A polyacetylenic compound, a preparation method and application thereof, a pharmaceutical composition, and a whitening and freckle-removing cosmetic

Abstract

This invention provides a polyacetylenic compound, its preparation method and application, a pharmaceutical composition, and a skin-whitening and spot-fading daily chemical product, relating to the field of pharmaceutical technology. The polyacetylenic compound provided by this invention exhibits excellent inhibitory effects on melanin production in cells, demonstrating high skin-whitening activity and showing promising application prospects in the preparation of skin-whitening and spot-fading drugs, drugs inhibiting melanin production, and skin-whitening and spot-fading daily chemical products. Furthermore, the polyacetylenic compound provided by this invention is a natural compound from the traditional Chinese medicine Platycodon grandiflorus, exhibiting high safety.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a polyacetylenic compound and its preparation method and application, pharmaceutical compositions, and skin whitening and spot-fading daily chemical products. Background Technology

[0002] Environmental pollution, irregular diet and sleep patterns, and various stressors have combined to cause a decline in skin condition, leading to increased attention to skin health and a greater desire for clear, smooth skin. Excessive melanin production in different parts of the body can cause severe pigmentation and related skin problems. Therefore, inhibiting melanin synthesis is a safe and effective treatment for improving pigmented skin conditions.

[0003] Traditional ingredients such as hydroquinone and retinoic acid have significant adverse reactions, leading research to focus on natural alternatives. Natural active ingredients obtained from traditional Chinese medicine not only effectively inhibit and eliminate melanin accumulation in the skin, but also offer higher safety compared to chemical products. With increasing consumer demand for safety and beauty, the demand for traditional Chinese medicine skin whitening products will continue to grow, demonstrating a huge market potential for their development.

[0004] Platycodon grandiflorus is a plant belonging to the Campanulaceae family. Platycodon grandif1orum The root of *Platycodon grandiflorus* (Jacq.) A.DC is a food and medicinal substance. Studies have shown that the main chemical components of *Platycodon grandiflorus* include saponins, flavonoids, phenylpropanoids, and terpenoids. Research reports indicate that it has certain whitening effects, but these are mostly concentrated in its saponin components. Currently, there are no research reports on the inhibitory activity of polyacetylenes in *Platycodon grandiflorus* on melanin production in cells. Obtaining polyacetylenes with whitening activity from *Platycodon grandiflorus* is of great significance. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a polyacetylene compound, its preparation method and application, a pharmaceutical composition, and a skin-whitening and blemish-reducing daily chemical product. The polyacetylene compound provided by this invention is a natural compound from the traditional Chinese medicine Platycodon grandiflorus, which has a good inhibitory effect on melanin production in cells and high skin-whitening activity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a polyacetylenic compound, comprising any one of compounds 1 to 5: .

[0007] This invention also provides a method for preparing the polyyne compounds described above, comprising the following steps: The roots of Platycodon grandiflorus were extracted using an extractant to obtain an extract. The extract was then concentrated and mixed with water and ethyl acetate for extraction, yielding an ethyl acetate phase and an aqueous phase, respectively. The extractant comprised an aqueous solution of a lower monohydric alcohol with a volume fraction of 70-95%. The lower monohydric alcohol in the aqueous solution comprised methanol and / or ethanol. (a) Preparation methods of compounds 1-3 The ethyl acetate phase was concentrated and then separated by first silica gel column chromatography to obtain component G7. The conditions for the first silica gel column chromatography separation included: the eluent included dichloromethane and methanol, the elution method was first gradient elution, and the volume ratio of dichloromethane to methanol during the first gradient elution was 1:0 to 0:1. Component G7 was separated by first reversed-phase chromatography to obtain component G7.6. The conditions for the first reversed-phase chromatography separation included: mobile phase A comprising mobile phase A and mobile phase B, mobile phase A comprising methanol and mobile phase B comprising water; the elution method was second gradient elution; the second gradient elution program included: 0.00~2.00 h, the volume fraction of mobile phase A being 10~20%; 2.00~2.01 h, the volume fraction of mobile phase A uniformly increasing from 10~20% to 30~40%; 2.01~4.00 h, the volume fraction of mobile phase A being 30~40%; 4.00 h... From 4.01 to 4.01 h, the volume fraction of mobile phase A uniformly increases from 30-40% to 50-60%; from 4.01 to 6.00 h, the volume fraction of mobile phase A is 50-60%; from 6.00 to 6.01 h, the volume fraction of mobile phase A uniformly increases from 50-60% to 65-75%; from 6.01 to 8.00 h, the volume fraction of mobile phase A is 65-75%; from 8.00 to 8.01 h, the volume fraction of mobile phase A uniformly increases from 65-75% to 95-100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A is 95-100%. The component G7.6 was separated by first gel column chromatography to obtain component G7.6.1; the mobile phase for the first gel column chromatography separation included methanol. The components G7.6.1 were separated by first high-performance liquid chromatography (HPLC) to obtain compounds 2, 3, and 1, respectively. The conditions for the first HPLC separation included: mobile phase A comprising acetonitrile and mobile phase B comprising water; elution mode being third gradient elution; and the third gradient elution program including: 0.00–0.01 min, with the volume fraction of mobile phase A being 35–45%; and 0.01–40.0 min, with the volume fraction of mobile phase A uniformly increasing from 35–45% to 50–60%. (b) Preparation methods of compounds 4-5 The aqueous phase was extracted with n-butanol to obtain the n-butanol phase. The n-butanol phase was concentrated and then separated by second silica gel column chromatography to obtain component J4. The conditions for the second silica gel column chromatography separation included: the eluent included dichloromethane and methanol, the elution method was fourth gradient elution, and the volume ratio of dichloromethane to methanol during the fourth gradient elution was 40:1 to 1:1. Component J4 was separated by a second reversed-phase chromatography to obtain component J4.6. The conditions for the second reversed-phase chromatography separation included: mobile phase A comprising mobile phase A and mobile phase B, mobile phase A comprising methanol and mobile phase B comprising water; the elution method was fifth gradient elution, and the fifth gradient elution program included: 0.00~2.00 h, the volume fraction of mobile phase A being 15~25%; 2.00~2.01 h, the volume fraction of mobile phase A uniformly increasing from 15~25% to 35~45%; 2.01~4.00 h, the volume fraction of mobile phase A being 35~45%; 4.00 h... From 4.01 to 4.01 h, the volume fraction of mobile phase A uniformly increases from 35-45% to 55-65%; from 4.01 to 6.00 h, the volume fraction of mobile phase A is 55-65%; from 6.00 to 6.01 h, the volume fraction of mobile phase A uniformly increases from 55-65% to 75-85%; from 6.01 to 8.00 h, the volume fraction of mobile phase A is 75-85%; from 8.00 to 8.01 h, the volume fraction of mobile phase A uniformly increases from 75-85% to 95-100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A is 95-100%. The component J4.6 was separated by a second high-performance liquid chromatography (HPLC) to obtain compounds 4 and 5, respectively. The conditions for the second HPLC separation included: mobile phase A comprising acetonitrile and mobile phase B comprising water; elution mode being a sixth gradient elution; and the sixth gradient elution program including: 0.00–0.01 min, the volume fraction of mobile phase A being 30–40%; and 0.01–40.0 min, the volume fraction of mobile phase A uniformly increasing from 30–40% to 45–55%.

