Tiopronin-flavantriol derivative as well as preparation method and application thereof

Abstract

The invention discloses tiopronin-flavantriol derivatives as well as a preparation method and application thereof, relates to the technical field of chemical conversion and pharmaceutical activity research of natural products, and aims at solving the problems that high-polymerization-degree procyanidine is difficult to absorb, high in monomer acquisition cost and low in efficiency and hinders biological activity research. A degradation reaction system is constructed based on a functionalized nucleophilic reagent oriented transformation strategy, and a series of novel tiopronin-flavantriol derivatives are prepared by degrading a procyanidine polymer in an acid medium by virtue of free sulfydryl of tiopronin. The invention discloses a one-step separation method for preparing eight derivatives and application potential of the derivatives in the aspect of resisting alcoholic liver injury, and provides new basis and guidance for development and utilization of procyanidine.

Description

Technical Field

[0001] This invention relates to the field of natural product chemical transformation and drug activity research technology, specifically to a method based on a functionalized nucleophilic reagent-directed transformation strategy to degrade proanthocyanidins in plants, construct a structure-activity-guided degradation reaction system to obtain eight thiopronine-flavonoid triol derivatives, and study the activity of the derivatives in resisting alcoholic liver injury. Background Technology

[0002] Proanthocyanidins are a class of highly polymerized polyphenolic compounds composed of different flavanol units linked by C-C bonds. They possess unique chemical structures and significant biological activities, demonstrating important value in the health field, and their food sources are extremely wide and diverse. Tea, a widely consumed beverage globally, is one of the important sources of proanthocyanidins; cocoa beans and their chocolate products also contain a certain amount of proanthocyanidins. In terms of fruits, grapes, blackberries, and other berries, as well as common apples, are all good carriers of proanthocyanidins. Among nuts and legumes, almonds, pistachios, and various beans also contain these polyphenolic compounds. Furthermore, red wine, as a fermented beverage, is also rich in proanthocyanidins due to the grapes it is made from. Therefore, proanthocyanidins are widely distributed in various food categories, including plant-based foods, fruits, nuts, and fermented beverages. This richness of sources provides diverse pathways for humans to obtain these polyphenolic compounds through diet and provides strong support for subsequent research on their health benefits and the development of functional foods.

[0003] The bioactivity of proanthocyanidins is influenced by a combination of factors, among which the characteristics of monomeric compounds, the linkage between monomers, and the degree of polymerization play a crucial role. Monomer flavantriols and di, tri, and tetrameric proanthocyanidins have shown good bioactivity in antioxidation, anti-inflammation, and antibacterial activities. Low-polymer proanthocyanidins, with their excellent water solubility, have broad application potential in food, pharmaceuticals, and cosmetics. However, research shows that proanthocyanidins in plants usually exist in high-polymer forms. Currently, the main challenges in researching high-polymer proanthocyanidins are: firstly, their large molecular weight and complex structure make them difficult for the human body to effectively absorb and utilize; secondly, obtaining their monomeric compounds is extremely difficult, costly, and inefficient. These factors severely hinder in-depth research into the bioactivity of high-polymer proanthocyanidins, leading to the frequent disposal of many of these potentially valuable substances as waste, resulting in a serious waste of plant resources.

[0004] Thiopronine, a novel glycine derivative containing free sulfhydryl groups, has demonstrated significant clinical value, widely used in the treatment of viral hepatitis, alcoholic hepatitis, drug-induced hepatitis, heavy metal poisoning hepatitis, fatty liver, and early-stage cirrhosis. The free sulfhydryl groups in its molecule act as nucleophiles in acidic environments, degrading proanthocyanidin polymers to generate flavanol thiopronine derivatives. For example, in patent CN110372656A, entitled "Flavonol Thiopronine Derivatives and Their Preparation Methods and Uses," thiopronine was used to degrade grape seed proanthocyanidins, successfully obtaining three novel flavanol-thiopronine derivatives (CT, EC-T, and ECG-T), and their antibacterial and antioxidant bioactivities were experimentally verified. This successful case provides new ideas and methods for the research on the degradation and transformation of proanthocyanidins.

[0005] Based on the medicinal value of thiopronine and successful precedents of its degradation, this study focuses on proanthocyanidins in *Persimmon simmon*. Given the lack of reports on proanthocyanidin degradation methods in *Persimmon simmon*, and the absence of research on the activity of novel thiopronine-flavonoid triol derivatives in combating alcoholic liver injury, this study proposes a strategy for the directional transformation of proanthocyanidins using functionalized nucleophilic reagents. This involves constructing a structure-activity-guided degradation reaction system to explore efficient degradation methods for proanthocyanidins in *Persimmon simmon* and to investigate the activity of novel derivatives in combating alcoholic liver injury, providing new theoretical basis and practical guidance for the further development and utilization of proanthocyanidins. Summary of the Invention

[0006] One of the objectives of this invention is to provide a series of thiopronine-flavonoid triol derivatives or pharmaceutically acceptable salts thereof derived from proanthocyanidins in persimmon.

[0007] The second objective of this invention is a method for preparing thiopronine-flavonoid triol derivatives using proanthocyanidins from persimmon as raw material.

[0008] The third objective of this invention is to provide a one-step method for the separation and preparation of eight thiopronine-flavonoid triol derivatives.

[0009] The fourth objective of this invention is to provide the application of the above-mentioned thiopronine-flavantriol derivatives or the eight thiopronine-flavantriol derivatives prepared by the above method in the treatment of alcoholic liver injury.

[0010] The names of the eight thiopronine-flavonoid triol derivatives are: (1) (–)-Gallocatechin-4 β -tiopronin methyl ester (GC-T, (–)-galactoatechin-4)β - Thiopronine methyl ester); (2) (–)-Gallocatechin-3 -O- gallate-4 β- tiopronin methyl ester (GCG-T,(–)-galactocatechin-3) -O- Gallate-4 β - Thiopronine methyl ester); (3) (+)-Catechin-4 β- tiopronin methyl ester (CT, (–)-catechin-4) β - Thiopronine methyl ester); (4) (–)-Epigallocatechin-4 β -tiopronin methyl ester (EGC-T, (–)-epigallocatechin-4) β - Thiopronine methyl ester); (5) (+)-Catechin-3 -O- gallate-4 β- tiopronin methyl ester (CG-T, (–)-catechin-3) -O- Gallate-4 β - Thiopronine methyl ester); (6) (–)-Epigallocatechin-3 -O- gallate-4 β- tiopronin methyl ester (EGCG-T, (–)-epigallocatechin-3) -O- Gallate-4 β - Thiopronine methyl ester); (7) (–)-Epicatechin-4 β- tiopronin methyl ester (EC-T, (–)-epicatechin-4) β - Thiopronine methyl ester); (8) (–)-Epicatechin-3 -O- gallate-4 β- tiopronin methyl ester (ECG-T,(–)-epicatechin-3) -O- Gallate-4 β - Thiopronine methyl ester).

