Eugenyl glycoside, and preparation method and application thereof

Abstract

The present application relates to the field of cosmetics, pharmaceutical technology and synthesis, in particular to a syringyl acyl glycoside, the structure of which is shown as formula A; the syringyl acyl glycoside can inhibit 5alpha-reductase and reduce the damage of dihydrotestosterone to hair follicle cells, and can be used for preparing a medicine for preventing, treating or assisting in treating alopecia or promoting hair growth, especially androgenic alopecia; and a cosmetic for preventing or relieving alopecia or promoting hair growth.

Description

Technical Field

[0001] This invention relates to the fields of cosmetics, pharmaceutical technology, and synthesis, specifically to a eugenol glycoside, its preparation method, and its application in the preparation of drugs or cosmetics that inhibit 5α-reductase and treat androgenetic alopecia (AGA). Background Technology

[0002] With changing lifestyles, the incidence of hair loss is rising year by year, becoming a widespread health problem globally and causing significant distress. Studies indicate that by 2025, the number of people experiencing hair loss in China will exceed 250 million, meaning that on average, one in six people will suffer from hair loss. Among various types of hair loss, the most common is androgenetic alopecia (AGA), also known as seborrheic alopecia, accounting for over 80% of all cases, and the affected population has been gradually expanding and becoming younger in recent years.

[0003] The main clinical manifestation of AGA is progressive follicular miniaturization, leading to progressive thinning, softening, and decreased density of hair. This means the hair follicles atrophy and shrink, resulting in slower and thinner hair growth; the progression is slow but irreversible. Many factors contribute to AGA, including hormones, genetics, and inflammation, with excessive activation of the androgen signaling pathway considered a core pathogenic factor. Studies have shown that androgens in the body include two types: testosterone (T) and dihydrotestosterone (DHT), with DHT being approximately five times more potent than T. 5α-reductase can convert low-activity T into high-activity DHT; in this context, 5α-reductase becomes an effective target for the treatment of AGA.

[0004] Currently, commonly used 5α-reductase inhibitors in clinical practice mainly include finasteride and dutasteride. However, these drugs are only effective in male patients, and their efficacy is minimal for patients with severe hair loss where hair follicles have completely atrophied. Furthermore, finasteride and dutasteride exert their effects by regulating androgen metabolism, resulting in a slow onset of action, and long-term oral administration can easily lead to male sexual dysfunction. Therefore, there is an urgent clinical need to develop 5α-reductase inhibitors with excellent in vitro and in vivo activity and good safety profiles.

[0005] Natural products are an important source of drug lead compounds. Among them, tannins, with their good safety profile and diverse biological activities, have shown promising applications in the treatment and intervention of various types of chronic diseases. However, limited natural sources and extraction difficulties restrict their further application.

[0006] 1,2,3,4,6-Five- O -Galloyl β -D - Glucose, with a structural formula such as S1, has the effect of alleviating hair loss and can inhibit 5α-reductase in vitro. However, it can be toxic to hair follicle cells at certain concentrations. Therefore, it is necessary to develop more types of natural or natural phenolic acyl glycoside tannins that have good 5α-reductase inhibitory activity while having low toxicity to hair follicle cells. These tannins can be used to prepare drugs and cosmetics that promote hair growth and alleviate or treat hair loss. Summary of the Invention

[0007] This invention discloses a eugenol glycoside, its synthesis method, and its applications, specifically 1,2,3,4-tetra- O - Eugenoyl glycoside, and revealed its novel applications in inhibiting 5α-reductase, repairing DHT-damaged hair follicle cells, and treating AGA.

[0008] The first technical solution of this invention is a eugenol glycoside, which is a 1,2,3,4-tetra- O -Syringoglycoside; specifically, 1,2,3,4-tetra- O -Cynoyl- β - L - Rhamnose, having the structural formula shown in Formula A.

[0009] The second aspect of this invention provides a method for preparing the above-mentioned eugenol glycoside, comprising the following steps: (1) The carboxyl and hydroxyl groups of syringic acid are protected with PX to obtain compound b; where P is a hydroxyl protecting group and X is a halogen. (2) Compound b reacts with a base and an alcohol to produce compound c; (3) Compound c of formula reacts with rhamnose through esterification to produce 1,2,3,4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl β L Rhamnose; (4) 1,2,3,4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl β L Rhamnose is reduced to obtain eugenol glycoside of formula A.

