Whitening and spot-fading composition based on molecular docking technology and preparation method therefor

Abstract

A whitening and spot-fading composition based on molecular docking technology and a preparation method therefor. The composition comprises atractylenolide I, pachymic acid, paeoniflorin and glabridin, wherein the mass ratio of the atractylenolide I, pachymic acid, paeoniflorin and glabridin is 50-600:100-700:600-2000:10-200. On the basis of the docking binding energy of active ingredients in four plants Atractylodes macrocephala, Paeonia lactiflora, poria cocos and licorice to a key target molecule of a disease, it is found by screening that compounding the atractylenolide I, pachymic acid, paeoniflorin and glabridin as active ingredients can obtain the composition having a remarkable whitening and spot-fading effect, the melanin synthesis inhibition rate of the composition reaches 69.4%-83.6%, and the tyrosinase activity inhibition rate of the composition reaches 76.8%-91.3%.

Description

A skin-whitening and freckle-removing composition based on molecular docking technology and its preparation method Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a whitening and freckle-removing composition based on molecular docking technology and its preparation method. Background Technology

[0002] Most East Asians are of yellow skin, and women generally desire fairer skin. Therefore, cosmetics with whitening and spot-removing effects are popular among women, and the demand for such products is constantly increasing. Traditional ingredients for treating skin pigmentation, such as hydroquinone, mercuric chloride, kojic acid, arbutin, and vitamin C, achieve whitening and spot-removing effects by inhibiting or interfering with melanin production. However, these ingredients have problems such as instability, skin irritation, and adverse reactions like contact dermatitis. In recent years, more and more natural plant active ingredients have been added to cosmetics as whitening ingredients. However, plant extracts have issues such as unclear active ingredients and lack of understanding of their mechanisms of action.

[0003] Atractylodes macrocephala, the dried rhizome of the plant Atractylodes macrocephala Koidz. (family Asteraceae), has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. Atractylodes macrocephala mainly contains volatile components, polysaccharides, polyphenols, and lactones, among other active ingredients. Studies have shown that the chemical components in Atractylodes macrocephala have an inhibitory effect on tyrosinase activity.

[0004] Poria cocos (Schw.) Wolf, a fungus belonging to the Polyporaceae family, is a dried sclerotium with diuretic, spleen-strengthening, and heart-calming effects. Poria cocos mainly contains polysaccharides and triterpenoids, among other active ingredients. The total triterpenoids in Poria cocos exhibit a significant inhibitory effect on tyrosinase and can also inhibit the growth of mouse melanoma cells.

[0005] White peony root is the dried root of Paeonia lactiflora Pall., a plant in the Ranunculaceae family. It has the effects of nourishing blood and regulating menstruation, astringing yin and stopping sweating, soothing the liver and relieving pain, and calming liver yang. The main chemical components of white peony root are monoterpenes and monoterpene glycosides, as well as triterpenes, tannins, polysaccharides, and flavonoids. Studies have shown that the crude extract of white peony root has a good inhibitory effect on tyrosinase.

[0006] Licorice is the dried root and rhizome of *Glycyrrhiza uralensis* Fisch., *Glycyrrhiza inflata* Bat., or *Glycyrrhiza glabra* L., all belonging to the legume family. It possesses various medicinal properties, including tonifying the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, alleviating spasms and pain, and harmonizing other herbs. The main components of licorice include triterpenoids, flavonoids, and alkaloids. Studies have shown that flavonoids such as glycyrrhizin and glycyrrhizin have good inhibitory effects on tyrosinase activity.

[0007] Modern biology suggests that skin pigmentation is caused by excessive melanin synthesis. A key step in melanin synthesis involves the regulation of the substrate L-tyrosine in the SCF-KIT pathway. By acting on estrogen receptor 1 (ESR1) and progesterone receptor (PGR) targets, L-tyrosine regulates the estrogen pathway, thereby modulating the SCF-KIT signaling pathway to reduce TYR synthesis, lower TYR concentration, and inhibit melanin synthesis. Therefore, effective whitening and spot-removing ingredients can be screened by utilizing three key targets: TYR, ESR1, and PGR.

[0008] Therefore, given the problems of unclear active ingredients and lack of mechanism of action in plant extracts, it is necessary to conduct research to identify the active ingredients in plants that have whitening and spot-removing effects using molecular docking technology, and to provide a preparation method and application of plant extracts with whitening effects based on clear indicators of active ingredients. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the purpose of this invention is to provide a whitening and freckle-removing composition based on molecular docking technology and its preparation method.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention provides a skin whitening and freckle-removing composition based on molecular docking technology, comprising atractylodes lactone I, pachymic acid, paeoniflorin, and glycyrrhizin; wherein the mass ratio of atractylodes lactone I, pachymic acid, paeoniflorin, and glycyrrhizin is 50-600:100-700:600-2000:10-200.

