Asiatic-acid-derived ceramide, and synthesis method therefor and use thereof

Abstract

The present invention belongs to the technical field of biomedicine. Disclosed is an asiatic-acid-derived ceramide, which has the structure of general formula (I) or an isomer of general formula I, wherein R1 is selected from one of the following structures in (II), and R2 is hydroxyl or a hydrogen atom. Further disclosed in the present invention are a method for synthesizing the asiatic-acid-derived ceramide and the use thereof. By means of the present invention, the solubility of ceramide is improved by means of introducing an asiatic acid or madecassic acid fragment with good fat solubility, thereby facilitating the use of ceramide in formulations. The compound of the present invention exhibits a significant inhibitory effect on inflammatory cytokine interleukin-6 (IL-6), and can also suppress the overexpression of TRPV1 receptors in sensitive skin and significantly reduce the expression of hyaluronidase, thereby achieving a good soothing effect.

Description

Ceramide derived from asiatic acid and its synthesis method and application TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to ceramide derived from asiatic acid and its synthesis method and application. BACKGROUND

[0002] Snow lotus is also known as Leigonggen and old tiger grass, and its Roman name is CICA (from the word root of cicatrisation). The main producing areas are Guangxi, Yunnan, Guizhou and Madagascar. These areas have long sunshine time, large diurnal temperature difference, unique climate and geographical environment, and are the producing areas of high-quality snow lotus. In China, snow lotus has been used as a medicinal herb for a long time, and its components such as asiaticoside, madecassoside, asiatic acid and hydroxyl asiatic acid are used to eliminate scars and resist oxidation.

[0003] Asiatic acid is a natural pentacyclic triterpenoid compound extracted from snow lotus, is a polyhydroxy acid, and is one of the effective components of snow lotus. Hydroxyl asiatic acid is one of the secondary components in snow lotus, and its structure contains a hydroxyl group based on asiatic acid. Studies have shown that other active substances in snow lotus, such as asiaticoside, mediate its therapeutic effect by being converted into asiatic acid through hydrolytic cleavage of the sugar moiety in the body; hydroxyl asiaticoside also exerts its biological activity by being converted into the aglycone hydroxyl asiatic acid through a similar conversion.

[0004] Ceramide is a class of compounds composed of long-chain sphingosine base and fatty acid, and the carbon chain length, unsaturation and number of hydroxyl groups of the sphingosine base and fatty acid parts can be varied. Ceramide is the main component of lipids in the extracellular matrix of the epidermal layer of human skin, accounting for about 50%. Ceramide, together with cholesterol and saturated fatty acid, produces a water-impermeable protective structure to prevent excessive water evaporation and also prevent the entry of microorganisms.

[0005] Due to the importance of ceramide, there is a wide demand for functional ceramide in the market. However, the existing ceramide oil is insoluble in water, which poses certain difficulties in its application in formulations. In many products, only a small amount of ceramide is added conceptually, which is difficult to exert the true function of ceramide. Therefore, it is of great development value to prepare new ceramide derivatives in order to improve the solubility of ceramide, make its application more convenient, and improve its other biological activities. SUMMARY

[0006] The purpose of the present application is to provide a novel ceramide, i.e. ceramide derived from asiatic acid.

[0007] Another object of the present application is to provide a synthetic method of the Gynostemma pentaphyllum acid-derived ceramide.

[0008] Another object of the present application is to provide the use of the Gynostemma pentaphyllum acid-derived ceramide.

[0009] To achieve one of the above objects, the present application adopts the following technical solutions:

[0010] In the first aspect of the present application, the Gynostemma pentaphyllum acid-derived ceramide has the structure of general formula I or isomers of general formula I:

[0011] wherein R 1 is selected from one of the following structures:

[0012] R 2 is a hydroxyl group or a hydrogen atom.

[0013] Further, the Gynostemma pentaphyllum acid-derived ceramide is one of the following structures:

[0014] Gynostemma pentaphyllum acid phytosphingosine Gynostemma pentaphyllum acid phytosphingosine

[0015] Gynostemma pentaphyllum acid phytosphingosine Gynostemma pentaphyllum acid phytosphingosine Gynostemma pentaphyllum acid phytosphingosine Gynostemma pentaphyllum acid phytosphingosine Gynostemma pentaphyllum acid phytosphingosine Gynostemma pentaphyllum acid phytosphingosine Gynostemma pentaphyllum acid phytosphingosine

[0016] Gynostemma pentaphyllum acid phytosphingosine

[0017] In the second aspect of the present application, a synthetic method of the Gynostemma pentaphyllum acid-derived ceramide comprises the following steps:

[0018] P1, reacting compound M1 and acetic anhydride, an organic base to obtain compound M2;

[0019] P2, reacting compound M2 and sphingosine base, a condensing agent, an organic base to obtain compound M3;

[0020] P3, reacting compound M3 and methanolic ammonia solution to obtain compound I.

[0021] Further, the organic base of P1 and P2 is diisopropylethylamine.

[0022] Further, the condensing agent is EDCI and HOBT.

[0023] Further, the molar ratio of the compound M1, acetic anhydride, and diisopropylethylamine is 1:(4-6):(4-6).