[0010] Preferably, in step (1), P is benzyl, triphenylmethyl, p-methoxybenzyl, acetyl or benzoyl; more preferably, PX is benzyl halide; in some preferred embodiments of the present invention, PX is benzyl bromide (BnBr).

[0011] In step (1), the conditions for the protective reaction are: eugenol and PX react under alkaline and anaerobic conditions for 6-16 hours; preferably, the reaction temperature is 40-80℃. The alkaline is potassium carbonate, sodium carbonate, potassium bicarbonate, or sodium bicarbonate, and the molar ratio of PX to eugenol is 2-8:1, preferably 3-4. The molar ratio of PX to eugenol is 2-4:1.

[0012] In step (1), after the reaction is complete, the solid is cooled, filtered, washed, filtered, and dried.

[0013] The reaction solvent in step (1) is N,N-dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane (DCM), or acetonitrile.

[0014] The alkali mentioned in step (2) is sodium hydroxide or potassium hydroxide, and the alcohol is preferably methanol. The molar ratio of the alkali to syringic acid is 3-6:1.

[0015] In step (2), the reaction time is 6-16 hours. After the reaction, acid is added and the precipitate is filtered off. After washing, compound of formula c is obtained. Preferably, acid is added to adjust the pH to 4-5.

[0016] In step (3), rhamnose is preferably L-rhamnose, and the molar ratio of rhamnose to compound c is 1:3-8, preferably 1:5-8.

[0017] In step (3), N,N'-dicyclohexylcarbodiimide (DCC) is used as a condensing agent and 4-dimethylaminopyridine (DMAP) is used as a catalyst; the molar ratio of rhamnose to DCC and DMAP is 1:8-10:7-10.

[0018] The reaction solvent in step (3) is dichloromethane (DCM), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), or acetonitrile.

[0019] In step (3), after the reaction is completed, the filtrate is filtered, dried, and purified by column chromatography. The eluent is petroleum ether (PE) / ethyl acetate (EA) with a volume ratio of 2-5:1.

[0020] In step (3), column chromatography is used to purify and separate the α- and β-glycosyl anomeric configurations.

[0021] The reduction reaction in step (4) is a hydrogenation reaction, preferably using palladium on carbon as a catalyst.

[0022] After the reaction, the solution is filtered and the solvent is removed from the filtrate. The solution is then recrystallized or subjected to column chromatography. The crystallization solvent is 1:1 acetone / water. For column chromatography, a silica gel column or an ODS column is used, with the eluent being 1:20 methanol / dichloromethane (DCM) or 3:7 methanol / water.

[0023] The third embodiment of the present invention is the above 1, 2, 3, 4-four- O -Cynoyl- β - L - Applications of rhamnose.

[0024] 1,2,3,4-Four- O -Cynoyl- β - L Rhamnose exhibits strong inhibitory activity against 5α-reductase, taking effect even at low concentrations, and is superior to the control drug finasteride; furthermore, it has low toxicity to hair follicle cells and can be applied over a wide range of concentrations.

[0025] Therefore, the eugenol glycoside of the present invention is used to prepare 5α-reductase inhibitors; it can reduce the level of dihydrotestosterone (DHT) and can be used to prepare drugs or cosmetics that inhibit DHT levels.

[0026] The eugenol glycoside (1,2,3,4-tetra-) of this invention O -Cynoyl- β - L -Rhamnose can be used to prepare drugs or pharmaceutical ingredients for the prevention, relief, treatment, or adjunctive treatment of hair loss, or for the preparation of drugs or pharmaceutical ingredients that promote hair growth. Preferably, the eugenol glycoside is used to prepare drugs or pharmaceutical ingredients for the prevention, relief, treatment, or adjunctive treatment of androgenetic alopecia.

[0027] The medications include topical or oral dosage forms. Topical dosage forms are suitable for application to the skin (such as the scalp or body skin) and include, but are not limited to, ointments, creams, gels, sprays, films, transdermal agents, solutions, oil solutions, tinctures, lotions, powders, and foams.

