Coriolic acid-derived ceramide, its synthesis method and application

Abstract

The present invention belongs to the technical field of biomedicine, and discloses a ceramide derived from asiatic acid, which has the structure of general formula I or an isomer of general formula I: wherein, R<supgt;1< / supgt> is selected from one of the following structures: R<supgt;2< / supgt> is a hydroxyl group or a hydrogen atom. The present invention also discloses a synthesis method and application of the ceramide derived from asiatic acid. By introducing a lipophilic asiatic acid or asiaticoside fragment, the solubility of the ceramide is improved, making its application in formulations more convenient; the compound of the present invention has an obvious inhibitory effect on the interleukin 6 (IL-6) inflammatory factor, can also inhibit the overexpression of the TRPV1 receptor in sensitive skin, and can significantly reduce the expression of hyaluronidase, thus having a good soothing effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a ceramide derived from asiatic acid and its synthesis method and application. Background Art

[0002] Centella asiatica is also known as gotu kola and tiger grass, and its Roman name is CICA (derived from the root word cicatrisation for scar healing). In China, Centella asiatica has a long history of being used as a medicinal herb, mainly using its asiaticoside, madecassoside, asiatic acid, madecassic acid and other components to eliminate scars and antioxidant.

[0003] Asiatic acid is a natural pentacyclic triterpenoid compound extracted from Centella asiatica, which is a polyhydroxy acid and one of the active components of Centella asiatica; madecassic acid is one of the minor components in Centella asiatica, and its structure contains a hydroxyl group on the basis of asiatic acid. Some studies have shown that other active substances contained in Centella asiatica, such as asiaticoside, its therapeutic effect is mediated by hydrolysis and cleavage of the sugar moiety in the body to convert into asiatic acid; madecassoside also exerts its biological activity by a similar conversion to become the aglycone madecassic acid.

[0004] Ceramide is a class of compounds composed of long-chain sphingosine bases and fatty acids, and the carbon chain length, unsaturation degree and number of hydroxyl groups of the sphingosine base part and the fatty acid part can all be changed. Ceramide is the main component of lipids in the extracellular matrix of the epidermal layer of human skin, accounting for about 50%. Together with cholesterol and saturated fatty acids, ceramide 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 current insoluble in oil and water properties of ceramide make it difficult to apply in formulations. In many products, only a very small amount of ceramide is added conceptually, making it difficult to exert the true role of ceramide. Therefore, preparing novel ceramide derivatives to improve the solubility of ceramide, make its application more convenient, and improve its other aspects of biological activity has important development value. Summary of the Invention

[0006] The purpose of the present invention is to provide a novel-structured ceramide, namely a ceramide derived from asiatic acid.

[0007] Another purpose of the present invention is to provide a synthesis method of the ceramide derived from asiatic acid.

[0008] Another purpose of the present invention is to provide the use of the ceramide derived from asiatic acid.

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

[0010] In the first aspect of the present invention, a ceramide derived from asiatic acid has a structure of general formula I or an isomer of general formula I:

[0011]

[0012] Wherein, R 1 is selected from one of the following structures:

[0013]

[0014] R 2 is a hydroxyl group or a hydrogen atom.

[0015] Furthermore, the ceramide derived from asiatic acid is one of the following structures:

[0016]

[0017]

[0018]

[0019] In the second aspect of the present invention, a method for synthesizing a ceramide derived from asiatic acid includes the following steps:

[0020]

[0021] P1. Reacting compound M1 with acetic anhydride and an organic base to obtain compound M2;

[0022] P2. Reacting compound M2 with a sphingosine base, a condensing agent, and an organic base to obtain compound M3;

[0023] P3. Reacting compound M3 with an ammonia-methanol solution to obtain compound I.

[0024] Furthermore, the organic base in P1 and P2 is diisopropylethylamine.

[0025] Furthermore, the condensing agent is EDCI and HOBT.

[0026] Furthermore, the molar ratio of compound M1, acetic anhydride, and diisopropylethylamine is 1:(4 - 6):(4 - 6).

[0027] Furthermore, 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).