[0011] The above eight thiopronine-flavonoid triol derivatives each have the following structures:

[0012] This invention also provides a method for preparing thiopronine-flavonoid triol derivatives by degradation of high-polymerization-degree polyphenols (PPPs) from persimmon, the specific steps of which are as follows: 1) Take the polyphenol extract of persimmon and the nucleophilic reagent thioproline, add acidified methanol, and prepare a reaction solution with a weight-volume ratio of 1 mg / mL to 5 mg / mL. 2) Place the above reaction solution in a sealed reaction flask and carry out acid-catalyzed degradation to obtain the degradation reaction solution; 3) Add water to the degradation reaction solution obtained in step 2) to adjust the pH of the system to 7.0, remove the organic solvent by rotary evaporation, reconstitute with water, separate using an open YMC-ODS-A-HG column, elute with water, ethyl acetate and methanol in sequence, collect the ethyl acetate eluent, add anhydrous sodium sulfate to remove water, remove the organic solvent by rotary evaporation, and freeze dry for later use.

[0013] The preparation method of the polyphenol extract of *Persimmon pulveratum* in step 1) is as follows: *Persimmon pulveratum* fruit powder is heated sequentially with methanol-water (80 / 20, v / v) and acetone-water (70 / 30, v / v) in a water bath at 50℃~60℃ for 1 h~2 h. The filtrate is obtained by filtration, and the organic solvent is removed by rotary evaporation to obtain a crude polyphenol extract solution of *Persimmon pulveratum* fruit. The crude polyphenol extract solution of *Persimmon pulveratum* fruit is loaded onto AB-8 macroporous adsorption resin. After adsorption, sugars and other insoluble substances are first eluted with purified water, followed by elution with anhydrous ethanol. The anhydrous ethanol eluent of *Persimmon pulveratum* fruit is collected, and the anhydrous ethanol is removed by rotary evaporation and then freeze-dried to obtain the polyphenol extract of *Persimmon pulveratum* fruit.

[0014] In step 1), the acidified methanol is a methanol solution that has been treated with hydrochloric acid.

[0015] In step 1), the mass ratio of the persimmon polyphenol extract to thioproline is 1:1 to 1:5, and more preferably 1:3.

[0016] In step 2), the acid-catalyzed degradation time is 20 min to 100 min, more preferably 60 min; the acid used is a conventional acid such as hydrochloric acid or sulfuric acid, preferably hydrochloric acid, with a concentration of 0.1 M to 0.5 M, more preferably 0.3 M; the acid-catalyzed degradation temperature is 30℃ to 70℃, more preferably 60℃.

[0017] In step 3), a YMC-ODS-A-HG (200×30 mm id, 25-40 μm) column was used for separation. After pretreatment with deionized water, the degradation reaction solution was loaded onto the column. Interfering components such as inorganic salts were eluted with 300 mL of distilled water, followed by elution with 250 mL of ethyl acetate to obtain the total thioproline degradation products. Undegraded polyphenols and other impurities were eluted with 250 mL of methanol. The ethyl acetate eluent was rotary evaporated at 40℃~45℃ to remove organic solvents, reconstituted with distilled water, and lyophilized for later use.

[0018] Furthermore, the degradation solution obtained from the acidification and degradation of the polyphenols in the above-mentioned persimmon was separated using a one-step method to obtain the monomeric compounds. The operation steps included: The lyophilized powder obtained in step 3) of the above degradation method is reconstituted with a water-methanol solution and then separated by a preparative reversed-phase high-performance liquid chromatography column to obtain the monomeric compounds of the eight thiopronine-flavonoid triol derivatives of the present invention.

[0019] The chromatographic column used was a reversed-phase C18 (250×10 mm, 5 μm) column, model YMC-Pack ODS-A (250×10 mm, 5 μm) (YMC Corporation, Japan); mobile phase A: 0.2% formic acid + 99.8% water, mobile phase B: 0.2% formic acid + 99.8% acetonitrile, flow rate 3.0 mL / min; column temperature 30℃; detection wavelength 280 nm; injection volume 2 mL. Gradient elution was used.

[0020] Specifically, the water-methanol solution is a 25% methanol aqueous solution.

[0021] The specific gradient elution program was as follows: 0 min → 10 min (i.e., from 0 minutes to 10 minutes, the rest is the same), 12% → 15% B (i.e., the concentration of phase B increases from 12% to 15%, the rest is the same); 10 min → 25 min, 15% → 20% B; 25 min → 35 min, 20% → 26% B; 35 min → 48 min, 26% → 32% B; 48 min → 55 min, 32% → 40% B; followed by column flushing and column equilibration. The obtained fractions were collected and rotary evaporated at 40℃~45℃, then lyophilized to obtain the powders of the above eight thiopronine-flavonoid triol derivatives.

[0022] A pharmaceutical composition comprising one or more of the following: a thiopronine-flavanol derivative prepared by degradation of high-polymerization-degree polyphenols (PPPs) of *Persimmon argentea* as described in this invention, or a thiopronine-flavanol derivative obtained by the "one-step" separation method, or a pharmaceutically acceptable salt thereof; and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients include fillers, binders, disintegrants, lubricants, solvents / diluents, cosolvents / sorponents, stabilizers, and pH adjusters. The dosage forms of the pharmaceutical composition include clinically commonly used oral formulations, injectable formulations, and topical formulations. Oral formulations include tablets (including ordinary tablets, film-coated tablets, enteric-coated tablets, and chewable tablets), capsules (including hard capsules and soft capsules), granules, powders, oral suspensions, oral solutions, syrups, etc.; injectable formulations include sterile powders for injection (which need to be dissolved in a suitable solvent before use), and injection solutions (including small-volume injections and large-volume infusions); topical formulations include ointments, creams, gels, lotions, eye drops, nasal drops, etc.

