HIF-1α signaling pathway, exerting anti-photoaging and barrier repair effects. Ginsenoside CK is known for its gentleness in skincare, avoiding the common irritation risks associated with retinol, making it suitable for sensitive skin repair.

[0003] Aescin is a natural herbal medicine extracted from the dried, mature seeds of plants in the Aesculaceae family (such as *Aesculus cusia*). Its sodium salt form is called sodium aescinate, with the molecular formula […]. (Aescin) or Sodium aescinate, usually appearing as a white powder, has pharmacological effects such as anti-inflammatory, anti-exudative, increasing venous tone, improving blood circulation, and correcting brain dysfunction.

[0004] Given their unique effects in the skincare field, they are often used in cosmetics. However, their transdermal absorption capacity is not good, and they cannot be absorbed by human skin effectively. Summary of the Invention

[0005] This invention solves the problems in related technologies and proposes a ginsenoside CK-aescin supramolecular and its preparation method. A stable supramolecular is obtained by using an appropriate ratio, and the transdermal absorption capacity of the obtained ginsenoside CK-aescin supramolecular is significantly improved.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: a ginsenoside CK-aescin supramolecular, wherein the ginsenoside CK-aescin supramolecular is formed by ginsenoside CK and aescin through non-covalent bonds, and the mass ratio of ginsenoside CK to aescin is 1:(1~25).

[0007] As a preferred option, the mass ratio of ginsenoside CK to aescin is 1:(10~20).

[0008] As a preferred option, the UV spectrum of the ginsenoside CK-aescin supramolecular structure shows a characteristic peak at 192 nm.

[0009] As a preferred embodiment, in the XRD pattern of the ginsenoside CK-aescin supramolecular structure, when the mass ratio of ginsenoside CK to aescin is 1:10, a characteristic peak is observed at a 2θ angle of 14.108°; when the mass ratio of ginsenoside CK to aescin is 1:15, characteristic peaks are observed at 2θ angles of 6.11° and 14.24°; and when the mass ratio of ginsenoside CK to aescin is 1:20, characteristic peaks are observed at 2θ angles of 6.166° and 14.40°.

[0010] As a preferred embodiment, in the TEM spectrum of ginsenoside CK-aescin, the supramolecular form of ginsenoside CK-aescin is spherical particles.

[0011] Another aspect of the present invention provides a method for preparing ginsenoside CK-aescinate supramolecular molecules, comprising the following steps:

[0012] S1. Disperse aescin in an aqueous solution of an organic solvent with a mass concentration of 8%~30% to prepare a suspension A with a mass concentration of 0.1%~1%, and dissolve ginsenoside CK in ethanol to prepare a solution D with a mass concentration of 5%-20% for later use.

[0013] S2. Stir suspension A for 3-10 minutes to obtain suspension B;

[0014] S3. Sonicate suspension B for 10-60 min to obtain emulsion C;

[0015] S4. Slowly add emulsion C to solution D under stirring, and stir for 45 min to 120 min to obtain crude product of ginsenoside CK-aescin supramolecular solution, wherein the mass ratio of ginsenoside CK to aescin is 1:(1~25).

[0016] S5. Dialyze the crude ginsenoside CK-aescin supramolecular solution in a dialysis bag for 12-24 hours to obtain an aqueous solution of ginsenoside CK-aescin supramolecular solution.

[0017] As a preferred embodiment, the organic solvent in step S1 is at least one of ethanol, DMSO, hexanediol, and butanediol.

[0018] As a preferred embodiment, magnetic stirring is used in steps S2 and S4, and the stirring speed is 300 rpm to 1000 rpm.

[0019] As a preferred embodiment, in step S3, the frequency of the ultrasound is 200W~600W, and the temperature of the ultrasound is 25℃~50℃.

[0020] As a preferred option, in step S5, the molecular weight of the dialysis bag is 8000-14000.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention first uses ultrasonic treatment to form aescin into nanospheres, and then stirs and interweaves ginsenoside CK into them. Through a suitable mass ratio of aescin to ginsenoside CK, a stable supramolecular system is finally formed. IVPT test shows that the transdermal absorption capacity of the prepared ginsenoside CK-aescin supramolecular is significantly improved, so that it can be better absorbed by human skin and help to exert its efficacy. Attached Figure Description

[0022] Figure 1 These are ultraviolet (UV) spectra of the supramolecular material and raw materials prepared in Example 1 of this invention.

