Asiatic acid derived ceramide as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses ceramide derived from asiatic acid, which has a structure shown in a general formula I or an isomer shown in the general formula I: # imgabs0, in which R1 is selected from one of the following structures: # imgabs1 # R2 is a hydroxyl group or a hydrogen atom. The invention further discloses a synthesis method and application of the ceramide derived from the asiatic acid, the asiatic acid or madecassic acid fragment with good fat solubility is introduced, the solubility of the ceramide is improved, and the ceramide is more convenient to apply in a formula; the compound disclosed by the invention has an obvious inhibiting effect on interleukin 6 (IL-6) inflammatory factors, can also inhibit the overexpression of a TRPV1 receptor of sensitive skin, and can obviously reduce the expression of hyaluronidase, so that the compound has a very good soothing effect.

Description

Technical Field

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

[0002] Centella asiatica is also known as Gotu Kola and tiger grass. Its Roman name is CICA (from the root word cicatrisation, which means scar healing). In China, Centella asiatica has a long history of use as a medicinal herb. Its ingredients, such as asiatica glycoside, madecassoside, asiatic acid, and madecassoside, are mainly used to eliminate scars and provide anti-oxidation effects.

[0003] Asiatic acid is a natural pentacyclic triterpenoid compound extracted from Centella asiatica. It is a polyhydroxy acid and one of the effective ingredients of Centella asiatica. Madecasic acid is one of the minor components of Centella asiatica. Its structure contains hydroxyl groups on the basis of madecasic acid. Studies have shown that the therapeutic effects of other active substances contained in Centella asiatica, such as asiaticoside, are mediated by the hydrolysis and cleavage of the sugar part in the body to convert it into asiatic acid. Madecasic acid also exerts its biological activity through similar transformation to become the aglycone madecasic acid.

[0004] Ceramide is a class of compounds composed of long-chain sphingoid bases and fatty acids, in which the carbon chain length, unsaturation and number of hydroxyl groups of the sphingoid base part and the fatty acid part can vary. Ceramide is the main component of lipids in the extracellular matrix of the epidermis of human skin, accounting for about 50%. Together with cholesterol and saturated fatty acids, ceramide produces an 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 is insoluble in oil and water, so it is difficult to use it in formulations. Many products only conceptually add a very small amount of ceramide, which makes it difficult to play the real role 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 of the invention

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

[0007] Another object of the present invention is to provide a method for synthesizing ceramide derived from Centella asiatica acid.

[0008] Another object of the present invention is to provide the use of ceramide derived from Centella asiatica acid.

[0009] In order to achieve one of the above purposes, the present invention adopts the following technical solutions:

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

[0011]

[0012] Among them, R 1 Select one of the following structures:

[0013]

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

[0015] Further, the asiatic acid-derived ceramide is one of the following structures:

[0016]

[0017]

[0018]

[0019] The second aspect of the present invention is a method for synthesizing ceramide derived from Centella asiatica acid, comprising the following steps:

[0020]

[0021] P1, compound M1, acetic anhydride and an organic base react to obtain compound M2;

[0022] P2, compound M2, sphingoid base, condensing agent, and organic base react to obtain compound M3;

[0023] P3, compound M3 and ammonia methanol solution are reacted to obtain compound I.

[0024] Furthermore, the organic bases of P1 and P2 are both diisopropylethylamine.

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

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

[0027] Furthermore, the molar ratio of the compound M2, sphingoid base, EDCI, HOBT and diisopropylethylamine is 1:(1.1-1.5):(1.2-2):(1.2-2):(1.2-2).

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

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

[0030] In a third aspect of the present invention, ceramide derived from Centella asiatica has anti-inflammatory and soothing effects and can be used in cosmetics, health products or in the preparation of medicines.

[0031] A composition comprising asiatic acid-derived ceramide, its isomer, its pharmaceutically acceptable salt, its hydrate or its solvate as an active ingredient.