[0023] The present invention also provides the use of thiopronine-flavanol derivatives prepared by degradation of the high degree of polymerization polyphenols (PPPs) of *Persimmon argentea*, or thiopronine-flavanol derivatives obtained by the "one-step" separation method, or pharmaceutical compositions containing one or more of the thiopronine-flavanol derivatives or pharmaceutically acceptable salts thereof, in the preparation of medicaments for treating liver injury. More preferably, the use is in the preparation of medicaments for treating alcoholic liver injury.

[0024] The beneficial effects of the preparation method and uses of the series of thiopronine-flavonoid triol derivatives of the present invention are as follows: This invention provides a series of thiopronine-flavantriol derivatives and their preparation methods, achieving the in-depth development and efficient utilization of natural resources. On the one hand, it increases the added value of resources, bringing new economic growth points to related industries; on the other hand, it reduces dependence on traditional raw materials, helping to alleviate resource shortages and demonstrating good economic and social benefits.

[0025] The proposed one-step separation and preparation method is a major technological breakthrough of this invention. This method simplifies the operation process, shortens the production cycle, improves production efficiency, and reduces production costs. Simultaneously, by reducing operational steps and intermediate processes, it effectively avoids the loss of the target compound, improves product purity and quality, and provides strong technical support for large-scale industrial production.

[0026] The significant potential of thiopronine-flavantriol derivatives in combating alcoholic liver injury offers new directions and insights for drug development. Using highly active derivatives such as GCG-T, ECG-T, CG-T, and EGCG-T as lead compounds, in-depth structural optimization and pharmacodynamic studies hold promise for developing novel anti-alcoholic liver injury drugs with independent intellectual property rights. Furthermore, these derivatives may possess other biological activities, such as antioxidant, anti-inflammatory, and anti-tumor effects, demonstrating the potential for further development into therapeutics for various diseases, with a very broad application prospect. Attached Figure Description

[0027] Figure 1 This is a chromatogram of crude extract of polyphenols from persimmon provided in an embodiment of the present invention; Figure 2 The degradation time (A), degradation temperature (B), acid concentration (C), and mass concentration ratio (D) provided for embodiments of the present invention affect the total yield of thiopronine degradation products. Y T The impact of ) Figure 3 The acid concentration provided in the embodiments of the present invention ( X 1 ), degradation temperature ( X 2 ) and the mass concentration ratio of PPPs to thioproline ( X 3 ) on the total yield of proline degradation products ( Y T The three-dimensional response surface plot of ). Figure 4 Chromatograms of eight thiopronine-flavanotriol derivatives provided in embodiments of the present invention; Figure 5 The (–)-galactocatechin-4 in this invention β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 6 The (–)-galactocatechin-4 in this invention β - Thiopronine methyl ester 13 C-NMR spectrum; Figure 7 The (–)-galactocatechin-3 in this invention -O- Gallate-4 β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 8 The (–)-galactocatechin-3 in this invention -O- Gallate-4 β - Thiopronine methyl ester13 C-NMR spectrum; Figure 9 The (+)-catechin-4 in this invention β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 10 In this invention, (–)-epigallocatechin-4 β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 11 In this invention, (–)-epigallocatechin-4 β - Thiopronine methyl ester 13 C-NMR spectrum; Figure 12 The (+)-catechin-3 in this invention -O- Gallate-4 β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 13 The (+)-catechin-3 in this invention -O- Gallate-4 β - Thiopronine methyl ester 13 C-NMR spectrum; Figure 14 In this invention, (–)-epigallocatechin-3 -O- Gallate-4 β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 15 In this invention, (–)-epigallocatechin-3 -O- Gallate-4 β - Thiopronine methyl ester 13 C-NMR spectrum; Figure 16 In this invention, (–)-epicatechin-4 β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 17 In this invention, (–)-epicatechin-3 -O- Gallate-4 β - Thiopronine methyl ester 1 H-NMR spectrum; Figure 18This is a cell viability diagram of a series of thioproline-flavantriol derivatives in ethanol-induced alcoholic liver damage in BRL-3A cells, as described in this invention; wherein, GC: (–)-gallicatechin, GCG: (–)-gallicatechin gallate, Cat.: (+)-catechin, EGC: (–)-epigallocatechin, CG: (+)-catechin gallate, EGCG: (–)-epigallocatechin gallate, EC: (–)-epicatechin, ECG: (–)-epicatechin-3- O - Gallic acid esters, T: Tiopronin; ### p <0.001, * p <0.05,** p <0.01, *** p <0.001. Detailed Implementation

[0028] The following examples help those skilled in the art to better understand the technical content of the present invention, but do not limit the present invention in any way.

[0029] This invention addresses the problems of high-polymerization-degree proanthocyanidins being difficult to absorb, having high monomer acquisition costs and low efficiency, which hinder bioactivity research. Taking proanthocyanidins as the research object, a degradation reaction system was constructed based on a functionalized nucleophilic reagent-directed conversion strategy. By utilizing the free thiol groups of thiopronin in an acidic medium to degrade proanthocyanidin polymers, a series of thiopronin-flavonoid triol derivatives were prepared.

[0030] First, the reaction conditions for the degradation process were optimized.

[0031] Example 1

[0032] A method for preparing thiopronine-flavonoid triol derivatives by degradation of high-polymerization-degree polyphenols (PPPs) from persimmon: (1) Accurately transfer 8.3 mL of 37% hydrochloric acid solution into a 1L volumetric flask, add methanol to the mark, and you will get a 0.1M hydrochloric acid methanol solution.

[0033] (2) Preparation and column chromatography separation of polyphenol extracts from *Persimmon pulveratum*: *Persimmon pulveratum* fruit powder was successively heated by reflux in a water bath at 60℃ for 1 h with 1000 mL each of methanol-water (80 / 20, v / v) and acetone-water (70 / 30, v / v). The filtrate was filtered, and the organic solvent was removed by rotary evaporation to obtain a crude polyphenol extract solution from *Persimmon pulveratum* fruit. 100 mL of the crude polyphenol extract solution from *Persimmon pulveratum* fruit was loaded onto AB-8 macroporous adsorption resin. After adsorption, sugars and other insoluble substances were first eluted with 3V purified water, followed by elution with 3V anhydrous ethanol. The anhydrous ethanol eluent from *Persimmon pulveratum* fruit was collected, and the anhydrous ethanol was removed by rotary evaporation and then lyophilized to obtain polyphenol extracts (PPPs) from *Persimmon pulveratum* fruit (see [reference]). Figure 1 ).