[0023] Figure 2 These are DSC test images of the supramolecular material and raw materials prepared in Example 1 of this invention;

[0024] Figure 3 These are XRD patterns of the supramolecular material and raw materials prepared in Example 1 of this invention;

[0025] Figure 4 These are XRD patterns of the supramolecular material and raw materials prepared in Example 2 of this invention;

[0026] Figure 5 These are XRD patterns of the supramolecular material and raw materials prepared in Example 3 of this invention;

[0027] Figure 6 This is a comparison chart of 24-hour IVPT of the supramolecular protein prepared in Example 1 of this invention and ginsenoside CK.

[0028] Figure 7 This is a comparison chart of 24-hour IVPT of the supramolecular and aescin obtained in Example 1 of this invention;

[0029] Figure 8 This is the supramolecular particle size distribution obtained in Example 1 of this invention;

[0030] Figure 9 This is an electron micrograph of the supramolecular material prepared in Example 1 of this invention. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0033] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0034] Example 1

[0035] Accurately weigh 1g of aescin and place it in a clean beaker. Add 20mL of anhydrous ethanol and 180mL of ultrapure water sequentially. Stir magnetically at 500rpm for 5min to ensure uniform mixing. After stirring, transfer the mixture to an ultrasonic water bath and sonicate at 300W, 40kHz, and 35℃ for 25min. After sonication, transfer the system back to a magnetic stirrer and slowly add 0.5mL of a 100mg / mL ginsenoside CK-ethanol solution (containing 50mg ginsenoside CK). Stir magnetically for 1h to complete the supramolecular self-assembly process. Finally, dialyze the solution in a dialysis bag with a molecular weight of 10000 for 18h to remove anhydrous ethanol and unencapsulated free drug, yielding an aqueous solution of aescin-ginsenoside CK supramolecular solution.

[0036] Example 2

[0037] Weigh 0.5g of aescin and place it in a beaker. Add 63.4mL of anhydrous ethanol and 450mL of pure water. Stir magnetically at 500rpm for 5min to mix the system evenly. After stirring, transfer the mixture to an ultrasonic water bath and sonicate at 300W, 40kHz, and 35℃ for 10min. After sonication, transfer the drug mixture back to a magnetic stirrer and slowly add 1mL of a 50mg / mL ginsenoside CK-ethanol solution (containing 50mg ginsenoside CK). Stir for 1h to complete the preparation. Finally, dialyze the solution in a dialysis bag with a molecular weight of 10000 for 18h to remove anhydrous ethanol and unencapsulated free drug, thus obtaining aescin-ginsenoside CK supramolecular aqueous solution.

[0038] Example 3

[0039] Weigh 0.75g of aescin and place it in a beaker. Add 66.5mL of anhydrous ethanol and 22.5mL of pure water. Stir magnetically at 500rpm for 5min to mix the system evenly. After stirring, transfer the mixture to an ultrasonic water bath and sonicate at 300W, 40kHz, and 35℃ for 10min. After sonication, transfer the drug mixture back to a magnetic stirrer and slowly add 0.25mL of a 200mg / mL ginsenoside CK-ethanol solution (containing 50mg ginsenoside CK). Stir for 1h to complete the preparation. Finally, dialyze the solution in a dialysis bag with a molecular weight of 10000 for 18h to remove anhydrous ethanol and unencapsulated free drug, thus obtaining aescin-ginsenoside CK supramolecular aqueous solution.

[0040] Example 4

[0041] Unlike Example 1, in this example, the organic solvent used is DMSO.

[0042] Example 5

[0043] Unlike Example 1, in this example, the organic solvent used is hexanediol.

[0044] Example 6

[0045] Unlike Example 1, in this example, butanediol is used as the organic solvent.

