Glycyrrhetinic acid lutein ester compound as well as preparation method and application thereof

Abstract

The invention is suitable for the technical field of medicine health, and particularly relates to a glycyrrhetinic acid lutein ester compound, a preparation method and application thereof. The glycyrrhetinic acid lutein ester compound provided by the invention is effective in inflammation relief, tissue repair and protection and treatment of ultraviolet injury, has good anti-inflammatory and antioxidant activity and repair capability, not only has good effects on anti-inflammatory, repair, anti-aging and blue light prevention of eyes, but also has good anti-inflammatory and anti-oxidation effects, and has good application prospects. Moreover, the composition has the effects equivalent to or even superior to those of special medicines in the aspects of skin state improvement, whitening, sunscreen, anti-aging and the like, and has good application prospects and potential.

Description

Technical Field

[0001] The present invention belongs to the field of medical health technology, and in particular relates to a glycyrrhetinic acid lutein ester compound, a preparation method and an application thereof. Background Art

[0002] Glycyrrhetinic acid itself and licorice root extracts containing glycyrrhetinic acid as a key ingredient can be used for anti-inflammatory, antibacterial and antioxidant effects. Licorice is often used in traditional Chinese medicine to blend various medicines to achieve synergistic and toxicity-reducing effects. Glycyrrhetinic acid is also commonly used as a pharmaceutical intermediate in the medical and health field. It is modified to produce products such as stearyl glycyrrhetinate for use in the pharmaceutical and cosmetic fields, playing an anti-allergic and soothing role.

[0003] Lutein has a recognized role in eye health. As a nutritional pigment widely found in the retina, lutein is a recognized safe substance that can protect the eyes by absorbing blue light, neutralizing harmful free radicals, and inhibiting intraocular inflammation. It has a protective and alleviating effect on eye diseases such as myopia, diabetic retinopathy, and cataracts. Its eye protection mechanism: The macula in the center of the retina is the most sensitive part of the vision, responsible for color recognition, motion detection, and visual contrast sensitivity; the lutein rich in the macula is essential for protecting and improving visual function. It mainly maintains visual health through three mechanisms: as a blue light filter, antioxidant, and anti-inflammatory agent. Lutein can reduce the radiation damage of ultraviolet rays, has high antioxidant activity and vasodilation function, can improve the nutrient supply of the eye, improve the microcirculation of the eye, and prevent oxidative damage to the eye by free radicals. Lutein has a preventive and ameliorative effect on eye diseases such as retinitis pigmentosa, macular degeneration, and cataracts. It can reduce the inflammatory response of the retina by blocking the expression of inflammatory mediators. A higher content of lutein in the diet can reduce the risk of eye diseases. In human skin, lutein and its antioxidant and anti-inflammatory activities protect against UV-induced skin damage; a high-lutein diet is associated with a reduced risk of wrinkles and cancer; lutein prevents UV-induced carcinogenesis in animals and improves physiological parameters of human skin, including antioxidant protection against UV irradiation.

[0004] Lutein is easily oxidized due to its structural characteristics, so its stability is poor. Therefore, when it is used in the market, lutein is usually esterified to form lutein esters to improve its stability. The lutein esters currently circulating in the market are usually lutein fatty acid esters. Although the stability of lutein fatty acid esters is improved compared to lutein, the pharmacological activity of fatty acids is weak and cannot play a role in synergistically improving the activity of lutein. Summary of the invention

[0005] The purpose of the present invention is to provide a glycyrrhetinic acid lutein ester compound, aiming to solve the problem that although the stability of lutein fatty acid ester is improved relative to lutein, the pharmacological activity of fatty acids is weak and cannot play a role in synergistically enhancing the activity of lutein.

