Menthol furan derivative of epigallocatechin gallate as well as preparation method and application of menthol furan derivative

Abstract

The invention provides a menthofuran derivative of epigallocatechin gallate, the structure of the derivative is shown as follows: # imgabs0 #. The preparation method of the derivative comprises the following steps: S1, dissolving persimmon tannin PT40 in a hydrochloric acid ethanol solution to obtain a reaction mother solution I; s2, dissolving menthofuran in a hydrochloric acid ethanol solution to obtain a reaction mother solution II; and S3, mixing the reaction mother liquor I and the reaction mother liquor II, reacting under a water bath condition, and separating by using a preparative reversed-phase high-performance liquid phase separation column after the reaction is completed, so as to obtain the EGCG-MF. According to the invention, the menthofuran is used as a nucleophilic reagent to degrade persimmon tannin PT40, the prepared EGCG-MF has a treatment effect in mice suffering from alcoholic gastric ulcer, high-yield EGCG-MF is obtained by optimizing the preparation process, and a new thought and a new method are provided for procyanidine in the fields of food and medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of plant functional component degradation technology and product application, and specifically relates to a menthofuran derivative of epigallocatechin gallate and a preparation method and application thereof. Background Art

[0002] Persimmon tannin is a highly polymerized polyphenolic compound with a very special structure compared to other tannins: highly polymerized, with an average degree of polymerization of 23.6%, a galloylated structure of up to 72%, and containing type A connections. Although persimmon tannin has functional activities such as anti-oxidation, lipid-lowering and weight-loss, and regulating sugar metabolism, its application is greatly limited due to its low solubility and low bioavailability. Degrading polymers into oligomers is an effective way to expand its application.

[0003] At present, the methods used to degrade proanthocyanidin polymers mainly include acid degradation, metal-catalyzed hydrogenolysis, nucleophilic substitution degradation and microbial degradation. The acid degradation method is to break the CC bond between flavan-3-ols under hot acidic conditions to complete the depolymerization of the polymer, but this method has low degradation efficiency, complex and unstable products, and the degradation products are difficult to be effectively utilized. The acid concentration is high, the corrosion is strong, and the environment is not friendly. Metal-catalyzed hydrogenolysis is to select a suitable hydrogenation catalyst to depolymerize the polymer under high temperature hydrogenation conditions. It has high requirements for equipment, harsh reaction conditions, and limited use. Microbial degradation is a technology that converts organic matter into inorganic matter by means of the growth and metabolism of microorganisms, but it is difficult to cultivate or find suitable microorganisms for degradation. Nucleophilic substitution degradation is to dissociate the extended unit of the polymer into a carbon cation intermediate under acidic conditions. The nucleophilic reagent will capture the carbon cation to generate the corresponding conjugate. It is a common method for the structural analysis of tannin compounds. Commonly used nucleophilic reagents include phloroglucinol and benzyl mercaptan. However, these reagents not only have low efficiency in degrading persimmon tannins with unique structure, but also have defects such as toxicity and strong irritating odor, making the products difficult to use in the food field.

[0004] Therefore, finding a new edible and efficient nucleophilic bond breaker to achieve efficient and environmentally friendly degradation of persimmon tannins, while obtaining a high yield of active degradation products, and applying them in the food or medical fields is a key technical problem that needs to be urgently solved in this field. Summary of the invention

[0005] In view of this, the present invention provides a nucleophilic bond cleaving agent for safely and efficiently degrading persimmon tannin, and simultaneously obtains a high yield of degradation product epigallocatechin gallate-menthofuran derivatives with anti-alcoholic gastric ulcer effect.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A menthofuran derivative of epigallocatechin gallate, the structure of the derivative is shown below:

[0008]

[0009] A method for preparing the above derivative comprises the following steps:

[0010] S1, dissolving persimmon tannin PT40 in hydrochloric acid ethanol solution to obtain reaction mother solution I;

[0011] S2, dissolving menthofuran in hydrochloric acid ethanol solution to obtain reaction mother solution II;

[0012] S3. Mix the reaction mother liquor I and the reaction mother liquor II, place them in a water bath for reaction, and after the reaction is completed, separate them using a preparative reversed-phase high performance liquid separation column to obtain the menthofuran derivative of epigallocatechin gallate.