[0034] (3) PPPs and thiopronine were placed in a 350 mL high-pressure reaction flask at a mass concentration ratio of 1:3. 100 mL of acidified methanol solution containing 0.3 M concentrated hydrochloric acid (2.46 mL) was added. The mixture was sealed and mixed evenly. The final concentrations of PPPs and thiopronine were 1 mg / mL and 3 mg / mL, respectively. The solutions were reacted at 50 °C for different times (20, 40, 60, 80, 100 min). The reaction was terminated by ice bath (0 °C) to obtain degradation reaction solution.

[0035] (4) The above reaction solutions were rotary evaporated at 30°C to obtain degradation reaction product powder (see Figure 2 A).

[0036] Depend on Figure 2 The results of the single-factor experiment show that when the acid-catalyzed degradation time is 20 min and 40 min, as the reaction time increases, Y T The degradation gradually increased, reaching a maximum at 60 min, but as the degradation time was extended to 80 min and 100 min, Y T The gradual decrease in polyphenols is likely due to the fact that, after the polyphenols in persimmons degrade over time, they may combine with other substances to form other byproducts. Y T The time decreased. Therefore, 60 minutes was identified as the optimal condition.

[0037] Example 2

[0038] The method of Example 1 was used to degrade the high degree of polymerization of persimmon polyphenols to obtain thioproline-flavonoid triol derivatives, with the following difference: Step (3): PPPs and thiopronine were placed in a 350 mL high-pressure reaction flask at a mass concentration ratio of 1:3. 100 mL of acidified methanol solution containing 2.46 mL of 0.3 M concentrated hydrochloric acid was added, and the mixture was sealed and thoroughly mixed. The final concentrations of PPPs and thiopronine were 1 mg / mL and 3 mg / mL, respectively. The solutions were reacted at different degradation temperatures (30, 40, 50, 60, 70 °C) for 60 min, and the reaction was terminated in an ice bath (0 °C) to obtain the degradation reaction solution. The rest was the same as in Example 1; the results are shown below. Figure 2 B.

[0039] Depend on Figure 2 The results of the single-factor experiment (B) show that when the degradation temperature is below 60℃, as the reaction temperature increases, Y T It gradually increases, reaching its maximum at 60℃, but as the degradation temperature continues to rise to 70℃, Y T The decrease is likely due to the fact that at excessively high temperatures, the degradation products of polyphenols in persimmons may combine with other substances to form byproducts, leading to [the decrease in temperature]. Y T The temperature drops. Therefore, 60°C is considered the optimal condition.

[0040] Example 3

[0041] The method of Example 1 was used to degrade the high degree of polymerization of persimmon polyphenols to obtain thioproline-flavonoid triol derivatives, with the following difference: Step (3): PPPs and thiopronine were placed in a 350 mL high-pressure reaction flask at a mass concentration ratio of 1:3. 100 mL of acidified methanol solution containing different hydrochloric acid concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 M) was added, and the mixture was sealed and mixed thoroughly. The final concentrations of PPPs and thiopronine were 1 mg / mL and 3 mg / mL, respectively. The solutions were reacted at 50°C for 60 min, and the reaction was terminated by an ice bath (0°C) to obtain the degradation reaction solution. The rest was the same as in Example 1; the results are shown below. Figure 2 C.

[0042] Depend on Figure 2 The results of the single-factor experiment (C) show that when the hydrochloric acid concentration is 0.1 M and 0.2 M, as the hydrochloric acid concentration increases, Y T Gradually increase the concentration until the hydrochloric acid concentration reaches 0.3 M. Y T At its maximum, when the hydrochloric acid concentration was further increased to 0.4 M and 0.5 M, Y T Gradually decrease the acid concentration; higher acid concentrations may cause severe damage to the polyphenol structure of persimmon.Y T The trend is downward. Therefore, 0.3 M is identified as the optimal condition.

[0043] Example 4

[0044] The method of Example 1 was used to degrade the high degree of polymerization of persimmon polyphenols to obtain thioproline-flavonoid triol derivatives, with the following difference: Step (3): PPPs and thiopronine were placed in 350 mL high-pressure reaction flasks at different mass concentration ratios (1:1, 1:2, 1:3, 1:4, and 1:5). 100 mL of acidified methanol solution containing 2.46 mL of 0.3 M concentrated hydrochloric acid was added. The mixture was sealed and thoroughly mixed. The solutions with different mass ratios were reacted at 50 °C for 60 min. The reaction was terminated by an ice bath (0 °C) to obtain the degradation reaction solution. The rest was the same as in Example 1; the results are shown below. Figure 2 D.

[0045] Depend on Figure 2 The results of the single-factor experiment (D) show that when the mass concentration ratio of PPPs to thioproline is 1:1 and 1:2, as the mass concentration ratio increases, Y T Gradually increase. When the concentration ratio of PPPs to thiopronine is 1:3, Y T Reaching the maximum. Further increasing the concentration ratio to 1:4 and 1:5, Y T Instead of continuing to increase, the concentration of PPPs decreased. This is likely because the nucleophile required at the C4 position of flavanols has reached saturation. Further addition of the nucleophile would interfere with the normal binding of flavanols and would also lead to waste. Therefore, a mass concentration ratio of PPPs to thioproline of 1:3 was determined to be the optimal condition.

[0046] Example 5

[0047] The method is the same as in Example 1. Based on Examples 1-4 above, the degradation time is... Y T The effect of was relatively small, while the other three factors had a smaller impact within the experimental range. Y T The impact is significant. Therefore, the degradation time was initially determined to be 60 min, based on the acid concentration ( X 1 ), degradation temperature ( X 2 ), the mass concentration ratio of PPPs to thioproline ( X 3 The main factor considered was the total yield of thiopronine degradation products. Y TThe degradation process was optimized using a 3-factor, 3-level Box-Behnken Design (BBD) with the response value as the factor. Factor codes and levels are shown in Table 1, and the results of the 17 groups of experiments are shown in Table 2.