[0008] Preferably, the extraction is performed 3 to 4 times, and the extraction time for each extraction is 40 to 50 hours. The ratio of the dry weight of the Platycodon grandiflorus root to the volume of the extractant used for a single extraction is 1 kg: 2-3 L.

[0009] Preferably, both the first and second reversed-phase chromatographic separations use ODS columns. The first gel column chromatography separation uses a hydroxypropyl dextran gel chromatography column. Both the first and second high-performance liquid chromatography (HPLC) separations use C18 reversed-phase columns.

[0010] Preferably, the volume ratio of dichloromethane to methanol during the first gradient elution process is 1:0, 98:2, 95:5, 9:1, 8:1, 5:1, 4:1, 2:1, 1:1 and 0:1, respectively. The volume ratios of dichloromethane and methanol in the fourth gradient elution process are 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 2:1 and 1:1, respectively.

[0011] The present invention also provides the application of the polyacetylenic compounds described in the above technical solutions or the polyacetylenic compounds prepared by the above technical solutions in the preparation of skin whitening and spot-fading drugs or in skin whitening and spot-fading daily chemical products.

[0012] Preferably, the skin-whitening and spot-fading drug includes a drug that inhibits melanin production.

[0013] The present invention also provides a pharmaceutical composition comprising an active component and pharmaceutically acceptable excipients, wherein the active component comprises a polyacetylene compound as described in the above-described technical solution or a polyacetylene compound prepared by the preparation method described in the above-described technical solution.

[0014] The present invention also provides a whitening and spot-fading daily chemical product, comprising a whitening active ingredient and excipients acceptable in the daily chemical industry, wherein the whitening active ingredient comprises the polyacetylene compound described in the above technical solution or the polyacetylene compound prepared by the preparation method described in the above technical solution.

[0015] Preferably, the excipients acceptable in the daily chemical industry may include at least one of solvents, humectants, thickeners, pH adjusters, oils, emulsifiers, skin conditioning agents, antioxidants, and chelating agents.

[0016] The polyacetylenic compounds provided by this invention exhibit excellent inhibitory effects on melanin production in cells, demonstrating high whitening activity and promising application prospects in the preparation of whitening and spot-fading drugs and daily whitening and spot-fading chemical products. These polyacetylenic compounds not only provide candidate components for inhibiting melanin production but also offer a reference for research on other medicinal plants and the development of new drugs, possessing significant economic benefits and practical application value. Furthermore, the polyacetylenic compounds provided by this invention are natural compounds from the traditional Chinese medicine Platycodon grandiflorus, exhibiting high safety.

[0017] This invention uses Platycodon grandiflorus as raw material. The process involves extracting Platycodon grandiflorus with a 70-95 vol% lower monohydric alcohol aqueous solution. The resulting extract is then mixed with water and ethyl acetate for extraction, yielding ethyl acetate and aqueous phases respectively. The ethyl acetate phase is then separated by silica gel column chromatography, reversed-phase chromatography, gel column chromatography, and high-performance liquid chromatography to obtain compounds 1-3. The aqueous phase is then extracted with n-butanol, and the resulting n-butanol phase is concentrated and further separated by silica gel column chromatography, reversed-phase chromatography, and high-performance liquid chromatography to obtain compounds 4-5. The preparation method provided by this invention can prepare five polyacetylenic compounds with different structures in a single step, with high product purity, simple preparation method, strong operability and reproducibility, low production cost, and is environmentally friendly, making it suitable for industrial production. Attached Figure Description

[0018] Figure 1 The hydrogen spectrum (600 MHz) of compound 1 prepared in Example 1. Figure 2 The carbon spectrum (150 MHz) of compound 1 prepared in Example 1. Figure 3 The heteronuclear single quantum correlation (HSQC) spectrum of compound 1 prepared in Example 1. Figure 4 The heteronuclear single-quantum coherence correlation spectrum of compound 1 prepared in Example 1 ( 1 H- 1 H COSY); Figure 5 The heteronuclear multibond correlation spectrum (HMBC) of compound 1 prepared in Example 1. Figure 6 Here is a high-resolution mass spectrum of compound 1 prepared in Example 1; Figure 7 The ultraviolet spectrum of compound 1 prepared in Example 1; Figure 8 The circular dichroism spectrum of compound 1 prepared in Example 1; Figure 9 The high-performance liquid chromatogram of compound 1 prepared in Example 1; Detailed Implementation

[0019] This invention provides a polyacetylenic compound having the structure shown in any one of formulas 1 to 5: .