[0013] Furthermore, the hydrochloric acid concentration in the hydrochloric acid ethanol solution in steps S1 and S2 is 0.1-0.4M.

[0014] In some specific embodiments, preferably, the hydrochloric acid concentration in the hydrochloric acid ethanol solution in steps S1 and S2 is 0.2M.

[0015] Furthermore, in the mixed solution formed by reaction mother solution I and reaction mother solution II in step S3, the mass ratio of menthol furan to persimmon tannin PT40 is (0.6-1.8):1.

[0016] In some specific embodiments, preferably, the mass ratio of the menthol furan to the persimmon tannin PT40 is 1:1.

[0017] Furthermore, the water bath reaction conditions in step S3 are: temperature 50-80° C., time 0.5-2 h; after the reaction is completed, the reaction is placed in an ice bath at 0° C. to terminate the reaction.

[0018] In some specific embodiments, preferably, the water bath reaction conditions in step S3 are: temperature 60° C., time 1.5 h.

[0019] In some specific embodiments, preferably, the separation conditions in step S3 are: the chromatographic column is a reverse phase C18 chromatographic column, the column temperature is 30°C; the mobile phase is a 0.13% trifluoroacetic acid aqueous solution, the injection volume is 200 μL; the eluent is a 0.1% trifluoroacetic acid acetonitrile solution, the gradient elution flow rate is 4.0 mL / min; the detection wavelength is 280 nm.

[0020] Application of the above derivatives in preventing alcoholic gastric ulcer.

[0021] The above-mentioned alcoholic gastric ulcer indicators include: gastric mucosal damage index, gastric tissue pathological damage, oxidative stress substance level, and inflammatory factor level.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention innovatively uses menthol furan as a nucleophilic reagent to degrade persimmon tannin PT40, and utilizes the special structure of persimmon tannin PT40 to obtain a large number of carbon cation extension units containing epigallocatechin gallate, thereby synthesizing highly active epigallocatechin gallate-menthol furan derivatives (EGCG-MF).

[0024] (2) The degradation method of persimmon tannin proposed in the present invention can obtain a high yield of EGCG-MF (the yield is 246 mg / g calculated based on the total mass of EGCG and MF) by optimizing the preparation parameters, which can realize the development and utilization of persimmon tannin resources.

[0025] (3) The EGCG-MF prepared by the present invention has a therapeutic effect in mice with alcoholic gastric ulcer, and has a good effect at both high doses (100 mg / kg) and low doses (50 mg / kg), which provides new ideas and methods for proanthocyanidins in the food and medical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The liquid chromatogram of persimmon tannin PT40 (Figure A) and the liquid chromatogram of the degradation reaction liquid (Figure B).

[0027] Figure 2 This is a graph showing the degradation efficiency of persimmon tannin at different reaction temperatures in Example 1.

[0028] Figure 3 This is a graph showing the degradation efficiency of persimmon tannin at different reaction times in Example 2.

[0029] Figure 4 This is a graph showing the degradation efficiency of persimmon tannin at different hydrochloric acid ethanol concentrations in Example 3.

[0030] Figure 5 This is a graph showing the degradation efficiency of persimmon tannin under different mass ratios of MF to PT40 in Example 4.

[0031] Figure 6 HPLC-MS spectrum of the prepared EGCG-MF, wherein A is the primary mass spectrum and B is the secondary mass spectrum.

[0032] Figure 7 This is the cleavage pathway map of EGCG-MF.