[0012] Preferably, the mass ratio of atractylodes lactone I, pamoatenic acid, paeoniflorin, and glycyrrhizin is 100-500:100-500:800-1800:10-120.

[0013] More preferably, the mass ratio of atractylodes lactone I, pamoateic acid, paeoniflorin, and glycyrrhizin is 300:400:1000:100.

[0014] Preferably, the preparation steps of the atractylodes lactone I are as follows:

[0015] A1. Weigh out Atractylodes macrocephala, add 8-12 times the amount of 75-85wt.% ethanol, soak and extract twice at 45-55℃, combine the filtrates and concentrate, then dilute with water;

[0016] A2. Load the diluted solution prepared in step A1 onto the macroporous resin, elute with 25-35 wt.% ethanol and water respectively, and discard the eluent; then elute with 75-85 wt.% ethanol, collect the eluent, concentrate and dry it;

[0017] A3. The dried material collected in step A2 was separated by silica gel column chromatography using a silica gel dry mixing method. Gradient elution was performed with a mixed solution of petroleum ether and ethyl acetate. The fraction containing atractylodes lactone I was concentrated, left to stand, and after white crystals precipitated, filtered and dried to obtain atractylodes lactone I extract. The HPLC content of atractylodes lactone I in the obtained atractylodes lactone I extract was above 80%.

[0018] Preferably, the preparation steps of the pachymic acid are as follows:

[0019] B1. Weigh out Poria cocos, add 18-22 times the amount of 75-85wt.% ethanol, reflux extract twice, combine the filtrates and concentrate, then dilute with water.

[0020] B2. Load the diluted solution prepared in step B1 onto the macroporous resin, elute with 25-35 wt.% ethanol and water respectively, and discard the eluent; then elute with 75-85 wt.% ethanol, collect the eluent, concentrate and dry it;

[0021] B3. The dried material collected in step B2 was separated by silica gel column chromatography using a silica gel dry mixing method. Gradient elution was performed with a mixed solution of dichloromethane and methanol. The fraction containing pachymic acid was concentrated, allowed to stand, and after white crystals precipitated, it was filtered and dried to obtain the pachymic acid extract. The HPLC content of pachymic acid in the obtained pachymic acid extract was above 80%.

[0022] Preferably, the preparation steps of paeoniflorin are as follows:

[0023] C1. Weigh out white peony root, add 8-12 times the amount of 65-75 wt.% ethanol, reflux extract twice, combine the filtrates and concentrate, then dilute with water.

[0024] C2. Load the diluted solution prepared in step C1 onto the macroporous resin, elute with water, and discard the eluent; then elute with 55-65 wt.% ethanol, collect the eluent, concentrate and dry it;

[0025] C3. The dried material collected in step C2 was separated by silica gel column chromatography using a silica gel dry mixing method. Gradient elution was performed with a mixed solution of dichloromethane and methanol. The fraction containing paeoniflorin was concentrated, allowed to stand, and after white crystals precipitated, it was filtered and dried to obtain the paeoniflorin extract. The HPLC content of paeoniflorin in the obtained paeoniflorin extract was above 80%.

[0026] Preferably, the preparation steps of the glycyrrhizin are as follows:

[0027] D1. Weigh out licorice, add 18-22 times the amount of 75-85wt.% ethanol, reflux extract twice, combine the filtrates and concentrate, then dilute with water.

[0028] D2. Load the diluted solution prepared in step D1 onto the macroporous resin, elute with 25-35 wt.% ethanol and water respectively, and discard the eluent; then elute with 75-85 wt.% ethanol, collect the eluent, concentrate and dry it;

[0029] D3. The dried material collected in step D2 was separated by silica gel dry mixing and silica gel column chromatography. Gradient elution was performed with a mixed solution of petroleum ether and ethyl acetate. The fraction containing glycyrrhizin was concentrated, left to stand, and after white crystals precipitated, filtered and dried to obtain glycyrrhizin extract. The HPLC content of glycyrrhizin in the obtained glycyrrhizin extract was more than 80%.

[0030] Preferably, in steps A1, B1, C1, and D1, the extraction time for each step is 1.5-2.5 hours.

[0031] Preferably, in steps A2, B2, C2, and D2, the flow rate of each diluent loading is 0.4-0.6 BV / h.