[0032] As used herein, the compound of "the structure of Formula I" has multiple chiral centers and thus includes chiral compounds of the structure, i.e., enantiomers and diastereomers, such as Centella asiaticacid phytosphingosine including

[0033]

[0034] wait.

[0035] The "isomer" used herein means that the compound of the present invention or a salt thereof has the same chemical formula or molecular formula but different optical properties or steric properties, including enantiomers, diastereomers and cis-trans isomers.

[0036] As used herein, "pharmaceutically acceptable salt" means a salt of one aspect of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts formed with inorganic acids or organic 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, hydroxysuccinic 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-hydroxyethylsulfonic 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, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid and muconic acid; or (2) salts formed when the acidic proton present in the parent compound is substituted.

[0037] As used herein, "hydrate" refers to a compound that is bound to water. The binding between the compound and water includes non-covalent binding.

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

[0039] Unless otherwise indicated, the term "compound of the present invention" or "ceramide" includes the compound itself, its pharmaceutically acceptable salt, its hydrate, its solvate, and its isomer.

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

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

[0042] 2. The present invention improves the solubility of ceramide by introducing asiatic acid or hydroxymadecassic acid fragments with good fat solubility, making it more convenient to use in the formulation. Compared with traditional ceramide NP, the compound of the present invention has better solubility.

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

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

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

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

[0047] Figure 4 This is a bar graph of the hyaluronidase inhibition rate test results in Example 13. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments.

[0049] Unless otherwise stated, chemicals were purchased from commercial products and were not further purified. Dichloromethane and tetrahydrofuran used in the experiments were anhydrous solvents. Thin layer chromatography (TLC) used 60F254 silica gel plates. Silica gel column chromatography used Qingdao Ocean Silica Gel (particle size 0.040-0.063 mm). TLC color development used UV light (254 nm) or iodine. NMR spectra were characterized using a Bruker DPX 400 nuclear magnetic resonance instrument. 1H NMR is at 400 MHz, the solvent is deuterated methanol or deuterated chloroform, and tetramethylsilane (TMS) is used as the internal standard. The unit of chemical shift is ppm, and the unit of coupling constant is Hz. 1 In H NMR, δ represents chemical shift, s represents singlet, d represents doublet, t represents triplet, q represents quartet, and m represents 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, 1.0eq (40mmol) of asiatic acid and 4.5eq (180mmol) of DIPEA were dissolved in 150ml of dichloromethane, and 4.5eq (180mmol) of acetic anhydride was dissolved in 50ml of dichloromethane, and added dropwise to the above reaction solution at room temperature. The reaction was carried out under nitrogen protection at room temperature for 8h until the reaction of asiatic acid was completed by TCL detection. Post-treatment: 150mL of water was added to extract twice, and 150mL of saturated brine was extracted twice. The organic phase was collected, dried with anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain the crude product S2, which was directly used in the next step reaction.

[0055] Under nitrogen protection, S2 1.0eq (10mmol), EDCI 1.5eq (15mmol), HOBT 1.5eq (15mmol) and DIPEA 1.5eq (15mmol) were added to a 250mL round-bottom flask, and 100mL tetrahydrofuran was added. The mixture was stirred at room temperature for 1 hour, and then phytosphingosine 1.2eq (12mmol) was added to the reaction system. The mixture was stirred at room temperature for 28 hours until S2 completely disappeared by TLC. Post-treatment: 60mL of water was added to quench, and 60mL of saturated brine was used for extraction twice. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain a crude product of S3.

[0056] S3 1.0eq (7mmol) and ammonia methanol solution 5.0eq (35mmol) were added to a 100mL round-bottom flask and reacted at room temperature for 2 hours until S3 was completely consumed by TLC. Post-treatment: 50mL tetrahydrofuran was added, and 60mL water was added to extract once, and 60mL saturated brine was added to extract once, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated in vacuo to obtain a crude product, which was purified by column chromatography to obtain the product Centella asiaticacid phytosphingosine.