[0048] Table 1. Coding and Levels of Each Factor

[0049] Table 2 Box-Behnken Design Experiment and Results

[0050] Box-Behnken Design model predictions and statistical analysis results: The experimental results were fitted using a quadratic multiple regression analysis using Design Expert V8.0.6.1 software to establish the total yield of thiopronine derivatives ( Y T ) on hydrochloric acid concentration ( X 1 ), degradation temperature ( X 2 ), the mass concentration ratio of PPPs to thioproline ( X 3 The quadratic multinomial regression numerical model equation is as follows: Y T =83.20+0.1538 X 1 +2.57 X 2 +2.87 X 3-0.4250 X 1 X 2 +1.52 X 1 X 3 +2.23 X 2 X 3 -4.09 X 1 2 -6.49 X 2 2 -7.19 X 3 2 Analysis of variance (ANOVA) was performed on the quadratic multinomial numerical model, and the results are shown in Table 3. The reliability of the model was evaluated based on ANOVA and correlation coefficients. FThe significance of the regression equation is determined by a probability test. P The smaller the value, the more significant the model. F= 45.64, P <0.0001 indicates that the experimental model is highly significant and the regression equation has a high degree of fit; the model determination coefficient R 2 = 0.9833, R 2 (adjusted) = A value of 0.9617 indicates a good model fit; the coefficient of variation is less than 5%. CV The percentage (1.70%) indicates that the model has good repeatability; the lack of fit term F= 2.06, P= 0.2489 > 0.05, indicating that the lack-of-fit term is not significant and can be used for predictive analysis of the degradation and conversion reaction of persimmon polyphenol thiopronin. Among the three factors affecting the degradation yield of thiopronin, the regression model... P It can be seen that the impact of the first-order term on the total yield is X 3 > X 2 > X 1 ,and X 3 and X 2 It is significant in the interaction terms. X 1 X 3 , X 2 X 3 It is significant in the quadratic term. X 1 2 , X 2 2 , X 3 2 All were significant, as shown in Table 3.

[0051] Table 3. Factor analysis results of the binomial regression equation.

[0052] A three-dimensional surface plot of the interactions of various factors can be found here. Figure 3 As shown, the surface exhibits a steep ellipse shape, opening downwards. As the factor level increases, Y TThe content showed a trend of first increasing and then decreasing. The optimal degradation process conditions obtained from single-factor and response surface optimization experiments were: acid concentration of 0.30 M, temperature of 62.37℃, PPPs to thiopronine mass concentration ratio of 1:3.24, and the highest yield of thiopronine degradation products was 83.85%.

[0053] Example 6

[0054] Based on the optimal conditions obtained above, the high-polymerization degree polyphenols of *Persimmon argentea* were degraded to obtain total thioproline-flavonoid triol derivatives: (1) Accurately transfer 8.3 mL of 37% hydrochloric acid solution into a 1L volumetric flask, add methanol to the mark, and you will get a 0.1M hydrochloric acid methanol solution.

[0055] (2) Preparation and column chromatography separation of polyphenol extract from Diospyros kaki: Diospyros kaki fruit powder was heated in a water bath at 60℃ for 1 h with 1000 mL each of methanol-water (80 / 20, v / v) and acetone-water (70 / 30, v / v). The filtrate was filtered, and the organic solvent was removed by rotary evaporation to obtain crude polyphenol extract of Diospyros kaki fruit. 100 mL of crude polyphenol extract of Diospyros kaki fruit was loaded onto AB-8 macroporous adsorption resin. After adsorption, sugars and other insoluble substances were removed by elution with 3V purified water, followed by elution with 3V anhydrous ethanol. The anhydrous ethanol eluent of Diospyros kaki fruit was collected, and the anhydrous ethanol was removed by rotary evaporation and then lyophilized to obtain polyphenol extract of Diospyros kaki (PPPs).

[0056] (3) PPPs and thiopronine were placed in a 350 mL high-pressure reaction flask at a mass concentration ratio of 1:3. 100 mL of acidified methanol solution containing 0.3 M concentrated hydrochloric acid (2.46 mL) was added. The mixture was sealed and mixed evenly. The final concentrations of PPPs and thiopronine were 1 mg / mL and 3 mg / mL, respectively. The solution was reacted at 60 °C for 60 min. The reaction was terminated by ice bath (0 °C) to obtain the degradation reaction solution. The experiment was repeated three times.

[0057] (4) Add 100 mL of water to the above degradation reaction solution and adjust the pH of the system to 7.0 with an appropriate amount of NaOH. Remove methanol by rotary evaporation and reconstitute with distilled water for later use. Separate the solution using a YMC-ODS-A-HG (200×30 mm id, 25-40 μm) column. After pretreatment with deionized water, load the degradation reaction solution onto the column. Elute with 300 mL of distilled water to remove interfering components such as inorganic salts, 250 mL of ethyl acetate to obtain the total thiopronine degradation product, and 250 mL of methanol to remove undegraded polyphenols and other impurities. Remove the organic solvent from the ethyl acetate eluent by rotary evaporation at 40 °C, reconstitute with distilled water, and freeze-dry for later use. The freeze-dried powder is the ethyl acetate layer powder of the thiopronine-flavonoid triol derivative.

[0058] (5) The powder obtained above was dissolved in 25% methanol aqueous solution, and the compound was separated and purified by semi-preparative liquid chromatography. The chromatographic column was a reversed-phase YMC-Pack ODS-A (250×10 mm, 5 μm) column (YMC Corporation, Japan); mobile phase A: 0.2% formic acid + 99.8% water, B: 0.2% formic acid + 99.8% acetonitrile, flow rate 3.0 mL / min; column temperature 30℃; detection wavelength 280 nm; injection volume 2 mL. Elution was performed by gradient elution, and the specific gradient elution program was as follows: 0→10 min, 12%→15% B; 10→25 min, 15%→20% B; 25→35 min, 20%→26% B; 35→48 min, 26%→32% B; 48→55 min, 32%→40% B; subsequent column flushing and equilibration were performed. The collected components were rotary evaporated at 40°C and freeze-dried to obtain eight thiopronine-flavonoid triol derivative powders (see [link to product]). Figure 4 ).