[0032] Preferably, in steps A2, B2, C2, and D2, the volume of elution with 25-35 wt.% ethanol is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h; the volume of elution with water is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h.

[0033] Preferably, in steps A2 and D2, the volume of elution using 75-85 wt.% ethanol is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h.

[0034] Preferably, in steps B2 and C2, the volume of elution using 55-65 wt.% ethanol is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h.

[0035] Preferably, in step A3, when gradient elution is performed using a mixed solution of petroleum ether and ethyl acetate, the mixing ratio of petroleum ether and ethyl acetate is 75:1, 50:1, and 25:1, respectively.

[0036] Preferably, in steps B3 and C3, when gradient elution is performed using a mixed solution of dichloromethane and methanol, the mixing ratio of dichloromethane and methanol is 50:1, 20:1, and 10:1, respectively.

[0037] Preferably, in step D3, when gradient elution is performed using a mixed solution of petroleum ether and ethyl acetate, the mixing ratio of petroleum ether and ethyl acetate is 5:1, 1:1, and 1:2, respectively.

[0038] Secondly, the present invention provides a method for preparing a whitening and freckle-removing composition based on molecular docking technology, wherein the components are mixed in a weight ratio to obtain the composition.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1) This invention screens the active ingredients of four plants—Atractylodes macrocephala, Paeonia lactiflora, Poria cocos, and Glycyrrhiza uralensis—and their binding energies to the core target molecules of diseases. It was found that when the active ingredients are Atractylodes macrocephala lactone I, pachymic acid, paeoniflorin, and glycyrrhizin, a composition with significant whitening and spot-removing effects can be obtained. The melanin synthesis inhibition rate reaches 69.4%-83.6%, and the tyrosinase activity inhibition rate reaches 76.8%-91.3%.

[0041] 2) This invention further enhances the whitening and spot-removing effects by specifically selecting atractylodes lactone I, pamoatein, paeoniflorin, and glycyrrhiza glabra in a mass ratio of 100-500:100-500:800-1800:10-120, with a melanin synthesis inhibition rate of 76.4%-83.6% and a tyrosinase activity inhibition rate of 86.8%-91.3%. Detailed Implementation

[0042] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0043] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0044] It should be understood that, except in any operational instance or otherwise indicated, all figures representing the amounts of ingredients used, for example, in the specification and claims, should be understood to be modified in all cases by the term "about". Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximations varying with the desired performance to be obtained according to the invention. It is not at all an attempt to limit the application of the doctrine of equivalents to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0045] Although the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​listed in the specific examples are reported as precisely as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation found in their respective test measurements.

[0046] In a specific embodiment of the present invention, the atractylenolide I, with the English name Atractylenolide I, CAS: 73069-13-3, and molecular formula C0.05, has the molecular formula C0.05. 15 H 18 O2, molecular weight 230.30, molecular structure formula is as follows:

[0047] Atractylodes lactone I is a lactone component of Atractylodes macrocephala, which has strong functions such as enhancing salivary amylase activity, promoting intestinal absorption, regulating the intestines, and inhibiting tumor growth. In a specific embodiment of the present invention, the atractylodes lactone I used can be obtained by extracting Atractylodes macrocephala, or it can be obtained by purchasing a commercially available product (purchased from Shanghai Shidande Standard Technical Service Co., Ltd., product name: Atractylodes lactone I, CAS No.: 73069-13-3). The present invention does not impose a specific limitation on its source.

[0048] In a specific embodiment of the present invention, the paeoniflorin, CAS: 23180-57-6, has the molecular formula Cp. 23 H 28 O 11 With a molecular weight of 480.46, its molecular structure is as follows:

[0049] Paeoniflorin is an important physiologically active substance with anti-inflammatory, immune system-enhancing, hepatoprotective, and analgesic effects. In the specific embodiments of this invention, the paeoniflorin used can be obtained by extracting from white peony root, or by purchasing commercially available products (purchased from Shanghai Shidande Standard Technology Service Co., Ltd., product name: paeoniflorin, CAS No.: 23180-57-6). This invention does not impose specific limitations on its source.

[0050] In a specific embodiment of the present invention, the pachymic acid, CAS: 29070-92-6, has the molecular formula C2. 33 H 52 O5, molecular weight 528.76, molecular structure formula is as follows:

[0051] Pachymic acid is a triterpenoid compound naturally found in traditional Chinese medicines such as Ganoderma lucidum and Poria cocos. It has a significant inhibitory effect on the invasion and proliferation of tumor cells such as lung cancer, pancreatic cancer, colon cancer, prostate cancer, and breast cancer. In the specific embodiments of this invention, the pachymic acid used can be obtained by extracting from Poria cocos or by purchasing commercially available products (purchased from Shanghai Shidande Standard Technology Service Co., Ltd., product name: pachymic acid, CAS number: 29070-92-6). This invention does not impose specific limitations on its source.

