A menthofuran derivative of epigallocatechin gallate and a preparation method and application thereof

Abstract

The present application provides a kind of EGCG-mint furan derivative, the derivative structure is as shown in the following: the preparation steps of above-mentioned derivative are as follows: S1, persimmon tannin PT40 is dissolved in hydrochloric acid ethanol solution, and reaction mother liquor I is obtained;S2, mint furan is dissolved in hydrochloric acid ethanol solution, and reaction mother liquor II is obtained;S3, reaction mother liquor I and reaction mother liquor II are mixed, and are reacted under water bath condition, after reaction is completed, separation is carried out using preparative reverse phase high performance liquid separation column, and EGCG-MF is obtained.The persimmon tannin PT40 of the present application is degraded using mint furan as nucleophilic reagent, the prepared EGCG-MF has therapeutic effect in alcoholic gastric ulcer mice, and high yield of EGCG-MF is obtained by optimizing preparation process, which provides new ideas and new methods for proanthocyanidin in food field and medical field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant functional component degradation and product application, and particularly relates to a menthofuran derivative of epigallocatechin gallate and a preparation method and application thereof. BACKGROUND

[0002] Persimmon tannin is a kind of polyphenol compound with high polymerization degree, which has a very special structure compared with other tannins: high polymerization, average polymerization degree up to 23.6%, gallolylated structure up to 72%, containing A type connection. Although persimmon tannin has functions and activities such as antioxidant, lipid-lowering and weight-reducing, regulating sugar metabolism, etc., its application is greatly limited due to its low solubility, low bioavailability, etc. Degradation of high polymer into low polymer is an effective way to expand its application.

[0003] At present, the methods for degrading procyanidin high polymer mainly include acid degradation, metal catalytic hydrogenolysis, nucleophilic substitution degradation and microbial degradation. The acid degradation method is to break the C-C bond between flavan-3-ols under hot acidic conditions, thereby completing the depolymerization of high polymer, but this method has low degradation efficiency, complex product, instability, difficult effective utilization of degradation product, high acid concentration, strong corrosion and environmental unfriendliness. The metal catalytic hydrogenolysis is to depolymerize the high polymer by selecting a suitable hydrogenation catalyst under high-temperature hydrogenation conditions, which has high requirements for equipment and harsh reaction conditions, and is limited in use. The microbial degradation is a technology for converting organic matter into inorganic matter by means of growth and metabolism of microorganisms, but it is difficult to cultivate the microorganisms for degradation or to find suitable microorganisms. The nucleophilic substitution degradation is to dissociate the extension unit of high polymer into a carbonium ion intermediate under acidic conditions, and a nucleophilic reagent will capture the carbonium ion to generate a corresponding conjugate, which is a commonly used method for structural analysis of tannin compounds. Commonly used nucleophilic reagents such as m-benzenetriol and benzyl mercaptan have the defects of toxicity, strong irritating odor and the like, and the product is difficult to be used in the field of food.

[0004] Therefore, it is a key technical problem to be solved in the field to find a new type of edible and efficient nucleophilic bond-breaking agent for realizing efficient and environmentally friendly degradation of persimmon tannin, obtaining high-yield active degradation products, and applying them in the fields of food and medicine. SUMMARY

[0005] In view of this, the present application provides a nucleophilic bond-breaking agent for safely and efficiently degrading persimmon tannin, and simultaneously obtaining a high-yield degradation product, epigallocatechin gallate-menthofuran derivative, which has anti-alcoholic gastric ulcer activity.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] A menthofuran derivative of epigallocatechin gallate, the derivative structure is as follows:

[0008]

[0009] A preparation method of the above derivative, comprising the following steps:

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

[0011] S2, take menthofuran and dissolve it in hydrochloric acid ethanol solution to obtain reaction mother liquor II;

[0012] S3, mix reaction mother liquor I and reaction mother liquor II, and react under water bath condition, and after the reaction is completed, separate by using a preparative reverse phase high performance liquid separation column to obtain the menthofuran derivative of epigallocatechin gallate.