[0024] Further, the molar ratio of the compound M2, sphingosine base, EDCI, HOBT, diisopropylethylamine is 1:(1.1-1.5):(1.2-2):(1.2-2):(1.2-2).

[0025] Further, the solvent of P1 is dichloromethane.

[0026] Further, the solvent of P2 is tetrahydrofuran.

[0027] In a third aspect of the present application, the ceramide derived from asiatic acid has anti-inflammatory and soothing effects, and can be used in cosmetics, health products, or in the preparation of pharmaceutical products.

[0028] A composition comprising, as an active ingredient, a ceramide derived from asiatic acid, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof.

[0029] As used herein, the "compound of structure of Formula I" has multiple chiral centers, and thus includes chiral compounds of the structure, i.e., enantiomers and diastereomers, such as the asiatic acid phytosphingosine includes , etc.

[0030] As used herein, "isomers" means that the compounds of the present application or salts thereof have the same chemical formula or molecular formula but have different optical or spatial properties, including enantiomers, diastereomers, and cis-trans isomers.

[0031] As used herein, "pharmaceutically acceptable salt" means a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, hydroxymalonic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methyldicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; or (2) salts formed when an acidic proton present in the parent compound is replaced by a substituent such as alkali metal ions, for example, sodium and potassium; alkaline earth metal ions, for example, calcium and magnesium; and aluminum and zinc; or organic amine ions, for example, N-methyl-glucamine, 1-ephenamine, and 1 -hydroxy- ethylidene-1, 1 -aminoethane-3-carboxylic acid.

[0032] As used herein, "hydrate" means a compound that is combined with water. The combination between the compound and water includes non-covalent combination.

[0033] As used herein, "solvate" means a complex formed by a solute molecule or ion and a solvent molecule or ion.

[0034] Unless otherwise indicated, the term "compound of the present application" or "ceramide" includes the compound itself, its pharmaceutically acceptable salt, its hydrate, its solvate, its isomer.

[0035] The present application has the following beneficial effects:

[0036] 1. The present application reacts centella asiatica acid, hydroxyl centella asiatica acid with sphingosine, phytosphingosine, dihydrosphingosine, 6-hydroxy sphingosine to obtain a novel structure of ceramide compound.

[0037] 2. The present application improves the solubility of ceramide by introducing the centella asiatica acid or hydroxyl centella asiatica acid segment with better fat solubility, making it more convenient to use in the formula. Compared with traditional ceramide NP, the compound of the present application has better solubility.

[0038] 3. The compound of the present application has obvious inhibitory effect on interleukin 6 (IL-6) inflammatory factor, can also inhibit the overexpression of sensitive skin TRPV1 receptor, can obviously reduce the expression of hyaluronidase, and has good soothing effect. BRIEF DESCRIPTION OF DRAWINGS

[0039] Fig. 1 is a column chart of cell proliferation activity detection results of Example 10;

[0040] Fig. 2 is a column chart of IL-6 factor expression amount detection results of Example 11;

[0041] Fig. 3 is a column chart of TRPV1 factor expression amount detection results of Example 12;

[0042] Fig. 4 is a column chart of hyaluronidase inhibition rate detection results of Example 13. DETAILED DESCRIPTION

[0043] The present application will be further described below in combination with specific examples.

[0044] Unless otherwise indicated, chemicals were purchased from commercial suppliers and used without further purification. Dichloromethane and tetrahydrofuran used in the experiments were anhydrous solvents. Thin layer chromatography (TLC) was performed on 60F254 silica gel plates. Silica gel column chromatography was performed using Qingdao Marine silica gel (particle size 0.040-0.063 mm). TLC was visualized using UV light (254 nm) or iodine. NMR spectra were recorded using a Bruker DPX 400 NMR spectrometer, 1 H NMR was 400 MHz, the solvent was deuterated methanol or deuterated chloroform, and tetramethylsilane (TMS) was used as an internal standard. The unit of chemical shift was ppm, and the unit of coupling constant was Hz. In 1 In H NMR, δ represents chemical shift, s represents singlet, d represents doublet, t represents triplet, q represents quartet, and m represents multiplet.

[0045] EDCI refers to 1-ethyl-(3-dimethylaminopropyl)carbodiimide, HOBt refers to 1-hydroxybenzotriazole, DCM refers to dichloromethane, THF refers to tetrahydrofuran, and DIPEA refers to N,N-diisopropylethylamine.

[0046] Example 1

[0047] Synthesis of phytosphingosine with asiatic acid

[0048] Under nitrogen protection, asiatic acid 1.0 eq (40 mmol) and DIPEA 4.5 eq (180 mmol) were dissolved in 150 mL of dichloromethane, and acetic anhydride 4.5 eq (180 mmol) was dissolved in 50 mL of dichloromethane, which was added dropwise to the above reaction solution at room temperature. The reaction was carried out at room temperature for 8 h under nitrogen protection until TLC detection showed that the reaction of asiatic acid was complete. Post-treatment: extraction with 150 mL of water twice, extraction with 150 mL of saturated brine twice, collection of the organic phase, drying with anhydrous magnesium sulfate, filtration, and vacuum concentration to obtain S2 crude product, which was directly used in the next step reaction.