[0057]

[0058] Asiatic acid phytosphingosine, 45% yield.

[0059] 1 H NMR (400MHz, Methanol-d4) δ5.36 (t, J=4.1Hz, 1H), 4.06 (q, J=4.6Hz, 1H), 3.72 (qd, J=10. 9,4.6Hz,3H),3.64–3.55(m,2H),3.53(d,J=11.1Hz,1H),3.38(d,J=9.6Hz,1H),3.28(d,J= 11.0Hz,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 Sphingosine Asiatic Acid

[0062]

[0063] Under nitrogen protection, S2 1.0eq (10mmol), EDCI 1.6eq (16mmol), HOBT 1.6eq (16mmol) and DIPEA 1.6eq (16mmol) were added to a 250mL round-bottom flask, and 100mL tetrahydrofuran was added. The mixture was stirred at room temperature for 1 hour, and then sphingosine 1.4eq (14mmol) was added to the reaction system. The mixture was stirred at room temperature for 24 hours until S2 completely disappeared by TLC. Post-treatment: 60mL of water was added to quench, and 60mL of saturated brine was used for extraction twice. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain a crude product of S4.

[0064] S4 1.0eq (6mmol) and ammonia methanol solution 5.0eq (30mmol) were added to a 100mL round-bottom flask and reacted at room temperature for 2 hours until S4 was completely consumed by TLC. Post-treatment: 50mL of tetrahydrofuran was added, and 50mL of water was added to extract once, and 50mL of saturated brine was added to extract once, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated in vacuo to obtain a crude product, which was purified by column chromatography to obtain the product asiatic acid sphingosine.

[0065]

[0066] Sphingosine asiaticacid, 40% yield.

[0067] 1 H NMR (400MHz, Methanol-d4) δ6.87(d,J=7.7Hz,1H),5.80–5.69(m,1H),5.52(ddt,J=15.3,6.7,1.5Hz,1H),5.33(t,J=3.6Hz ,1H),4.18(t,J=6.3Hz,1H),3.87–3.66(m,3H),3.66–3.58(m,1H),3.53(d,J=11.1Hz,1H),3.38(d,J=9.6Hz,1H),3.29(d,J =11.0Hz,1H),2.18(d,J=10.5Hz,1H),2.15–1.94(m,6H),1.88(td,J=13.7,4.3Hz,1H),1.68(ddt,J=13.2,9.7,4.6Hz,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=6.3Hz,6H),0.90(d,J=5.4Hz,4H),0.72(s,3H).

[0068] Example 3

[0069] Synthesis of Sphinganine from Centella asiatica

[0070]

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

[0072] S5 1.0eq (5mmol) and ammonia methanol solution 5.0eq (25mmol) were added to a 100mL round-bottom flask and reacted at room temperature for 2 hours until S5 was completely consumed by TLC. Post-treatment: 50mL of tetrahydrofuran was added, and 50mL of water was added to extract once, and 50mL of saturated brine was added to extract once, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated in vacuo to obtain a crude product, which was purified by column chromatography to obtain the product Centella asiatica dihydrosphingosine.

[0073]

[0074] Centella asiaticacid dihydrosphinganine, 33% yield.

[0075] 1 H NMR(400MHz,Methanol-d4)δ6.99(d,J=8.0Hz,1H),5.35(t,J=3.7Hz,1H),3.84– 3.61(m,5H),3.53(d,J=11.1Hz,1H),3.38(d,J=9.5Hz,1H),3.29(d,J=11.0Hz,1 H),2.25–1.83(m,6H),1.81–1.37(m,15H),1.32(d,J=6.8Hz,26H),1.18(s,3H), 1.07(s,5H),0.99(d,J=5.8Hz,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, S2 1.0eq (10mmol), EDCI 1.8eq (18mmol), HOBT 1.8eq (18mmol) and DIPEA 1.8eq (18mmol) were added to a 250mL round-bottom flask, and 100mL tetrahydrofuran was added. The mixture was stirred at room temperature for 1 hour, and then 6-hydroxysphingosine 1.4eq (14mmol) was added to the reaction system. The mixture was stirred at room temperature for 24 hours until S2 completely disappeared by TLC. Post-treatment: 60mL of water was added to quench, and 60mL of saturated brine was used for extraction twice. The organic layer was separated, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain a crude product of S6.