[0028] Further, the solvent of P1 is dichloromethane.

[0029] Further, the solvent of P2 is tetrahydrofuran.

[0030] In the third aspect of the present invention, asiatic acid-derived ceramide has anti-inflammatory and soothing effects and can be used in cosmetics, health products or for preparing medicines.

[0031] A composition comprising asiatic acid-derived ceramide, its isomers, its pharmaceutically acceptable salts, its hydrates or its solvates as active ingredients.

[0032] The compound of "Structure of Formula I" used herein has multiple chiral centers, and thus includes chiral compounds of this structure, namely enantiomers and diastereomers. For example, asiatic acid phytosphingosine includes

[0033]

[0034] etc.

[0035] As used herein, "isomers" means that the compounds or salts of the present invention have the same chemical formula or molecular formula but different optical properties or spatial properties, including enantiomers, diastereomers and cis-trans isomers.

[0036] As used herein, "pharmaceutically acceptable salts" means salts of the present invention that are pharmaceutically acceptable and have the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts formed with inorganic acids or organic acids. Inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., and organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, hydroxybutyric 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-methylbicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptanoic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxy naphthoic acid, salicylic acid, stearic acid and mucic acid; or (2) salts formed when the acidic protons present in the parent compound are replaced.

[0037] As used herein, "hydrates" means a compound combined with water. The combination between the compound and water includes non-covalent binding.

[0038] As used herein, "solvates" means a complex formed by solute molecules or ions and solvent molecules or ions.

[0039] Unless otherwise specified, the term "compound of the present invention" or "ceramide" includes the compound itself, its pharmaceutically acceptable salts, its hydrates, its solvates, and its isomers.

[0040] The present invention has the following beneficial effects:

[0041] 1. The present invention reacts asiatic acid, hydroxyasiatic acid with sphingosine, phytosphingosine, dihydrosphingosine, 6-hydroxysphingosine to obtain a novel class of ceramide compounds.

[0042] 2. By introducing the asiatic acid or hydroxyasiatic acid fragment with better liposolubility, the present invention improves the solubility of ceramide, making its application in formulations more convenient. Compared with traditional ceramide NP, the compounds of the present invention have better solubility.

[0043] 3. The compounds of the present invention have a significant inhibitory effect on interleukin 6 (IL-6) inflammatory factors, can also inhibit the overexpression of TRPV1 receptors in sensitive skin, and can significantly reduce the expression of hyaluronidase, thus having a good soothing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a bar chart of the detection results of cell proliferation viability in Example 10;

[0045] Figure 2 is a bar chart of the detection results of the expression level of IL-6 factor in Example 11;

[0046] Figure 3 is a bar chart of the detection results of the expression level of TRPV1 factor in Example 12;

[0047] Figure 4 is a bar chart of the detection results of the hyaluronidase inhibition rate in Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0048] The following further describes the present invention with specific examples.

[0049] Unless otherwise specified, chemicals are purchased from commercial products and are not further purified. Dichloromethane and tetrahydrofuran used in the experiments are anhydrous solvents. Thin layer chromatography (TLC) uses 60F254 silica gel plates. Silica gel column chromatography uses Qingdao Marine silica gel (particle size 0.040 - 0.063 mm). TLC color development uses UV light (254 nm) or iodine. NMR spectra are characterized using a Bruker DPX 400 nuclear magnetic resonance instrument, 11H NMR was measured at 400 MHz using deuterated methanol or deuterated chloroform as the solvent and tetramethylsilane (TMS) as the internal standard. The chemical shift is in ppm and the coupling constant is in Hz. In 1 1H NMR, δ represents the chemical shift, s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, and m represents a multiplet.

[0050] 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.

[0051] Example 1

[0052] Synthesis of asiatic acid phytosphingosine

[0053]

[0054] Under nitrogen protection, asiatic acid 1.0 eq (40 mmol) and DIPEA 4.5 eq (180 mmol) were dissolved in 150 ml of dichloromethane. Acetic anhydride 4.5 eq (180 mmol) was dissolved in 50 ml of dichloromethane and 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 the reaction of asiatic acid was completed as detected by TLC. Post-treatment: Extracted twice with 150 mL of water and twice with 150 mL of saturated brine. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product of S2, which was directly used for the next reaction.