[0028] The eugenol glycoside (1,2,3,4-tetra-) of this invention O -Cynoyl-β - L -Rhamnose can be used to prepare raw materials for skin care products, cosmetics, or daily chemical products that prevent hair loss, alleviate hair loss, or promote hair growth. Preferably, the eugenol glycoside is used to prepare raw materials for skin care products, cosmetics, or daily chemical products that prevent or alleviate androgenetic alopecia. The dosage forms of skin care products or cosmetics include ointments, creams, lotions, oil solutions, aqueous solutions, gels, powders, or aerosols, etc., and product types include, but are not limited to, shampoos, shampoo pastes, shampoo bars, scalp treatments, conditioners, cleansing gels, hair oils, hair masks, and serums.

[0029] Another technical solution of the present invention is a drug, pharmaceutical raw material, cosmetic or topical skin agent containing the above-mentioned eugenol glycoside (1,2,3,4-tetra- O -Cynoyl- β - L - Rhamnose). The above-mentioned eugenol glycoside is an active ingredient in drugs, pharmaceutical raw materials, cosmetics, or topical skin preparations.

[0030] The beneficial effect of this invention lies in obtaining a novel natural phenolic acyl glycoside-like tannin, 1,2,3,4-tetra-, by selecting the phenolic acyl group and sugar structure. O -Cynoyl- β - L Rhamnose, with its 5α-reductase inhibitory activity and low toxicity to hair follicle cells, lays the foundation for further development of AGA treatment and intervention drugs. Attached Figure Description

[0031] Figure 1 The 1H NMR spectrum of compound S1; Figure 2 The carbon NMR spectrum of compound S1; Figure 3 The low-resolution mass spectrum of compound S1; Figure 4 The 1H NMR spectrum of compound A; Figure 5 The carbon NMR spectrum of compound A; Figure 6 High-resolution mass spectra of compound A; Figure 7 The 1H NMR spectrum of compound IV; Figure 8 The carbon NMR spectrum of compound IV; Figure 9 High-resolution mass spectra of compound IV; Figure 10 The 1H NMR spectrum of compound V; Figure 11 The carbon NMR spectrum of compound V; Figure 12 This is the high-resolution mass spectrum of compound V. Detailed Implementation

[0032] The structural formulas of eugenol glycosides or other phenolic acid glycosides in Examples 1-4 are shown below.

[0033] Example 1: Synthesis of compound S1: Gallic acid 1a (1.70 g, 10.0 mmol, 1.0 eq.) and K₂CO₃ (8.28 g, 60.0 mmol, 6.0 eq.) were dissolved in 20.0 mL DMF. BnBr (4.76 mL, 40.0 mmol, 4.0 eq.) was added with stirring. After N₂ replacement, the mixture was reacted overnight at 60 °C. After the reaction was complete, the mixture was cooled to room temperature, filtered, and EA was added. The mixture was washed with 1 M HCl and saturated brine, dried, filtered, and then the solvent was removed by rotary evaporation under reduced pressure to obtain benzyl 3,4,5-tris(benzyloxy)benzoate 2a. Then, 20.0 mL MeOH and 20.0 mL of 2.3 M NaOH aqueous solution were added, and the mixture was refluxed overnight. After the reaction was completed, 1 M HCl was added to adjust the pH to 4-5 while stirring. After a large amount of white solid appeared, the mixture was filtered through a Buchner funnel and the filter cake was washed with H2O to obtain 3,4,5-tribenzyloxybenzoic acid 3a (3.49 g, 7.92 mmol).

[0034] Compound 3a (3.49 g, 7.92 mmol, 7.2 eq.) was added to the flask. D - Glucose 4a (198 mg, 1.10 mmol, 1.0 eq.), DCC (2.03 g, 9.90 mmol, 9.1 eq.), DMAP (1.11 g, 9.10 mmol, 8.4 eq.), and DCM (100 mL) were refluxed overnight, filtered with diatomaceous earth, evaporated to dryness under reduced pressure, and purified by silica gel column chromatography (PE:EA = 5:1~2:1) to obtain 1,2,3,4,6-penta- O -[3,4,5-tris(benzyloxy)benzoyl]- β - D - Glucose 6a (1.83 g, 0.799 mmol).