[0049] Under nitrogen protection, S2 1.0 eq (10 mmol), EDCI 1.5 eq (15 mmol), HOBT 1.5 eq (15 mmol), and DIPEA 1.5 eq (15 mmol) were added to a 250 mL round-bottom flask, 100 mL of tetrahydrofuran was added, and stirring was carried out at room temperature for 1 h. Then phytosphingosine 1.2 eq (12 mmol) was added to the reaction system, and stirring was carried out at room temperature for 28 h until TLC detection showed that S2 completely disappeared. Post-treatment: quenching with 60 mL of water, extraction with 60 mL of saturated brine twice, separation of the organic layer, drying with anhydrous magnesium sulfate, filtration, and vacuum concentration to obtain S3 crude product.

[0050] S3 1.0 eq (7 mmol), ammonium methanol solution 5.0 eq (35 mmol) were added to a 100 mL round bottom flask and allowed to react at room temperature for 2 hours until TLC indicated complete consumption of S3. Work-up: 50 mL of tetrahydrofuran were added, extracted once with 60 mL of water and once with 60 mL of saturated brine, separated the organic layer, dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum to obtain the crude product, which was purified by column chromatography to obtain the product, asparagus acid phytosphingosine.

[0051] Asparagus acid phytosphingosine, 45% yield.

[0052] 1 H NMR (400 MHz, Methanol-d4) δ 5.36 (t, J = 4.1 Hz, 1H), 4.06 (q, J = 4.6 Hz, 1H), 3.72 (qd, J = 10.9, 4.6 Hz, 3H), 3.64 - 3.55 (m, 2H), 3.53 (d, J = 11.1 Hz, 1H), 3.38 (d, J = 9.6 Hz, 1H), 3.28 (d, J = 11.0 Hz, 1H), 2.20 - 1.94 (m, 5H), 1.90 - 1.82 (m, 1H), 1.79 - 1.60 (m, 6H), 1.60 - 1.23 (m, 32H), 1.17 (s, 3H), 1.08 (s, 5H), 1.03 - 0.97 (m, 3H), 0.97 - 0.90 (m, 7H), 0.88 (s, 3H), 0.72 (s, 3H).

[0053] Example 2

[0054] Synthesis of asparagus acid phytosphingosine

[0055] S2 1.0 eq (10 mmol), EDCI 1.6 eq (16 mmol), HOBT 1.6 eq (16 mmol) and DIPEA 1.6 eq (16 mmol) were added to a 250 mL round bottom flask under nitrogen protection, 100 mL of tetrahydrofuran were added, stirred at room temperature for 1 hour, then phytosphingosine 1.4 eq (14 mmol) was added to the reaction system, stirred at room temperature for 24 hours until TLC indicated complete disappearance of S2. Work-up: 60 mL of water were added to quench, extracted twice with 60 mL of saturated brine, separated the organic layer, dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum to obtain the crude product S4.

[0056] S4 1.0 eq (6 mmol), ammoniacal methanol solution 5.0 eq (30 mmol) were added to a 100 mL round bottom flask and allowed to react at room temperature for 2 hours until TLC indicated complete consumption of S4. Work-up: 50 mL of tetrahydrofuran were added, extracted once with 50 mL of water and once with 50 mL of saturated brine, separated the organic layer, dried over anhydrous magnesium sulfate, filtered and concentrated under vacuum to obtain the crude, which was purified by column chromatography to obtain the product sphingosine bryonolic acid.

[0057] Sphingosine bryonolic acid, 40% yield.

[0058] 1 H NMR (400 MHz, Methanol-d4) δ 6.87 (d, J = 7.7 Hz, 1H), 5.80 - 5.69 (m, 1H), 5.52 (ddt, J = 15.3, 6.7, 1.5 Hz, 1H), 5.33 (t, J = 3.6 Hz, 1H), 4.18 (t, J = 6.3 Hz, 1H), 3.87 - 3.66 (m, 3H), 3.66 - 3.58 (m, 1H), 3.53 (d, J = 11.1 Hz, 1H), 3.38 (d, J = 9.6 Hz, 1H), 3.29 (d, J = 11.0 Hz, 1H), 2.18 (d, J = 10.5 Hz, 1H), 2.15 - 1.94 (m, 6H), 1.88 (td, J = 13.7, 4.3 Hz, 1H), 1.68 (ddt, J = 13.2, 9.7, 4.6 Hz, 4H), 1.61 - 1.25 (m, 31H), 1.17 (s, 3H), 1.13 - 1.06 (m, 4H), 0.99 (s, 3H), 0.93 (d, J = 6.3 Hz, 6H), 0.90 (d, J = 5.4 Hz, 4H), 0.72 (s, 3H).

[0059] Example 3

[0060] Synthesis of dihydro sphingosine bryonolic acid

[0061] S2 1.0 eq (10 mmol), EDCI 1.4 eq (14 mmol), HOBT 1.4 eq (14 mmol) and DIPEA 1.4 eq (14 mmol) were added to a 250 mL round bottom flask under nitrogen protection, 100 mL tetrahydrofuran was added, stirred at room temperature for 1 hour, then dihydrosphingosine 1.3 eq (13 mmol) was added to the reaction system, stirred at room temperature for 22 hours until TLC detection S2 completely disappeared. Post-processing: 70 mL water was added to quench, 70 mL saturated brine was extracted twice, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated under vacuum to obtain S5 crude product.

