A novel typha glycoside graft compound, its preparation method and application

Abstract

The application discloses a typhaloside grafted compound and a preparation method and application thereof. The typhaloside grafted compound has the following structural expression, and the preparation method comprises the following steps: dissolving epigallocatechin gallate and typhaloside, reacting under the action of an acid catalyst, and preparing the typhaloside grafted compound. The typhaloside grafted compound has improved uric acid reducing effect.

Description

Technical Field

[0001] This invention relates to a grafted compound, and more particularly to a new typha glycoside grafted compound, its preparation method, and its application, belonging to the field of pharmaceutical synthesis technology. Background Technology

[0002] Uric acid (UA) is a chemical waste product (excreted by the kidneys) and a byproduct of purine metabolism. Normal UA concentrations in urine and serum range from 1.40 to 4.40 mmol / L and 0.30 to 0.5 mmol / L, respectively. Elevated UA levels can lead to various diseases, such as gout, hyperuricemia, Lesch-Nyhan syndrome, kidney disease, and cardiovascular disease. Therefore, the treatment of hyperuricemia is of significant clinical importance.

[0003] Currently, commonly used drugs in clinical practice include allopurinol and febuxostat, xanthine oxidase inhibitors that inhibit uric acid synthesis, and probenecid and benzbromarone, which promote uric acid excretion. Although these drugs are highly effective, they have varying degrees of adverse reactions. Allopurinol can cause severe and even fatal skin damage in some patients; febuxostat carries unpredictable cardiovascular risks and can increase the risk of death; and benzbromarone can cause severe hepatotoxicity. For these reasons, there is a high demand for the development of drugs to treat hyperuricemia.

[0004] Typhaneoside is one of the main flavonoid components in Typha pollen, and it has effects such as improving microcirculation, reducing oxygen consumption in the heart and brain, dilating blood vessels, promoting coagulation, anti-inflammation, inducing labor, lowering blood lipids, and anti-atherosclerosis. Furthermore, patent CN116196323B first reported the application of typhaneoside in lowering uric acid. However, through in-depth research, the inventors found that directly using typhaneoside as the active ingredient at low concentrations still cannot achieve a satisfactory uric acid-lowering effect. Summary of the Invention

[0005] To address the above technical problems, this invention proposes a new typha glycoside graft compound, its preparation method, and its application.

[0006] First, this invention provides a new typha glycoside graft compound having the following structural expression:

[0007]

[0008] Secondly, the present invention also provides a method for preparing a new typha glycoside graft compound, comprising: dissolving epigallocatechin gallate and typha glycoside, reacting them under the action of an acid catalyst to obtain the new typha glycoside graft compound.

[0009] The reaction process expression above is as follows:

[0010]

[0011] In the preparation method of this invention, epigallocatechin gallate (EGCG) can be derived from Wuyi rock tea extract. Wuyi rock tea is an oolong tea with the characteristic "rocky charm" (rocky essence and floral aroma), and long-term consumption has anti-obesity and lipid-lowering effects, exhibiting good biocompatibility. Typhain is one of the main flavonoid components in Typha pollen, also derived from plants, and has almost zero biotoxicity. This invention, through chemical modification, reacts EGCG with typhain, and the resulting product unexpectedly possesses a significant uric acid-lowering effect, thus completing this invention. This invention greatly increases the market prospects of EGCG-modified products and is expected to contribute to the development of future uric acid-lowering drugs.

[0012] As a preferred embodiment of the preparation method of the present invention, the acid catalyst is an inorganic acid, preferably one or more of hydrochloric acid, sulfuric acid, and nitric acid, with hydrochloric acid being the most preferred.

[0013] As a preferred embodiment of the preparation method of the present invention, the epigallocatechin gallate is dissolved in an aqueous solution of an acid catalyst, and then typha glycoside is added for reaction.

[0014] Preferably, the concentration of the aqueous solution of the acid catalyst is 2-10 mol / L, more preferably 5-8 mol / L, such as 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, etc.