[0035] Compound 6a (1.83 g, 0.799 mmol, 1.0 eq.), 10% palladium on carbon, and 30.0 mL THF were added to a two-necked flask. After reacting overnight at 40 °C, the mixture was filtered with diatomaceous earth, and the solvent was removed under reduced pressure. The mixture was then crystallized from the acetone-H2O system to give a white solid S1 (616 mg, 0.660 mmol), with a yield of 82%. 1 H NMR (400 MHz, methanol-) d 4) δ 7.03 (s, 2H), 6.97 (s, 2H), 6.90 (s, 2H), 6.87 (s, 2H), 6.82 (s, 2H), 6.14 (d, J = 8.3 Hz, 1H), 5.81 (t, J = 9.7 Hz, 1H), 5.56 – 5.47 (m, 2H), 4.43 (d, J = 10.8 Hz, 1H),4.34 – 4.25 (m, 2H). 13 C NMR (150 MHz, methanol-) d 4) δ 168.0, 167.3, 167.1, 167.0,166.3, 146.6, 146.5, 146.5, 146.4, 146.3, 141.3, 140.7, 140.7, 140.4, 140.2,120.8, 120.1, 119.9, 119.9, 119.3, 110.6, 110.6, 110.4, 110.4, 110.3, 110.3,93.8, 74.4, 74.1, 72.2, 69.8, 63.1. LRMS (ESI-TOF) m / z: [MH] - Calculated value C 41 H 31 O 26 - 939.1, measured value 939.1. The proton NMR spectrum, carbon NMR spectrum, and low-resolution mass spectra of compound S1 are as follows: Figure 1-3 As shown.

[0036] Example 2 Synthesis of Compound A: Eugenol 1e (19.7 g, 0.1 mol, 1.0 eq.) and K₂CO₃ (41.4 g, 0.3 mol, 3.0 eq.) were dissolved in 200 mL DMF. BnBr (23.8 mL, 0.2 mol, 2.0 eq.) was added with stirring. After N₂ replacement, the mixture was reacted overnight at 60 °C. After the reaction was complete, the mixture was cooled to room temperature, filtered, and EA was added. The mixture was washed with 1 M HCl and saturated brine, dried, filtered, and then rotary evaporated under reduced pressure to remove all solvent, yielding 3,5-dimethoxy-4- O 2e-Benzylbenzoate. Then, 200 mL of MeOH and 200 mL of 2.3 M NaOH aqueous solution were added, and the mixture was refluxed overnight. After the reaction was complete, 1 M HCl was added to adjust the pH to 4-5 with stirring. After a large amount of white solid appeared, the mixture was filtered through a Buchner funnel, and the filter cake was washed with H₂O to obtain 3,5-dimethoxy-4- O 3e-benzylbenzoic acid (22.0 g, 76.5 mmol).

[0037] Compound 3e (2.28 g, 7.92 mmol, 7.2 eq.) was added to the flask. L Rhamnose 5a (198 mg, 1.10 mmol, 1.0 eq.), DCC (2.03 g, 9.90 mmol, 9.1 eq.), DMAP (1.11 g, 9.10 mmol, 8.4 eq.), and DCM (100 mL) were refluxed overnight, filtered with diatomaceous earth, evaporated to dryness under reduced pressure, and purified by silica gel column chromatography (PE:EA = 5:1~2:1) to obtain 1,2,3,4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl α L Rhamnose 7a (531 mg, 0.427 mmol) and 1,2,3,4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl β L Rhamnose 7b (509 mg, 0.409 mmol).