[0046] Characterization results:

[0047] 1. The supramolecular structure of ginsenoside CK-aescinate obtained in Example 1 was subjected to ultraviolet (UV) spectrophotometry, as follows:

[0048] Instruments: UV-2600 Shimadzu UV spectrophotometer (Shimadzu, Japan); PR224ZH / E electronic balance (accuracy 0.01g, Ohaus Instruments Co., Ltd.);

[0049] Reagents: Anhydrous ethanol; pure water

[0050] Methods: Measurement method: absorbance; slit width: 2 nm; scanning range: 150 nm - 350 nm; scanning speed: medium.

[0051] Results Analysis: Ultraviolet (UV) absorption spectra analysis of ginsenoside CK, aescin, and the supramolecular ginsenoside CK-aescin formed by the two showed that: Figure 1 As shown, the maximum absorption peaks of the two raw materials correspond to 203 nm (ginsenoside CK) and 206 nm (aescinate), respectively. However, the maximum absorption peak of the supramolecular component blue-shifts to 192 nm. This wavelength change reflects that during supramolecular formation, the molecular aggregation state of aescin and ginsenoside CK may be disrupted due to non-covalent interactions (such as inclusion and hydrogen bonding), leading to an increased energy barrier and significantly enhanced absorption in the ultraviolet region. The difference in UV spectral characteristics provides spectroscopic evidence for the successful construction of the aescin-ginsenoside CK supramolecular component, proving that the two are not simply physically blended, but rather a newly formed supramolecular component.

[0052] 2. The supramolecular sample obtained in Example 1 was subjected to DSC testing. The test parameters were as follows: temperature 100~400℃, heating rate 10.0℃ / min, Ar 20.0mL / min. The test results are as follows. Figure 2 As shown, from Figure 2 It can be seen from this:

[0053] 1) Ginsenoside CK: It exhibits a strong endothermic peak at 243.58~252.69℃, corresponding to its characteristic phase transition process;

[0054] 2) Aescin: It exhibits a two-step endothermic behavior (196.33~202.36℃, 253.10~267.56℃), reflecting its own multi-step thermal change pattern;

[0055] 3) Ginsenoside CK-Aescin supramolecular: The supramolecular product formed by the two did not exhibit the characteristic endothermic peaks of the above-mentioned raw materials, and only a weak endothermic peak appeared at 197.51~202.29℃, with a significant reduction in the intensity of the thermal effect.

[0056] The results indicate that in the supramolecular product, ginsenoside CK and aescin bind through non-covalent interactions, altering the original molecular aggregation state and crystal structure of the active pharmaceutical ingredient, and thus exhibiting thermal behavior characteristics different from those of the active pharmaceutical ingredient.

[0057] 3. The supramolecules obtained in Examples 1, 2, and 3 were subjected to XRD tests, and the test parameters are as follows:

[0058] Voltage and current: 40kV, 40mA

[0059] Detector: DteX250(H)

[0060] Test range: 5-40°

[0061] Step size: 0.01°

[0062] Scan speed: 10.00 deg / min

[0063] Diverging slit: 10mm;

[0064] The test results are as follows:

[0065] like Figure 3 As shown, ginsenoside CK in Example 1 exhibits a distinct characteristic diffraction peak (2θ=15.23°), indicating a certain degree of crystallinity; aescin diffraction peak (2θ=14.40°) has extremely low intensity, showing an amorphous state; while the supramolecular product formed by the two has a significantly higher diffraction peak intensity than the two raw materials, and exhibits unique diffraction behavior (2θ=6.11°, 14.24°). This result indicates that after aescin and ginsenoside are combined, they do not form a physical mixture of the two raw materials, but rather construct a new crystal phase structure, and the degree of crystallinity of the supramolecular product is significantly improved compared to the raw materials. This is consistent with the conclusion of the DSC spectrum, further proving the successful construction of the supramolecular system.

[0066] like Figure 4As shown, in Example 2, ginsenoside CK exhibits a distinct characteristic diffraction peak (2θ=15.23°), indicating a certain degree of crystallinity; aescin's diffraction peak (2θ=14.40°) has extremely low intensity, showing an amorphous state; while the supramolecular product formed by the two has a significantly higher diffraction peak intensity than the two raw materials and exhibits unique diffraction behavior (2θ=14.108°). This result indicates that after aescin and ginsenoside combine, they do not form a physical mixture of the two raw materials, but rather construct a new crystal phase structure, and the degree of crystallinity of the supramolecular product is significantly improved compared to the raw materials. This corroborates the conclusions of the DSC spectrum, further proving the successful construction of the supramolecular system.