[0020] This invention also provides a method for preparing the polyyne compounds described in the above technical solution, comprising the following steps: The roots of Platycodon grandiflorus were extracted using an extractant to obtain an extract. The extract was then concentrated and mixed with water and ethyl acetate for extraction, yielding an ethyl acetate phase and an aqueous phase, respectively. The extractant comprised a 70-95 vol% aqueous solution of a lower monohydric alcohol, wherein the lower monohydric alcohol in the aqueous solution comprised methanol and / or ethanol. (a) Preparation methods of compounds 1-3 The ethyl acetate phase was concentrated and then separated by first silica gel column chromatography to obtain component G7. The conditions for the first silica gel column chromatography separation included: the eluent included dichloromethane and methanol, the elution method was first gradient elution, and the volume ratio of dichloromethane to methanol during the first gradient elution was 1:0 to 0:1. Component G7 was separated by first reversed-phase chromatography to obtain component G7.6. The conditions for the first reversed-phase chromatography separation included: mobile phase A comprising mobile phase A and mobile phase B, mobile phase A comprising methanol and mobile phase B comprising water; the elution method was second gradient elution; the second gradient elution program included: 0.00~2.00 h, the volume fraction of mobile phase A being 10~20%; 2.00~2.01 h, the volume fraction of mobile phase A uniformly increasing from 10~20% to 30~40%; 2.01~4.00 h, the volume fraction of mobile phase A being 30~40%; 4.00 h... From 4.01 to 4.01 h, the volume fraction of mobile phase A uniformly increases from 30-40% to 50-60%; from 4.01 to 6.00 h, the volume fraction of mobile phase A is 50-60%; from 6.00 to 6.01 h, the volume fraction of mobile phase A uniformly increases from 50-60% to 65-75%; from 6.01 to 8.00 h, the volume fraction of mobile phase A is 65-75%; from 8.00 to 8.01 h, the volume fraction of mobile phase A uniformly increases from 65-75% to 95-100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A is 95-100%. The component G7.6 was separated by first gel column chromatography to obtain component G7.6.1; the mobile phase for the first gel column chromatography separation included methanol. The components G7.6.1 were separated by first high-performance liquid chromatography (HPLC) to obtain compounds 2, 3, and 1, respectively. The conditions for the first HPLC separation included: mobile phase A comprising acetonitrile and mobile phase B comprising water; elution mode being third gradient elution; and the third gradient elution program including: 0.00–0.01 min, with the volume fraction of mobile phase A being 35–45%; and 0.01–40.0 min, with the volume fraction of mobile phase A uniformly increasing from 35–45% to 50–60%. (b) Preparation methods of compounds 4-5 The aqueous phase was extracted with n-butanol to obtain the n-butanol phase. The n-butanol phase was concentrated and then separated by second silica gel column chromatography to obtain component J4. The conditions for the second silica gel column chromatography separation included: the eluent included dichloromethane and methanol, the elution method was fourth gradient elution, and the volume ratio of dichloromethane to methanol during the fourth gradient elution was 40:1 to 1:1. Component J4 was separated by a second reversed-phase chromatography to obtain component J4.6. The conditions for the second reversed-phase chromatography separation included: mobile phase A comprising mobile phase A and mobile phase B, mobile phase A comprising methanol and mobile phase B comprising water; the elution method was fifth gradient elution, and the fifth gradient elution program included: 0.00~2.00 h, the volume fraction of mobile phase A being 15~25%; 2.00~2.01 h, the volume fraction of mobile phase A uniformly increasing from 15~25% to 35~45%; 2.01~4.00 h, the volume fraction of mobile phase A being 35~45%; 4.00 h... From 4.01 to 4.01 h, the volume fraction of mobile phase A uniformly increases from 35-45% to 55-65%; from 4.01 to 6.00 h, the volume fraction of mobile phase A is 55-65%; from 6.00 to 6.01 h, the volume fraction of mobile phase A uniformly increases from 55-65% to 75-85%; from 6.01 to 8.00 h, the volume fraction of mobile phase A is 75-85%; from 8.00 to 8.01 h, the volume fraction of mobile phase A uniformly increases from 75-85% to 95-100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A is 95-100%. The component J4.6 was separated by a second high-performance liquid chromatography (HPLC) to obtain compounds 4 and 5, respectively. The conditions for the second HPLC separation included: mobile phase A comprising acetonitrile and mobile phase B comprising water; elution mode being a sixth gradient elution; and the sixth gradient elution program including: 0.00–0.01 min, with the volume fraction of mobile phase A being 30–40%; and 0.01–40.0 min, with the volume fraction of mobile phase A uniformly increasing from 30–40% to 45–55%.

[0021] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0022] This invention uses an extractant to extract Platycodon grandiflorus roots to obtain an extract. The extract is then concentrated and mixed with water and ethyl acetate for extraction, yielding an ethyl acetate phase and an aqueous phase, respectively.

[0023] In this invention, the Platycodon root is Platycodon grandiflorus (a plant in the Campanulaceae family). Platycodon grandiflorusThe root of *Platycodon grandiflorus* (Jacq.) A. DC. is used. In this invention, the *Platycodon grandiflorus* can be cut and crushed before use to obtain coarse powder. The particle size of the coarse powder can be 10-30 mesh.

[0024] In this invention, the extractant comprises an aqueous solution of a lower monohydric alcohol with a volume fraction of 70-95%, wherein the volume fraction of methanol in the aqueous solution of the lower monohydric alcohol is 70-95%, or may be 75-90%, or may be 80-85%; the lower monohydric alcohol in the aqueous solution of the lower monohydric alcohol includes methanol and / or ethanol.

[0025] In this invention, the extraction temperature can be room temperature (15~30℃), and the number of extractions can be 3~4 times; the extraction time for a single extraction can be 40~50 hours, or 42~48 hours, or even 44~46 hours, specifically 45 hours. In this invention, the ratio of the dry weight of the Platycodon grandiflorus root to the volume of the extractant used for a single extraction (material-liquid ratio) can be 1 kg: 2~3 L, or 1 kg: 2.2~2.8 L, or even 1 kg: 2.4~2.6 L, specifically 1 kg: 3 L or 1 kg: 2.5 L.

[0026] The specific method and conditions of concentration described in this invention are not particularly limited. Any concentration method known to those skilled in the art that can remove the solvent can be used.

[0027] In this invention, the extraction of the concentrated extract followed by mixing with water and ethyl acetate can be specifically performed as follows: the extract is concentrated to obtain a paste (denoted as the first paste), which is then suspended in water, and ethyl acetate is added for extraction. In this invention, the material-to-liquid ratio of the first paste to water (i.e., water for mixed extraction or water for suspension) can be 1 kg: 5-15 L, or 1 kg: 8-12 L, specifically 1 kg: 10 L. In this invention, the extraction can be performed 3-4 times. In this invention, the material-to-liquid ratio of the first paste to ethyl acetate used for a single extraction can be 1 kg: 5-15 L, or 1 kg: 8-12 L, specifically 1 kg: 10 L.

[0028] The preparation methods of compounds 1-3 are described in detail below.

[0029] After obtaining the ethyl acetate phase, the present invention concentrates the ethyl acetate phase and then separates it by first silica gel column chromatography to obtain component G7. The specific concentration method and conditions described in the present invention are not particularly limited; any method well-known to those skilled in the art that can remove the solvent is acceptable, such as vacuum concentration. The concentration yields an extract (denoted as the second extract). In the present invention, the conditions for the first silica gel column chromatography separation include: the silica gel particle size can be 100-200 mesh; the eluent includes dichloromethane and methanol; the elution method is first gradient elution; and the volume ratio of dichloromethane to methanol during the first gradient elution process is 1:0 to 0:1, specifically 1:0, 98:2, 95:5, 9:1, 8:1, 5:1, 4:1, 2:1, 1:1, and 0:1. In this invention, during the first silica gel column chromatography separation process, a thin-layer silica gel plate can be used for development and identification, combining identical elution components to obtain a total of 9 components, which are sequentially denoted as component G1, component G2, component G3, component G4, component G5, component G6, component G7, component G8, and component G9. In this invention, the developing solvent used for the thin-layer silica gel plate development and identification may include dichloromethane and methanol, and the volume ratio of dichloromethane to methanol may be 4:1 to 1:1, or 3:1 to 1.5:1, specifically 2:1.