[0038] Compound 7b (509 mg, 0.409 mmol, 1.0 eq.), 10% palladium on carbon, and 15.0 mL THF were added to a two-necked flask. After reacting overnight at 40 °C, the mixture was filtered through diatomaceous earth, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (DCM:MeOH = 20:1, R...). f = 0.25), yielding white solid A (318 mg, 0.360 mmol), yield 88%. 1 H NMR (400 MHz, acetone- d 6) δ 8.14 (s, 4H), 7.54 (s, 2H), 7.33 (s, 2H), 7.25 (s, 2H), 7.12 (s, 2H), 6.50 (d, J = 1.2 Hz, 1H), 6.06 (dd, J = 3.3, 1.2 Hz, 1H), 5.85 (dd, J = 10.1, 3.2Hz, 1H), 5.74 (t, J = 9.8 Hz, 1H), 4.36 – 4.25 (m, 1H), 3.95 (s, 6H), 3.85 (s, 6H), 3.72 (s, 6H), 3.65 (s, 6H), 1.43 (d, J = 6.1 Hz, 3H). 13 C NMR (150 MHz, acetone- d 6) δ 166.4, 166.1, 165.6, 164.2, 148.6, 148.3, 148.2, 142.3, 142.2,142.1, 141.9, 120.5, 120.3, 120.0, 119.7, 108.2, 108.2, 108.1, 107.8, 91.9,72.6, 72.4, 71.6, 71.3, 56.7, 56.6, 56.3, 56.3, 18.1. HRMS (ESI-TOF) m / z: [MH] - Calculated value C 42 H 43 O 21 -883.2302, measured value 883.2294. The proton NMR spectrum, carbon NMR spectrum, and low-resolution mass spectra of compound A are as follows: Figure 4-6 As shown.

[0039] Example 3 Synthesis of Compound IV: Compound 3e (2.28 g, 7.92 mmol, 7.2 eq.) was added to the flask. D Xylose 5c (198 mg, 1.10 mmol, 1.0 eq.), DCC (2.03 g, 9.90 mmol, 9.1 eq.), DMAP (1.11 g, 9.10 mmol, 8.4 eq.), and DCM (100 mL) were refluxed overnight, filtered with diatomaceous earth, evaporated to dryness under reduced pressure, and purified by silica gel column chromatography (PE:EA = 5:1~2:1) to obtain 1,2,3,4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl α D Xylose 7d (496 mg, 0.403 mmol) and 1, 2, 3, 4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl β D Xylose 7e (544 mg, 0.442 mmol).

[0040] Compound 7d (496 mg, 0.403 mmol, 1.0 eq.), 10% palladium on carbon, and 15.0 mL THF were added to a two-necked flask. After reacting overnight at 40 °C, the mixture was filtered through diatomaceous earth, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (DCM:MeOH = 20:1, R...). f = 0.25), to give white solid IV (312 mg, 0.359 mmol), yield 89%.1 H NMR (400 MHz, acetone- d 6) δ 8.12 (s, 4H), 7.50 (s, 2H), 7.30 (d, J = 7.9 Hz, 4H), 7.16 (s, 2H), 6.76(d, J = 3.6 Hz, 1H), 6.31 (t, J = 10.0 Hz, 1H), 5.63 (dd, J = 10.1, 3.5 Hz, 1H), 5.53 (td, J = 10.3, 5.8 Hz, 1H), 4.36 (dd, J = 11.2, 5.8 Hz, 1H), 4.20 (t, J = 11.0Hz, 1H), 3.95 (s, 6H), 3.85 (s, 6H), 3.81 (s, 6H), 3.67 (s, 6H). 13 C NMR (150MHz, acetone-) d 6) δ 166.6, 165.9, 165.6, 164.8, 148.6, 148.3, 148.2, 142.5,142.0, 120.2, 120.0, 120.0, 119.7, 108.4, 108.2, 108.1, 107.9, 91.2, 71.5,71.2, 70.3, 62.2, 56.7, 56.6, 56.6, 56.3. HRMS (ESI-TOF) m / z: [MH] - Calculated value C 41 H 41 O 21 - 869.2146, measured value 869.2141. The proton NMR spectrum, carbon NMR spectrum, and low-resolution mass spectra of compound IV are as follows: Figure 7-9 As shown.