[0055] 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 the mixture was stirred at room temperature for 1 h. Subsequently, phytosphingosine 1.2 eq (12 mmol) was added to the reaction system and stirred at room temperature for 28 h until S2 completely disappeared as detected by TLC. Post-treatment: Quenched with 60 mL of water, extracted twice with 60 mL of saturated brine, the organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product of S3.

[0056] Add S3 1.0 eq (7 mmol) and ammonia methanol solution 5.0 eq (35 mmol) into a 100 mL round-bottom flask, and react at room temperature for 2 hours until S3 is completely consumed as detected by TLC. Work-up: Add 50 mL of tetrahydrofuran, extract once with 60 mL of water and once with 60 mL of saturated brine, separate the organic layer, add anhydrous magnesium sulfate for drying, filter and concentrate in vacuo to obtain the crude product, which is purified by column chromatography to obtain the product asiatic acid phytosphingosine.

[0057]

[0058] Asiatic acid phytosphingosine, with a yield of 45%.

[0059] 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).

[0060] Example 2

[0061] Synthesis of asiatic acid sphingosine

[0062]

[0063] Under nitrogen protection, add 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) into a 250 mL round-bottom flask, add 100 mL of tetrahydrofuran, stir at room temperature for 1 hour, then add sphingosine 1.4 eq (14 mmol) into the reaction system, and stir at room temperature for 24 hours until S2 completely disappears as detected by TLC. Work-up: Quench with 60 mL of water, extract twice with 60 mL of saturated brine, separate the organic layer, add anhydrous magnesium sulfate for drying, filter and concentrate in vacuo to obtain the crude product of S4.

[0064] Add S4 1.0 eq (6 mmol) and ammonia methanol solution 5.0 eq (30 mmol) into a 100 mL round-bottom flask, and react at room temperature for 2 hours until S4 is completely consumed as detected by TLC. Post-treatment: Add 50 mL of tetrahydrofuran, extract once with 50 mL of water and once with 50 mL of saturated brine, separate the organic layer, add anhydrous magnesium sulfate for drying, filter and concentrate under vacuum to obtain the crude product, which is purified by column chromatography to obtain the product asiatic acid sphingosine.

[0065]

[0066] Asiatic acid sphingosine, with a yield of 40%.

[0067] 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).

[0068] Example 3

[0069] Synthesis of dihydrosphingosine asiatic acid

[0070]

[0071] Under nitrogen protection, 1.0 eq (10 mmol) of S2, 1.4 eq (14 mmol) of EDCI, 1.4 eq (14 mmol) of HOBT, and 1.4 eq (14 mmol) of DIPEA were added to a 250 mL round-bottom flask. 100 mL of tetrahydrofuran was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, 1.3 eq (13 mmol) of dihydrosphingosine was added to the reaction system, and the mixture was stirred at room temperature for 22 hours until S2 completely disappeared as detected by TLC. Post-treatment: 70 mL of water was added to quench the reaction, and the mixture was extracted twice with 70 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum to obtain the crude product of S5.

[0072] 1.0 eq (5 mmol) of S5 and 5.0 eq (25 mmol) of ammonia in methanol solution were added to a 100 mL round-bottom flask and reacted at room temperature for 2 hours until S5 was completely consumed as detected by TLC. Post-treatment: 50 mL of tetrahydrofuran was added, and the mixture was extracted once with 50 mL of water and once with 50 mL of saturated brine. The organic layer was separated, 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 dihydrosphingosine asiaticate.

[0073]

[0074] Dihydrosphingosine asiaticate, with a yield of 33%.

[0075] 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).