[0052] In a specific embodiment of the present invention, the glycyrrhizin, known in English as Glabridin, CAS: 59870-68-7, has the molecular formula C0. 20 H 20 O4, with a molecular weight of 324.37, has the following molecular structure:

[0053] Glycyrrhizin possesses immunomodulatory, lipid-lowering, blood glucose-lowering, estrogen-like, antibacterial, antioxidant, and anti-inflammatory effects. It exhibits strong anti-free radical oxidation activity in the cytochrome P450 / NADPH oxidation system, significantly inhibiting free radicals generated during metabolism to prevent oxidative damage to oxidatively sensitive biomolecules (low-density lipoprotein, DNA) and cell walls, thereby preventing certain pathological changes related to free radical oxidation. In specific embodiments of this invention, the glabridin used can be obtained through extraction from licorice or by purchasing commercially available products (purchased from Shanghai Shidande Standard Technology Service Co., Ltd., product name: glabridin, CAS No.: 59870-68-7). This invention does not specifically limit its source.

[0054] In the following examples, all other raw materials and reagents used are commercially available products.

[0055] Example 1

[0056] Literature searches were conducted using Atractylodes macrocephala, Paeonia lactiflora, Poria cocos, and Glycyrrhiza uralensis as keywords to identify the chemical components contained in each plant. Targets with well-defined mechanisms related to melanin synthesis, estrogen signaling pathways, and tyrosine metabolism, particularly relevant to skin whitening and freckle removal, were selected for screening. Molecular docking was performed using three key proteins: TYR, ESR1, and PGR. The specific steps are as follows:

[0057] (1) Protein structure processing: PyMol software was used to perform preprocessing operations on the three key proteins TYR, ESR1, and PGR, including removing small molecules and completing amino acid residues.

[0058] (2) Receptor-ligand pretreatment: Using AutoDock Tools software, the treated protein was pretreated by removing water molecules, adding hydrogen, adding a force field, and distributing charges, and then selected as a receptor to export a pdbqt file; the chemical components were pretreated by adding hydrogen, selecting as ligands, detecting ligand centers and spin bonds, and then exported as pdbqt files.

[0059] (3) Molecular docking: Using AutoDock Tools software, import the receptor and ligand files, set the active pocket parameters, and run AutoDock4 to perform molecular docking; the binding energy between molecules reflects the affinity between the ligand and the receptor. The lower the binding energy, the greater the docking activity and the more stable the structure.

[0060] The specific binding energies of the active components to the targets are shown in Table 1. As can be seen from Table 1, the binding energies of the active components to the targets are all <-1.2 kcal / mol, and hydrogen bond interactions exist between them and the proteins of the three target sites.

[0061] Table 1. Binding energy (kcal / mol) between active ingredients and core disease target molecules

[0062] Example 2

[0063] Based on the results of Example 1, a combination of the active ingredients atractylodes lactone I, pamoateic acid, paeoniflorin, and glycyrrhizin was selected as the test substance. The preparation steps of each active ingredient are as follows:

[0064] 1. Preparation of Atractylodes lactone I extract

[0065] 1.1 Weigh 250g of Atractylodes macrocephala, add 2500ml of 80% ethanol, and extract at 50℃ for 2 hours each time, for 2 extractions; combine the two filtrates, concentrate until there is no alcohol odor, and dilute with purified water to 1000ml for later use;

[0066] 1.2 Take 250g of the treated macroporous resin (D101) and fill it into the resin (column volume BV is 300ml); take the extract obtained in step 1.1 and load it at a flow rate of 0.5 BV / h; then elute with 3 BV of 30% ethanol and 3 BV of purified water at a flow rate of 1.0 BV / h, respectively, and discard the eluent; then elute with 3 BV of 80% ethanol at a flow rate of 1.0 BV / h, collect the eluent, concentrate and dry it;

[0067] 1.3 The dried product obtained in step 1.2 was separated by silica gel dry mixing and silica gel (200-300 mesh) column chromatography. Different ratios of petroleum ether-ethyl acetate (75:1, 50:1, 25:1) were used for gradient elution. When the ratio of petroleum ether to ethyl acetate was 25:1, the resulting fraction was detected by thin-layer chromatography.