[0041] Example 4 Synthesis of Compound V Compound 7e (544 mg, 0.442 mmol 1.0 eq.), 10% palladium on carbon, and 15.0 mL THF were added to a two-necked flask. After reacting overnight at 40 °C, the mixture was filtered through diatomaceous earth, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (DCM:MeOH = 20:1, R...). f= 0.25), to give a white solid V (315 mg, 0.362 mmol), yield 82%. 1 H NMR (400 MHz, acetone- d 6) δ8.14 (s, 4H), 7.34 (s, 2H), 7.29 (d, J = 5.7 Hz, 4H), 7.25 (s, 2H), 6.24 (d, J =7.7 Hz, 1H), 6.06 (t, J = 9.4 Hz, 1H), 5.71 (dd, J = 9.7, 7.7 Hz, 1H), 5.40 (td, J = 9.4, 5.4 Hz, 1H), 4.51 (dd, J = 11.6, 5.4 Hz, 1H), 4.08 – 3.95 (m, 1H), 3.88(s, 6H), 3.85 (s, 6H), 3.80 (d, J = 5.1 Hz, 12H). 13 C NMR (150 MHz, acetone- d 6) δ166.4, 166.1, 166.1, 165.1, 148.6, 148.5, 148.4, 142.6, 142.3, 142.3, 120.3,120.2, 120.1, 119.7, 108.4, 108.3, 94.4, 73.1, 72.0, 71.2, 68.1, 64.3, 56.7,26.2. HRMS (ESI-TOF) m / z: [MH] - Calculated value C 41 H 41 O 21 - 869.2146, measured value 869.2141. The proton NMR spectrum, carbon NMR spectrum, and low-resolution mass spectra of compound V are as follows: Figure 10-12 As shown.

[0042] Example 5 The in vitro 5α-reductase inhibitory activity of the acyl glycoside tannins synthesized in Examples 1-4 was detected. The experimental steps and results are as follows: 1. Instruments and Materials Dimethyl sulfoxide (DMSO); phosphate-buffered saline (PBS); 5α-reductase; NADPH solution; testosterone solution; multi-wavelength microplate reader; 96-well plate; centrifuge tubes, etc.

[0043] 2. Experimental Methods: The compounds synthesized in the above examples were dissolved in DMSO. Based on the molecular weight of the compounds, sample working solutions were prepared sequentially (the concentrations of compounds S1, A, IV, V, and the positive control finasteride were all 0.02 wt%). 1 mL of enzyme solution, 1 mL of NADPH solution, and 1 mL of testosterone solution were added to a test tube, which was then divided into sample tubes and enzyme tubes. 1 mL of the corresponding sample working solution and PBS solution were added to each tube, and the mixture was gently shaken. 200 μL of each solution was added to a 96-well plate and placed in a microplate reader for detection. The absorbance was measured at 340 nm and recorded as A. 样品0 Or A 酶0 After incubating at 37 ℃ for 20 min, the sample was tested again, and A was obtained. 样品20 Or A 酶20 Calculate the inhibition rate.

[0044] Table 1. 5α-Reductase Inhibitory Activity of Phenolic Acyl Glycosides The results of 5α-reductase inhibitory activity are shown in Table 1. It can be seen that: 1, 2, 3, 4, 6-5- O -Galloyl- β - D - Glucose (natural, S1), 1,2,3,4-tetra- O -Cynoyl- β - L Rhamnose (non-natural, A) exhibits significantly superior inhibitory activity against 5α-reductase compared to the clinically used drug finasteride.

[0045] Example 6 This embodiment evaluates the safety of the eugenol glycoside tannin molecules selected in Example 5 at the cellular level. The experimental steps are as follows: 1. Instruments and Materials Cell lines: Human dermal papilla cells (HDPCs); HDPCs complete culture medium; trypsin; phosphate-buffered saline (PBS); dimethyl sulfoxide (DMSO); CCK-8 reagent; multi-wavelength microplate reader; 96-well plate; centrifuge tubes, etc.

[0046] 2. Experimental Methods (1) Take HDPCs in the logarithmic growth phase with a confluence of 70% to 80%, discard the culture medium, and wash twice with PBS. Add an appropriate amount of trypsin and digest in a 37 ℃ incubator. When the cells become round and begin to detach, add HDPCs-specific complete culture medium to stop the digestion. Repeatedly pipette to prepare a single-cell suspension, transfer to a centrifuge tube and centrifuge. Discard the supernatant, add HDPCs-specific complete culture medium to resuspend the cells, pipette to mix well, and prepare a single-cell suspension of appropriate concentration.