[0033] Figure 8 The figure is a direct diagram of the damage of EGCG-MF to gastric tissue in mice with alcoholic gastric ulcer, where A is a direct comparison diagram of the damage, B is the damage index score, and C is the damage inhibition rate.

[0034] Fig. 9 This is the HE staining of EGCG-MF on gastric tissue of mice with alcoholic gastric ulcer.

[0035] Fig.10 This is the PAS staining of EGCG-MF on gastric tissue of mice with alcoholic gastric ulcer.

[0036] Fig.11 These are the levels of oxidative stress indicators of gastric tissue in mice with alcoholic gastric ulcer induced by EGCG-MF, where A is the MDA indicator, B is the SOD indicator, and C is the GSH indicator.

[0037] Fig.12 The above are the levels of inflammatory factors in gastric tissue of mice with alcoholic gastric ulcer induced by EGCG-MF, where A is the TNF-α index, B is the IL-6 index, and C is the IL-10 index. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0039] Sources and physical and chemical parameters of key test materials:

[0040] Persimmon tannin PT40 was prepared in the laboratory: persimmon pulp was mixed with 1% hydrochloric acid-methanol mixture at a solid-liquid ratio of 1:3, condensed and refluxed at 80℃ for 40min, repeated three times, and the extracts were combined and allowed to stand overnight. The extract was filtered, concentrated to 50mL (per 2000ml) by rotary evaporation at 35℃, and the extract was extracted with AB-8 multiporous resin. Sugar and other soluble impurities were first removed with distilled water, low molecular weight phenolic compounds were washed with 10% ethanol / water (v / v), and tannins were finally eluted with anhydrous ethanol. The eluate was collected, the solvent was removed with a vacuum rotary evaporator, and the residue was freeze-dried. The freeze-dried PTs were dissolved in methanol and equilibrated on a Toyopearl HW-50F column (70×350mm, id, Tokyo, Japan) with a 20% acetone / water mixture (v / v). Finally, the target tannin was eluted with a 40% acetone / water mixture (v / v), and the collected fractions were evaporated under reduced pressure at 35°C, freeze-dried, and stored at -20°C.

[0041] Example 1

[0042] This embodiment provides a menthofuran derivative of epigallocatechin gallate, and its preparation is as follows:

[0043] S1. Take hydrochloric acid and add it to ethanol to prepare a hydrochloric acid ethanol solution with a hydrochloric acid concentration of 0.1 M.

[0044] S2. Weigh persimmon tannin PT40 and menthofuran (MF) in a mass ratio of 1:1, and then add the hydrochloric acid ethanol solution in step S1 to obtain persimmon tannin PT40 reaction mother liquor (persimmon tannin PT40 concentration is 10 mg / mL) and menthofuran reaction mother liquor (menthofuran concentration is 10 mg / mL) in turn.

[0045] S3. Mix equal volumes of persimmon tannin PT40 reaction mother liquor and menthol furan reaction mother liquor, place them in a water bath set according to a temperature gradient (50°C, 60°C, 70°C, 80°C) in turn to react for 2 hours. After the reaction is completed, place them in an ice bath at 0°C to terminate the reaction, and obtain a degradation reaction liquid.

[0046] S4. Separating the obtained degradation reaction liquid by using a preparative reverse-phase high performance liquid separation column to obtain epigallocatechin gallate-menthofuran derivative (EGCG-MF).

[0047] The separation conditions are as follows: the chromatographic column is a reverse phase C18 chromatographic column, the column temperature is 30°C; the mobile phase is a 0.13% trifluoroacetic acid aqueous solution, the injection volume is 200 μL; the eluent is a 0.1% trifluoroacetic acid acetonitrile solution, the gradient elution flow rate is 4.0 mL / min; the detection wavelength is 280 nm.

[0048] The results of EGCG-MF prepared under temperature gradient are shown in Figure 2 ,Depend on Figure 2 It can be seen that the peak area at 60°C increased significantly compared with 50°C, but when comparing the sum of the peak areas at each temperature, we found that there was no significant difference between 60, 70 and 80°C. Therefore, from the perspective of energy saving, we chose 60°C as the optimal temperature.