[0076] Example 4

[0077] Synthesis of asiatic acid-6-hydroxysphingosine

[0078]

[0079] Under nitrogen protection, 1.0 eq (10 mmol) of S2, 1.8 eq (18 mmol) of EDCI, 1.8 eq (18 mmol) of HOBT and 1.8 eq (18 mmol) of DIPEA were added to a 250 mL round-bottom flask, 100 mL of tetrahydrofuran was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, 1.4 eq (14 mmol) of 6-hydroxysphingosine was added to the reaction system, and the mixture was stirred at room temperature for 24 hours until S2 completely disappeared as detected by TLC. Post-treatment: The reaction was quenched by adding 60 mL of water, and the mixture was extracted twice with 60 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain the crude product of S6.

[0080] 1.0 eq (6 mmol) of S6 and 5.0 eq (30 mmol) of ammonia in methanol solution were added to a 100 mL round-bottom flask, and the reaction was carried out at room temperature for 2 hours until S6 was completely consumed as detected by TLC. Post-treatment: 50 mL of tetrahydrofuran was added, and the mixture was extracted once with 50 mL of water and once with 50 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain the crude product, which was purified by column chromatography to obtain the product asiatic acid-6-hydroxysphingosine.

[0081]

[0082] Asiatic acid-6-hydroxysphingosine, with a yield of 38%.

[0083] 1 1H 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).

[0084] Example 5

[0085] Synthesis of Madecassoside Phytosphingosine

[0086]

[0087] Under nitrogen protection, 1.0 eq (30 mmol) of madecassoside and 5.5 eq (165 mmol) of DIPEA were dissolved in 150 ml of dichloromethane. 5.5 eq (165 mmol) of acetic anhydride was dissolved in 50 ml of dichloromethane and 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 reaction of madecassoside was completed as detected by TLC. Post-treatment: Extracted twice with 150 mL of water and twice with 150 mL of saturated brine, the organic phase was collected, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain the crude product of S7, which was directly used for the next reaction.

[0088] Under nitrogen protection, 1.0 eq (8 mmol) of S7, 1.5 eq (12 mmol) of EDCI, 1.5 eq (15 mmol) of HOBT and 1.5 eq (15 mmol) of DIPEA were added to a 250 mL round-bottom flask, 100 mL of tetrahydrofuran was added, and the mixture was stirred at room temperature for 1 h. Subsequently, 1.2 eq (9.6 mmol) of phytosphingosine was added to the reaction system and stirred at room temperature for 20 h until S7 completely disappeared as detected by TLC. Post-treatment: Quenched with 60 mL of water, extracted twice with 60 mL of saturated brine, the organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain the crude product of S8.

[0089] 1.0 eq (5 mmol) of S8 and 6.0 eq (30 mmol) of ammonia methanol solution were added to a 100 mL round-bottom flask and reacted at room temperature for 2 h until S8 was completely consumed as detected by TLC. Post-treatment: 50 mL of tetrahydrofuran was added, extracted once with 60 mL of water and once with 60 mL of saturated brine, the organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain the crude product, which was purified by column chromatography to obtain the product madecassoside phytosphingosine.

[0090]

[0091] Madecassoside Phytosphingosine, with a yield of 35%.

[0092] 11H 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).

[0093] Example 6

[0094] Synthesis of hydroxylated asiatic acid sphingosine

[0095]

[0096] Under nitrogen protection, 1.0 eq (8 mmol) of S7, 1.5 eq (12 mmol) of EDCI, 1.5 eq (12 mmol) of HOBT, and 1.5 eq (15 mmol) of DIPEA were added to a 250 mL round-bottom flask. 100 mL of tetrahydrofuran was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, 1.4 eq (11.2 mmol) of sphingosine was added to the reaction system, and the mixture was stirred at room temperature for 30 hours until S7 completely disappeared as detected by TLC. Post-treatment: The reaction was quenched by adding 60 mL of water, and the mixture was extracted twice with 60 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product of S9.

[0097] 1.0 eq (5 mmol) of S9 and 6.0 eq (30 mmol) of ammonia methanol solution were added to a 100 mL round-bottom flask and reacted at room temperature for 2 hours until S9 was completely consumed as detected by TLC. Post-treatment: 50 mL of tetrahydrofuran was added, and the mixture was extracted once with 50 mL of water and once with 50 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product, which was purified by column chromatography to obtain the product hydroxylated asiatic acid sphingosine.