[0015] As a preferred embodiment of the preparation method of the present invention, the molar ratio of epigallocatechin gallate and typhaein is (1-1.4):1, preferably (1.2-1.3):1, for example 1.1:1, 1.15:1, 1.2:1, 1.25:1, 1.3:1, 1.35:1, etc.

[0016] As a preferred embodiment of the preparation method of the present invention, the reaction is carried out under reflux with stirring for 3-5 hours, for example, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, etc. Preferably, the reflux temperature is 75-120℃, and more preferably 80℃, 85℃, 90℃, 95℃, 100℃, 105℃, 110℃, 115℃, etc.

[0017] As a preferred embodiment of the preparation method of the present invention, after the reaction is completed, the reaction solution is cooled and neutralized with alkali to a pH of 6.5-7, and the typha glycoside graft compound is purified to obtain the new typha glycoside.

[0018] The base used for neutralization can be any inert organic or inorganic base. Considering the need for simpler post-processing, inorganic bases are preferred, and more preferably one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.

[0019] More preferably, the concentration of the inorganic base can be 1-5 mol / L.

[0020] In this invention, the specific purification method for the product is not limited in any way, and can be extraction purification, crystallization purification, column chromatography purification, or a combination of at least two of them, which are conventionally used in the art. For extraction purification, suitable extractants are ethyl acetate and water. In order to improve the purity and yield of the extract, the product can be recovered by multiple extractions. After extraction, conventional vacuum distillation and crystallization / column chromatography methods can be used for further purification.

[0021] Furthermore, the present invention also provides the use of the typha-noside grafted compound as described above or the typha-noside grafted compound prepared by the method described above as a uric acid-lowering drug.

[0022] This invention reports for the first time a novel typha-based glycoside graft compound and its synthesis. The bio-based raw material is widely available and has almost zero biotoxicity. It has good human adaptability and is expected to reduce adverse reactions and other intolerances in humans while lowering high uric acid levels. Moreover, compared with typha-based glycoside, it still has a significant uric acid-lowering effect at a lower dose. Detailed Implementation

[0023] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention.

[0024] Unless otherwise specified, all raw materials and reagents used in this invention can be purchased commercially; all solvents are of analytical grade. Epigallocatechin gallate (CAS 989-51-5) was purchased from Shanghai Maclean, and typhaoside (CAS 104472-68-6) was purchased from Shanghai Aladdin.

[0025]

Example 1

[0026] Epigallocatechin gallate (531 mg, 1.2 mmol) was dissolved in 20 mL of 3 mol / L hydrochloric acid. The mixture was heated and stirred at 100 °C for 30 min until completely dissolved. Then, typhain (770 mg, 1 mmol) was added, and the mixture was heated under reflux and stirred for another 4 hours to complete the reaction. The reaction solution was cooled to room temperature and neutralized to pH 7.0 with 2 mol / L sodium hydroxide aqueous solution. After cooling, the solution was extracted three times with 200 mL of ethyl acetate. The extractant was then washed with 100 mL of saturated sodium chloride solution, and excess water was removed with anhydrous sodium sulfate. After drying, the solvent was evaporated, and the solution was purified by column chromatography (methanol:dichloromethane, volume ratio 1:100) to obtain the product, namely the typhain graft compound (yield 39%).

[0027] 1H NMR (500MHz, Chloroform-d): δ8.13(d,J=2.2Hz,1H),7.73(dd,J=9.2,2.1Hz,1H),7.66(s,2H),7.47(s,1H),7.27(s,1H),7.09(d,J=9.0Hz ,3H),6.88(d,J=2.2Hz,1H),6.68(d,J=2.4Hz,1H),5.57(d,J=7.7Hz,1H),5.17(d,J=6.4Hz,2H),5.04(dt,J=6.8,3.8Hz,1H),4.98(d,J=5.8 Hz,2H),4.63–4.52(m,2H),4.42(d,J=6.0Hz,1H),4.24(d,J=6.4Hz,1H),3.92–3.78(m,9H),3.73–3.62(m,4H),3.53(tdd,J=9.1,6.4,2.4Hz ,1H),3.46(tdt,J=9.3,6.4,3.9Hz,2H),3.30(d,J=6.6Hz,1H),3.27(ddt,J=8.9,4.5,2.3Hz,1H),2.20(s,1H),1.25(dd,J=6.2,1.1Hz,6H).