[0047] (2) Seed the above cell suspension into 96-well culture plates, 100 μL per well. Also include control wells (without drug) and blank wells containing only culture medium. During plating, intermittently mix the cell suspension. After plating, gently shake the plate, add PBS to the outermost ring to prevent edge effects, and incubate overnight at 37 ℃ in a 5% CO2 incubator for 24 hours.

[0048] (3) Calculate and weigh appropriate amounts of each compound based on its molecular weight, and add DMSO to prepare a stock solution. The next day, dilute the stock solution with HDPCs-specific complete culture medium to prepare drug working solutions of different concentrations.

[0049] (4) Remove the culture plate and discard the old culture medium (operate row by row to prevent cells from drying out). Add HDPCs-specific complete culture medium and different concentrations of drug working solution respectively. Place the culture plate back into a 37 ℃, 5% CO2 incubator and continue culturing for 72 hours.

[0050] (5) After culturing for 72 hours, observe the cell state under an inverted microscope. Prepare CCK-8 working solution by mixing CCK-8 reagent and HDPCs-specific complete culture medium at a volume ratio of 1:10. Discard the old culture medium, add CCK-8 working solution to each well (add an equal amount of working solution to the blank zeroing well), and incubate in the dark for 1.5 to 2 hours. After the colorimetric reaction is complete, measure the absorbance (OD value) of each well at a wavelength of 450 nm using a microplate reader and record the experimental data.

[0051] 3. Specific experimental results The results are shown in Table 2. Compounds S1 and A exhibited concentration-dependent cytotoxicity against human dermal papilla cells in the range of 2–16 μM. At a low concentration of 0.1 μM, cell viability in all groups was close to 100%, with no significant cell damage. Cell viability decreased significantly from 2 μM onwards, and the toxicity continued to increase with increasing concentration. Among them, the natural phenolic acyl glycoside S1 was more toxic, with a significant decrease in cell viability at 16 μM. In contrast, the eugenol rhamnoside A of this invention showed weaker cytotoxicity and superior safety.

[0052] Table 2 Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

[0053] This invention designs and synthesizes a natural syringoyl glycoside tannin by forming a covalent ester bond between syringoyl and rhamnose, and evaluates its 5α-reductase inhibitory activity and cytotoxicity, laying the foundation for further development of AGA treatment and intervention drugs.

Claims

1. A eugenol glycoside, characterized in that, For 1, 2, 3, 4 - four - O -Syringoglycoside, having the following structural formula: 。 2. The method for preparing the eugenol glycoside according to claim 1, characterized in that, Includes the following steps: (1) The carboxyl and hydroxyl groups of syringic acid are protected with PX to obtain compound b; where P is a hydroxyl protecting group and X is a halogen. (2) Compound b reacts with a base in an alcohol solvent to produce compound c; (3) Compound c of formula reacts with rhamnose through esterification to produce 1,2,3,4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl β L Rhamnose; (4) 1,2,3,4 Four O (3,5 Dimethoxy 4 O Benzylbenzoyl β L Rhamnose is reduced to obtain the eugenol glycoside described in claim 1.

3. The method for preparing eugenol glycoside according to claim 2, characterized in that, The reduction reaction in step (4) is a catalytic hydrogenation reaction.

4. The method for preparing eugenol glycoside according to claim 2, characterized in that, The hydroxyl protecting group P mentioned in step (1) is benzyl, triphenylmethyl, p-methoxybenzyl, acetyl or benzoyl.

5. The use of the eugenol glycoside according to claim 1 in the preparation of 5α-reductase inhibitors or in the preparation of drugs that inhibit dihydrotestosterone levels.

6. The use of the eugenol glycoside according to claim 1 in the preparation of a medicament for treating or adjuvant treatment of hair loss, promoting hair follicle cell proliferation, or promoting hair growth.

7. The use of the eugenol glycoside according to claim 1 in the preparation of raw materials for skin care products, cosmetics or daily chemical products that slow down hair loss, alleviate hair loss or promote hair growth.

8. A drug, pharmaceutical ingredient, cosmetic, or topical skin agent, characterized in that, It contains the eugenol glycoside as described in claim 1.

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