[0059] In addition, based on actual conditions, the experiment was repeated three times with an acid concentration of 0.30 M, a temperature of 60℃, and a PPPs to thiopronine mass concentration ratio of 1:3. The total yield of thiopronine degradation products was measured to be 85.20 ± 0.60%. The results showed that the difference from the predicted value was not significant, indicating that the method is accurate and reliable.

[0060] Ultra-high performance liquid chromatography was used to... Figure 4 As can be seen from the purity analysis of the chromatographic peaks 1-8 (i.e., the eight thiopronine-flavonoid triol derivatives), the eight thiopronine-flavonoid triol derivatives are denoted as: GC-T, GCG-T, CT, CG-T, EGC-T, EGCG-T, EC-T and ECG-T, and the purity of these derivatives is all above 93%.

[0061] The structures of GC-T, GCG-T, CT, CG-T, EGC-T, EGCG-T, EC-T and ECG-T compounds were identified.

[0062] Each degradation product, 10 mg, was dissolved in deuterated methanol (Methanol-d4) as the effective solvent and then used for nuclear magnetic resonance detection. 1 H-NMR, 13 The structure of degradation products was confirmed by methods such as C-NMR. Figures 5-17 The partial attributions of carbon and hydrogen signals are as follows: (1) (–)-gallocatechin-4 β -tiopronin methyl ester (GC-T): C 21 H 23 NO 10 S. 1 H-NMR (600 MHz, CD3OD) δ 6.55 (s, 2H, C (2′, 6′)-H), 5.99 (d, J = 2.1 Hz, 1H, C (8)-H), 5.92 (d, J = 2.3 Hz, 1H, C (6)-H), 5.14 (s, 1H, C(2)-H), 4.15 (d, J = 2.4 Hz, 1H, C (4)-H), 4.07 (d, J = 2.4 Hz, 1H, C (3)-H),4.07-3.97 (m, 2H, C (13)-H), 3.85 (m, 1H, C (11)-H), 3.71 (s, 3H, C (15)-H),1.55 (d, J = 7.2 Hz, 3H, C (16)-H). 13 C-NMR (151 MHz, CD3OD) δ173.93 (C-14),168.73 (C-12), 156.19 (C-9), 155.84 (C-5), 154.06 (C-7), 143.71 (C-3′, C-5′),130.59 (C-4′), 128.31 (C-1′), 103.87 (C-2′, C-6′), 97.16 (C-10), 93.72 (C-6),92.70 (C-8), 72.63 (C-2), 69.29 (C-3), 49.68 (C-15), 43.33 (C-4), 41.28 (C-13), 39.15 (C-11), 16.01 (C-16)。

[0063] (2)(–)-gallocatechin-3 -O- gallate-4 β -tiopronin methyl ester (GCG-T): C 28 H 27 NO 14 S 1 H-NMR (600 MHz, CD3OD) δ 6.77 (s, 2H, C (2′′, 6′′)-H), 6.43(s, 2H, C (2′, 6′)-H), 5.87 (d, J = 2.3 Hz, 1H, C (8)-H), 5.84 (d, J = 2.3 Hz,1H, C (6)-H), 5.31 (s, 1H, C (2)-H), 5.28 (s, 1H, C (3)-H), 4.16 (d, J = 2.4Hz, 1H, C (4)-H), 3.95 (m, 2H, C (13)-H), 3.85 (m, 1H, C (11)-H), 3.56 (s,3H, C (15)-H), 1.49 (d, J = 7.2 Hz, 3H, C (16)-H). 13 C-NMR (151 Hz, CD3OD) δ175.01 (C-14), 170.19 (C-12), 165.69 (C-7′′), 157.97 (C-9), 157.07 (C-5),155.46 (C-7), 145.26 (C-3′, C-5′), 144.76 (C-3′′, C-5′′), 138.42 (C-4′′),132.32 (C-4′), 128.85 (C-1′), 119.58 (C-1′′), 108.79 (C-2′′, C-6′′), 105.37(C-2′, C-6′), 98.08 (C-10), 95.42 (C-6), 94.18 (C-8), 73.20 (C-2), 72.23 (C-3), 51.12 (C-15), 44.95 (C-4), 40.94 (C-13), 40.22 (C-11), 17.60 (C-16)。

[0064] (3)(+)-catechin-4 β -tiopronin methyl ester (C-T): C 21 H 23 NO9S. 1 H-NMR (600 MHz, CD3OD) δ 6.88 (d, J = 2.1 Hz, 1H, C (2′)-H),6.73 (dd, J = 8.2, 2.1 Hz, 1H, C (6′)-H), 6.67 (d, J = 8.1 Hz, 1H, C (5′)-H),5.87 (d, J = 2.4 Hz, 1H, C (8)-H), 5.80 (d, J = 2.3 Hz, 1H, C (6)-H), 5.09 (s,1H, C (2)-H), 4.03 (d, J = 2.5 Hz, 1H, C (4)-H), 3.96 (d, J = 2.5 Hz, 1H, C(3)-H), 3.87 (m, 2H, C (13)-H), 3.73 (m, 1H, C (11)-H), 3.58 (s, 3H, C (15)-H), 1.42 (d, J = 7.2 Hz, 3H, C (16)-H)。

[0065] (4)(–)-epigallocatechin-4 β -tiopronin methyl ester (EGC-T): C 21 H 23 NO 10 S. 1 H-NMR (600 MHz, CD3OD) δ 6.57 (s, 2H, C (2′, 6′)-H), 5.99 (d, J = 2.3 Hz, 1H, C (8)-H), 5.95 (d, J = 2.3 Hz, 1H, C (6)-H), 5.20 (s, 1H, C(2)-H), 4.09 (d, J = 2.5 Hz, 1H, C (4)-H), 4.06 (m, 2H, C (13)-H), 4.01 (d, J = 2.5 Hz, 1H, C (3)-H), 3.78 (m, 1H, C (11)-H), 3.75 (s, 3H, C (15)-H), 1.52(d, J = 7.4 Hz, 3H, C (16)-H). 13 C-NMR (151 MHz, CD3OD) δ 174.27 (C-14), 170.02(C-12), 158.01 (C-9), 157.33 (C-5), 156.04 (C-7), 145.31 (C-3′, C-5′), 132.20(C-4′), 129.64 (C-1′), 105.45 (C-2′, C-6′), 97.54 (C-10), 95.38 (C-6), 94.54(C-8), 74.16 (C-2), 70.23 (C-3), 51.20 (C-15), 42.90 (C-4), 42.17 (C-13),40.76 (C-11), 17.28 (C-16)。