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

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

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

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

[0017] Further, the water bath reaction condition in step S3 is: temperature 50-80℃, time 0.5-2h; after the reaction is completed, place in 0℃ ice bath to terminate the reaction.

[0018] In some specific embodiments, preferably, the water bath reaction condition in step S3 is: temperature 60℃, time 1.5h.

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

[0020] The above derivative is applied to anti-alcoholic gastric ulcer.

[0021] The above alcoholic gastric ulcer indexes include: gastric mucosa damage index, gastric tissue pathology damage, oxidative stress substance level, and inflammatory factor level.

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

[0023] (1) The present application innovatively uses menthofuran as a nucleophile to degrade persimmon tannin PT40, and a large number of carbon cation extension units containing epigallocatechin gallate are obtained by using the special structure of persimmon tannin PT40, and a high-activity epigallocatechin gallate-menthofuran derivative (EGCG-MF) is synthesized.

[0024] (2) The degradation method of persimmon tannin proposed in the present application can obtain high yield of EGCG-MF (the yield reaches 246 mg / g based on the total mass of EGCG and MF) through optimization of preparation parameters, and can realize development and utilization of persimmon tannin resources.

[0025] (3) The EGCG-MF prepared in the present application has a therapeutic effect on alcoholic gastric ulcer mice, and has a good effect at a high dose (100 mg / kg) and a low dose (50 mg / kg), which provides a new idea and a new method for procyanidins in the fields of food and medicine. BRIEF DESCRIPTION OF DRAWINGS

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

[0027] Figure 2 is a result graph of persimmon tannin degradation efficiency under different reaction temperatures in Example 1.

[0028] Figure 3 is a result graph of persimmon tannin degradation efficiency under different reaction times in Example 2.

[0029] Figure 4 is a result graph of persimmon tannin degradation efficiency under different concentrations of hydrochloric acid ethanol in Example 3.

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

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

[0032] Figure 7 is a cleavage pathway spectrum of EGCG-MF.

[0033] Figure 8 is a direct view of the gastric tissue damage of alcoholic gastric ulcer mice treated with EGCG-MF, wherein A is a direct view comparison graph of the damage, B is an injury index score, and C is an injury inhibition rate.

[0034] Figure 9 EGCG-MF on the gastric tissue of alcoholic gastric ulcer mice.

[0035] Figure 10 EGCG-MF on the gastric tissue of alcoholic gastric ulcer mice.

[0036] Figure 11 EGCG-MF on the gastric tissue of alcoholic gastric ulcer mice.

[0037] Figure 12 EGCG-MF on the gastric tissue of alcoholic gastric ulcer mice. DETAILED DESCRIPTION

[0038] The application will be further described in conjunction with specific embodiments so that those skilled in the art better understand the present application.

[0039] Key test material sources and physicochemical parameters:

[0040] Persimmon tannin PT40 was prepared in the laboratory: persimmon pulp was mixed with 1% hydrochloric acid-methanol mixed solution at a solid-liquid ratio of 1:3, and was condensed and refluxed at 80°C for 40 min, repeated three times, and the extraction liquid was combined and left overnight. The extraction liquid was filtered, and concentrated to 50 mL (2000 ml each) at 35°C. The extract was extracted with AB-8 macroporous resin. First, distilled water was used to remove sugar and other soluble impurities, 10% ethanol / water (v / v) was used to wash low molecular weight phenolic compounds, and finally anhydrous ethanol was used to elute tannin. 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 (70×350 mm, 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), the collected fraction was evaporated under reduced pressure at 35°C, freeze-dried, and stored at -20°C.

[0041] Example 1

[0042] This example provides a thin mint furan derivative of epigallocatechin gallate, which is prepared as follows:

[0043] S1, hydrochloric acid was added to ethanol to prepare a 0.1M hydrochloric acid ethanol solution.

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

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

[0046] S4. The obtained degradation reaction solution is separated using a preparative reversed-phase high-performance liquid chromatography column to obtain epigallocatechin gallate-menthol furan derivative (EGCG-MF).