[0030] After obtaining component G7, the present invention performs a first reversed-phase chromatographic separation on component G7 to obtain component G7.6. In the present invention, the conditions for the first reversed-phase chromatographic separation include: the chromatographic column may include an ODS column; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A includes methanol and mobile phase B is water; the elution method is a second gradient elution, and the program for the second gradient elution includes: 0.00~2.00 h, the volume fraction of mobile phase A is 10~20%, specifically 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%; 2.00~2.01 h, the volume fraction of mobile phase A is uniformly increased from 10~20% to 30~40%. Specifically, the volume fraction of mobile phase A can be uniformly increased from any one of the following values: 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, and 20% to any one of the following values: 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, and 40%; from 2.01 to 4.00 h, the volume fraction of mobile phase A is 30% to 40%, specifically 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%; from 4.00 to 4.01 h, the volume fraction of mobile phase A varies from 30% to 40%. The mobile phase A is uniformly increased to 50-60%, specifically from any one of the following values: 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, and 40%, to any one of the following values: 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%; from 4.01 to 6.00 h, the volume fraction of the mobile phase A is 50-60%, specifically 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%; from 6.00 to 6.01 h, the volume fraction of the mobile phase A... The volume fraction is uniformly increased from 50-60% to 65-75%, specifically from any one of the values ​​of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% to any one of the values ​​of 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, and 75%; 6.01-8.00h, the volume fraction of the mobile phase A is 65-75%, specifically 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, and 75%; 8.00-8.From 8.01 to 10.00 h, the volume fraction of the mobile phase A is uniformly increased from 65-75% to 95-100%, specifically from any one of the following values: 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, and 80% to any one of the following values: 95%, 96%, 97%, 98%, 99%, and 100%; from 8.01 to 10.00 h, the volume fraction of the mobile phase A is 95-100%, specifically 95%, 96%, 97%, 98%, 99%, or 100%. In this invention, during the first reversed-phase chromatographic separation process, a thin-layer silica gel plate can be used for development and identification. Identical eluting components are combined, resulting in a total of eight components, sequentially designated as component G7.1, component G7.2, component G7.3, component G7.4, component G7.5, component G7.6, component G7.7, and component G7.8. In this invention, the developing solvent used for the thin-layer silica gel plate development and identification may include dichloromethane and methanol. The volume ratio of dichloromethane to methanol can be 4:1 to 1:1, or 3:1 to 1.5:1, specifically 2:1.

[0031] After obtaining component G7.6, this invention performs first gel column chromatography separation on component G7.6 to obtain component G7.6.1. In this invention, the conditions for the first gel column chromatography separation include: the chromatographic column may include a hydroxypropyl dextran gel chromatography column; the mobile phase includes methanol; and the elution method is isocratic elution. In this invention, thin-layer silica gel plate development and identification can be used during the first gel column chromatography separation process, and identical eluted components are combined to obtain a total of 3 components, sequentially denoted as component G7.6.1, component G7.6.2, and component G7.6.3. In this invention, the chromatographic peak of component G7.6.1 in HPLC exhibits characteristic absorption of polyacetylenic components, which facilitates differentiation from other components. In this invention, the developing solvent used for thin-layer silica gel plate development and identification may include dichloromethane and methanol, and the volume ratio of dichloromethane to methanol may be 4:1 to 1:1, or 3:1 to 1.5:1, specifically 2:1.

[0032] After obtaining component G7.6.1, the present invention performs a first high-performance liquid chromatography (HPLC) separation on component G7.6.1 to obtain compounds 2, 3, and 1, respectively. In the present invention, the conditions for the first HPLC separation include: the chromatographic column may include a C18 reversed-phase column; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A includes acetonitrile and mobile phase B is water; the flow rate of the mobile phase can be 6-9 mL / min, specifically 6 mL / min, 7 mL / min, 8 mL / min, or 9 mL / min; the elution method is third-gradient elution, the third-gradient elution program includes: 0.00-0.01 min, the volume fraction of mobile phase A is 35-45%, specifically 3... 5%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%; 0.01~40.0 min, the volume fraction of the mobile phase A is uniformly increased from 35~45% to 50~60%, specifically from any one of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45% to any one of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%.

[0033] The preparation methods of compounds 4 and 5 are described in detail below.

[0034] After obtaining the aqueous phase, the present invention extracts the aqueous phase with n-butanol to obtain the n-butanol phase. The n-butanol phase is then concentrated and separated by a second silica gel column chromatography to obtain component J4. The specific concentration method and conditions described in this invention are not particularly limited; any method well-known to those skilled in the art that removes the solvent is acceptable, such as vacuum concentration. The concentrated product is a paste (denoted as the third paste). In this invention, the conditions for the second silica gel column chromatography separation include: silica gel particle size of 100-200 mesh; eluents including dichloromethane and methanol; and elution using a fourth gradient elution method. During the fourth gradient elution, the volume ratio of dichloromethane to methanol is 40:1 to 1:1, specifically 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 2:1, and 1:1. In this invention, during the second silica gel column chromatography separation process, a thin-layer silica gel plate can be used for development and identification to combine identical elution fractions, resulting in a total of 5 fractions, sequentially denoted as fraction J1, fraction J2, fraction J3, fraction J4, and fraction J5. In this invention, the developing solvent used for the thin-layer silica gel plate development and identification may include dichloromethane and methanol, and the volume ratio of dichloromethane to methanol may be 3:1 to 1:1, or 2:1 to 1:1, specifically 1:1.

[0035] After obtaining component J4, the present invention performs a second reversed-phase chromatographic separation on component J4 to obtain component J4.6. In the present invention, the conditions for the second reversed-phase chromatographic separation include: the chromatographic column can be an ODS column; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A includes methanol and mobile phase B is water; the elution method is fifth gradient elution, and the fifth gradient elution program includes: 0.00~2.00 h, the volume fraction of mobile phase A is 15~25%, specifically 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%; 2.00~2.01 h, the volume fraction of mobile phase A is uniformly increased from 15~25% to 35~45%, which can be... Specifically, the mobile phase A is uniformly increased from any one of the following values: 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25% to any one of the following values: 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%; from 2.01 to 4.00 h, the volume fraction of the mobile phase A is 35% to 45%, specifically 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%; from 4.00 to 4.01 h, the volume fraction of the mobile phase A is uniformly increased from 35% to 45%. The volume fraction of mobile phase A is uniformly increased to 55-65%, specifically from any one of the following values: 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%, to any one of the following values: 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, and 65%; from 4.01 to 6.00 h, the volume fraction of mobile phase A is 55-65%, specifically 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%; from 6.00 to 6.01 h, the volume fraction of mobile phase A... The volume fraction is uniformly increased from 55-65% to 75-85%, specifically from any one of the following values: 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, and 65%, to any one of the following values: 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and 85%; 6.01-8.00h, the volume fraction of the mobile phase A is 75-85%, specifically 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%; 8.00-8.From 8.01 to 10.00 h, the volume fraction of mobile phase A is uniformly increased from 75-85% to 95-100%, specifically from any one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and 85% to any one of 95%, 96%, 97%, 98%, 99%, and 100%. From 8.01 to 10.00 h, the volume fraction of mobile phase A is 95-100%, specifically 95%, 96%, 97%, 98%, 99%, or 100%. In this invention, during the second reversed-phase chromatographic separation process, a thin-layer silica gel plate can be used for development and identification, and identical elution components can be combined to obtain a total of 7 components, sequentially denoted as component J4.1, component J4.2, component J4.3, component J4.4, component J4.5, component J4.6, and component J4.7. In this invention, the developing agent used for the thin-layer silicone plate unfolding and identification may include dichloromethane and methanol. The volume ratio of methanol to dichloromethane may be 1:1 to 1:3, or 2:1 to 1:1, specifically 1:1.