[0006] The present invention is achieved by providing a glycyrrhetinic acid lutein ester compound, wherein the glycyrrhetinic acid lutein ester compound is:

[0007]

[0008] Another object of the present invention is to provide a method for preparing a glycyrrhetinic acid lutein ester compound, the method comprising:

[0009] Lutein and triethylamine are dissolved in dichloromethane, and a sulfonylating agent is added in an ice bath to generate a sulfonated intermediate of lutein;

[0010] The lutein sulfonylation intermediate reacts with glycyrrhetinic acid under alkaline conditions to undergo a nucleophilic substitution reaction to generate a glycyrrhetinic acid lutein ester compound.

[0011] Preferably, the sulfonylating agent is p-toluenesulfonyl chloride, methanesulfonyl chloride or benzenesulfonyl chloride.

[0012] Preferably, the synthesis route of glycyrrhetinic acid lutein ester compound is:

[0013]

[0014] Preferably, the step of dissolving lutein and triethylamine in dichloromethane, adding a sulfonylation reagent in an ice bath, and generating a lutein sulfonylation intermediate comprises:

[0015] Dissolve lutein in dichloromethane, place in an ice bath, add triethylamine, slowly drop p-toluenesulfonyl chloride solution into the reaction solution, stir, and detect by TLC until the reaction is complete. Add water, extract and separate the liquids, wash with sodium bicarbonate solution, water and brine in sequence, dry the organic phase with anhydrous sodium sulfate, filter, add anhydrous ethanol, concentrate under reduced pressure, distill to remove dichloromethane, refrigerate in a refrigerator for crystallization, and filter to obtain lutein sulfonylation intermediate.

[0016] Preferably, the step of subjecting the lutein sulfonylated intermediate to a nucleophilic substitution reaction with glycyrrhetinic acid under alkaline conditions to generate a glycyrrhetinic acid-lutein ester compound comprises:

[0017] The lutein sulfonylation intermediate was dissolved in a mixed solvent of dichloromethane:acetonitrile, glycyrrhetinic acid and potassium carbonate were added, and the mixture was stirred at room temperature for 3 hours. The mixture was monitored by TLC thin-layer chromatography. After the reaction was complete, water was added, and the liquids were separated by extraction. The aqueous layer was extracted with dichloromethane, and the organic phases were combined and washed with sodium bicarbonate solution, water and brine in sequence. The organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 10 mL. The mixture was refrigerated and placed for crystallization. The mixture was filtered and washed with a small amount of dichloromethane to obtain a glycyrrhetinic acid lutein ester compound.

[0018] Another object of the present invention is to provide a glycyrrhetinic acid lutein ester compound for use in the preparation of anti-inflammatory and antioxidant products.

[0019] The present invention provides a glycyrrhetinic acid lutein ester compound, which is an effective ingredient in relieving inflammation, tissue repair and protection and treatment of ultraviolet damage. The compound has good anti-inflammatory, antioxidant activity and repair ability. It not only has good effects on anti-inflammatory, repair, anti-aging and blue light protection of the eyes, but also shows effects comparable to or even exceeding those of special drugs in improving skin condition, whitening, sun protection, anti-aging, etc., and has good application prospects and potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the effect of a glycyrrhetinic acid lutein ester compound provided in an embodiment of the present invention on wound healing;

[0021] Figure 2 This is a diagram showing the protective and therapeutic effects of the glycyrrhizic acid lutein ester compound provided in an embodiment of the present invention on an acute ultraviolet damaged mouse skin model. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] A glycyrrhetinic acid lutein ester compound, wherein the glycyrrhetinic acid lutein ester compound is:

[0024]

[0025] In this example, (1) lutein and triethylamine are dissolved in dichloromethane, and a sulfonylating agent (including p-toluenesulfonyl chloride, methanesulfonyl chloride, benzenesulfonyl chloride, etc.) is added in an ice bath to generate a lutein sulfonyl intermediate.

[0026] (2) The intermediate obtained in (1) is reacted with glycyrrhetinic acid under alkaline conditions to undergo a nucleophilic substitution reaction to generate a glycyrrhetinic acid xanthophyll compound of formula I.