[0068] 1.4 Combine the fraction containing atractylodes lactone I detected by thin-layer chromatography in step 1.3, concentrate, let stand, precipitate white crystals, filter, dry, and obtain the atractylodes lactone I extract. Its content was determined to be 83.5% by HPLC.

[0069] 2. Preparation of paeoniflorin extract

[0070] 2.1 Weigh 250g of white peony root, add 2500ml of 70% ethanol, reflux for extraction, 2 hours each time, extract twice; combine the two filtrates, concentrate until there is no alcohol odor, dilute with purified water to 1000ml, and set aside;

[0071] 2.2 Take 250g of the treated macroporous resin (AB-8) and fill it into the resin (column volume BV is 300ml); take the extract obtained in step 2.1 and load it at a flow rate of 0.5 BV / h; elute with 3 BV of purified water at a flow rate of 1.0 BV / h, and discard the eluent; then elute with 3 BV of 60% ethanol at a flow rate of 1.0 BV / h, collect the eluent, concentrate and dry it;

[0072] 2.3 The dried product obtained in step 2.2 was separated by silica gel dry mixing and silica gel (200-300 mesh) column chromatography. Different ratios of dichloromethane-methanol (50:1, 20:1, 10:1) were used for gradient elution. When dichloromethane:methanol = 10:1 elution, the resulting fraction was detected by thin-layer chromatography.

[0073] 2.4 Combine the fraction containing paeoniflorin detected by thin-layer chromatography in step 2.3, concentrate, let stand, precipitate white crystals, filter, dry, and obtain paeoniflorin extract. Its content is determined by HPLC to be 86.9%.

[0074] 3. Preparation of Poria cocos acid extract

[0075] 3.1 Weigh 250g of Poria cocos, add 5000ml of 80% ethanol, reflux for extraction, 2 hours each time, extract twice; combine the two filtrates, concentrate until there is no alcohol taste, dilute with purified water to 1000ml, and set aside;

[0076] 3.2 Take 250g of the treated macroporous resin (HPD100) and fill it into the resin (column volume BV is 300ml); take the extract obtained in step 3.1 and load it at a flow rate of 0.5 BV / h; then elute with 3 BV of 30% ethanol and 3 BV of purified water at a flow rate of 1.0 BV / h, respectively, and discard the eluent; then elute with 3 BV of 80% ethanol at a flow rate of 1.0 BV / h, collect the eluent, concentrate and dry it;

[0077] 3.3 The dried product obtained in step 3.2 was separated by silica gel dry mixing and silica gel (200-300 mesh) column chromatography. Different ratios of dichloromethane-methanol (50:1, 20:1, 10:1) were used for gradient elution. When dichloromethane:methanol = 10:1 elution, the obtained fraction was detected by thin-layer chromatography.

[0078] 3.4 Combine the fraction containing pachymic acid detected by thin-layer chromatography in step 3.3, concentrate, let stand, precipitate white crystals, filter, dry, and obtain pachymic acid extract. Its content is determined by HPLC to be 84.3%.

[0079] 4. Preparation of glycyrrhizin extract

[0080] 4.1 Weigh 250g of licorice, add 5000ml of 80% ethanol, reflux and extract, 2h each time, extract twice; combine the two filtrates, concentrate until there is no alcohol taste, dilute with purified water to 1000ml, and set aside;

[0081] 4.2 Take 250g of the treated macroporous resin (D101) and fill it into the resin (column volume BV is 300ml); take the extract obtained in step 4.1 and load it at a flow rate of 0.5 BV / h; then elute with 3 BV of 30% ethanol and 3 BV of purified water at a flow rate of 1.0 BV / h, respectively, and discard the eluent; then elute with 3 BV of 80% ethanol at a flow rate of 1.0 BV / h, collect the eluent, concentrate and dry it;

[0082] 4.3 The dried product obtained in step 4.2 was separated by silica gel dry mixing and silica gel (200-300 mesh) column chromatography. Different ratios of petroleum ether-ethyl acetate (5:1, 1:1, 1:2) were used for elution. When the ratio of petroleum ether to ethyl acetate was 1:2, the resulting fraction was detected by thin-layer chromatography.

[0083] 4.4 Combine the fraction containing glycyrrhizin detected by thin-layer chromatography in step 4.3, concentrate, let stand, precipitate white crystals, filter, dry, and obtain glycyrrhizin extract. Its content is determined by HPLC to be 81.6%.