[0062] S5 1.0 eq (5 mmol), ammonia methanol solution 5.0 eq (25 mmol) were added to a 100 mL round bottom flask, reacted at room temperature for 2 hours until TLC detection S5 was completely consumed. Post-processing: 50 mL tetrahydrofuran was added, extracted with 50 mL water once, 50 mL saturated brine once, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated under vacuum to obtain crude product, purified by column chromatography to obtain the product dihydrosphingosine of asiatic acid.

[0063] Dihydrosphingosine of asiatic acid, 33% yield.

[0064] 1 H NMR (400 MHz, Methanol-d4) δ 6.99 (d, J = 8.0 Hz, 1H), 5.35 (t, J = 3.7 Hz, 1H), 3.84 - 3.61 (m, 5H), 3.53 (d, J = 11.1 Hz, 1H), 3.38 (d, J = 9.5 Hz, 1H), 3.29 (d, J = 11.0 Hz, 1H), 2.25 - 1.83 (m, 6H), 1.81 - 1.37 (m, 15H), 1.32 (d, J = 6.8 Hz, 26H), 1.18 (s, 3H), 1.07 (s, 5H), 0.99 (d, J = 5.8 Hz, 3H), 0.97 - 0.89 (m, 7H), 0.88 (s, 3H), 0.72 (s, 3H).

[0065] Example 4

[0066] Synthesis of asiatic acid-6-hydroxysphingosine

[0067] S2 1.0 eq (10 mmol), EDCI 1.8 eq (18 mmol), HOBT 1.8 eq (18 mmol) and DIPEA 1.8 eq (18 mmol) were added to a 250 mL round bottom flask under nitrogen protection, 100 mL tetrahydrofuran was added, stirred at room temperature for 1 hour, then 6-hydroxysphingosine 1.4 eq (14 mmol) was added to the reaction system, stirred at room temperature for 24 hours until TLC detection S2 completely disappeared. Post-processing: 60 mL water was added to quench, 60 mL saturated brine was extracted twice, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated under vacuum to obtain S6 crude product.

[0068] S6 1.0 eq (6 mmol), 5.0 eq (30 mmol) of methanolic ammonia solution was added to a 100 mL round bottom flask, reacted at room temperature for 2 hours until TLC detection S6 was completely consumed. Post-processing: 50 mL tetrahydrofuran was added, extracted with 50 mL water once, extracted with 50 mL saturated brine once, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated under vacuum to obtain crude product, purified by column chromatography to obtain the product asiatic acid-6-hydroxysphingosine.

[0069] Asiatic acid-6-hydroxysphingosine, 38% yield.

[0070] 1 H NMR (400 MHz, Methanol-d4) δ 6.82 (d, J = 7.7 Hz, 1H), 5.90 - 5.82 (m, 1H), 5.72 - 5.66 (m, 1H), 5.33 (t, J = 3.6 Hz, 1H), 4.34 - 4.29 (m, 1H), 4.10 (t, J = 6.1 Hz, 1H), 3.92 - 3.76 (m, 3H), 3.62 - 3.53 (m, 1H), 3.48 (d, J = 10.8 Hz, 1H), 3.30 (d, J = 9.4 Hz, 1H), 3.26 (d, J = 10.8 Hz, 1H), 2.14 (d, J = 10.2 Hz, 1H), 2.11 - 1.97 (m, 6H), 1.82 - 1.74 (m, 1H), 1.68 - 1.52 (m, 4H), 1.51 - 1.28 (m, 29H), 1.14 (s, 3H), 1.10 - 1.04 (m, 4H), 0.96 (s, 3H), 0.88 (d, J = 6.4 Hz, 6H), 0.86 (d, J = 5.4 Hz, 4H), 0.69 (s, 3H).

[0071] Example 5

[0072] Synthesis of hydroxyl asiatic acid phytosphingosine

[0073] Under nitrogen protection, hydroxyl asiatic acid 1.0 eq (30 mmol), DIPEA 5.5 eq (165 mmol) were dissolved in 150 ml dichloromethane, acetic anhydride 5.5 eq (165 mmol) was dissolved in 50 ml dichloromethane, and then added dropwise to the above reaction solution at room temperature. The reaction was carried out at room temperature for 10 h under nitrogen protection until the hydroxyl asiatic acid reaction was completed by TCL detection. Post-processing: 150 mL of water was added and extracted twice, 150 mL of saturated brine was added and extracted twice, the organic phase was collected, dried with anhydrous magnesium sulfate, filtered and concentrated under vacuum to obtain S7 crude product, which was directly used in the next step reaction.

[0074] Under nitrogen protection, S7 1.0 eq (8 mmol), EDCI 1.5 eq (12 mmol), HOBT 1.5 eq (15 mmol) and DIPEA 1.5 eq (15 mmol) were added to a 250 mL round-bottom flask, 100 mL of tetrahydrofuran was added, and stirred at room temperature for 1 h. Then phytosphingosine 1.2 eq (9.6 mmol) was added to the reaction system, and stirred at room temperature for 20 h until the S7 completely disappeared by TLC detection. Post-processing: 60 mL of water was added to quench, 60 mL of saturated brine was extracted twice, the organic layer was separated, dried with anhydrous magnesium sulfate, filtered and concentrated under vacuum to obtain S8 crude product.