[0098]

[0099] Hydroxylated asiatic acid sphingosine, 40% yield.

[0100] 11H 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).

[0101] Example 7

[0102] Synthesis of Dihydrosphingosine Hydroxyasiaticate

[0103]

[0104] Under nitrogen protection, 1.0 eq (8 mmol) of S7, 1.5 eq (12 mmol) of EDCI, 1.5 eq (12 mmol) of HOBT, and 1.5 eq (15 mmol) of DIPEA were added to a 250 mL round-bottom flask. 100 mL of tetrahydrofuran was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, 1.3 eq (10.4 mmol) of dihydrosphingosine was added to the reaction system, and the mixture was stirred at room temperature for 32 hours until S7 completely disappeared as detected by TLC. Post-treatment: The reaction was quenched by adding 60 mL of water, and the mixture was extracted twice with 60 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product of S10.

[0105] 1.0 eq (5 mmol) of S10 and 6.0 eq (30 mmol) of ammonia methanol solution were added to a 100 mL round-bottom flask and reacted at room temperature for 2 hours until S10 was completely consumed as detected by TLC. Post-treatment: 50 mL of tetrahydrofuran was added, and the mixture was extracted once with 50 mL of water and once with 50 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product, which was purified by column chromatography to obtain the product dihydrosphingosine hydroxyasiaticate.

[0106]

[0107] Dihydrosphingosine Hydroxyasiaticate, 30% yield.

[0108] 11H 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).

[0109] Example 8

[0110] Synthesis of Madecassic acid-6-hydroxysphingosine

[0111]

[0112] Under nitrogen protection, 1.0 eq (8 mmol) of S7, 1.5 eq (12 mmol) of EDCI, 1.5 eq (12 mmol) of HOBT, and 1.5 eq (15 mmol) of DIPEA were added to a 250 mL round-bottom flask, 100 mL of tetrahydrofuran was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, 1.4 eq (11.2 mmol) of 6-hydroxysphingosine was added to the reaction system, and the mixture was stirred at room temperature for 30 hours until S7 completely disappeared as detected by TLC. Post-treatment: The reaction was quenched by adding 60 mL of water, and the mixture was extracted twice with 60 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product of S11.

[0113] 1.0 eq (5 mmol) of S11 and 6.0 eq (30 mmol) of ammonia in methanol solution were added to a 100 mL round-bottom flask and reacted at room temperature for 2 hours until S11 was completely consumed as detected by TLC. Post-treatment: 50 mL of tetrahydrofuran was added, and the mixture was extracted once with 50 mL of water and once with 50 mL of saturated brine. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo to obtain the crude product, which was purified by column chromatography to obtain the product madecassic acid-6-hydroxysphingosine.

[0114]

[0115] Hydroxy asiatic acid - 6 - hydroxy sphingosine, 41% yield.

[0116] 1 H 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).

[0117] Example 9

[0118] Comparison of the solubility of ceramide NP and asiatic acid ceramide

[0119] Octyldodecanol and isocetyl alcohol commonly used in cosmetic formulation systems were respectively selected as the test solvents to compare the solubility of ceramide NP and asiatic acid ceramide. Weigh 0.1 g of the sample and add it to 9.9 g of the solvent to prepare a 1% sample solution, and weigh 0.2 g of the sample and add it to 9.8 g of the solvent to prepare a 2% sample solution. Test their solubility at 25 °C and 50 °C respectively.