[0049] Example 2

[0050] This embodiment provides a menthofuran derivative of epigallocatechin gallate, and its preparation is basically the same as that of Example 1, except that: in step S3, the reaction temperature is 60°C, but the reaction time is sequentially reacted according to a time gradient (0.5h, 1h, 1.5h, 2h), and the rest remain unchanged.

[0051] The results of EGCG-MF prepared under time gradient are shown in Figure 3 ,Depend on Figure 3 It can be seen that as the reaction time increases, the yield increases first and then decreases. This is because proanthocyanidins will also undergo side reactions during the degradation process. Prolonging the reaction time may first be beneficial to side reactions rather than depolymerization, and vice versa, it may be beneficial to depolymerization rather than side reactions. Therefore, the reaction time of 1.5h is selected as the optimal reaction time.

[0052] Example 3

[0053] This embodiment provides a menthofuran derivative of epigallocatechin gallate, and its preparation is basically the same as that of Example 1, except that: in step S1, hydrochloric acid ethanol solution is sequentially prepared according to a concentration gradient (0.1M, 0.2M, 0.3M, 0.4M); in step S3, the reaction temperature is kept constant at 60°C, and the reaction temperature is 1.5h; and the rest remain unchanged.

[0054] The results of EGCG-MF prepared in a concentration gradient hydrochloric acid ethanol solution are shown in Figure 4 ,Depend on Figure 4 It can be seen that: with the increase of hydrochloric acid concentration, the content of proanthocyanidin oligomers produced by the reaction of PT40 and MF first increases and then decreases. When the hydrochloric acid concentration is 0.2M, the peak area of ​​the new substance is the largest. The explanation for this phenomenon is that a higher concentration of H will promote the breakage of the inter-flavan bond of proanthocyanidin, generate more carbon cations to accelerate the forward reaction; and under higher acidity conditions, proanthocyanidin itself will also undergo a certain degree of degradation, resulting in a decrease in the content of newly generated products, which may be the reason for the decrease in degradation efficiency due to excessive acid concentration. Therefore, the optimal reaction acidity is selected as the reaction hydrochloric acid ethanol concentration of 0.2M.

[0055] Example 4

[0056] The present embodiment provides a menthofuran derivative of epigallocatechin gallate, and its preparation is basically the same as that of Example 1, except that: the concentration of hydrochloric acid in the hydrochloric acid ethanol solution in step S1 is 0.2M; in step S2, persimmon tannin PT40 and menthofuran are weighed in sequence in a mass ratio gradient (MF:PT40=0.6:1, 1:1, 1.4:1, 1.8:1); in step S3, the reaction temperature is kept constant at 60°C, and the reaction temperature is 1.5h; the rest remain unchanged.

[0057] The results of EGCG-MF prepared by the gradient of MF and PT40 mass ratio are shown in Figure 5 ,Depend on Figure 5 It can be seen that the area of ​​the new peak increases with the increase of MF mass because the higher the nucleophile dosage, the more it can promote the forward reaction. When the mass ratio of the two is 1:1, the total area of ​​the new peak does not continue to increase when the amount of menthofuran is continued to increase, indicating that the depolymerization has been completed. From the perspective of saving raw materials, the reaction mass ratio of 1:1 is selected as the best reaction mass ratio.

[0058] It can be seen from the above examples that the target product has the highest yield when the water bath temperature is 60°C, the reaction time is 1.5h, the concentration of ethanol hydrochloride is 0.2M, and the mass ratio of MF to PT40 is 1:1. The products prepared under the best conditions in the above examples 1-4 were collected together, and the organic solvent was removed by rotary evaporation at 30°C and then vacuum freeze-dried to obtain EGCG-MF powder. The yield of EGCG-MF was calculated to be 246mg / g (calculated based on the total mass of EGCG and MF added).