[0027] (3) The method for synthesizing a glycyrrhetinic acid derivative according to claim 2, wherein the reaction process of steps (1) and (2) is as follows:

[0028]

[0029] The glycyrrhetinic acid lutein ester provided by the present invention has a synergistic improvement effect on pharmacological activity, and its anti-inflammatory activity, anti-ultraviolet damage activity and the like are significantly better than the use of lutein and glycyrrhetinic acid alone.

[0030] The technical solution of the present invention is as follows:

[0031] A new, safe and effective glycyrrhetinic acid lutein ester and a preparation method thereof, wherein the compound has a structure shown in Formula I:

[0032]

[0033] In addition to the active ingredient or pharmaceutically acceptable carrier, the compound of the present invention can be prepared as an excipient, including but not limited to topical preparations such as tablets, capsules, soft candies, creams, ointments (semisolid topical preparations), microemulsions, gels, pastes, (TTS) transdermal preparations (such as patches, bandages, etc.), etc. Pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch), cellulose, and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, etc.), malt, gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, etc.), alginic acid, emulsifiers (such as Tween), humectants (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer, etc. Such carriers enable the pharmaceutical composition of the present invention to be appropriately selected according to the preparations that can be used to select one or more of the present invention. The excipient formulation of the pharmaceutical composition of the present invention can be appropriately selected according to the available materials. For example, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose hydrochloride, sodium alginate and polyvinyl pyrrolidone can be used as suspending agents or dispersing agents.

[0034] The present invention also provides the use of the glycyrrhetinic acid lutein ester in anti-inflammatory and anti-oxidation.

[0035] Example 1

[0036] S1: Dissolve lutein (III, 2.0 mmol) in dichloromethane (50 mL), place in an ice bath, add triethylamine (5 eq), slowly drop a solution of p-toluenesulfonyl chloride (2.5 eq) in dichloromethane (30 mL) into the reaction solution, stir and react for 2-3 hours, detect by TLC until the reaction is complete, add water (100 mL), extract and separate the liquids, extract the aqueous layer with dichloromethane (2×30 mL), wash with sodium bicarbonate solution, water, and brine in sequence, dry the organic phase with anhydrous sodium sulfate, filter, add anhydrous ethanol (10 mL), concentrate under reduced pressure, distill off dichloromethane, refrigerate for crystallization, and filter to obtain lutein intermediate III.

[0037] S2: The lutein intermediate III obtained in the previous step was dissolved in a mixed solvent (60 mL) of dichloromethane:acetonitrile (1:2), and glycyrrhetinic acid (1.1 eq) and potassium carbonate (2 eq) were added. The mixture was stirred at room temperature for 3 hours and monitored by TLC thin layer chromatography. After the reaction was complete, water (100 mL) was added, and the mixture was extracted and separated. The aqueous layer was extracted with dichloromethane (2×30 mL), and the organic phases were combined and washed with sodium bicarbonate solution, water, and brine in sequence. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to about 10 mL, refrigerated in a refrigerator for crystallization, filtered, and washed with a small amount of dichloromethane to obtain compound III.

[0038] Structural data characterization:

[0039] 13 C NMR (151MHz, DMSO) δ197.60,178.93,170.68,147.51,144.59,141.61,135.02,134.05(2C),133.51,133.30,132.72,131.69,129.13,128 .98,127.38,127.17,125.87(2C),125.72,125.38,113.95,72.36,63 .34,59.46,57.92,57.50,56.04,54.86,54.57,53.62,52.95,52.45, 52.12,48.14,47.01,43.98,41.93,36.02,35.69,35.11,33.54,31.3 1,30.12,29.35,29.02(2C),27.31,26.80(2C),26.58,25.71,24.77, 24.75,24.51,24.20,24.02,23.87,23.81,23.44,22.83(2C),22.25,22.11,21.31,18.53(2C),14.86,13.92,13.32.HRMS(ESI):m / z[M+H] +Calculate for C 70 H 101 O5:1021.7644; found:1021.7640.