[0120] Table 1 Comparison test results of solubility in octyldodecanol

[0121] Sample 1％(25℃) 1％(50℃) 2％(50℃) Ceramide NP Very slightly soluble Slightly soluble Slightly soluble Madecassic acid phytosphingosine Sparingly soluble Soluble Soluble Hydroxy madecassic acid phytosphingosine Sparingly soluble Soluble Soluble Madecassic acid sphingosine Soluble Soluble Soluble Hydroxy madecassic acid sphingosine Soluble Soluble Soluble Madecassic acid dihydrosphingosine Sparingly soluble Soluble Soluble Hydroxy madecassic acid dihydrosphingosine Sparingly soluble Soluble Soluble Madecassic acid - 6 - hydroxy - sphingosine Soluble Soluble Soluble Hydroxy madecassic acid - 6 - hydroxy - sphingosine Soluble Soluble Soluble

[0122] Table 2 Comparison test results of solubility in isocetyl alcohol

[0123]

[0124]

[0125] The results show that the asiatic acid ceramide obtained by introducing the lipophilic asiatic acid fragment exhibits better solubility than the traditional ceramide NP, which is beneficial for high - content addition applications in formulations.

[0126] Example 10

[0127] MTT method to detect the proliferation activity of compounds on cells

[0128] Seed human keratinocyte HaCaT cells at 1×104 The density of cells per well was seeded in a 96-well plate and incubated overnight in an incubator. After 24 h, the supernatant was discarded, and 100 μL of samples with different concentrations (asiatic acid phytosphingosine of Example 1) diluted with DMEM medium or blank DMEM medium was added (three groups were set in parallel for each concentration). After continuing to incubate for 24 h, the medium was removed, 100 μL of thiazolyl blue (MTT) was added to each well, the absorbance at 450 nm was measured, and the cell survival rate was calculated as A 给药孔 / A 空白孔 × 100%.

[0129] Table 3 Test results of cell proliferation activity of asiatic acid phytosphingosine

[0130]

[0131] The results are shown in Table 3, Figure 1 As shown, asiatic acid phytosphingosine has no toxic or side effects on human keratinocytes at low concentrations and shows good biosafety.

[0132] Example 11

[0133] Detect the anti-inflammatory and repair efficacy of asiatic acid phytosphingosine by LPS-induced cell method

[0134] Interleukin 6 (IL-6) is the most typical cytokine related to inflammation. It plays an important role in host defense by regulating immune and inflammatory responses. Inflammation can affect the skin barrier, enhance epidermal water loss, and also affect the growth of keratinocytes. It is difficult to recover after the barrier is damaged. At the same time, it will decompose the extracellular matrix, cause skin collapse, and inhibit the synthesis of collagen, making the skin loose and wrinkled. Therefore, effectively reducing the generation of interleukin IL-6 in keratinocytes and fibroblasts caused by external damage and ultraviolet rays, and reducing the inflammatory response are crucial for restoring the skin barrier and protecting the elastic stability of the skin.

[0135] Macrophage RAW was seeded in a 96-well plate at a density of 1 × 10 4 cells per well, placed in an incubator to adhere overnight. After 24 h, the supernatant was discarded, and 100 μL of samples with different concentrations (asiatic acid phytosphingosine) diluted with DMEM medium was added. The blank control group was DMEM medium without samples. There were 4 replicate wells in each group and they were incubated in an environment of 5 wt% CO2 and 37 °C. After 2 h of drug administration, 10 μg / mL LPS was added to the lipopolysaccharide model group and the experimental group and they were co-incubated until 24 h. After the reaction ended, 50 μL of cell supernatant was taken and the mRNA expression level of IL-6 factor was detected on a fluorescence quantitative PCR instrument.

[0136] Table 4 Anti-inflammatory test results of asiatic acid phytosphingosine on IL-6

[0137]

[0138] The results are shown in Table 4, Figure 2 as shown. The IL-6 expression level in the blank control group was set to 0.02. Under the stimulation of LPS with a working concentration of 10 μg / mL, the IL-6 level was 5000 times the basal level. Under the action of phytosphingosine asiaticate at concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L respectively, the relative mRNA expression levels of IL-6 were 74.72, 69.2, and 60.19 respectively.

[0139] One-way ANOVA was used for statistical analysis. When comparing the blank control group with other concentrations, if the P value > 0.05, it indicates no significant difference; if the P value < 0.0001, it indicates a significant difference. Statistical analysis showed that when comparing the LPS model group with other concentrations, the P values of 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 a significant synergistic effect.