[0036] After obtaining component J4.6, the present invention performs a second high-performance liquid chromatography (HPLC) separation on component J4.6 to obtain compounds 4 and 5, respectively. In the present invention, the conditions for the second HPLC separation include: the chromatographic column may include a C18 reversed-phase column; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A includes acetonitrile and mobile phase B is water; the flow rate of the mobile phase can be 6-9 mL / min, specifically 6 mL / min, 7 mL / min, 8 mL / min, or 9 mL / min; the elution method is sixth gradient elution, the program of which includes: 0.00-0.01 min, the volume fraction of mobile phase A being 30-40%, specifically 3... 0%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%; 0.01~40.0 min, the volume fraction of mobile phase A is uniformly increased from 30~40% to 45~55%, specifically from any one of 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, and 40% to any one of 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, and 55%.

[0037] The present invention provides separation and purification conditions for polyyne monomers from *Platycodon grandiflorus*. The chemical structures of the obtained compounds were determined by NMR, MS, and ECD. This method is simple, easy to perform, and controllable. The polyyne compounds prepared by this invention were all determined by high-performance liquid chromatography using the area normalization method, and their purity was greater than 98%. The method for preparing polyyne compounds provided by this invention is simple, rapid, has high purity, and good reproducibility.

[0038] This invention also provides the application of the polyacetylenic compounds described in the above-described technical solutions or the polyacetylenic compounds prepared by the above-described technical solutions in the preparation of skin whitening and spot-fading drugs or in skin whitening and spot-fading daily chemical products. In this invention, the skin whitening and spot-fading drugs may include drugs that inhibit melanin production. The polyacetylenic compounds prepared in this invention were found by the CCK8 assay to have no effect on the normal growth of mouse B16F10 cells at the tested concentration, demonstrating good safety. These compounds have a significant inhibitory effect on melanin production in B16F10 cells, possessing strong practicality in skin whitening cosmetics and drug development, and can be used for the prevention of skin melanin deposition and skin whitening effects.

[0039] This invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients. The active ingredient comprises a polyacetylene compound as described in the above-described technical solution or a polyacetylene compound prepared by the method described in the above-described technical solution. This invention does not specifically limit the pharmaceutically acceptable excipients; any pharmaceutically acceptable excipient well known to those skilled in the art can be used, such as one or more of stearic acid, petrolatum, glycerin, liquid paraffin, cyclodextrin, microcrystalline cellulose, and croscarmellose sodium. This invention does not specifically limit the dosage form of the pharmaceutical composition; any dosage form well known to those skilled in the art can be used, such as creams, tablets, capsules, or granules.

[0040] This invention also provides a whitening and spot-fading daily chemical product, comprising a whitening active ingredient and excipients acceptable in the daily chemical industry. The whitening active ingredient includes the polyacetylene compounds described in the above-described technical solution or polyacetylene compounds prepared by the preparation method described in the above-described technical solution. In this invention, the excipients acceptable in the daily chemical industry may include at least one of solvents, humectants, thickeners, pH adjusters, oils, emulsifiers, skin conditioning agents, antioxidants, and chelating agents. This invention does not specifically limit the types and amounts of the solvents, humectants, thickeners, pH adjusters, oils, emulsifiers, skin conditioning agents, antioxidants, and chelating agents; any excipients known to those skilled in the art that can be used in whitening and spot-fading daily chemical products can be used, specifically one or more of hyaluronic acid, glycerin, disodium diethylaminetetraacetate, Tween 20, PEG-60 hydrogenated castor oil, and juniper flower oil. In this invention, the dosage form of the whitening and spot-fading daily chemical product may include an aqueous solution, an emulsion, or an ointment.

[0041] To further illustrate the present invention, the following detailed descriptions, in conjunction with embodiments, illustrate the polyacetylenic compounds, their preparation methods and applications, pharmaceutical compositions, and skin-whitening and spot-fading daily chemical products provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0042] In the following embodiments, the materials and instruments are as follows: Platycodon grandiflorus is a plant belonging to the Campanulaceae family. Platycodon grandiflorus The dried root of (Jacq.) A. DC. was collected in Taihe County, Anhui Province. The medicinal material sample (No. 20210612) is preserved in the Laboratory of Traditional Chinese Medicine Chemistry, School of Pharmacy, Anhui University of Traditional Chinese Medicine. Thermo Fisher Scientific Orbitrap Exploris 120 liquid chromatography-mass spectrometry (LC-MS) system (Germany); Bruker AVⅢ-600MHz nuclear magnetic resonance spectrometer (solvents include deuterated methanol, etc.); Waters 1525 analytical / preparative high-performance liquid chromatograph (HPLC) from the USA; One-dimensional and two-dimensional nuclear magnetic resonance (1D / 2D) spectra were measured on a Bruker 600MHz NMR spectrometer (deuterated methanol was used as the deuterated reagent). High-resolution mass spectrometry (ESI-HRMS) was performed using a Thermo Fisher Scientific Orbitrap Exploris 120 liquid chromatography-mass spectrometry system. Mouse skin melanoma cell line B16F10 (Wuhan Pronosai Life Science Technology Co., Ltd.); Bio-Tek SpectraMax i3X Multifunctional Microplate Reader (USA) Fetal bovine serum (Nanjing Shenghang Biotechnology Co., Ltd.); Arbutin (batch number: K621084), α-melanocyte stimulating hormone (α-MSH, batch number: K372809, Kekuller Guangzhou Biomedical Co., Ltd.).

[0043] Example 1 20 kg of dried Platycodon grandiflorus roots were pulverized to a particle size of 10-30 mesh, and then extracted four times at 25°C for 45 hours each time. The extracts were combined, and the extractant was recovered by vacuum concentration to obtain 1.5 kg of the first extract. The ratio of dried Platycodon grandiflorus roots to the ethanol aqueous solution used for each extraction was 1 kg: 3 L.

[0044] The first extract was suspended in 15 L of water and extracted four times with ethyl acetate (15 L each time), yielding an ethyl acetate phase and an aqueous phase. The ethyl acetate phase was concentrated under reduced pressure to remove ethyl acetate (ethyl acetate can be reused), yielding 416 g of the second extract. The aqueous phase was extracted with n-butanol, and the n-butanol extract was collected and concentrated under reduced pressure to remove n-butanol (n-butanol can be reused), yielding 320 g of the third extract.

[0045] The second extract was separated by silica gel column chromatography (100-200 mesh silica gel, with volume ratios of dichloromethane and methanol of 1:0, 98:2, 95:5, 9:1, 8:1, 5:1, 4:1, 2:1, 1:1, and 0:1, respectively). Thin-layer silica gel plate development and identification were performed (developing solvent: dichloromethane and methanol volume ratio = 2:1). Identical elution fractions were combined to obtain 9 fractions, which were named fraction G1 (3.1g), fraction G2 (2.2g), fraction G3 (5.2g), fraction G4 (63.9g), fraction G5 (22.4g), fraction G6 (12.6g), fraction G7 (14.2g), fraction G8 (26.4g), and fraction G9 (13.8g).