[0040] Embodiment 2:

[0041] Anti-inflammatory activity experiment of glycyrrhetinic acid lutein ester:

[0042] In order to verify that the glycyrrhetinic acid lutein ester compound disclosed in the present invention has anti-inflammatory activity, the glycyrrhetinic acid lutein ester compound prepared in the above example was selected for activity experiment.

[0043] 1. Experimental principle: Lipopolysaccharide (LPS) is one of the components of the cell wall of Gram-negative bacteria. LPS is a pathogen existing in nature. Its toxic component is mainly lipid A, which is the main pathogenic part that triggers inflammatory response. Nitric oxide (NO) plays an important role in the immune system and is involved in the pathogenesis and control of infectious diseases, tumors, chronic degenerative diseases and autoimmune processes. RAW264.7 cells are mouse mononuclear macrophages. LPS induces RAW264.7 cells to produce NO. Griess reagent was used to detect the level of LPS-induced NO release in RAW264.7 cells to evaluate the anti-inflammatory activity of glycyrrhetinic acid lutein ester compounds.

[0044] 2. The experimental steps are as follows:

[0045] (1) Weigh the compound and dissolve it in DMSO.

[0046] (2) RAW264.7 cells were plated in 48-well plates at 5 × 10 4 Cells were cultured overnight at a density of 100 μl / well, with 300 μl of cell suspension in each well.

[0047] (3) After 24 hours, 300 μL of 20 μM glycyrrhetinic acid lutein ester, lutein, and glycyrrhetinic acid solutions, as well as 300 μL of solutions of the three compounds at different concentration gradients (2.5, 5, 10, 20, and 40 μM) were added to the drug wells in the 48-well plate, respectively. The control wells and stimulation wells were replaced with culture medium. One hour later, LPS (500 ng / mL) was used to stimulate the cells in the 48-well plate. LPS did not need to be added to the control wells, but it was added to the remaining wells.

[0048] (3) After 24 hours, the NO content was detected using a Griess kit. First, 50 μL of the supernatant of the cell culture medium was placed in a 96-well plate, and then 50 μL of Griess reagents I and II were respectively placed in the 96-well plate containing the supernatant, and the mixed solution was measured at 540 nm using an ELISA reader. The NO inhibition rate was calculated according to the formula:

[0049] NO inhibition rate (%) = (A (刺激孔) -A (给药孔) )÷(A (刺激孔) -A (对照孔) )×100

[0050] 3. Experimental results: At a concentration of 20 μM, the NO inhibition rate of lutein was 55.89±10.22, the NO inhibition rate of glycyrrhetinic acid was only 16.79±6.18, and the NO inhibition rate of glycyrrhetinic acid lutein ester (I) was 109.02±3.2, and its anti-inflammatory activity was significantly enhanced. At the same time, its IC 50 The value is 3.92μM.

[0051] Example 3

[0052] Wound healing experiment of glycyrrhetinic acid lutein ester:

[0053] In order to verify that the glycyrrhetinic acid lutein ester disclosed in the present invention has the ability to heal wounds, the glycyrrhetinic acid lutein ester prepared in the above example was selected to perform a cell scratch experiment.

[0054] 1. Principle: The cell scratch assay, also known as the wound healing assay, is an experimental technique widely used in cell biology research, mainly used to evaluate the migration ability, repair ability and cell-to-cell interaction of cells. This experimental technique is of great value in studying cell migration and invasion, the wound healing process, and the effect of drugs on cell migration. The principle of the cell scratch assay is based on artificially creating a "scratch" or blank area on the cell monolayer, and then observing and measuring how the cells fill this blank area. When the cell monolayer is fused, a scratch is made on the cell layer using a gun tip or other tool, and the cells at the edge of the scratch will fill this blank area by migrating. By capturing images at different time points and measuring the changes in the scratch spacing, the migration speed and repair ability of the cells can be quantitatively analyzed.