[0084] Example 3

[0085] This embodiment provides a skin-whitening and freckle-removing composition based on molecular docking technology. The atractylodes lactone I extract, paeoniflorin extract, pamoate extract, and glycyrrhizin extract, prepared according to the method of Example 2, are formulated into corresponding compositions using ethanol (or polyols such as butylene glycol, propylene glycol, or dipropylene glycol can be used instead) as the solvent, according to the concentrations of the active ingredients shown in Table 2. For example, taking atractylodes lactone I in experimental group 1 as an example, the HPLC content of the atractylodes lactone extract prepared according to Example 2 is 83.5%, and the concentration of atractylodes lactone I extract corresponding to a concentration of 300 ug / ml is 351.7 ug / ml. The composition is then prepared by mixing the atractylodes lactone I extract concentration with ethanol.

[0086] Table 2

[0087] Example 4

[0088] This embodiment provides a skin whitening and freckle-removing composition based on molecular docking technology (experimental group 12), which is prepared by adding commercially available atractylodes lactone I, paeoniflorin, pamoatenic acid, and glycyrrhizin to a solvent (ethanol is used in this embodiment) at the concentrations of experimental group 1 in Table 1 above.

[0089] Comparative Example 1

[0090] This comparative example provides a whitening and freckle-removing composition based on molecular docking technology (experimental group 13), which is basically the same as the composition of Example 4 (experimental group 12) and the preparation method, except that commercially available glycyrrhizic acid (purchased from Shanghai Shidande Standard Technical Service Co., Ltd., product name: glycyrrhizic acid, CAS number: 1405-86-3) is used in an equal amount to replace glycyrrhizin.

[0091] Comparative Example 2

[0092] This comparative example provides a whitening and freckle-removing composition based on molecular docking technology (experimental group 14), which is basically the same as the composition of Example 4 (experimental group 12) and the preparation method, except that: commercially available paeoniflorin (purchased from Shanghai Shidande Standard Technical Service Co., Ltd., product name: paeoniflorin, CAS number: 39011-90-0) is used in an equal amount to replace paeoniflorin.

[0093] Comparative Example 3

[0094] This comparative example provides a whitening and freckle-removing composition based on molecular docking technology (experimental group 15), which is basically the same as the composition and preparation method of experimental group 9 in Example 3, except that commercially available atractylodes lactone II (purchased from Shanghai Shidande Standard Technical Service Co., Ltd., product name: atractylodes lactone II, CAS number: 73069-14-4) is used in an equal amount to replace atractylodes lactone I.

[0095] Evaluation experiment on the whitening and spot-removing efficacy of plant extract compositions:

[0096] (1) Sample preparation

[0097] The compositions prepared in Examples 3-4 and Comparative Examples 1-3 were used as test samples (Experimental Groups 1-15).

[0098] (2) Cell Culture

[0099] B16 cells were cultured in RPMI-1640 complete medium (containing 10% fetal bovine serum) at 37°C under 5% CO2 (the CO2 concentration in the incubator air was 5%).

[0100] (3) Evaluation test of the effect of inhibiting tyrosinase activity

[0101] B16 cells in the exponential growth phase (purchased from Beina Chuanglian Biotechnology Co., Ltd.) were digested and dispersed to prepare a single-cell suspension. The cell density was adjusted to 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 12 hours until complete cell adhesion. The supernatant was then discarded. PBS solution containing each test sample (final concentration of each test sample was 5%, serving as the test group) or an equal volume of PBS solution (serving as the control group) was added to each well, with three replicates. After culturing for 72 hours, the supernatant was discarded, and the cells were washed twice with DPBS. 100 μl of DPBS buffer containing 0.5% Triton X-100 was added to each well, and the plates were immediately placed at -80°C for 2 hours. The plates were then thawed at room temperature and incubated at 37°C for 5 minutes. A blank control group (100 μl of DPBS buffer containing 0.5% Triton X-100) was also set up. After incubation in each group, 50 μL of 10 mmol / L L-DOPA was added, and the reaction was carried out at 37°C for 2 hours. The absorbance at 475 nm was measured using a microplate reader. Enzyme activity is calculated using the following formula: Tyrosinase activity inhibition rate = (OD value of control group - OD value of test group) / (OD value of control group - OD value of blank group) × 100%.