[0046] Component G7 (14.2 g) was separated by reversed-phase chromatography. Thin-layer silica gel plate development and identification were performed (developing solvent: dichloromethane and methanol, volume ratio = 2:1). Identical elution fractions were combined to obtain eight components, which were subsequently designated as components G7.1, G7.2, G7.3, G7.4, G7.5, G7.6 (533 mg), G7.7, and G7.8. The reversed-phase chromatographic conditions were as follows: ODS column, mobile phase A: methanol, mobile phase B: water, gradient elution program: 0.00–2.00 h, mobile phase A volume fraction 15%; 2.00–2.01 h, mobile phase A volume fraction uniformly increased from 15% to 35%; 2.01–4.00 h, mobile phase A volume fraction 35%; 4.00–4.01 h, mobile phase A volume fraction increased from 35% to... The volume fraction of mobile phase A was uniformly increased to 55% from 4.01 to 6.00 h; from 6.00 to 6.01 h, the volume fraction of mobile phase A was uniformly increased from 55% to 70%; from 6.01 to 8.00 h, the volume fraction of mobile phase A was 70%; from 8.00 to 8.01 h, the volume fraction of mobile phase A was uniformly increased from 70% to 100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A was 100%.

[0047] Fraction G7.6 (533 mg) was separated by hydroxypropyl dextran gel chromatography with anhydrous methanol as the mobile phase and isocratic elution. The fractions were then analyzed by thin-layer silica gel plate development (the developing solvent was dichloromethane and methanol in a volume ratio of 2:1). The same elution fractions were combined to obtain three fractions, which were named fractions G7.6.1 (103 mg), G7.6.2, and G7.6.3, respectively.

[0048] Component G7.6.1 (103 mg) was separated by high performance liquid chromatography to obtain compound 2 (1.9 mg, t). R =17.4min), compound 3 (12.4mg, t R =20.9min) and compound 1 (36.0mg, tR =22.2 min). The high-performance liquid chromatography (HPLC) separation conditions were as follows: the chromatographic column was a C18 reversed-phase column, mobile phase A was acetonitrile, mobile phase B was water, and the gradient elution program was: 0.00~0.01 min, with the volume fraction of mobile phase A being 40%; 0.01~40.0 min, with the volume fraction of mobile phase A increasing uniformly from 40% to 55%), and the mobile phase flow rate was 8 mL / min.

[0049] The third extract was separated by silica gel column chromatography (100-200 mesh silica gel, with volume ratios of dichloromethane and methanol of 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 2:1, and 1:1, respectively). Thin-layer silica gel plate development and identification were performed (developing solvent: dichloromethane and methanol volume ratio = 1:1). Identical elution fractions were combined to obtain 5 fractions, which were named fraction J1, fraction J2, fraction J3, fraction J4 (63.9 g), and fraction J5, respectively.

[0050] Component J4 (63.9 g) was separated by reversed-phase chromatography using thin-layer silica gel plate development (developing solvent: dichloromethane and methanol, volume ratio = 1:1). Identical eluting fractions were combined to obtain seven components, designated as J4.1, J4.2, J4.3, J4.4, J4.5, J4.6 (80.4 mg), and J4.7. The reversed-phase chromatographic conditions were as follows: ODS column, mobile phase A: methanol, mobile phase B: water, gradient elution program: 0.00–2.00 h, mobile phase A volume fraction 20%; 2.00–2.01 h, mobile phase A volume fraction uniformly increased from 20% to 40%; 2.01–4.00 h, mobile phase A volume fraction 40%; 4.00–4.01 h, mobile phase A volume fraction increased from 40% to... The volume fraction of mobile phase A was uniformly increased to 60% from 4.01 to 6.00 h; from 6.00 to 6.01 h, the volume fraction of mobile phase A was uniformly increased from 60% to 80%; from 6.01 to 8.00 h, the volume fraction of mobile phase A was 80%; from 8.00 to 8.01 h, the volume fraction of mobile phase A was uniformly increased from 80% to 100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A was 100%.

[0051] Component J4.6 (80.4 mg) was separated by high performance liquid chromatography to obtain compound 4 (12.9 mg, t). R =22.8min) and compound 5 (5.5mg, t R=27.7 min). The high-performance liquid chromatography (HPLC) separation conditions were as follows: the chromatographic column was a C18 reversed-phase column, mobile phase A was acetonitrile, mobile phase B was water, and the gradient elution program was: 0.00~0.01 min, with the volume fraction of mobile phase A being 35%; 0.01~40.0 min, with the volume fraction of mobile phase A increasing uniformly from 35% to 50%; and the mobile phase flow rate was 8 mL / min.

[0052] The chemical structures of compounds 1–5 were determined by 1D / 2D NMR and ESI-HRMS, respectively. The NMR spectral data are shown in Tables 1–3. Specifically, the proton NMR spectrum (deuterated methanol), carbon NMR spectrum (deuterated methanol), heteronuclear single-quantum correlation spectrum (HSQC spectrum), and heteronuclear single-quantum coherent correlation spectrum (HSQC spectrum) of compound 1 are also shown. 1 H- 1 The H COSY spectrum, heteronuclear multibond correlation spectrum (HMBC spectrum), high-resolution mass spectrum, ultraviolet spectrum, circular dichroism spectrum (CD spectrum), and high-performance liquid chromatography (HPLC) chromatogram are shown below. Figures 1-9 As shown. By Figures 1-9 As shown in Tables 1-3, the present invention successfully extracted compounds 1-5 with the described structure, and the purity of the compounds was above 98%.

[0053] Table 1. 1H NMR (600MHz) and 1C NMR (150MHz) data for compound 1 (deuterated methanol)

[0054] Table 2. 1H NMR (600MHz) and 1C NMR (150MHz) data for compounds 2 and 3 (deuterated methanol)

[0055] Table 3. 1H NMR (600MHz) and 1C NMR (150MHz) data for compounds 4 and 5 (deuterated methanol)

[0056] The physicochemical data of compounds 1-5 are as follows: Compound 1: Molecular formula is C 16 H 20 O5; a brownish-yellow oily substance, readily soluble in methanol; UV (CH3OH) λ max :215nm, 267nm, 284nm. [α] + 34.09 (c 0.088, CH3OH); HR-ESI-MS m / z 315.1195 [M+Na] + (Calculated value: 315.1203). The active hydrogen atoms of the three hydroxyl groups in compound 1 do not show a signal in the proton spectrum.

[0057] Compound 2: Molecular formula is C 22 H 30 O9; a brownish-yellow oily substance, readily soluble in methanol; UV (CH3OH) λ max : 213nm, 266nm, 282nm. α ] -129.41 (c 0.034, CH3OH); HR-ESI-MS m / z 461.1778 [M+Na] + (Calculated value: 461.1782).

[0058] Compound 3: Molecular formula is C 22 H 30 O9; a brownish-yellow oily substance, readily soluble in methanol; UV (CH3OH) λ max : 214nm, 267nm, 283nm. α ] -91.73 (c 0.045, CH3OH); HR-ESI-MS m / z 461.1776 [M+Na] + (Calculated value: 461.1782).