[0080] S6 1.0eq (6mmol) and ammonia methanol solution 5.0eq (30mmol) were added to a 100mL round-bottom flask and reacted at room temperature for 2 hours until S6 was completely consumed by TLC. Post-treatment: 50mL of tetrahydrofuran was added, 50mL of water was added to extract once, 50mL of saturated brine was added to extract once, the organic layer was separated, anhydrous magnesium sulfate was added to dry, filtered and concentrated in vacuo to obtain a crude product, which was purified by column chromatography to obtain the product Centella asiaticacid-6-hydroxysphingosine.

[0081]

[0082] Centella asiaticacid-6-hydroxysphingosine, 38% yield.

[0083] 1 H NMR(400MHz, Methanol-d4)δ6.82(d,J=7.7Hz,1H),5.90–5.82(m,1H),5.72–5.66(m,1H),5.33(t,J=3.6Hz,1H),4.34 –4.29(m,1H),4.10(t,J=6.1Hz,1H),3.92–3.76(m,3H),3.62–3.53(m,1H),3.48(d,J=10.8Hz,1H),3.30(d,J=9.4Hz, 1H),3.26(d,J=10.8Hz,1H),2.14(d,J=10.2Hz,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=6.4Hz,6H),0.86(d,J=5.4Hz,4H),0.69(s,3H).

[0084] Example 5

[0085] Synthesis of Madecassic Acid Phytosphingosine

[0086]

[0087] Under nitrogen protection, 1.0eq (30mmol) of madecassoic acid and 5.5eq (165mmol) of DIPEA were dissolved in 150ml of dichloromethane, and 5.5eq (165mmol) of acetic anhydride were dissolved in 50ml of dichloromethane, and added dropwise to the above reaction solution at room temperature. The reaction was carried out under nitrogen protection at room temperature for 10h until TCL detected that the reaction of madecassoic acid was complete. Post-treatment: 150mL of water was added to extract twice, and 150mL of saturated brine was extracted twice. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo to obtain crude S7, which was directly used in the next step.

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

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

[0090]

[0091] Madecassic acid phytosphingosine, 35% yield.

[0092] 1H NMR(400MHz, Methanol-d4)δ5.41(t,J=3.6Hz,1H),4.61(s,1H),4.40(s,1H),4.07(p ,J=4.7,4.2Hz,1H),3.73(ddt,J=20.3,9.4,5.6Hz,3H),3.65–3.53(m,3H),3.46(d,J =11.1Hz,1H),3.30(d,J=2.5Hz,1H),2.21–1.48(m,16H),1.42(d,J=6.3Hz,4H),1.31 (s,24H),1.25–1.16(m,3H),1.13(d,J=3.3Hz,5H),1.08(s,4H),1.01–0.88(m,10H).

[0093] Example 6

[0094] Synthesis of Madecassic Acid Sphingosine

[0095]

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

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

[0098]

[0099] Madecassic acid sphingosine, 40% yield.

[0100] 1H NMR(400MHz, Methanol-d4)δ6.89(dd,J=15.1,7.8Hz,1H),5.80–5.68(m,1H),5.52(d dd,J=15.4,6.9,4.3Hz,1H),5.38–5.36(m,1H),4.40(s,1H),4.17(dt,J=12.3,6.5Hz ,1H),3.91–3.54(m,5H),3.45(d,J=11.1Hz,1H),3.30(d,J=2.7Hz,1H),2.22–1.47(m ,16H),1.42(d,J=6.6Hz,5H),1.31(s,23H),1.21–1.06(m,10H),1.01–0.85(m,11H).