[0075] S8 1.0 eq (5 mmol) and ammonia methanol solution 6.0 eq (30 mmol) were added to a 100 mL round-bottom flask, and reacted at room temperature for 2 h until the S8 was completely consumed by TLC detection. Post-processing: 50 mL of tetrahydrofuran was added, extracted with 60 mL of water once and 60 mL of saturated brine once, the organic layer was separated, dried with anhydrous magnesium sulfate, filtered and concentrated under vacuum to obtain the crude product, which was purified by column chromatography to obtain the product hydroxyl asiatic acid phytosphingosine.

[0076] Hydroxyl asiatic acid phytosphingosine, 35% yield.

[0077] 1H NMR (400 MHz, Methanol-d4) δ 5.41 (t, J = 3.6 Hz, 1H), 4.61 (s, 1H), 4.40 (s, 1H), 4.07 (p, J = 4.7, 4.2 Hz, 1H), 3.73 (ddt, J = 20.3, 9.4, 5.6 Hz, 3H), 3.65 - 3.53 (m, 3H), 3.46 (d, J = 11.1 Hz, 1H), 3.30 (d, J = 2.5 Hz, 1H), 2.21 - 1.48 (m, 16H), 1.42 (d, J = 6.3 Hz, 4H), 1.31 (s, 24H), 1.25 - 1.16 (m, 3H), 1.13 (d, J = 3.3 Hz, 5H), 1.08 (s, 4H), 1.01 - 0.88 (m, 10H).

[0078] Example 6

[0079] Synthesis of hydroxyl asiatic acid sphingosine

[0080] S7 1.0 eq (8 mmol), EDCI 1.5 eq (12 mmol), HOBT 1.5 eq (12 mmol) and DIPEA 1.5 eq (15 mmol) were added into a 250 mL round bottom flask, 100 mL tetrahydrofuran was added, stirred at room temperature for 1 hour, then sphingosine 1.4 eq (11.2 mmol) was added into the reaction system, stirred at room temperature for 30 hours until TLC detection S7 completely disappeared. Post-processing: 60 mL water was added to quench, 60 mL saturated brine was extracted twice, the organic layer was separated, anhydrous magnesium sulfate was added for drying, filtered and concentrated under vacuum to obtain S9 crude product.

[0081] S9 1.0 eq (5 mmol), methanolic ammonia solution 6.0 eq (30 mmol) were added into a 100 mL round bottom flask, reacted at room temperature for 2 hours until TLC detection S9 was completely consumed. Post-processing: 50 mL tetrahydrofuran was added, 50 mL water was extracted once, 50 mL saturated brine was extracted once, the organic layer was separated, anhydrous magnesium sulfate was added for drying, filtered and concentrated under vacuum to obtain crude product, which was purified by column chromatography to obtain the product hydroxyl asiatic acid sphingosine.

[0082] Hydroxyl asiatic acid sphingosine, 40% yield.

[0083] 1H NMR (400 MHz, Methanol-d4) δ 6.89 (dd, J = 15.1, 7.8 Hz, 1H), 5.80 - 5.68 (m, 1H), 5.52 (ddd, J = 15.4, 6.9, 4.3 Hz, 1H), 5.38 - 5.36 (m, 1H), 4.40 (s, 1H), 4.17 (dt, J = 12.3, 6.5 Hz, 1H), 3.91 - 3.54 (m, 5H), 3.45 (d, J = 11.1 Hz, 1H), 3.30 (d, J = 2.7 Hz, 1H), 2.22 - 1.47 (m, 16H), 1.42 (d, J = 6.6 Hz, 5H), 1.31 (s, 23H), 1.21 - 1.06 (m, 10H), 1.01 - 0.85 (m, 11H).

[0084] Example 7

[0085] Synthesis of hydroxyl asiatic acid dihydrosphingosine

[0086] S7 1.0 eq (8 mmol), EDCI 1.5 eq (12 mmol), HOBT 1.5 eq (12 mmol) and DIPEA 1.5 eq (15 mmol) were added into a 250 mL round bottom flask, 100 mL tetrahydrofuran was added, stirred at room temperature for 1 hour, then dihydrosphingosine 1.3 eq (10.4 mmol) was added into the reaction system, stirred at room temperature for 32 hours until TLC detection S7 completely disappeared. Post-processing: 60 mL water was added to quench, 60 mL saturated brine was extracted twice, the organic layer was separated, anhydrous magnesium sulfate was added for drying, filtered and concentrated under vacuum to obtain S10 crude product.

[0087] S10 1.0 eq (5 mmol), methanolic ammonia solution 6.0 eq (30 mmol) were added into a 100 mL round bottom flask, reacted at room temperature for 2 hours until TLC detection S10 was completely consumed. Post-processing: 50 mL tetrahydrofuran was added, extracted with 50 mL water once, extracted with 50 mL saturated brine once, the organic layer was separated, anhydrous magnesium sulfate was added for drying, filtered and concentrated under vacuum to obtain a crude product, which was purified by column chromatography to obtain the product hydroxyl asiatic acid dihydrosphingosine.