[0059] Compound 4: Molecular formula is C 26 H 40 O 13 Brownish-yellow oily substance, readily soluble in methanol; UV (CH3OH) λ max :214nm, 264nm, 283nm. [α] -73.08 (c 0.078, CH3OH); HR-ESI-MS m / z 583.2351 [M+Na] + (Calculated value: 583.2361).

[0060] Compound 5: Molecular formula is C 26 H 38 O 13 Brownish-yellow oily substance, readily soluble in methanol; UV (CH3OH) λ max : 214nm, 267nm, 283nm. α ] -65.22 (CH3OH; c 0.046); HR-ESI-MS m / z : 581.2192 [M+Na]+ (Calculated value: 581.2205).

[0061] Test Example 1 Compounds 1-5 inhibit melanin production in cells. Materials and reagents: Mouse skin melanoma cell line B16F10 cells, RPMI-1640 basal medium and DMEM high glucose medium, fetal bovine serum, trypsin digestion solution, arbutin, α-MSH.

[0062] Principle: B16F10 cells are an immortalized melanocyte cell line derived from non-tumor mouse melanocytes. Due to their relatively easy in vitro culture and the fact that their melanin production mechanism is largely the same as that of normal human cells, they are widely used in melanin-related research. α-Melanocyte-stimulating hormone (α-MSH) is a polypeptide derived from promelanin (POMC), which can activate the expression of the microophthalmic transcription factor (MITF) gene via the cyclic adenosine monophosphate (cAMP) pathway. MITF can activate enzymes related to melanin synthesis, thereby promoting melanin production. Therefore, α-MSH-stimulated B16F10 cells can be selected as a cell model to evaluate the inhibitory effect of compounds on melanin production.

[0063] (1) B16F10 melanoma cell viability assay of compounds 1-5 The cytotoxicity of polyacetylenes was assessed using the CCK-8 assay. B16F10 cells were seeded in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. Then, 100 μL of DMEM medium containing the compound sample solution (50 µmol / L) was added to each well, and the cells were incubated for another 24 h. Finally, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 1.5 h. The absorbance was measured at 450 nm using a microplate reader. A control group without drug treatment and a cell-free blank group were included, with three replicates per group.

[0064] Cell viability calculation formula: Cell viability = (OD) 加药 -OD 空白 ) / (OD 0加药 -OD 空白 ) × 100%. Wherein, OD 加药 : Absorbance values ​​of wells containing cells, culture medium, CCK-8 solution, and the drug solution to be tested. OD 空白 : Absorbance values ​​of wells containing culture medium, CCK-8 solution, and cells-free samples. OD 0加药 Absorbance values ​​for wells containing cells, culture medium, CCK-8 solution, and no test drug solution. Cell viability results are shown in Table 4.

[0065] Table 4. Effects of compounds 1-5 on the survival rate of B16F10 cells ( ±s, )

[0066] Table 4 shows that compounds 1-5 all achieved a B16F10 cell survival rate of over 90% at a concentration of 50 µmol / L, indicating that compounds 1-5 had no significant cytotoxicity to B16F10 cells and were safe.

[0067] (2) Detection of melanin content in B16F10 melanoma cells of compounds 1-5 The melanin content in B16F10 melanoma cells was determined using the NaOH lysis method. B16F10 cells were seeded in 6-well cell culture plates using DMEM medium (2 mL per well) and incubated at 37°C with 5% CO2 for 24 h. After discarding the DMEM medium, the cells were divided into four groups: a blank control group, an α-MSH (100 nmol / L) model group, an α-MSH (100 nmol / L) + arbutin (500 μg / mL) group, and an α-MSH (100 nmol / L) + compound group (concentrations of 2.5 µmol / L, 5 µmol / L, 10 µmol / L, 25 µmol / L, and 50 µmol / L, respectively). The cells were then incubated in a cell culture incubator for another 72 h. Cells were washed twice with PBS, digested with trypsin, and collected in centrifuge tubes after centrifugation at 900 rpm for 4 min. 400 μL of 1 mol / L NaOH solution containing 10% dimethyl sulfoxide was added to each sample to lyse the cells. The cells were incubated at 80℃ for 1 h, then transferred to 96-well plates (100 μL per well). Absorbance was measured at 405 nm. A blank control was used as a reference, and three replicates were set up for each group to compare the relative melanin content in the cells. Results are shown in Table 5.

[0068] Formula for calculating melanin content: Melanin content = (OD) 药物处理组 ) / (OD 空白组 ) × 100%.

[0069] Table 5. Effects of different test concentrations of compounds 1-5 on melanin content in B16F10 cells ( - x ± s , n = 3)

[0070] Note: This indicates a comparison with the model group (α-MSH). P <0.001.

[0071] Table 5 shows that compounds 1-5 all have a certain inhibitory effect on melanin production in B16F10 cells at different concentration ranges, and the inhibitory effect is better than that of the positive control drug arbutin at some test concentrations.

[0072] (3) Detection of melanin content in B16F10 melanoma cells of compounds 6-8 Compounds 6-8 were tested according to the melanin content of B16F10 melanoma cells containing compounds 1-5 in (2). The test concentration of compounds 6-8 was 50 µmol / L. The test results are shown in Table 6.

[0073] Compound 6; Compound 7; Compound 8.

[0074] Table 6. Effects of compounds 6-8 on melanin content in B16F10 cells ( - x ± s , n = 3)

[0075] As shown in Table 6, compared with the model group (α-MSH), compounds 6-8 did not significantly inhibit melanin production in cells. However, compounds 1-5 in this application showed significant inhibitory effects on melanin production in cells.

[0076] (4) Tyrosinase activity assay in melanoma cells of compounds 1-5 The relative activity of intracellular tyrosinase was determined using the dopa oxidation method. B16F10 cells were seeded in 6-well cell culture plates using DMEM medium (2 mL per well) and incubated at 37°C with 5% CO2 for 24 h. After discarding the DMEM medium, the cells were divided into four groups: a blank control group, an α-MSH (100 nmol / L) model group, an α-MSH (100 nmol / L) + arbutin (500 μg / mL) group, and an α-MSH (100 nmol / L) + compound group (concentrations of 2.5 µmol / L, 5 µmol / L, 10 µmol / L, 25 µmol / L, and 50 µmol / L, respectively). The cells were then incubated in a cell culture incubator for another 72 h. After trypsin digestion, the cell pellet was collected in centrifuge tubes and washed twice with PBS. Add 200 μL of PBS buffer containing 1% Triton X-100 (v / v), freeze at -80℃ for 30 min, thaw at room temperature, centrifuge at 12000 g for 20 min, and the supernatant is the crude tyrosinase extract. Take 50 μL of the supernatant and add 150 μL of 1 mg / mL L-DOPA solution to a 966-well plate, react at 37℃ for 1 h, and measure the absorbance at 475 nm. Using the blank group as a control, compare the relative intracellular tyrosinase activities of each group, with three replicates for each group. The results are shown in Table 7.

[0077] Table 7. Effects of different test concentrations of compounds 1-5 on the relative activity of intracellular tyrosinase in B16F10 cells. ± s, )

[0078] Note: This indicates a comparison with the model group (α-MSH). P <0.001.