[0066] (5)(+)-catechin-3 -O- gallate-4 β -tiopronin methyl ester (CG-T): C 28 H27 NO 13 S. 1 H-NMR (600 MHz, CD3OD) δ 6.85 (d, J = 2.0 Hz, 1H, C (2′)-H),6.78 (s, 2H, C (2′′, 6′′)-H), 6.74 (dd, J = 8.3, 2.0 Hz, 1H, C (6′)-H), 6.61(d, J = 8.2 Hz, 1H, C (5′)-H), 5.87 (d, J = 2.3 Hz, 1H, C (8)-H), 5.84 (d, J = 2.3 Hz, 1H, C (6)-H), 5.35 (s, 1H, C (2)-H), 5.31 (s, 1H, C (3)-H), 4.16 (d, J = 2.4 Hz, 1H, C (4)-H), 3.93 (m, 2H, C (13)-H), 3.83 (m, 1H, C (11)-H), 3.56(s, 3H, C (15)-H), 1.49 (d, J = 7.2 Hz, 3H, C (16)-H). 13 C-NMR (151 MHz, CD3OD) δ 175.00 (C-14), 170.15 (C-12), 165.65 (C-7′′), 157.97 (C-9), 157.08 (C-5),155.49 (C-7), 144.80 (C-3′′, C-5′′), 144.54 (C-3′), 144.51 (C-4′), 138.45 (C-4′′), 129.45 (C-1′), 119.51 (C-1′′), 117.91 (C-6′), 114.54 (C-5′), 113.66 (C-2′), 108.73 (C-2′′, C-6′′), 98.05 (C-10), 95.42 (C-6), 94.18 (C-8), 73.21 (C-2), 72.21 (C-3), 51.10 (C-15), 44.96 (C-4), 40.91 (C-13), 40.18 (C-11), 17.58(C-16)。

[0067] (6)(–)-epigallocatechin-3 -O- gallate-4 β -tiopronin methyl ester (EGCG-T): C 28 H 27 NO 14 S. 1 H-NMR (600 MHz, CD3OD) δ 6.81 (s, 2H, C (2′′, 6′′)-H), 6.45(s, 2H, C (2′, 6′)-H), 5.88 (d, J = 2.3 Hz, 1H, C (8)-H), 5.87 (d, J = 2.3 Hz,1H, C (6)-H), 5.29 (s, 1H, C (2)-H), 5.24 (s, 1H, C (3)-H), 4.11 (d, J = 2.0Hz, 1H, C (4)-H), 3.93 (m, 2H, C (13)-H), 3.91 (m, 1H, C (11)-H), 3.48 (s,3H, C (15)-H), 1.44 (d, J = 7.4 Hz, 3H, C (16)-H). 13 C-NMR (151 MHz, CD3OD) δ 173.99 (C-14), 169.86 (C-12), 165.96 (C-7′′), 158.26 (C-9), 157.15 (C-5),155.85 (C-7), 145.38 (C-3′, C-5′), 144.81 (C-3′′, C-5′′), 138.54 (C-4′′),132.46 (C-4′), 128.58 (C-1′), 119.43 (C-1′′), 108.81 (C-2′′, C-6′′), 105.34(C-2′, C-6′), 96.85 (C-10), 95.65 (C-6), 94.51 (C-8), 73.15 (C-2), 72.01 (C-3), 51.13 (C-15), 42.89 (C-4), 40.85 (C-13), 39.66 (C-11), 17.13 (C-16)。

[0068] (7)(–)-epiatechin-4 β -tiopronin methyl ester (EC-T): C 21 H 23 NO9S. 1 H-NMR (600 MHz, CD3OD) δ 6.91 (d, J = 2.0 Hz, 1H, C (2′)-H),6.74 (dd, J = 8.2, 2.0 Hz, 1H, C (6′)-H), 6.69 (d, J = 8.1 Hz, 1H, C (5′)-H),5.87 (d, J = 2.3 Hz, 1H, C (8)-H), 5.82 (d, J = 2.3 Hz, 1H, C (6)-H), 5.14 (s,1H, C (2)-H), 3.93 (d, J = 2.5 Hz, 1H, C (4)-H), 3.90 (m, 2H, C (13)-H), 3.92(d, J = 2.5 Hz, 1H, C (3)-H), 3.66 (m, 1H, C (11)-H), 3.63 (s, 3H, C (15)-H),1.40 (d, J = 7.4 Hz, 3H, C (16)-H)。

[0069] (8)(–)-epicatechin-3 -O- gallate-4 β -tiopronin methyl ester (ECG-T): C 28 H 27 NO 13 S. 1 H-NMR (600 MHz, CD3OD) δ 6.87 (d, J = 2.0 Hz, 1H, C (2′)-H),6.81 (s, 2H, C (2′′, 6′′)-H), 6.76 (dd, J = 8.2, 2.1 Hz, 1H, C (6′)-H), 6.64(d, J = 8.1 Hz, 1H, C (5′)-H), 5.88 (d,J = 2.4 Hz, 1H, C (8)-H), 5.87 (d, J = 2.3 Hz, 1H, C (6)-H), 5.35 (s, 1H, C (2)-H), 5.24 (s, 1H, C (3)-H), 4.13 (d, J = 2.1 Hz, 1H, C (4)-H), 3.94 (m, 2H, C (13)-H), 3.92 (m, 1H, C (11)-H), 3.49 (s, 3H, C (15)-H), 1.44 (d, J = 7.3 Hz, 3H, C (16)-H).