[0101] Example 7

[0102] Synthesis of Madecassic Acid Sphinganine

[0103]

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

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

[0106]

[0107] Madecassic acid sphinganine, 30% yield.

[0108] 1H NMR(400MHz, Methanol-d4)δ7.00(dd,J=14.1,7.9Hz,1H),5.43–5.35(m,1H),4.40(s,1H),3.86–3.71(m,3 H),3.67(dt,J=10.6,3.6Hz,2H),3.60(d,J=11.1Hz,1H),3.45(d,J=11.1Hz,1H),3.30(d,J=2.5Hz,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=5.6Hz,4H),1.31(s,2 6H), 1.21–1.17 (m, 2H), 1.16–1.11 (m, 5H), 1.08 (s, 5H), 0.99 (d, J = 7.0Hz, 4H), 0.93 (q, J = 6.9, 6.4Hz, 7H).

[0109] Example 8

[0110] Synthesis of Madecassic Acid-6-Hydroxysphingosine

[0111]

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

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

[0114]

[0115] Madecassic acid-6-hydroxysphingosine, 41% yield.

[0116] 1 H NMR(400MHz, Methanol-d4)δ6.86(dd,J=15.2,7.6Hz,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=12.1,6.6H z,1H),3.81–3.62(m,4H),3.41(d,J=11.2Hz,1H),3.30(d,J=2.7Hz,1H),2.22–1.47( m,17H),1.47(d,J=6.4Hz,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 isomeric hexadecyl alcohol, which are commonly used in cosmetic formulation systems, were selected as test solvents to compare the solubility of ceramide NP and asiatic acid ceramide. 0.1g of sample was weighed and added to 9.9g of solvent to prepare a 1% sample solution, and 0.2g of sample was weighed and added to 9.8g of solvent to prepare a 2% sample solution. The solubility was tested at 25℃ and 50℃ respectively.

[0120] Table 1 Comparative test results of solubility in octyl dodecanol

[0121] sample 1％(25℃) 1％(50℃) 2％(50℃) Ceramide NP Very slightly soluble Slightly soluble Slightly soluble Asiatic acid phytosphingosine Slightly soluble Dissolve Dissolve Madecassic acid phytosphingosine Slightly soluble Dissolve Dissolve Sphingosine asiaticacid Dissolve Dissolve Dissolve Madecassic acid sphingosine Dissolve Dissolve Dissolve Centella asiatica Sphinganine Slightly soluble Dissolve Dissolve Madecassate Sphinganine Slightly soluble Dissolve Dissolve Centella asiatica-6-hydroxy-sphingosine Dissolve Dissolve Dissolve Madecassic acid-6-hydroxy-sphingosine Dissolve Dissolve Dissolve

[0122] Table 2 Comparative test results of solubility in isomeric hexadecanol

[0123]

[0124]

[0125] The results showed that the asiatic acid ceramide obtained by introducing the fat-soluble asiatic acid fragment showed better solubility than the traditional ceramide NP, which is conducive to high-content addition application in the formula.

[0126] Example 10

[0127] MTT assay to detect the effects of compounds on cell proliferation

[0128] Human keratinocytes HaCaT cells were cultured at 1×104 The cells were seeded at a density of 100 μL / well in a 96-well plate and incubated overnight in an incubator. After 24 hours, the supernatant was discarded, and 100 μL of samples (Centella asiaticacid phytosphingosine in Example 1) diluted with DMEM medium or blank DMEM medium were added (three groups were set in parallel for each concentration), and the medium was removed after further incubation for 24 hours. 100 μL of MTT was added to each well, and the absorbance at 450 nm was measured to calculate the cell survival rate = A 给药孔 / A 空白孔 ×100%.