[0088] Hydroxyl asiatic acid dihydrosphingosine, 30% yield.

[0089] 1H NMR (400 MHz, Methanol-d4) δ 7.00 (dd, J = 14.1, 7.9 Hz, 1H), 5.43 - 5.35 (m, 1H), 4.40 (s, 1H), 3.86 - 3.71 (m, 3H), 3.67 (dt, J = 10.6, 3.6 Hz, 2H), 3.60 (d, J = 11.1 Hz, 1H), 3.45 (d, J = 11.1 Hz, 1H), 3.30 (d, J = 2.5 Hz, 1H), 2.23 - 2.04 (m, 3H), 2.04 - 1.77 (m, 4H), 1.75 - 1.66 (m, 3H), 1.63 - 1.48 (m, 5H), 1.42 (d, J = 5.6 Hz, 4H), 1.31 (s, 26H), 1.21 - 1.17 (m, 2H), 1.16 - 1.11 (m, 5H), 1.08 (s, 5H), 0.99 (d, J = 7.0 Hz, 4H), 0.93 (q, J = 6.9, 6.4 Hz, 7H).

[0090] Example 8

[0091] Synthesis of hydroxy asiatic acid-6-hydroxysphingosine

[0092] S7 1.0 eq (8 mmol), EDCI 1.5 eq (12 mmol), HOBT 1.5 eq (12 mmol) and DIPEA 1.5 eq (15 mmol) were added into a 250 mL round bottom flask, 100 mL tetrahydrofuran was added, stirred at room temperature for 1 hour, then 6-hydroxysphingosine 1.4 eq (11.2 mmol) was added into the reaction system, stirred at room temperature for 30 hours until TLC detection S7 completely disappeared. Post-processing: 60 mL water was added to quench, 60 mL saturated brine was extracted twice, the organic layer was separated, anhydrous magnesium sulfate was added for drying, filtered and concentrated under vacuum to obtain S11 crude product.

[0093] S11 1.0 eq (5 mmol), methanolic ammonia solution 6.0 eq (30 mmol) were added into a 100 mL round bottom flask, reacted at room temperature for 2 hours until TLC detection S11 was completely consumed. Post-processing: 50 mL tetrahydrofuran was added, 50 mL water was extracted once, 50 mL saturated brine was extracted once, the organic layer was separated, anhydrous magnesium sulfate was added for drying, filtered and concentrated under vacuum to obtain a crude product, which was purified by column chromatography to obtain the product hydroxy asiatic acid-6-hydroxysphingosine.

[0094] Hydroxy asiatic acid-6-hydroxysphingosine, 41% yield.

[0095] 1H NMR (400 MHz, Methanol-d4) δ 6.86 (dd, J = 15.2, 7.6 Hz, 1H), 5.88 - 5.71 (m, 1H), 5.62 - 5.56 (m, 1H), 5.43 - 5.36 (m, 1H), 4.40 (s, 1H), 4.32 - 4.28 (m, 1H), 4.15 (dt, J = 12.1, 6.6 Hz, 1H), 3.81 - 3.62 (m, 4H), 3.41 (d, J = 11.2 Hz, 1H), 3.30 (d, J = 2.7 Hz, 1H), 2.22 - 1.47 (m, 17H), 1.47 (d, J = 6.4 Hz, 4H), 1.31 (s, 21H), 1.23 - 1.09 (m, 11H), 1.04 - 0.82 (m, 10H).

[0096] Example 9

[0097] Comparison of solubility of ceramide NP and asiaticoside ceramide

[0098] The solubility of ceramide NP and asiaticoside ceramide was compared by using octyldodecanol and isohexadecanol commonly used in cosmetic formulation system as test solvents. 0.1 g of sample was added to 9.9 g of solvent to prepare a 1% sample solution, and 0.2 g of sample was added to 9.8 g of solvent to prepare a 2% sample solution, and the solubility was tested at 25°C and 50°C, respectively.

[0099] Table 1 Comparison test results of solubility in octyldodecanol

[0100] Table 2 Comparison test results of solubility in isohexadecanol

[0101] The results show that the asiaticoside ceramide obtained by introducing the liposoluble asiaticoside fragment exhibits more excellent solubility than the traditional ceramide NP, which is beneficial to high content addition application in the formulation.

[0102] Example 10

[0103] MTT method for detecting the proliferation activity of compounds on cells

[0104] Human keratinocyte HaCaT cells were seeded at 1 x 10 4Cells were seeded at a density of 1 cell / well in 96-well plates and incubated overnight. After 24 hours, the supernatant was discarded, and 100 μL of different concentrations of sample (Aspergillus asiatica phytosphingosine from Example 1) or blank DMEM medium diluted in DMEM medium (three parallel sets for each concentration) were added. After incubation for another 24 hours, the medium was removed, and 100 μL of thiazolyl blue (MTT) was added to each well. The absorbance at 450 nm was measured, and the cell viability was calculated as A. 给药孔 / A 空白孔 ×100%.

[0105] Table 3. Results of cell proliferation activity test of asiatic acid phytosphingosine.