[0028]

Example 2

[0029] Epigallocatechin gallate (619 mg, 1.4 mmol) was dissolved in 20 mL of 6 mol / L hydrochloric acid, heated and stirred at 90 °C for 30 min until completely dissolved. Typhain (770 mg, 1 mmol) was then added, and the mixture was heated under reflux and stirred for 5 hours to complete the reaction. The reaction solution was cooled to room temperature and neutralized to pH 7.0 with 2 mol / L sodium hydroxide aqueous solution. After cooling, the solution was extracted three times with 200 mL of ethyl acetate. The extractant was then washed with 100 mL of saturated sodium chloride solution, and excess water was removed with anhydrous sodium sulfate. After drying, the solvent was evaporated, and the solution was purified by column chromatography (methanol:dichloromethane, volume ratio 1:100) to obtain the product, the typhain graft compound (yield 43%).

[0030]

Example 3

[0031] Epigallocatechin gallate (486 mg, 1.1 mmol) was dissolved in 20 mL of 8 mol / L hydrochloric acid. The mixture was heated and stirred at 120 °C for 30 min until completely dissolved. Then, typhain (770 mg, 1 mmol) was added, and the mixture was heated under reflux and stirred for another 3 hours to complete the reaction. The reaction solution was cooled to room temperature and neutralized to pH 7.0 with 2 mol / L sodium hydroxide aqueous solution. After cooling, the solution was extracted three times with 200 mL of ethyl acetate. The extractant was then washed with 100 mL of saturated sodium chloride solution, and excess water was removed with anhydrous sodium sulfate. After drying, the solvent was evaporated, and the solution was purified by column chromatography (methanol:dichloromethane, volume ratio 1:100) to obtain the product, namely the typhain graft compound (yield 41%).

[0032]

Application Example

[0033] (1) Cytotoxicity test:

[0034] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing the typhain graft compound prepared in Example 1, while the control group was a culture medium containing typhain. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD value at 450 nm between the experimental group and the control group at the same concentration.

[0035] (2) Determination of uric acid-lowering effect

[0036] ① Animal Preparation: The experimental animals were adult male SD rats weighing 150-170g each, provided by Yangzhou University. During the experiment, the rats were kept at 21±2℃ with free access to food and water, and a 12-hour light / dark cycle was maintained. Animal welfare and experimental procedures were strictly carried out in accordance with the relevant provisions of the guidelines for the husbandry and use of experimental animals, and every effort was made to minimize animal suffering.

[0037] ② Animal grouping and feeding: Rats were divided into 7 groups, namely blank group, model group, low-dose experimental group, medium-dose experimental group, high-dose experimental group, positive control group, and raw material control group; each group consisted of 10 rats.

[0038] ③ Drug preparation: First, prepare a 0.8 wt% CMC-Na solution. Dissolve the typhain graft compound prepared in Example 1 in the CMC-Na solution to prepare low-dose experimental group solutions, medium-dose experimental group solutions, and high-dose experimental group solutions with drug concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. Then, dissolve benzbromarone in the CMC-Na solution to prepare a positive control group solution with a drug concentration of 1 mg / mL. Dissolve typhain in the CMC-Na solution to prepare a raw material control group solution with a drug concentration of 1 mg / mL.