[0129] Table 3 Cell proliferation activity test results of Centella asiaticacid phytosphingosine

[0130]

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

[0132] Embodiment 11

[0133] Detection of the anti-inflammatory and repairing effects of asiatic acid phytosphingosine using LPS-induced cell method

[0134] Interleukin 6 (IL-6) is the most typical cytokine associated with inflammation. It plays an important role in host defense by regulating immune and inflammatory responses. Inflammation affects the skin barrier, increases epidermal water loss, and affects the growth of keratinocytes. Once the barrier is damaged, it is difficult to recover. At the same time, it also decomposes the extracellular matrix, causing skin collapse, while inhibiting collagen synthesis, making the skin loose and wrinkled. Therefore, it is crucial to effectively reduce the production of interleukin IL-6 in keratinocytes and fibroblasts caused by external damage and ultraviolet rays, and reduce inflammatory responses, to restore the skin barrier and protect skin elasticity and stability.

[0135] RAW macrophages were cultured at a density of 1×10 4 Each well was seeded in a 96-well plate and placed in an incubator to adhere overnight. After 24 hours, the supernatant was discarded and 100 μL of samples (Centella asiaticacid phytosphingosine) of different concentrations diluted in DMEM medium were added. The blank control group was DMEM medium without samples. Each group had 4 replicates and was incubated in a 5wt% CO2, 37°C environment. 2 hours after administration, 10 μg / mL LPS was added to the lipopolysaccharide model group and the experimental group and incubated together for 24 hours. After the reaction, 50 μL of the cell supernatant was taken and the mRNA expression of IL-6 factor was detected on a fluorescent quantitative PCR instrument.

[0136] Table 4 Asiatic acid phytosphingosine IL-6 anti-inflammatory test results

[0137]

[0138] The results are shown in Table 4. Figure 2 As shown, the IL-6 expression level of the blank control group was set to 0.02. Under the stimulation of LPS at a working concentration of 10 μg / mL, the IL-6 level was 5000 times the basal level. Under the action of asiatic acid phytosphingosine at concentrations of 7.8125 mg / L, 15.625 mg / L, and 31.25 mg / L, 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. If the P value of the blank control group was > 0.05, it indicated no significant difference. If the P value was < 0.0001, it indicated a significant difference. Statistical analysis showed that compared with other concentrations, the P values ​​of the samples of the LPS model group at 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] Co-incubation of cells with phytosphingosine and asiatic acid can significantly reduce the expression of intracellular IL-6 inflammatory factors, with obvious dose dependence. Centella asiatica acid phytosphingosine has a significant inhibitory effect on IL-6, has good anti-inflammatory and soothing effects, and can promote the repair of inflammatory damaged skin.

[0141] Example 12

[0142] TRPV1 inhibition assay to detect the soothing and anti-allergic effects of sphingosine asiaticacid

[0143] HaCaT cells were cultured at a density of 1×10 4 The cells were seeded in 96-well plates and placed in an incubator overnight. After 24 hours, the supernatant was discarded and 100 μL of samples (Centella asiaticacid sphingosine) 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-butyl cyclohexanol. There were 3 replicates in each group and incubated in a 5wt% CO2, 37°C environment. 2 hours after administration, 50 μmol / L capsaicin was added to the experimental group and incubated together for 24 hours. After the reaction, cell RNA was extracted, reverse transcribed, and the mRNA expression of TRPV1 factor was detected on a fluorescent quantitative PCR instrument. The 2-ΔΔCq method was used for data processing and analysis.

[0144] Table 5 Test results of the soothing and anti-allergic effect of Centella asiatica Sphingosine

[0145]

[0146]

[0147] The results are shown in Table 5. Figure 3 As shown, 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 the positive control group 0.4% trans-tetra-tert-butyl cyclohexanol was 0.46 times that of the capsaicin model group, indicating that the method is reliable. Under the action of the compound 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. If the P value of the blank control group was compared with other concentrations, it indicated no significant difference, and if the P value was <0.0001, it indicated a significant difference. Statistical analysis showed that the P value of the capsaicin model group was 0.0367 for the sample with a concentration of 7.8125 mg / L compared with other concentrations, and the p values ​​of the positive control group, 15.625 mg / L and 31.25 mg / L samples were all less than 0.0001, indicating that the samples with higher concentrations had a significant synergistic effect.