[0070] Example 7

[0071] In this invention, a rat BRL-3A hepatocyte model of alcoholic liver injury was established. Cells were divided into a normal control group, an ethanol-damaged group, and an ethanol-treated group, with three replicates per group. Cells were spaced at a density of 4 × 10⁶ cells / well. 4 Cells / mL, 100 μL / well were seeded into 96-well plates. After stabilization for 24 h, the drug-treated groups were pre-protected for 4 h. After pre-protection, the normal control group was replaced with fresh complete culture medium, the ethanol-induced liver injury group was replaced with culture medium containing 400 mM ethanol, and the drug-treated groups were replaced with culture medium containing 10, 20, and 40 µM of drug in 400 mM ethanol. The plates were sealed with sealing film and incubated for 20 h. The MTT assay was then used to evaluate the protective effect of the drug on BRL-3A cells induced by alcoholic liver injury. The same experiment was repeated three times for data analysis.

[0072] See results Figure 18 Most thiopronine-flavantriol derivatives exhibited protective effects against ethanol-induced hepatocellular damage within a certain concentration range, showing a dose-dependent effect; that is, the protective effect against ethanol-induced liver injury weakened as the compound concentration decreased. Among them, GCG-T, CG-T, EGCG-T, and ECG-T showed highly significant protective effects at low concentrations, capable of reversing ethanol-induced alcoholic liver injury, with CG-T and ECG-T exhibiting stronger protective effects. From a chemical structural analysis perspective, compounds exhibiting significant hepatoprotective activity share a common characteristic to some extent: the presence of a galloyl group. Preliminary research suggests that the presence of a galloyl group may be an important factor affecting the hepatoprotective effect of the degradation products.

[0073] In addition, such as Figure 18 As shown, comparing the activities of the parent nuclei ECG / CG, EGCG / GCG, and their corresponding degradation products, it was found that the introduction of nucleophiles led to significantly stronger hepatoprotective activities of GCG-T, CG-T, EGCG-T, and ECG-T compared to their corresponding parent nuclei, indicating that the conjugation of nucleophiles can improve the survival rate of BRL-3A cells. Studies have shown that thioproline and reduced glutathione can reduce the degree of oxidative stress through mechanisms such as regulating redox balance and alleviating oxidative stress, thereby reducing hepatocyte damage. Therefore, the hepatoprotective activity of degradation products is not only closely related to their molecular structure but also to the biological activity of the nucleophiles themselves, providing valuable information for further improvement and design of new drugs.

Claims

1. A thiopronine-flavantriol derivative or a pharmaceutically acceptable salt thereof, characterized in that, The thiopronine-flavonoid triol derivative is selected from any one of the following compounds: 。 2. A method for preparing thiopronine-flavonoid triol derivatives by degradation of high-polymerization-degree polyphenols from persimmon, characterized in that, Includes the following steps: 1) Take the polyphenol extract of persimmon and the nucleophile thioproline, add acidified methanol, and prepare a reaction solution; 2) Place the reaction solution in a sealed reaction flask and carry out acid-catalyzed degradation to obtain the degradation reaction solution; 3) Add water to the degradation reaction solution to adjust the pH of the system to 7.0, remove the organic solvent, reconstitute with water, and separate using an open YMC-ODS-A-HG chromatographic column. Elute with water, ethyl acetate and methanol in sequence, collect the ethyl acetate eluent, remove water and organic solvent, and freeze dry for later use.

3. The method according to claim 2, characterized in that, Step 1) Preparation method of polyphenol extract of persimmon: Persimmon fruit powder is heated by reflux at 50℃~60℃ with methanol-water and acetone-water in sequence, filtered to remove organic solvent, and a crude polyphenol extract solution of persimmon fruit is obtained; the crude polyphenol extract solution of persimmon fruit is loaded onto AB-8 macroporous adsorption resin, and after adsorption, it is first eluted with pure water, and then eluted with anhydrous ethanol. The anhydrous ethanol eluent of persimmon fruit is collected, the anhydrous ethanol is removed and then freeze-dried to obtain the polyphenol extract of persimmon fruit.

4. The method according to claim 2, characterized in that, In step 1), the acidified methanol is a methanol solution treated with hydrochloric acid; the mass ratio of the persimmon polyphenol extract to thioproline is 1:1 to 1:

5.

5. The method according to claim 2, characterized in that, In step 2), the acid-catalyzed degradation time is 20 min to 100 min; the acid used is hydrochloric acid with a concentration of 0.1 M to 0.5 M; and the acid-catalyzed degradation temperature is 30℃ to 70℃.

6. The method according to any one of claims 2-5, characterized in that, The degradation solution obtained from the acidification and degradation of polyphenols in persimmon was separated using a one-step separation method. The operation steps included: The lyophilized powder obtained in claim 2 was reconstituted with a water-methanol solution, and then the compound was separated and purified by a semi-preparative liquid chromatograph to obtain the monomer compound. The chromatographic column used was a reversed-phase YMC-Pack ODS-A column, 250×10 mm, 5 μm; mobile phase A: 0.2% formic acid + 99.8% water, mobile phase B: 0.2% formic acid + 99.8% acetonitrile, flow rate 3.0 mL / min; column temperature 30℃; detection wavelength 280 nm; injection volume 2 mL.

7. The method according to claim 6, characterized in that, Elution was performed using a gradient elution program as follows: 0 min → 10 min, 12% → 15% B; 10 min → 25 min, 15% → 20% B; 25 min → 35 min, 20% → 26% B; 35 min → 48 min, 26% → 32% B; 48 min → 55 min, 32% → 40% B; followed by column flushing and equilibration; the obtained fractions were collected, the solvent was removed, and the fractions were lyophilized to obtain the thiopronine-flavonoid triol derivative described in claim 1.

8. A pharmaceutical composition, characterized in that, The product comprises one or more of the following: the thiopronine-flavantriol derivative of claim 1 or a pharmaceutically acceptable salt thereof; the thiopronine-flavantriol derivative obtained by the method of claim 2; or the thiopronine-flavantriol derivative monomeric compound obtained by the one-step separation method of claim 6; and a pharmaceutically acceptable excipient.

9. The use of a thiopronine-flavantriol derivative of claim 1 or a pharmaceutically acceptable salt thereof, or a thiopronine-flavantriol derivative obtained by the method of claim 2, or a thiopronine-flavantriol derivative monomer compound obtained by the one-step separation method of claim 6, or the pharmaceutical composition of claim 8, in the preparation of a medicament for treating liver injury.

10. The application according to claim 9, characterized in that, The use of the thiopronine-flavanotriol derivative or its pharmaceutically acceptable salt or monomeric compound or the pharmaceutical composition in the preparation of a drug for treating alcoholic liver injury.

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