[0039] ④ Drug administration experiment: Rats were administered the drug via gastric administration at a dose of 10 mL / kg per day for 7 consecutive days, based on their body weight. The specific administration methods were as follows: the blank group and the model group were given 0.8 wt% CMC-Na solution; the low-dose experimental group, the medium-dose experimental group, and the high-dose experimental group were given experimental drug solutions with concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively; the positive control group was given a positive control drug solution with a concentration of 1 mg / mL; and the raw material control group was given a raw material control drug solution with a concentration of 1 mg / mL.

[0040] ⑤ Sample processing and sampling: 30 minutes after the last administration on day 8, except for the control group, all other groups received an intraperitoneal injection of 50 mg / ml hypoxanthine solution (solvent: 0.8 wt% CMC-Na solution) to establish a hyperuricemia model. The dosage was 1000 mg / kg. Immediately 30 minutes after administration, the eyeballs were enucleated and blood was collected. After serum exudation, the sample was centrifuged at 15000 rpm for 4 minutes. The supernatant serum was collected and placed in a capped sample tube, diluted with physiological saline to the required volume, and the serum uric acid content was measured using a Beckman LX20 fully automated biochemical analyzer. Data are expressed as mean ± standard deviation, and the test results are shown in Table 1.

[0041] Table 1. Results of serum uric acid level test

[0042] Serum uric acid (μmol / L) Blank group 127.68±21.63 Model group 695.25±64.37 Low-dose experimental group 573.56±79.84 Medium-dose experimental group 531.04±74.93 High-dose experimental group 449.25±53.28 Positive control group 528.42±68.40 Raw material control group 649.10±58.42

[0043] The test results above show that the typha-based grafted compound provided by this invention can exert a uric acid-lowering effect in a short time at a dosage of 5-20 mg / kg, with almost zero biotoxicity. Moreover, the uric acid-lowering level at lower dosages is significantly better than that of the control group using typha-based grafts alone, producing unexpected effects compared to known technologies, demonstrating significant technical advantages.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A typhaneoside grafted compound, characterized in that, Has the following structural expression: 。 2. The method for preparing the typhaein grafted compound according to claim 1, characterized in that, Include: Dissolve epigallocatechin gallate and typhaneoside, and react under the action of acid catalyst to prepare typhaneoside grafted compound.

3. The method for preparing the typhaein grafted compound according to claim 2, characterized in that, The acid catalyst is inorganic acid.

4. The method for preparing the typhaein grafted compound according to claim 3, characterized in that, The acid catalyst is one or more of hydrochloric acid, sulfuric acid and nitric acid.

5. The method for preparing the typhaein grafted compound according to claim 3, characterized in that, The acid catalyst is hydrochloric acid.

6. The method for preparing the typhaein grafted compound according to claim 2, characterized in that, After the epigallocatechin gallate is dissolved in the aqueous solution of acid catalyst, the typhaneoside is added for reaction.

7. The method for preparing the typhaein grafted compound according to claim 6, characterized in that, The concentration of the aqueous solution of acid catalyst is 2-10 mol / L.

8. The method for preparing the typhaein grafted compound according to claim 7, characterized in that, The concentration of the aqueous solution of acid catalyst is 5-8 mol / L.

9. The method for preparing the typhaein grafted compound according to any one of claims 2-8, characterized in that, The molar ratio of epigallocatechin gallate to typhaneoside is (1-1.4):

1.

10. The method for preparing the typhaein grafted compound according to claim 9, characterized in that, The molar ratio of epigallocatechin gallate to typhaneoside is (1.2-1.3):

1.

11. A process for the preparation of a typhaneoside grafted compound according to any one of claims 2-8, characterized in that, The reaction is stirred under reflux condition for 3-5 h.

12. The method for preparing the typhaein grafted compound according to claim 11, characterized in that, The reflux temperature is 75-120 DEG C.

13. A process for the preparation of a typhaneoside grafted compound according to any one of claims 2-8, characterized in that, After the reaction is completed, the reaction solution is cooled, neutralized with alkali to pH 6.5-7, and purified to obtain typhaneoside grafted compound.

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