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

[0048] The results of EGCG-MF prepared according to the temperature gradient are shown below. Figure 2 ,Depend on Figure 2 It can be seen that the peak area increases significantly at 60℃ compared to 50℃, but comparing the sum of the peak areas at each temperature, we found no significant difference between 60, 70, and 80℃. Therefore, from the perspective of energy conservation, we chose 60℃ as the optimal temperature.

[0049] Example 2

[0050] This embodiment provides a peppermint furan derivative of epigallocatechin gallate, which is prepared in basically the same way as in Example 1, except that the reaction temperature in step S3 is 60°C, but the reaction time is carried out in sequence according to the time gradient (0.5h, 1h, 1.5h, 2h), while the rest remain unchanged.

[0051] Results of EGCG-MF prepared according to time gradient are shown below. Figure 3 ,Depend on Figure 3 It can be seen that the yield first increases and then decreases with the extension of reaction time. This is because proanthocyanidins undergo side reactions during degradation. Extending the reaction time may initially favor side reactions rather than depolymerization, and vice versa. Therefore, a reaction time of 1.5 hours is selected as the optimal reaction time.

[0052] Example 3

[0053] This embodiment provides a peppermint furan derivative of epigallocatechin gallate, which is prepared in basically the same way as in Example 1, except that: in step S1, hydrochloric acid ethanol solutions are prepared sequentially according to the concentration gradient (0.1M, 0.2M, 0.3M, 0.4M); in step S3, the reaction temperature is kept constant at 60℃ and the reaction time is 1.5h; all other steps remain unchanged.

[0054] The results of EGCG-MF prepared by hydrochloric acid-ethanol solution with concentration gradient are shown below. Figure 4 ,Depend on Figure 4 It can be observed that as the hydrochloric acid concentration increases, the content of proanthocyanidin oligomers produced by the reaction of PT40 and MF first increases and then decreases, with the peak area of ​​the new product being the largest at a hydrochloric acid concentration of 0.2M. This phenomenon can be explained by the fact that higher concentrations of H promote the breaking of the flavanine bonds in proanthocyanidins, generating more carbocations and accelerating the forward reaction. However, under higher acidity conditions, proanthocyanidins themselves also undergo a certain degree of degradation, leading to a decrease in the content of the newly formed product. This may be the reason why the degradation efficiency decreases at excessively high acid concentrations. Therefore, a hydrochloric acid-ethanol concentration of 0.2M is chosen as the optimal reaction acidity.

[0055] Example 4

[0056] This embodiment provides a menthol furan derivative of epigallocatechin gallate, the preparation of which is basically the same as in Example 1, except that: in step S1, the concentration of hydrochloric acid in the hydrochloric acid ethanol solution is 0.2M; in step S2, persimmon tannin PT40 and menthol furan are weighed in sequence according to the 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℃ and the reaction time is 1.5h; all other steps remain unchanged.

[0057] Results of EGCG-MF prepared according to the mass ratio gradient of MF to PT40 are shown below. 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 a higher dose of nucleophile can promote the reaction in the forward direction. However, when the mass ratio of MF and menthol is 1:1, further increasing the amount of menthol furan does not increase the total area of ​​the new peak, indicating that depolymerization is complete. From the perspective of saving raw materials, a reaction mass ratio of 1:1 is chosen as the optimal reaction mass ratio.

[0058] As demonstrated in the above examples, the highest yield of the target product was obtained under the following conditions: a water bath temperature of 60°C, a reaction time of 1.5 h, a hydrochloric acid-ethanol concentration of 0.2 M, and a mass ratio of MF to PT40 of 1:1. The products prepared under the optimal conditions in Examples 1-4 were collected, and after removing the organic solvent by rotary evaporation at 30°C, they were freeze-dried under vacuum to obtain EGCG-MF powder. The calculated yield of EGCG-MF was 246 mg / g (based on the total mass of added EGCG and MF).