[0059] The degradation reaction solution prepared by the optimal process was subjected to HPLC-MS analysis. Four new flavan-3-ol menthofuran conjugates were formed by the degradation reaction, and the MS analysis results of the compound with the highest response value were analyzed (see Figure 6 ) are as follows: retention time is 24.664min, [MH] - The fragment ions are 605, and the main fragment ions are 455, 453, 435, 409, 391, 327, 309, 285, 177, and 125.

[0060] Among them, m / z 453 is the loss of galloyl group [MH-152] - The fragment ions have different fragmentation pathways:

[0061] First, it can lose a water molecule to form a fragment ion of m / z 435 ([MMH-152-18]-), and the fragment molecule continues to undergo HRF fission, followed by RDA fission, losing 126Da to form 309 ([MH-152-18-126]-) (Hui-Jing L, LM D. Tandem mass spectrometry for sequencing proanthocyanidins. [J]. Analytical chemistry, 2007, 79 (4): 1739-48.).

[0062] Secondly, m / z 435 can also undergo QM cleavage, losing part of menthane to form m / z 285, and then m / z 285 undergoes 1,4 a-ring cleavages to form a fragment ion of m / z 125 (Vivas N, Gaulejac DVN, Vitry C, et al. Impact of ethanol content on the scavenging activities of oak wood C-glycosidicella gitannins. Application to the evaluation of the nutritional status of spirits[J]. Journal of the Institute of Brewing, 2013, 119(3): 116-125.).

[0063] Then, RDA fission can also occur directly with m / z 453, with the loss of 126Da forming a fragment ion of m / z 327 ([MH-152-126]-) (Gu et al., 2003), followed by QM cleavage to lose the menthofuran fragment ion to form m / z 177 ([MH-152-126-150]-); m / z 453 can also first undergo RDA fission, losing 168Da to form a fragment ion of 285 ([MH-152-168]-) (APNeilson, A. Shopf, B. R. Cooper, M. A. Pereira, J. A. Bomser, Ferruzzi, M. G. Catechin degradation with concurrent formation of homo-andheterocatechin dimers during in vitro digestion. J Agric Food Chem, 2007, 55(22): 8941-8949.).

[0064] Finally, the possible fragmentation pathway of m / z 453 is to lose a part of the carbon dioxide fragment molecule to form m / z 409 ([MH-152-44]-)(Rodrigues, CM, Rinaldo, D., dos Santos, LC, Montoro, P., Piacente, S., Pizza, C., Hiruma-Lima, CA, Brito, AR, & Vilegas, W. (2007). Metabolic fingerprinting using direct flow injection electrospray ionization tandemmass spectrometry for the characterization of proanthocyanidins from the barks of Hancor nia speciosa. Rapid Communications in Mass Spectrometry, 21, 1907–1914), and then continue to lose a water molecule to form m / z 391 fragment ion ([MH-152-44-18]-)(Li, HJ, & Deinzer, ML (2008). The mass spectral analysis of isolated hops A-type proanthocyanidins by electrospray io nization tandem massspectrometry. Journal of Mass Spectrometry, 43, 1353–1363.).

[0065] The ion at m / z 605 can also undergo QM fragmentation, resulting in the direct loss of C4 menthol furan, leaving a fragment ion of m / z 455, indicating that the extended unit of the menthan modification is epigallocatechin gallate (Chen J, Xu Z, Zhu W, et al. Novel proanth ocyanidin dimer analogues with the C-ring-openeddiaryl-propan-2-gallate structural unit and enhanced antioxidant activities[J]. Journal of Functional Foods, 2016, 21290-300.).