[0067] like Figure 5 As shown, ginsenoside CK in Example 2 exhibits a distinct characteristic diffraction peak (2θ=15.23°), indicating a certain degree of crystallinity; aescin's diffraction peak (2θ=14.40°) has extremely low intensity, showing an amorphous state; while the supramolecular product formed by the two has a significantly higher diffraction peak intensity than the two raw materials, and exhibits unique diffraction behavior (2θ=6.166°, 14.40°). Although the diffraction peak values ​​are the same as those of aescin, the peak shape at this point is significantly changed after the formation of the supramolecular product, becoming sharper and more intense. This result indicates that after aescin and ginsenoside combine, they do not form a physical mixture of the two raw materials, but rather construct a new crystal phase structure, and the crystallinity of the supramolecular product is significantly improved compared to the raw materials. This corroborates the conclusions of the DSC spectrum, further proving the successful construction of the supramolecular system.

[0068] 4. The supramolecular sample in Example 1 was subjected to IVPT testing. The specific method is as follows:

[0069] Pigskin model: Select abdominal or back skin from 1-3 month old miniature pigs (such as Bama miniature pigs);

[0070] Supply fluid preparation:

[0071] Aescin solution: Propylene glycol is used as the solvent for preparation. The concentration is consistent with that of the supramolecular sample. After preparation, it is dissolved by sonication. It can only be used after complete dissolution.

[0072] Ginsenoside CK solution: Propylene glycol is used as the solvent for preparation. The concentration is consistent with that of the supramolecular sample. After preparation, it is dissolved by sonication. It can be used only after complete dissolution.

[0073] Ginsenoside CK-Aescin supramolecular solution: Generally, no treatment is required and it can be directly loaded. If the concentration is too high, it needs to be diluted with pure water to a suitable concentration before use (to prevent the sample amount from being too large and causing the skin surface sample results to exceed the standard curve range).

[0074] Sample supply solution addition: Add 400 μL of each of the following supramolecular supply solutions: aescin, ginsenoside CK, and ginsenoside CK-aescin;

[0075] Internal sampling and analysis of pig skin: The pig skin and filter membrane that have been rinsed clean were placed in a centrifuge tube, the centrifuge tube was weighed on a balance and zeroed, a certain amount of 80% methanol was added to the centrifuge tube, the weight was recorded, and the sample was ultrasonically extracted for 1 hour before being injected for analysis.

[0076] The test results are shown in the table below:

[0077]

[0078] like Figure 6 As shown in the table above, using ginsenoside CK as the target compound and miniature pig skin as a model, it was found that the transdermal absorption of the supramolecular ginsenoside CK-aescin was superior to that of the raw ginsenoside CK.

[0079]

[0080] like Figure 7 As shown in the table above, using aescin as the target compound and miniature pig skin as a model, it was found that the transdermal absorption of ginsenoside CK-aescin supramolecular was superior to that of the raw aescin.

[0081] The above results indicate that, compared with the two raw materials, the transdermal absorption effect is significantly enhanced after the formation of supramolecular structures.

[0082] 5. The particle size distribution of the supramolecular sample prepared in Example 1 was determined as follows: 100 μL of ginsenoside CK-aescin supramolecular sample was added to a cuvette, followed by dilution with 1 mL of ultrapure water. The particle size distribution was then measured using an Anton Paar laser particle size distribution analyzer. Figure 8 As shown, the supramolecular particle size of ginsenoside CK-aescin is 178.28 nm, the PDI is 0.215, and the dispersibility is good.

[0083] 6. Transmission electron microscopy (TEM) analysis of the supramolecular sample obtained in Example 1: 10 μL of the ginsenoside CK-aescin supramolecular sample was dropped onto a grid, forming droplets that covered the grid surface. The droplets were allowed to stand for 5 minutes, excess moisture was absorbed with filter paper, and the sample was allowed to air dry. The morphology and size of the completely dried sample were observed under a TEM microscope. The results are as follows: Figure 9 As shown, TEM results indicate that the supramolecular sample of ginsenoside CK-aescin is well dispersed, with a structure exhibiting alternating light and dark areas, spherical particles, no obvious agglomeration, and consistent size and particle size distribution.