[0102] (4) Tests to inhibit melanin synthesis in melanocytes

[0103] Take B16 cells in the exponential growth phase and adjust the cell density to 2 × 10⁶ cells per well. 5Cells were seeded in 6-well plates. After 12 hours, the supernatant was discarded, and PBS solution containing each test sample (final concentration of each test sample was 5%, as the test group) or an equal volume of PBS solution (as the control group) was added. Three replicates were set up. After 72 hours of culture, the cells were washed twice with DPBS, digested, and collected in centrifuge tubes. The cells were centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded. 2 ml of DPBS was added, and the cells were gently pipetted to prepare a cell suspension (20 μl of which was diluted to a certain factor for cell counting). 500 μl of ethanol-ether mixture (volume ratio 1:1) was added, and the suspension was incubated at room temperature for 30 minutes. The cells were then centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. 1 ml of 1 mol / L NaOH solution containing 10% DMSO was added, and the mixture was carefully mixed. The cells were incubated in an 80°C water bath for 45 minutes, and the absorbance at 405 nm was measured. Melanin synthesis inhibition rate = (OD value of test group / corresponding number of cells) / (OD value of control group / corresponding number of cells) × 100%. The results are shown in Table 3.

[0104] Table 3 Note: Compared with experimental group 1, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0105] The experimental results in Table 3 show that the compositions prepared in the embodiments of the present invention (experimental groups 1-9 and experimental group 12) have significant inhibitory effects on tyrosinase activity and melanin synthesis, with melanin synthesis inhibition rates of 69.4%-83.6% and tyrosinase activity inhibition rates of 76.8%-91.3%.

[0106] The results from experimental groups 1-5 and experimental group 12 show that by specifically selecting the content ratio of atractylodes lactone I, paeoniflorin, pamoate, and glycyrrhizin at 100-500:100-500:800-1800:10-120, the efficacy in inhibiting tyrosinase activity and melanin synthesis can be further improved, with melanin synthesis inhibition rate reaching 76.4%-83.6% and tyrosinase activity inhibition rate reaching 86.8%-91.3%. However, a comparison of the results from experimental groups 4 with experimental groups 6 and 8, and experimental groups 5 with experimental group 7, shows that when the content ratios in experimental groups 6 and 8, and experimental group 7, are not within the aforementioned ranges, the melanin synthesis inhibition rate and tyrosinase activity inhibition rate decrease compared to experimental groups 4 or 5.

[0107] A comparison of the results of experimental groups 10-11 with those of experimental group 1 shows that the absence of two active ingredients led to a significant decrease in the melanin synthesis inhibition rate and the tyrosinase activity inhibition rate (p<0.001 compared to experimental group 1). A comparison of the results of experimental groups 13-14 with those of experimental group 12, and of experimental group 15 with those of experimental group 9, shows that replacing the active ingredients with glycyrrhizic acid instead of glabridin, paeoniflorin instead of paeoniflorin, or atractylodes lactone II instead of atractylodes lactone I, all resulted in a significant decrease in the melanin synthesis inhibition rate and the tyrosinase activity inhibition rate of the prepared composition. This demonstrates that the present invention, by specifically selecting and combining atractylodes lactone I, paeoniflorin, pamoatenic acid, and glabridin, can achieve a significant inhibition of tyrosinase activity and melanin synthesis, thus possessing excellent whitening and freckle-removing effects.

[0108] Furthermore, a comparison of the results from experimental groups 1 and 12 shows that whether the active ingredient extracts with an HPLC content of over 80% were obtained using the method of Example 2 for formulation, or the active ingredient raw materials were directly purchased, the effects were comparable. This demonstrates that the active ingredients responsible for the whitening and freckle-removing effects of this invention are atractylodes lactone I, paeoniflorin, pamoatenic acid, and glycyrrhizin.

[0109] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A skin-whitening and freckle-removing composition based on molecular docking technology, characterized in that, It includes atractylodes lactone I, pamoate, paeoniflorin, and glycyrrhizin; the mass ratio of atractylodes lactone I, pamoate, paeoniflorin, and glycyrrhizin is 50-600:100-700:600-2000:10-200.

2. The whitening and freckle-removing composition based on molecular docking technology according to claim 1, characterized in that, The mass ratio of atractylodes lactone I, pamoate, paeoniflorin, and glycyrrhizin is 100-500:100-500:800-1800:10-120.

3. The whitening and freckle-removing composition based on molecular docking technology according to claim 1, characterized in that, The preparation steps of the atractylodes lactone I are as follows: A1. Weigh out Atractylodes macrocephala, add 8-12 times the amount of 75-85wt.% ethanol, soak and extract twice at 45-55℃, combine the filtrates and concentrate, then dilute with water; A2. Load the diluted solution prepared in step A1 onto the macroporous resin, elute with 25-35 wt.% ethanol and water respectively, and discard the eluent; then elute with 75-85 wt.% ethanol, collect the eluent, concentrate and dry it; A3. The dried material collected in step A2 was separated by silica gel column chromatography using a silica gel dry mixing method. Gradient elution was performed with a mixed solution of petroleum ether and ethyl acetate. The fraction containing atractylodes lactone I was concentrated, left to stand, and after white crystals precipitated, filtered and dried to obtain atractylodes lactone I extract. The HPLC content of atractylodes lactone I in the obtained atractylodes lactone I extract was above 80%.