[0055] 2. The experimental steps are as follows:

[0056] (1) Marking: On the bottom of the six-well plate, use a marker to draw five horizontal lines along the ruler as marking lines;

[0057] (2) Plating: Plating according to the group, using HaCaT cells, with a cell density of 5×10 5 About 100 cells / well, shake to spread the cells evenly;

[0058] (3) Scratching: After the cells have grown fully (about 24 hours later), use the tip of a white gun to draw a vertical line perpendicular to the well plate and the marked line so that the scratch and the marked line intersect, forming 7 areas. Select 3 areas as fixed detection points.

[0059] (4) Washing: Discard the old culture medium and gently rinse with PBS two to three times until the cells are washed clean;

[0060] (5) Adding liquid: According to the grouping, the drug administration group was added with the culture medium of compound I (6.25, 12.5 μM), and the control group was added with the same volume of serum-free culture medium;

[0061] (6) Photographing and observing: After scratching, washing, and adding liquid, take a 4X photo under a microscope as a 0h control. Place the cells in a 37°C, 5% CO2 incubator. Take out the cells at 24h and 48h, observe the width of the scratch at the same position under a microscope, and take photos;

[0062] (7) Data analysis: ImageJ software can be used to analyze the scratch area and calculate it using the formula.

[0063] 24h / 48h scratch area (%) = (0h scratch area - 24h / 48h scratch area) ÷ 0h scratch area × 100.

[0064] 3. Experimental results: The effect of compound I on enhancing the proliferation and migration of HaCaT cells was evaluated by the scratch method. Figure 1 As shown. The results showed that the scratches in the Normal group slowly narrowed within 48 hours. Through drug intervention, at concentrations of 6.25μM and 12.5μM, the scratches narrowed, the cell state was stable, and the scratch area 0h-24h and 24h-48h change ratios were higher than those in the normal group, indicating that compound I has good repair ability and has a certain therapeutic effect on wound healing.

[0065] Example 4

[0066] Protective and therapeutic effects of glycyrrhetinic acid lutein ester on acute ultraviolet damaged mouse skin model:

[0067] 1. Research background: Skin cancer is mainly caused by exposure to ultraviolet radiation. Ultraviolet radiation comes from artificial sunlight sources such as the sun or tanning beds. In 2020, excessive exposure to ultraviolet rays caused approximately 1.2 million new cases of squamous cell carcinoma and basal cell carcinoma (non-melanoma skin cancer) and 325,000 cases of skin melanoma, 64,000 premature deaths from non-melanoma skin cancer, and 57,000 premature deaths from skin melanoma. It is estimated that a total of 18 million people worldwide are blind due to cataracts, of which about 10% may be due to exposure to ultraviolet radiation. Excessive sun exposure in children and adolescents will lead to skin cancer in the future.

[0068] 2. Materials: The experimental model was 7-8 week old female BALB / c mice, provided by the Experimental Animal Center of Anhui Medical University, and kept in a purified and ventilated animal room at (25±2)℃, 12h light / dark conditions, 22-25℃ ambient temperature, with free access to food and water.

[0069] 3. Methods:

[0070] 3.1 Preparation of cream containing compound I:

[0071] The oil phase of stearic acid (2 g), vaseline (1 g), glyceryl monostearate (1 g) and liquid paraffin (3 g) was mixed and melted at 80° C. Then, different concentrations of Compound I (0.5%, 4% W / O) were mixed with the aqueous phase of Tween-80 (1 g), glycerol (2 g), and distilled water (50 mL), and finally the aqueous phase was mixed and stirred with the oil phase at 30° C. for emulsification.

[0072] 3.2 Animal experiments:

[0073] After the standard anesthesia procedure of intraperitoneal injection of sodium pentobarbital (60 mg / kg body weight) in mice, the back area below the back above the tail was shaved. Then the mice were divided into a control group, a model group, a 4% cream group containing compound I, and a 0.5% cream group containing compound I. Every other day after depilation, the depilatory area of ​​the mice was smeared with the same dose of cream, and the control group and the model group were smeared with a drug-free cream. After three days of medication, the mice were anesthetized again on the fourth day, and the depilatory area was irradiated with a 311nm ultraviolet lamp for 30 minutes in a circular area with a diameter of 1 cm, and the rest of the body was covered with tin foil. After irradiation, the cream was applied to the depilatory area for three consecutive days. The skin of the depilatory area of ​​the mice was photographed and recorded every day.