[0066] The ion at m / z 605 can also lose a gallic acid molecule to form a fragment ion at m / z 435 ([MH-152-170]-) before subsequent fragmentation (Sipowo Tala, VR, da Silva, VC, Rodrigues, CM, Nkengfack, AE, dos Santos, LC, & Vilegas, W. (2013). Characterization of proanthocyanidins from Parkia biglobosa (Jacq.) G. Don. (Fabaceae) by flow injection analysis-electrospray ionization ion trap tandem mass spectrometry and liquid chromatography / electrospray ionization mass spectrometry. Molecules, 18, 2803–2820.).

[0067] Based on the above mass spectrometry fragmentation information, it can be inferred that it is a menthofuran compound of epigallocatechin gallate (EGCG-MF). The fragmentation pathway is shown in Figure 7 .

[0068] Furthermore, in order to understand the therapeutic effect of the EGCG-MF prepared above on alcoholic gastric ulcer, the EGCG-MF prepared by the best process was selected and the following animal experiments were also carried out.

[0069] 1. Establishment of the mouse alcoholic gastric ulcer model

[0070] 30 healthy male C57BL / 6 mice, weighing 20-25g. Before the formal start of the experiment, the animals were placed in an environment with a temperature of 25±2℃, an environmental humidity of 56±5%, and a light-dark cycle of 12h light / 12h dark for one week for adaptive feeding. During this period, standard pellet feed and water were freely available. The mice were fasted for 12h before gavage, and then gavage with 95% anhydrous ethanol at a gavage volume of 10mL / kg was performed on the mice, and the drug was administered 12h after modeling.

[0071] 2. Experimental groups and treatment administration

[0072] The experiment set up a normal control group, a model group, a cimetidine group (100 mg / kg), a L-EGCG-MF (50 mg / kg) group, and a H-EGCG-MF (100 mg / kg) group. The drugs were administered continuously for 3 days. Both the control group and the model group were given 0.5% CMC-Na solution. 24 hours after the last administration, the mice were killed by cervical dislocation and the stomach was removed. The stomach was cut open along the greater curvature of the stomach, the contents of the stomach were cleaned with ice-cold saline, the surface of the stomach tissue was gently wiped dry with absorbent paper, and the damage to the stomach wall was recorded with a camera to evaluate the gastric ulcer. Subsequently, the stomach tissue was divided into two: one half was fixed in 4% paraformaldehyde solution for histological analysis, and the other half was stored at -80°C for subsequent experimental detection.

[0073] The specific results of subsequent experiments are as follows:

[0074] EGCG-MF improves gastric tissue damage in mice with alcoholic gastric ulcer

[0075] Experimental method: The injury index and ulcer inhibition rate of gastric tissue were calculated according to the GUTH scoring standard. The specific scoring indicators are as follows: 1 point for punctate bleeding or erosion; 2 points for linear or strip bleeding with a length of less than 1 mm; 3 points for a lesion length between 1-2 mm; 4 points for a lesion length between 2-4 mm; and 5 points for a lesion length greater than 4 mm. If the lesion width is greater than 1 mm, the score is multiplied by 2. The ulcer inhibition rate is calculated according to the following formula:

[0076] Ulcer inhibition rate = (A1-A2) / A1×100%

[0077] A1 represents the average gastric tissue injury index of mice in the model group, and A2 represents the average gastric tissue injury index of mice in the preventive medication group.

[0078] Experimental results: The blank group mice had no abnormal symptoms in the stomach, the internal tissue was smooth and flat, with obvious wrinkles, no bleeding and edema, and the color was light red; the model group mice had bloated stomachs, severe gastric mucosal damage, obvious tissue bleeding and edema in many places, and fewer gastric mucosal wrinkles; compared with the model group, the two compound administration groups had only a small number of white ulcer spots, and no large-scale ulcers and tissue bleeding. This shows that EGCG-MF can improve the ulcer damage caused by alcohol to the gastric tissue of mice ( Figure 8 ).