4. The whitening and freckle-removing composition based on molecular docking technology according to claim 1, characterized in that, The preparation steps of the poria cocos acid are as follows: B1. Weigh out Poria cocos, add 18-22 times the amount of 75-85wt.% ethanol, reflux extract twice, combine the filtrates and concentrate, then dilute with water. B2. Load the diluted solution prepared in step B1 onto the macroporous resin, elute with 25-35 wt.% ethanol and water respectively, and discard the eluent; then elute with 75-85 wt.% ethanol, collect the eluent, concentrate and dry it; B3. The dried material collected in step B2 was separated by silica gel column chromatography using a silica gel dry mixing method. Gradient elution was performed with a mixed solution of dichloromethane and methanol. The fraction containing pachymic acid was concentrated, allowed to stand, and after white crystals precipitated, it was filtered and dried to obtain the pachymic acid extract. The HPLC content of pachymic acid in the obtained pachymic acid extract was above 80%.

5. The whitening and freckle-removing composition based on molecular docking technology according to claim 1, characterized in that, The preparation steps of paeoniflorin are as follows: C1. Weigh out white peony root, add 8-12 times the amount of 65-75 wt.% ethanol, reflux extract twice, combine the filtrates and concentrate, then dilute with water. C2. Load the diluted solution prepared in step C1 onto the macroporous resin, elute with water, and discard the eluent; then elute with 55-65 wt.% ethanol, collect the eluent, concentrate and dry it; C3. The dried material collected in step C2 was separated by silica gel column chromatography using a silica gel dry mixing method. Gradient elution was performed with a mixed solution of dichloromethane and methanol. The fraction containing paeoniflorin was concentrated, allowed to stand, and after white crystals precipitated, it was filtered and dried to obtain the paeoniflorin extract. The HPLC content of paeoniflorin in the obtained paeoniflorin extract was above 80%.

6. The whitening and freckle-removing composition based on molecular docking technology according to claim 1, characterized in that, The preparation steps of the glycyrrhizin are as follows: D1. Weigh out licorice, add 18-22 times the amount of 75-85wt.% ethanol, reflux extract twice, combine the filtrates and concentrate, then dilute with water. D2. Load the diluted solution prepared in step D1 onto the macroporous resin, elute with 25-35 wt.% ethanol and water respectively, and discard the eluent; then elute with 75-85 wt.% ethanol, collect the eluent, concentrate and dry it; D3. The dried material collected in step D2 was separated by silica gel dry mixing and silica gel column chromatography. Gradient elution was performed with a mixed solution of petroleum ether and ethyl acetate. The fraction containing glycyrrhizin was concentrated, left to stand, and after white crystals precipitated, filtered and dried to obtain glycyrrhizin extract. The HPLC content of glycyrrhizin in the obtained glycyrrhizin extract was more than 80%.

7. The whitening and freckle-removing composition based on molecular docking technology according to any one of claims 3-6, characterized in that, In steps A2, B2, C2, and D2, the flow rate for loading each diluent is 0.4-0.6 BV / h.

8. The whitening and freckle-removing composition based on molecular docking technology according to any one of claims 3-6, characterized in that, In steps A2, B2, C2, and D2, the volume of elution with 25-35 wt.% ethanol is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h; the volume of elution with water is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h. In steps A2 and D2, the elution volume of 75-85 wt.% ethanol is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h. In steps B2 and C2, the elution volume of 55-65 wt.% ethanol is 2.5-3.5 BV and the flow rate is 0.5-1.5 BV / h.

9. The whitening and freckle-removing composition based on molecular docking technology according to any one of claims 3-6, characterized in that, In step A3, when gradient elution is performed using a mixed solution of petroleum ether and ethyl acetate, the mixing ratios of petroleum ether and ethyl acetate are 75:1, 50:1, and 25:1, respectively. In steps B3 and C3, when gradient elution is performed using a mixed solution of dichloromethane and methanol, the mixing ratio of dichloromethane and methanol is 50:1, 20:1, and 10:1, respectively. In step D3, when gradient elution is performed using a mixed solution of petroleum ether and ethyl acetate, the mixing ratios of petroleum ether and ethyl acetate are 5:1, 1:1, and 1:2, respectively.

10. A method for preparing a whitening and freckle-removing composition based on molecular docking technology according to any one of claims 1-9, characterized in that, The components are mixed according to their weight ratio to obtain the final product.

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