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

[0150] Embodiment 13

[0151] Hyaluronidase inhibition method to detect the soothing and anti-allergic effect of hydroxy-Centella asiatica sphingosine

[0152] Hyaluronic acid (HA) has strong water absorption and adhesion, can regulate the secretion of cytokines, and affect cell growth, proliferation, migration and differentiation. 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 cleavage enzyme of hyaluronic acid. Its excessive activity can lead to the degradation of HA, resulting in the disintegration of the extracellular matrix, the reduction of joint viscosity, skin aging, and the development and progression of inflammation. The hyaluronidase inhibition test is the most typical in vitro method for evaluating anti-allergic activity. The hyaluronidase inhibition rate is used as an indicator to evaluate the anti-allergic activity of the substance. The greater the hyaluronidase inhibition rate, the stronger the anti-allergic activity.

[0153] 125 μL of the corresponding concentration sample (madecassoside dihydrosphinganine) was evenly mixed with 125 μL of hyaluronidase and acetic acid buffer solution, and kept warm in a 37°C water bath for 20 min. 25 μL of 2.5 mmol / L CaCl2 solution was added, and the tube without enzyme was incubated at 37°C for 20 min. 125 μL of 0.4 g / L 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 incubated at 37°C for 20 min and then allowed 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 the reaction was carried out in a boiling water bath for 15 min, an ice water bath for 10 min, and then allowed to stand at room temperature for 10 min. 250 μL of P-DAB (EhrLich reagent) was added and fully shaken, and then 950 μL of anhydrous ethanol was added and allowed to stand at room temperature for 30 min. The corresponding absorbance value was measured at a wavelength of 530 nm, and the photometric value of the hyaluronidase activity inhibition rate of the sample was calculated. The hyaluronidase activity inhibition rate of the sample = 1-(CD) / (AB)×100% (A is the absorbance value of the system containing enzyme without sample; B is the absorbance value of the system without enzyme without sample; C is the absorbance value of the system containing enzyme with sample; D is the absorbance value of the system without enzyme with sample).

[0154] Table 6 Soothing effect test results of hydroxy-Centella asiaticacid dihydrosphingosine

[0155]

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

[0157] One-way ANOVA was used for statistical analysis. If the P value of the blank control group was compared with other concentrations, it indicated no significant difference if it was > 0.05, and if the P value was < 0.0001, it indicated a significant difference. Statistical analysis showed that compared with other concentrations, the P values ​​of the positive control group, 7.8125 mg / L, 15.625 mg / L and 31.25 mg / L samples were all less than 0.0001, indicating that the samples at the above concentrations had a significant synergistic effect.

[0158] Co-incubation of cells with hydroxy-Centella oxysporum fumarate can significantly reduce the expression of hyaluronidase in a dose-dependent manner, which proves that hydroxy-Centella oxysporum fumarate has a good soothing effect.

[0159] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Ceramide derived from Centella asiaticacid, which has the structure of general formula I or an isomer of general formula I: in, R 1 Select one of the following structures: R 2 A hydroxyl group or a hydrogen atom.

2. The ceramide derived from Centella asiatica 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: P1, compound M1, acetic anhydride and an organic base react to obtain compound M2; P2, compound M2, sphingoid base, condensing agent, and organic base react to obtain compound M3; 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 3, 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 3, characterized in that The molar ratio of the compound M2, sphingoid base, 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 any one of claims 1 or 2 in cosmetics, health products or preparation of medicines.

9. The use according to claim 8, characterized in that The asiatic acid-derived ceramide has anti-inflammatory or soothing effects.

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

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