[0079] Analysis of gastric histopathology of mice with alcoholic gastric ulcer induced by EGCG-MF

[0080] Experimental methods: Gastric tissue specimens fixed in 4% paraformaldehyde solution were obtained, and gastric tissue pathology was evaluated by hematoxylin-eosin staining (H&E) and periodic acid-Schiff staining (PAS). Pathological analysis. Briefly, according to the prescribed standards and procedures, the gastric tissue was treated with graded ethanol and xylene solutions, and the tissue was embedded in paraffin. The tissue was cut into 4-5 μm slices and stained. The staining results and imaging were then observed under a microscope (NIB610).

[0081] Experimental results: HE staining results showed ( Fig. 9 ), the glandular structure of mice in the blank control group was intact, the glandular epithelial cells were neatly arranged, and there was no inflammatory cell infiltration or bleeding; the gastric mucosal damage of mice in the model group was serious, with epithelial cell shedding, inflammatory cell infiltration and tissue congestion; the gastric tissues of the cimetidine group and the EGCG-MF group showed varying degrees of glandular destruction and tissue bleeding, but compared with the model group, they were alleviated to a certain extent. PAS staining results showed ( Fig.10 ), the blank group mice were stained dark purple, indicating that the mucus layer was not damaged; while the mucus of the model group mice was greatly damaged, and the color development was greatly weakened compared with the blank group; the mucus layer of the drug-treated group mice was mostly normal, and the color development was stronger than that of the model group, indicating that the mucus layer of the mice was repaired after treatment, proving that the degradation product has a protective effect on the gastric mucosal barrier.

[0082] EGCG-MF improves the production of oxidative stress factors in mice with alcoholic gastric ulcer

[0083] Experimental method: Accurately weigh a certain amount of gastric tissue and homogenize it with phosphate buffer solution (pH 7.4) 9 times the weight of the tissue, centrifuge it at 3000r / min for 15min at 4℃, and take the supernatant. According to the instructions of the kit, the SOD, MDA, GSH and other oxidative factor markers in the gastric tissue of mice were measured.

[0084] Experimental results: MDA is the end product of lipid peroxidation and can indirectly reflect the degree of gastric tissue damage. Compared with the blank group, the MDA content in the model group increased significantly. There was a significant difference in the MDA content between the cimetidine group and the EGCG-MF group and the model group (P < 0.05), and the MDA content decreased. GSH is an important antioxidant in humans and rodents. It can ensure that red blood cells can normally supply oxygen in the body without being affected by ROS. It also plays an important role in maintaining the integrity of the gastric mucosa. The GSH level in the gastric tissue of the treated mice was improved to a certain extent compared with the model group, and the content was significantly increased (P < 0.01). The GSH level in the model group was significantly decreased compared with the blank group (P < 0.01). EGCG-MF can restore the GSH level of gastric ulcer mice, indicating that it can enhance the ability of the endogenous antioxidant defense system. SOD can specifically remove superoxide anions, thereby protecting gastric mucosal cells from damage caused by oxygen free radicals as the first line of defense. The results showed that compared with the normal group, the SOD activity in the model group was significantly decreased. Compared with the model group, the SOD activity of the cimetidine group increased significantly (P < 0.001), and the SOD level of the EGCG-MF group was significantly higher than that of the model group, with significant differences (P < 0.05).

[0085] Biochemical results showed that EGCG-MF could improve the ulcer damage caused by alcohol to gastric tissue in mice by inhibiting oxidative stress. Fig.11 ).

[0086] EGCG-MF improves the production of inflammatory factors in mice with alcoholic gastric ulcer

[0087] EGCG-MF inhibited the production of pro-inflammatory factors TNF-α and IL-6 and promoted the production of anti-inflammatory factor IL-10 in the gastric tissue of mice with alcoholic gastric ulcer.

[0088] Experimental method: Accurately weigh a certain amount of gastric tissue and homogenize it with phosphate buffer solution (pH 7.4) 9 times the weight of the tissue, centrifuge it at 3000r / min for 15min at 4°C, and take the supernatant. According to the instructions of the ELISA kit, the inflammatory factor markers such as IL-6, IL-10, and TNF-α in gastric tissue were measured.