[0084] 7. The contents of ginsenoside CK and aescin in the supramolecular sample of Example 1 were tested as follows: The ginsenoside CK-aescin supramolecular sample was diluted 5 times, 200 mg of the test preparation was taken, 1 mL of methanol was added, the mixture was stirred and sonicated for 5 min, filtered through a 0.22 µm microporous membrane and added to a liquid chromatography vial. The contents of aescin and ginsenoside CK in the supramolecular sample were obtained by HPLC analysis.

[0085] The content of aescin in aescin is as follows:

[0086]

[0087] The ginsenoside CK content is as follows:

[0088]

[0089] By detecting the contents of ginsenoside CK and aescin in the supramolecular structure, the mass ratio of ginsenoside CK to aescin in the supramolecular structure was found to be approximately 1:19.4, which is consistent with the mass ratio of the two raw materials in Example 1 being 1:20.

[0090] The above are preferred embodiments of the present invention. Those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments described above. Any obvious improvements, substitutions or modifications made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. A supramolecular ginsenoside CK-aescinate, characterized in that: The ginsenoside CK-aescin supramolecular structure is formed by ginsenoside CK and aescin through non-covalent bonds, and the mass ratio of ginsenoside CK to aescin is 1:(1~25).

2. The ginsenoside CK-aescin supramolecular according to claim 1, characterized in that: The mass ratio of ginsenoside CK to aescin is 1:(10~20).

3. The ginsenoside CK-aescin supramolecular according to claim 1, characterized in that: In the UV spectrum of the ginsenoside CK-aescin supramolecular, there is a characteristic peak at 192 nm.

4. The ginsenoside CK-aescin supramolecular according to claim 2, characterized in that: In the XRD pattern of the ginsenoside CK-aescin supramolecular structure, when the mass ratio of ginsenoside CK to aescin is 1:10, a characteristic peak is observed at a 2θ angle of 14.108°; when the mass ratio of ginsenoside CK to aescin is 1:15, characteristic peaks are observed at 2θ angles of 6.11° and 14.24°; and when the mass ratio of ginsenoside CK to aescin is 1:20, characteristic peaks are observed at 2θ angles of 6.166° and 14.40°.

5. The ginsenoside CK-aescin supramolecular according to claim 1, characterized in that: In the TEM image of ginsenoside CK-aescin, the supramolecular form of ginsenoside CK-aescin is spherical particles.

6. A method for preparing the supramolecular ginsenoside CK-aescinate as described in claim 1, characterized in that: The steps are as follows: S1. Disperse aescin in an aqueous solution of an organic solvent with a mass concentration of 8%~30% to prepare a suspension A with a mass concentration of 0.1%~1%, and dissolve ginsenoside CK in ethanol to prepare a solution D with a mass concentration of 5%-30% for later use. S2. Stir suspension A for 3-10 minutes to obtain suspension B; S3. Sonicate suspension B for 10-60 min to obtain emulsion C; S4. Slowly add emulsion C to solution D under stirring, and stir for 45 min to 120 min to obtain crude product of ginsenoside CK-aescin supramolecular solution, wherein the mass ratio of ginsenoside CK to aescin is 1:(1~25). S5. Dialyze the crude ginsenoside CK-aescin supramolecular solution in a dialysis bag for 12-24 hours to obtain an aqueous solution of ginsenoside CK-aescin supramolecular solution.

7. The method for preparing ginsenoside CK-aescinate supramolecular according to claim 6, characterized in that: The organic solvent in step S1 is at least one of ethanol, DMSO, hexanediol, and butanediol.

8. The method for preparing ginsenoside CK-aescinate supramolecular according to claim 6, characterized in that: In steps S2 and S4, magnetic stirring is used at a speed of 300 rpm to 1000 rpm.

9. The method for preparing ginsenoside CK-aescinate supramolecular according to claim 6, characterized in that: In step S3, the frequency of the ultrasound is 200W~600W, and the temperature of the ultrasound is 25℃~50℃.

10. The method for preparing ginsenoside CK-aescinate supramolecular according to claim 6, characterized in that: In step S5, the molecular weight of the dialysis bag is 8000-14000.

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