[0140] The co-incubation of phytosphingosine asiaticate with cells can significantly reduce the expression of the intracellular IL-6 inflammatory factor, with obvious dose-dependence. Phytosphingosine asiaticate has an obvious inhibitory effect on IL-6, has good anti-inflammatory and soothing effects, and can promote the repair of inflamed and damaged skin.

[0141] Example 12

[0142] Detection of the soothing and anti-allergic effects of phytosphingosine asiaticate by TRPV1 inhibition method

[0143] HaCaT cells were seeded in 96-well plates at a density of 1×10 4 cells / well, placed in an incubator to adhere overnight. After 24 h, the supernatant was discarded, and 100 μL of samples (phytosphingosine asiaticate) at different concentrations diluted with DMEM medium were added. The blank control group was DMEM medium without samples, and the positive control group was 0.4% trans-4-tert-butylcyclohexanol. There were 3 replicate wells in each group and they were incubated in an environment of 5 wt% CO2 and 37 °C. After 2 h of drug administration, 50 μmol / L capsaicin was added to the experimental group and co-incubated until 24 h. After the reaction ended, cell RNA was extracted, reverse-transcribed, and the mRNA expression level of the TRPV1 factor was detected on a fluorescence quantitative PCR instrument, and data processing and analysis were performed by the 2-ΔΔCq method.

[0144] Table 5 Test results of the soothing and anti-allergic effects of phytosphingosine asiaticate

[0145]

[0146]

[0147] The results are shown in Table 5, Figure 3 as follows. The TRPV1 activity of the blank control group was set to 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 0.4% trans-tetra-tert-butylcyclohexanol in the positive control group was 0.46 times that of the capsaicin model group, indicating the reliability of the method. Under the action of asiatic acid sphingosine at concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L, the TRPV1 levels were 0.844, 0.65, and 0.50 times that of the capsaicin model group, respectively.

[0148] One-way ANOVA was used for statistical analysis. When comparing the blank control group with other concentrations, if the P value > 0.05, it indicates no significant difference; if the P value < 0.0001, it indicates a significant difference. The statistical analysis showed that when comparing the capsaicin model group with other concentrations, the P value of the sample at a concentration of 7.8125 mg / L was 0.0367, and the P values of the samples in the positive control group, 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.

[0149] TRPV1 belongs to a member of the transient receptor potential (TRP) superfamily in the skin. It is the switch for skin burning and stinging, and is closely related to the occurrence of skin sensitivity and stinging. It can achieve a soothing and anti-allergic effect by inhibiting the overexpression of TRPV1 receptors in sensitive skin, and help the skin build tolerance. Compared with the model group, asiatic acid sphingosine co-incubated with cells can significantly reduce the TRPV1 expression, and the dose-dependence is obvious. This proves that asiatic acid sphingosine has a good soothing effect and can soothe sensitive skin.

[0150] Example 13

[0151] Detection of the soothing and anti-allergic efficacy of dihydrosphingosine hydroxyasiatate by hyaluronidase inhibition method

[0152] Hyaluronic acid (HA) has strong water absorption capacity and adhesiveness, can regulate the secretion of cytokines, and affect the growth, proliferation, migration and differentiation of cells. Therefore, it plays a major role in maintaining skin moisture and elasticity, wound healing and angiogenesis. At the same time, hyaluronic acid is also involved in vasodilation and allergic reactions, so it is also closely related to skin sensitivity. Hyaluronidase is a specific lyase of hyaluronic acid. Its excessive activity will lead to the degradation of HA, resulting in the disintegration of the extracellular matrix, the reduction of joint viscosity, skin aging, and contribute to the development and progression of inflammation. The hyaluronidase inhibition experiment is the most typical in vitro method for evaluating anti-allergic activity. The anti-allergic activity of a substance is evaluated by the hyaluronidase inhibition rate. The greater the hyaluronidase inhibition rate, the stronger the anti-allergic activity.