[0106] The results are shown in Table 3 and Figure 1. At low concentrations, asiatic acid phytosphingosine showed no toxic side effects on human keratinocytes, demonstrating good biocompatibility.

[0107] Example 11

[0108] The anti-inflammatory and restorative effects of asiatic acid phytosphingosine were detected using the LPS-induced cell method.

[0109] Interleukin 6 (IL-6) is a cytokine most typically associated with inflammation. It plays a crucial role in host defense by regulating immune and inflammatory responses. Inflammation affects the skin barrier, increasing epidermal moisture loss and hindering keratinocyte growth, making barrier repair difficult once damaged. It also breaks down the extracellular matrix, causing skin collapse and inhibiting collagen synthesis, leading to loose skin and wrinkles. Therefore, effectively reducing IL-6 production in keratinocytes and fibroblasts induced by external damage and UV radiation, and lowering the inflammatory response, is essential for restoring the skin barrier and maintaining skin elasticity and stability.

[0110] Macrophages were RAW at a density of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and incubated overnight. After 24 hours, the supernatant was discarded, and 100 μL of different concentrations of asiatic acid phytosphingosine (Sphingosine) diluted in DMEM medium was added. The blank control group contained no sample in DMEM medium. Each group had four replicates, and the cells were incubated at 37°C with 5 wt% CO2. Two hours after drug administration, 10 μg / mL LPS was added to both the lipopolysaccharide model group and the experimental group, and they were co-incubated for 24 hours. After the reaction, 50 μL of cell supernatant was collected, and the mRNA expression level of IL-6 was detected using a real-time PCR instrument.

[0111] Table 4 Results of the anti-inflammatory test of Centella asiatica acid, phytosphingosine, and IL-6.

[0112] The results are shown in Table 4 and Fig. 2. The IL-6 expression level of the blank control group was set as 0.02. Under the stimulation of LPS at a working concentration of 10 μg / mL, the IL-6 level was 5000 times the basic level. Under the action of the GOS plant sphingosine at concentrations of 7.8125 mg / L, 15.625 mg / L and 31.25 mg / L, the mRNA relative expression of IL-6 was 74.72, 69.2 and 60.19, respectively.

[0113] Statistical analysis was performed by One-way ANOVA. If the P value was greater than 0.05, it indicated no significant difference. If the P value was less than 0.0001, it indicated significant difference. The statistical analysis showed that the P values of the LPS model group and the samples at concentrations of 7.8125 mg / L, 15.625 mg / L and 31.25 mg / L were all less than 0.0001, indicating that the samples at the above concentrations had significant synergistic effects.

[0114] The GOS plant sphingosine can significantly reduce the expression of IL-6 inflammatory factors in cells in a dose-dependent manner. The GOS plant sphingosine has a significant inhibitory effect on IL-6 and good anti-inflammatory soothing effect, and can promote the repair of inflammatory damaged skin.

[0115] Example 12

[0116] Detection of soothing and anti-sensitivity effect of GOS plant sphingosine by TRPV1 inhibition method

[0117] HaCaT cells were seeded in a 96-well plate at a density of 1×10 4 The cells were incubated in a 5wt% CO2, 37℃ environment. After 2h of administration, 50 μmol / L capsaicin was added to the experimental group and co-incubated for 24h. After the reaction, the cell RNA was extracted, reverse transcribed, and the mRNA expression of TRPV1 factor was detected on a fluorescence quantitative PCR instrument. The data were processed and analyzed by the 2-ΔΔCq method.

[0118] Table 5: Soothing and anti-sensitivity effect test results of GOS plant sphingosine

[0119] The results are shown in Table 5 and Figure 3. The TRPV1 activity of the blank control group was set as 0.3577. Under the stimulation of capsaicin at a working concentration of 50 μmol / L, the TRPV1 level was 2.99 times the basal level. The TRPV1 factor level of the positive control group of 0.4% trans-tetra-t-butylcyclohexanol was 0.46 times that of the capsaicin model group, indicating that the method was reliable. Under the action of the compound asiatic and sphingosine at concentrations of 7.8125 mg / L, 15.625 mg / L and 31.25 mg / L, the TRPV1 level was 0.844, 0.65 and 0.50 times that of the capsaicin model group, respectively.

[0120] Statistical analysis was performed by One-way ANOVA. If the P value was greater than 0.05, there was no significant difference between the blank control group and other concentrations. If the P value was less than 0.0001, there was a significant difference. Statistical analysis showed that the P value of the sample at a concentration of 7.8125 mg / L was 0.0367, and the P values of the positive control group and the samples at concentrations of 15.625 mg / L and 31.25 mg / L were all less than 0.0001, indicating that the samples at higher concentrations had a significant synergistic effect.

[0121] TRPV1 belongs to the TRP superfamily and is a switch for skin burning and stinging, and is closely related to the occurrence of skin sensitive stinging. The soothing and anti-sensitivity effect can be achieved by inhibiting the overexpression of sensitive skin TRPV1 receptors, helping the skin to establish tolerance. Compared with the model group, the co-incubation of asiatic and sphingosine with cells can significantly reduce the expression of TRPV1 in a dose-dependent manner, which proves that asiatic and sphingosine has good soothing effect and can soothe sensitive skin.