[0089] Experimental results: The results of ELISA experiments showed that the content of TNF-α in tissues after ethanol stimulation was significantly increased compared with the blank group (P < 0.01), and the mice after treatment were significantly improved compared with the model group (P < 0.01), which proved that EGCG-MF can improve gastric ulcers by inhibiting the production of this pro-inflammatory factor. The increase in TNF-α content will stimulate the production of IL-6 and aggravate the symptoms of inflammatory response. IL-6 then stimulates neutrophils and macrophages in the inflammatory site to produce various harmful substances that can damage gastric tissue. The IL-6 results showed that similar to the TNF-α results, the IL-6 content in the gastric tissue of mice in the model group was significantly increased (P < 0.01), and the IL-6 content in the gastric tissue of mice in the drug-treated group was significantly decreased compared with the model group (P < 0.01). The IL-10 level of mice in the model group was significantly different from that in the blank group (P < 0.01), the high-dose H-EGCG-MF group and the cimetidine group had significant improvement effects (P < 0.01), and the low-dose L-EGCG-MF group was significantly different from the model group (P < 0.05).

[0090] The results of ELISA experiments showed that EGCG-MF can effectively improve the ulcer damage caused by alcohol to the gastric tissue of mice by playing an anti-inflammatory role ( Fig.12 ).

[0091] The above experimental results show that the persimmon tannin degradation product epigallocatechin gallate-menthofuran derivative (EGCG-MF) provided by the present invention can effectively reduce the gastric tissue damage index caused by alcohol in mice, protect the gastric mucosal barrier, and effectively improve alcoholic gastric ulcers through antioxidant and anti-inflammatory effects.

[0092] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0093] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A menthofuran derivative of epigallocatechin gallate, characterized in that: The derivative structure is shown below:

2. A method for preparing the derivative according to claim 1, characterized in that: The following steps are involved: S1, dissolving persimmon tannin PT40 in hydrochloric acid ethanol solution to obtain reaction mother solution I; S2, dissolving menthofuran in hydrochloric acid ethanol solution to obtain reaction mother solution II; S3. Mix the reaction mother liquor I and the reaction mother liquor II, place them in a water bath for reaction, and after the reaction is completed, separate them using a preparative reversed-phase high performance liquid separation column to obtain the menthofuran derivative of epigallocatechin gallate.

3. The preparation method according to claim 2, characterized in that: The hydrochloric acid concentration in the hydrochloric acid ethanol solution in steps S1 and S2 is 0.1-0.4M.

4. The preparation method according to claim 3, characterized in that: The hydrochloric acid concentration in the hydrochloric acid ethanol solution in steps S1 and S2 is 0.2M.

5. The preparation method according to claim 2, characterized in that: In step S3, the mass ratio of menthol furan to persimmon tannin PT40 in the mixed solution formed by reaction mother liquor I and reaction mother liquor II is (0.6-1.8):

1.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the menthol furan to the persimmon tannin PT40 is 1:

1.

7. The preparation method according to claim 2, characterized in that: The water bath reaction conditions in step S3 are as follows: temperature 50-80° C., time 0.5-2 h; after the reaction is completed, the reaction is placed in an ice bath at 0° C. to terminate the reaction.

8. The preparation method according to claim 7, characterized in that: The water bath reaction conditions in step S3 are: temperature 60° C., time 1.5 h.

9. The preparation method according to claim 2, characterized in that: Separation conditions in step S3: the chromatographic column is a reverse phase C18 chromatographic column, the column temperature is 30°C; the mobile phase is a 0.13% trifluoroacetic acid aqueous solution, the injection volume is 200 μL; the eluent is a 0.1% trifluoroacetic acid acetonitrile solution, the gradient elution flow rate is 4.0 mL / min; the detection wavelength is 280 nm.

10. Use of the derivative according to claim 1 in treating alcoholic gastric ulcer.

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