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

[0060] Wherein, m / z453 is the [MH-152] formed by the loss of its galloyl group. - The resulting fragment ions (Liu H, Zou T, Gao J, et al. Depolymerization of cranberry procyanidins using (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin gallate as chain breakers[J]. Food Chemistry, 2013, 141(1):488-494.) exhibit different cleavage pathways:

[0061] First, it can lose a water molecule to form a fragment ion ([MMH-152-18]-) with m / z 435. The fragment molecule continues to undergo HRF fission, followed by RDA fission, losing 126 Da 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 a portion of the menthofuran to form m / z 285, which then undergoes 1,4 ring cleavage of the a ring to form the fragment ion at m / z 125 (Vivas N, Gaulejac D V N, Vitry C, et al. Impact of ethanol content on the scavenging activities of oak wood C-glycosidic ellagitannins. 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 cleavage can also occur directly from m / z 453, losing 126 Da to form the fragment ion at m / z 327 ([M-H-152-126]-) (Gu et al., 2003), which then undergoes QM cleavage to lose the menthofuran fragment ion to form m / z 177 ([M-H-152-126-150]-); m / z 453 can also first undergo RDA cleavage, losing 168 Da to form the fragment ion at 285 ([M-H-152-168]-) (A.P. Neilson, A.S. Hopf, B.R. Cooper, M.A. Pereira, J.A. Bomser, Ferruzzi, M.G. Catechin degradation with concurrent formation of homo- and heterocatechin dimers during in vitro digestion. J Agric Food Chem, 2007, 55(22): 8941-8949.).

[0064] Finally, the possible fragmentation pathway for m / z 453 is the loss of a fraction of carbon dioxide fragment molecule forming m / z 409 ([M-H-152-44]-) (Rodrigues, C. M., Rinaldo, D., dos Santos, L. C, Montoro, P., Piacente, S., Pizza, C, Hiruma-Lima, C. A., Brito, A. R., & Vilegas, W. (2007). Metabolic fingerprinting using direct flow injection electrospray ionization tandem mass spectrometry for the characterization of proanthocyanidins from the barks of Hancornia speciosa. Rapid Communications in Mass Spectrometry, 21, 1907-1914), followed by the loss of a water molecule forming m / z 391 fragment ion ([M-H-152-44-18]-) (Li, H. J., & Deinzer, M. L. (2008). The mass spectral analysis of isolated hops A-type proanthocyanidins by electrospray ionization tandem mass spectrometry. 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 the menthol furan at the C4 position, leaving a m / z 455 fragment ion, indicating that the menthanofuran-modified extension unit is epigallocatechin gallate (Chen J, Xu Z, Zhu W, et al. Novel proanthocyanidin dimer analogues with the C-ring-opened diaryl-propan-2-gallate structural unit and enhanced antioxidant activities [J]. Journal of Functional Foods, 2016, 21 290-300.).

[0066] The ion at m / z 605 can also lose a gallic acid molecule, forming a fragment ion at m / z 435 before subsequent fragmentation ([M-H-152-170]-) (Sipowo Tala, V. R., da Silva, V. C, Rodrigues, C. M., Nkengfack, A. E., dos Santos, L. C, & 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] According to the above mass spectrometry fragmentation information, it can be inferred that it is a menthofuran compound of epigallocatechin gallate (EGCG-MF), and the fragmentation pathway is as follows: Figure 7 .

[0068] Further, in order to understand the therapeutic effect of the above prepared EGCG-MF on alcoholic gastric ulcer, the EGCG-MF prepared by the optimal process was also subjected to the following animal experiments.

[0069] 1. Establishment of 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 adaptive feeding for one week. During this period, standard pellet feed and water can be freely used. Before gavage, the mice were fasted for 12h, then 95% anhydrous ethanol was gavaged to the mice according to the gavage volume of 10mL / kg, and the modeling was performed for 12h, and then the drug administration was started.