[0122] Example 13

[0123] Detection of soothing and anti-sensitivity effect of hydroxyl asiatic and sphingosine by hyaluronidase inhibition method

[0124] Hyaluronic acid (HA) has strong water absorption capacity and adhesion, can regulate the secretion of cytokines, and affects the growth, proliferation, migration and differentiation of cells, thus playing a major role in maintaining skin moisture and elasticity, wound healing and blood vessel formation. At the same time, hyaluronic acid is also involved in vasodilation and allergic reactions, so it has a great relationship with skin sensitivity. Hyaluronidase is a specific lytic enzyme of hyaluronic acid, and its excessive activity can lead to the degradation of HA, resulting in the disintegration of extracellular matrix, the reduction of joint viscosity, skin aging, and the development and progression of inflammation. Hyaluronidase inhibition experiment is the most typical in vitro method for evaluating anti-allergic activity. The anti-allergic activity is evaluated by the hyaluronidase inhibition rate as an index. The greater the hyaluronidase inhibition rate, the stronger the anti-allergic activity.

[0125] 125 μL of the sample corresponding to the concentration (hydroxyl asiatic acid dihydrosphingosine) was uniformly mixed with 125 μL of hyaluronidase, acetic acid buffer solution, and incubated in a 37°C water bath for 20 min. 25 μL of 2.5 mmol / L CaCl2 solution was added, and incubation was continued at 37°C for 20 min. 0.4 g / L of a sodium hyaluronate solution was added to the tube containing hyaluronidase, and 125 μL of pH 5.6 acetic acid buffer solution was added to the tube without enzyme, and incubation was carried out at 37°C for 20 min, and then the tube was left to stand at room temperature for 10 min; 1.0 mL of ultrapure water, 25 μL of 5.0 mol / L NaOH solution, and 125 μL of acetylacetone solution were added, and boiling water bath reaction was carried out for 15 min, ice water bath reaction was carried out for 10 min, and then the tube was left to stand at room temperature for 10 min. 250 μL of P-DAB (Ehrlich reagent) was added and shaken thoroughly, and then 950 μL of anhydrous ethanol was added and left to stand at room temperature for 30 min. The absorbance value was measured at 530 nm, and the hyaluronidase activity inhibition rate of the sample was calculated.

[0126] Table 6 Test results of the soothing effect of hydroxyl asiatic acid dihydrosphingosine

[0127] The results are shown in Table 6 and FIG. 4. The hyaluronidase inhibition rate of the positive control group was 80.56%. Under the action of the compound hydroxyl asiatic acid dihydrosphingosine at concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L, the hyaluronidase inhibition rates were 35.95%, 44.00%, and 57.74%, respectively.

[0128] Statistical analysis was performed by One-way ANOVA. If the P value was > 0.05, there was no significant difference, and if the P value was < 0.0001, there was a significant difference. Statistical analysis showed that the P values of the blank control group and other concentrations were all less than 0.0001, indicating that the above-mentioned concentrations had a significant synergistic effect.

[0129] Co-incubation of hydroxyl asiatic acid dihydrosphingosine with cells can significantly reduce the expression of hyaluronidase in a dose-dependent manner, which proves that hydroxyl asiatic acid dihydrosphingosine has good soothing effect.

[0130] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any changes or replacements within the technical scope disclosed by the present application, which can be easily thought by any person skilled in the art, should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A ceramide derived from Centella asiatica having the structure of Formula I or an isomer of Formula I: ###0001### Formula I wherein R 1 selected from one of the following structures: R 2 is a hydroxyl group or a hydrogen atom.

2. The Withaferin A derived ceramide according to claim 1, wherein, which is one of the following structures:

3. The method of synthesizing the ceramide derived from Centella asiatica acid according to claim 1 or 2, comprising the steps of: P1, reacting compound M1 with acetic anhydride and an organic base to obtain compound M2; P2, reacting compound M2 with sphingosine base, a condensing agent and an organic base to obtain compound M3; P3, reacting compound M3 with methanolic ammonia solution to obtain compound I.

4. The method of synthesis of claim 3, wherein, The organic base in P1 and P2 is diisopropylethylamine; the condensing agent is EDCI and HOBT.

5. The method of synthesis of claim 3, wherein, The molar ratio of compound M1, acetic anhydride and diisopropylethylamine is 1:(4-6):(4-6).

6. The method of synthesis of claim 3, wherein, The molar ratio of compound M2, sphingosine base, EDCI, HOBT and diisopropylethylamine is 1:(1.1-1.5):(1.2-2):(1.2-2):(1.2-2).

7. The method of synthesis of claim 3, wherein, The solvent in P1 is dichloromethane and the solvent in P2 is tetrahydrofuran.

8. Use of the centella asiatica acid-derived ceramide according to any one of claims 1 or 2 in cosmetics, nutraceuticals or in the manufacture of a medicament.

9. Use according to claim 8, characterized in that, The centella asiatica acid-derived ceramide has anti-inflammatory or soothing efficacy.

10. A composition comprising, as an active ingredient, the centella asiatica acid-derived ceramide according to claim 1 or 2, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof.

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