[0153] Mix 125 μL of the sample (hydroxyasiatic acid dihydrosphingosine) with the corresponding concentration with 125 μL of hyaluronidase and acetate buffer solution evenly, and incubate in a water bath at 37 °C for 20 min. Add 25 μL of 2.5 mmol / L CaCl2 solution, and continue to incubate at 37 °C for 20 min. Add 125 μL of 0.4 g / L sodium hyaluronate solution to the tube containing hyaluronidase, and add 125 μL of pH 5.6 acetate buffer solution to the tube without enzyme. After incubating at 37 °C for 20 min, let it stand at room temperature for 10 min; add 1.0 mL of ultrapure water, 25 μL of 5.0 mol / L NaOH solution and 125 μL of acetylacetone solution, react in a boiling water bath for 15 min and in an ice water bath for 10 min, and then let it stand at room temperature for 10 min. Add 250 μL of P-DAB (EhrLich reagent) and shake well, and then add 950 μL of absolute ethanol and let it stand at room temperature for 30 min. Measure the corresponding absorbance value at a wavelength of 530 nm, and calculate the hyaluronidase activity inhibition rate of the sample = 1 - (C - D) / (A - B) × 100% (A is the absorbance value of the enzyme-containing system without sample; B is the absorbance value of the enzyme-free system without sample; C is the absorbance value of the enzyme-containing system with sample; D is the absorbance value of the enzyme-free system with sample).

[0154] Table 6 Test results of the soothing effect of hydroxyasiatic acid dihydrosphingosine

[0155]

[0156] The results are shown in Table 6, Figure 4 As shown, the hyaluronidase inhibition rate of the positive control group is 80.56%. Under the action of the compound hydroxyasiatic acid dihydrosphingosine at concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L, the hyaluronidase inhibition rates are 35.95%, 44.00%, and 57.74% respectively.

[0157] One-way ANOVA was used for statistical analysis. When comparing the blank control group with other concentrations, if the P value > 0.05, it indicates no significant difference; if the P value < 0.0001, it indicates a significant difference. Statistical analysis showed that when comparing the blank control group with other concentrations, the P values of the positive control 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 a significant synergistic effect.

[0158] Co-incubation of hydroxylated asiatic acid with cells can significantly reduce the expression of hyaluronidase, with an obvious dose-dependence, which proves that hydroxylated asiatic acid has a good soothing effect.

[0159] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. Ceramide derived from Centella asiatica acid, having the structure of general formula I: in, R 1 Select one of the following structures: R 2 A hydroxyl group or a hydrogen atom.

2. The asiatic acid-derived ceramide according to claim 1, characterized in that It is one of the following structures:

3. The method for synthesizing ceramide derived from Centella asiatica acid according to claim 1 or 2, comprising the following steps: As R 2 is a hydrogen atom, As R 2 is hydroxyl group, P1, compound M1, acetic anhydride and an organic base react to obtain compound M2; P2, compound M2 and a sphingosine base compound, a condensing agent and an organic base are reacted to obtain compound M3; the sphingosine base compound is sphingosine, phytosphingosine, dihydrosphingosine or 6-hydroxysphingosine; P3, compound M3 and ammonia methanol solution are reacted to obtain compound I.

4. The synthesis method according to claim 3, characterized in that The organic bases of P1 and P2 are both diisopropylethylamine; the condensing agents are EDCI and HOBT.

5. The synthesis method according to claim 4, characterized in that The molar ratio of the compound M1, acetic anhydride and diisopropylethylamine is 1:(4-6):(4-6).

6. The synthesis method according to claim 4, characterized in that The molar ratio of the compound M2, the sphingoid base compound, EDCI, HOBT and diisopropylethylamine is 1:(1.1-1.5):(1.2-2):(1.2-2):(1.2-2).

7. The synthesis method according to claim 3, characterized in that The solvent of P1 is dichloromethane, and the solvent of P2 is tetrahydrofuran.

8. Use of the ceramide derived from Centella asiatica acid according to claim 1 or 2 in the preparation of cosmetics or medicines with anti-inflammatory or soothing effects.

9. A composition comprising the asiatic acid-derived ceramide or a pharmaceutically acceptable salt thereof according to claim 1 or 2 as an active ingredient.

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