[0074] 3.3 Experimental results: Figure 2 As shown, after UV irradiation on the fourth day, the skin of the model group on the fifth day showed obvious skin damage and redness compared with the normal group. The IL group of the drug administration showed slight skin damage, while the IH skin showed no obvious changes. The drug administration was continued for three days, and the skin changes of the mice on the eighth day were observed. The skin damage wound of the model group was increasing. In the drug administration group, IL showed obvious damage compared with the fifth day, and IH still showed no obvious changes, indicating that the compound has a good protective effect on the skin irradiated by ultraviolet rays, but the effect is worse at low concentrations than at high concentrations. At the concentration of 4%, compound I has a good protective and repairing effect.

[0075] Table 1. Anti-inflammatory activity of glycyrrhetinic acid lutein ester

[0076]

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A glycyrrhetinic acid lutein ester compound, characterized in that: The glycyrrhetinic acid lutein ester compound is:

2. A method for preparing a glycyrrhetinic acid lutein ester compound, characterized in that: The method comprises: Lutein and triethylamine are dissolved in dichloromethane, and a sulfonylating agent is added in an ice bath to generate a sulfonated intermediate of lutein; The lutein sulfonylation intermediate reacts with glycyrrhetinic acid under alkaline conditions to undergo a nucleophilic substitution reaction to generate a glycyrrhetinic acid lutein ester compound.

3. The method for preparing the glycyrrhetinic acid lutein ester compound according to claim 2, characterized in that: The sulfonylating agent is p-toluenesulfonyl chloride, methanesulfonyl chloride or benzenesulfonyl chloride.

4. The method for preparing the glycyrrhetinic acid lutein ester compound according to claim 2, characterized in that: The synthetic route of glycyrrhetinic acid lutein ester compound is:

5. The method for preparing the glycyrrhetinic acid lutein ester compound according to claim 2, characterized in that: The step of dissolving lutein and triethylamine in dichloromethane, adding a sulfonylation reagent in an ice bath, and generating a lutein sulfonylation intermediate comprises: Dissolve lutein in dichloromethane, place in an ice bath, add triethylamine, slowly drop p-toluenesulfonyl chloride solution into the reaction solution, stir, and detect by TLC until the reaction is complete. Add water, extract and separate the liquids, wash with sodium bicarbonate solution, water and brine in sequence, dry the organic phase with anhydrous sodium sulfate, filter, add anhydrous ethanol, concentrate under reduced pressure, distill to remove dichloromethane, refrigerate in a refrigerator for crystallization, and filter to obtain lutein sulfonylation intermediate.

6. The method for preparing the glycyrrhetinic acid lutein ester compound according to claim 2, characterized in that: The step of subjecting the lutein sulfonylation intermediate to a nucleophilic substitution reaction with glycyrrhetinic acid under alkaline conditions to generate a glycyrrhetinic acid-lutein ester compound comprises: The lutein sulfonylation intermediate was dissolved in a mixed solvent of dichloromethane:acetonitrile, glycyrrhetinic acid and potassium carbonate were added, and the mixture was stirred at room temperature for 3 hours. The mixture was monitored by TLC thin-layer chromatography. After the reaction was complete, water was added, and the liquids were separated by extraction. The aqueous layer was extracted with dichloromethane, and the organic phases were combined and washed with sodium bicarbonate solution, water and brine in sequence. The organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to 10 mL. The mixture was refrigerated and placed for crystallization. The mixture was filtered and washed with a small amount of dichloromethane to obtain a glycyrrhetinic acid lutein ester compound.

7. Use of a glycyrrhetinic acid lutein ester compound in the preparation of anti-inflammatory and antioxidant products.

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