[0079] The data in Table 7 show that compounds 1-5 may inhibit melanin production in cells by inhibiting tyrosinase activity.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polyacetylenic compound, characterized in that, Includes any one of compounds 1 through 5: 。 2. The method for preparing the polyyne compound according to claim 1, characterized in that, Includes the following steps: The roots of Platycodon grandiflorus were extracted using an extractant to obtain an extract. The extract was then concentrated and mixed with water and ethyl acetate for extraction, yielding an ethyl acetate phase and an aqueous phase, respectively. The extractant comprised an aqueous solution of a lower monohydric alcohol with a volume fraction of 70-95%. The lower monohydric alcohol in the aqueous solution comprised methanol and / or ethanol. (a) Preparation methods of compounds 1-3 The ethyl acetate phase was concentrated and then separated by silica gel column chromatography to obtain component G7; The conditions for the first silica gel column chromatography separation include: the eluent includes dichloromethane and methanol, the elution method is first gradient elution, and the volume ratio of dichloromethane to methanol during the first gradient elution process is 1:0 to 0:1; Component G7 was separated by first reversed-phase chromatography to obtain component G7.

6. The conditions for the first reversed-phase chromatography separation included: mobile phase A comprising mobile phase A and mobile phase B, mobile phase A comprising methanol and mobile phase B comprising water; the elution method was second gradient elution; the second gradient elution program included: 0.00~2.00 h, the volume fraction of mobile phase A being 10~20%; 2.00~2.01 h, the volume fraction of mobile phase A uniformly increasing from 10~20% to 30~40%; 2.01~4.00 h, the volume fraction of mobile phase A being 30~40%; 4.00 h... From 4.01 to 4.01 h, the volume fraction of mobile phase A uniformly increases from 30-40% to 50-60%; from 4.01 to 6.00 h, the volume fraction of mobile phase A is 50-60%; from 6.00 to 6.01 h, the volume fraction of mobile phase A uniformly increases from 50-60% to 65-75%; from 6.01 to 8.00 h, the volume fraction of mobile phase A is 65-75%; from 8.00 to 8.01 h, the volume fraction of mobile phase A uniformly increases from 65-75% to 95-100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A is 95-100%. The component G7.6 was separated by first gel column chromatography to obtain component G7.6.1; the mobile phase for the first gel column chromatography separation included methanol. The components G7.6.1 were separated by first high-performance liquid chromatography (HPLC) to obtain compounds 2, 3, and 1, respectively. The conditions for the first HPLC separation included: mobile phase A comprising acetonitrile and mobile phase B comprising water; elution mode being third gradient elution; and the third gradient elution program including: 0.00–0.01 min, with the volume fraction of mobile phase A being 35–45%; and 0.01–40.0 min, with the volume fraction of mobile phase A uniformly increasing from 35–45% to 50–60%. (b) Preparation methods of compounds 4-5 The aqueous phase was extracted with n-butanol to obtain the n-butanol phase. The n-butanol phase was concentrated and then separated by second silica gel column chromatography to obtain component J4. The conditions for the second silica gel column chromatography separation included: the eluent included dichloromethane and methanol, the elution method was fourth gradient elution, and the volume ratio of dichloromethane to methanol during the fourth gradient elution was 40:1 to 1:

1. Component J4 was separated by a second reversed-phase chromatography to obtain component J4.

6. The conditions for the second reversed-phase chromatography separation included: mobile phase A comprising mobile phase A and mobile phase B, mobile phase A comprising methanol and mobile phase B comprising water; the elution method was fifth gradient elution, and the fifth gradient elution program included: 0.00~2.00 h, the volume fraction of mobile phase A being 15~25%; 2.00~2.01 h, the volume fraction of mobile phase A uniformly increasing from 15~25% to 35~45%; 2.01~4.00 h, the volume fraction of mobile phase A being 35~45%; 4.00 h... From 4.01 to 4.01 h, the volume fraction of mobile phase A uniformly increases from 35-45% to 55-65%; from 4.01 to 6.00 h, the volume fraction of mobile phase A is 55-65%; from 6.00 to 6.01 h, the volume fraction of mobile phase A uniformly increases from 55-65% to 75-85%; from 6.01 to 8.00 h, the volume fraction of mobile phase A is 75-85%; from 8.00 to 8.01 h, the volume fraction of mobile phase A uniformly increases from 75-85% to 95-100%; from 8.01 to 10.00 h, the volume fraction of mobile phase A is 95-100%. The component J4.6 was separated by a second high-performance liquid chromatography (HPLC) to obtain compounds 4 and 5, respectively. The conditions for the second HPLC separation included: mobile phase A comprising acetonitrile and mobile phase B comprising water; elution mode being a sixth gradient elution; and the sixth gradient elution program including: 0.00–0.01 min, the volume fraction of mobile phase A being 30–40%; and 0.01–40.0 min, the volume fraction of mobile phase A uniformly increasing from 30–40% to 45–55%.

3. The preparation method according to claim 2, characterized in that, The extraction is performed 3 to 4 times, and the extraction time for each extraction is 40 to 50 hours. The ratio of the dry weight of the Platycodon grandiflorus root to the volume of the extractant used for a single extraction is 1 kg: 2-3 L.

4. The preparation method according to claim 2, characterized in that, Both the first and second reversed-phase chromatographic separations use ODS columns. The first gel column chromatography separation uses a hydroxypropyl dextran gel chromatography column; Both the first and second high-performance liquid chromatography (HPLC) separations use C18 reversed-phase columns.

5. The preparation method according to claim 2, characterized in that, During the first gradient elution process, the volume ratios of dichloromethane and methanol were 1:0, 98:2, 95:5, 9:1, 8:1, 5:1, 4:1, 2:1, 1:1, and 0:1, respectively. The volume ratios of dichloromethane and methanol in the fourth gradient elution process are 40:1, 30:1, 20:1, 10:1, 5:1, 4:1, 2:1 and 1:1, respectively.

6. The use of the polyacetylenic compound of claim 1 or the polyacetylenic compound prepared by any one of claims 2 to 5 in the preparation of skin whitening and spot-fading drugs or in skin whitening and spot-fading daily chemical products.

7. The application according to claim 6, characterized in that, The skin-whitening and spot-fading drugs include those that inhibit melanin production.

8. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes the polyyne compound of claim 1 or the polyyne compound prepared by the preparation method of any one of claims 2 to 5.

9. A whitening and spot-fading daily chemical product, characterized in that, It includes whitening active ingredients and excipients acceptable in the daily chemical industry, wherein the whitening active ingredients include the polyacetylene compound of claim 1 or the polyacetylene compound prepared by the preparation method of any one of claims 2 to 5.

10. The whitening and spot-fading daily chemical product according to claim 9, characterized in that, The excipients acceptable in the daily chemical industry may include at least one of solvents, humectants, thickeners, pH adjusters, oils, emulsifiers, skin conditioning agents, antioxidants, and chelating agents.

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