[0071] 2. Experimental grouping and treatment administration

[0072] The experimental setup normal control group, model group, cimetidine group (100 mg / kg), L-EGCG-MF (50 mg / kg) group, H-EGCG-MF (100 mg / kg) group. Continuous administration for 3 days. The control group and the model group were given 0.5% CMC-Na solution. 24 hours after the last administration, the mice were sacrificed by cervical dislocation, and the stomach was removed. The stomach was cut along the greater curvature, and the contents of the stomach were washed clean with ice-cold normal saline. The water on the surface of the gastric tissue was gently wiped off with a water-absorbing paper, and the damage to the inner wall of the stomach was recorded by a camera for evaluation of gastric ulceration. Subsequently, the gastric tissue was divided into two halves: 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 the subsequent experiments are as follows:

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

[0075] Experimental method: The gastric tissue was scored according to the GUTH scoring standard for damage index and ulcer inhibition rate calculation, and the specific scoring indicators are as follows: 1 point for pinpoint bleeding or erosion; linear, strip bleeding, length <1 mm, 2 points, damage length between 1-2 mm, 3 points; damage length between 2-4 mm, 4 points; damage length >4 mm, 5 points. If the damage width is >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 x 100%

[0077] A1 represents the average damage index of the gastric tissue of the model group mice, and A2 represents the average damage index of the gastric tissue of the mice in the drug prevention group.

[0078] Experimental results: The blank group mice had no abnormal symptoms in the stomach, the internal tissue was smooth and flat, the folds were obvious, there was no bleeding and edema, and the color was light red; the model group mice had gastric distension and gas, severe gastric mucosa damage, and obvious tissue bleeding and edema in multiple places, with reduced gastric mucosa folds; compared with the model group, only a small amount of white ulcer points appeared in the two compound administration groups, without large area of ulcer and tissue bleeding, etc. It is shown that EGCG-MF can improve the ulcer damage of the gastric tissue of mice caused by alcohol Figure 8 ).

[0079] EGCG-MF improves the pathological analysis of gastric tissue in alcoholic gastric ulcer mice

[0080] Experimental method: Take the fixed in 4% paraformaldehyde solution of gastric tissue samples, using hematoxylin-eosin staining (H&E) and periodic acid-Schiff staining (PAS) for gastric histopathology evaluation. Pathological analysis. Briefly, according to the specified standard and procedure, the gastric tissue was treated with graded ethanol and xylene solution, and the tissue was embedded in paraffin. The tissue was cut into 4-5 μm thin sections, and stained. Then the results of staining and imaging were observed under a microscope (NIB610).

[0081] Experimental results: HE staining results show that Figure 9 ), the glandular structure of the mice in the blank control group was complete, the glandular epithelial cells were arranged in order, and there was no inflammatory cell infiltration, bleeding, etc.; the gastric mucosa of the mice in the model group was severely damaged, and there were epithelial cell shedding, inflammatory cell infiltration, and tissue hyperemia, etc.; the gastric tissue of the mice in the cimetidine group and the EGCG-MF group showed different degrees of glandular destruction and tissue bleeding, etc., but compared with the model group, they were relieved to a certain extent. PAS staining results show that Figure 10 ), the mice in the blank group were stained dark purple, indicating that the mucus layer was not damaged; while the mucus of the mice in the model group was severely damaged, and the color development degree was greatly weakened compared with the blank group; the mucus layer of the mice in the drug groups was mostly normal, and the color development was intense compared with the model group, indicating that the mucus layer of the mice after treatment was repaired, proving that the degradation product had a protective effect on the gastric mucosal barrier.

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

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

[0084] Results: MDA is the end product of lipid peroxidation, which can indirectly reflect the degree of gastric tissue damage. Compared with the blank group, the MDA content of the model group was significantly increased. There was a significant difference in MDA content between the cimetidine group and the EGCG-MF group and the model group (P<0.05), and the MDA content was decreased. GSH is an important antioxidant in humans and rodents, which can ensure that red blood cells can normally play a role in supplying oxygen in the body without being affected by ROS, and also plays an important role in maintaining the integrity of the gastric mucosa. The GSH level of the treated mouse gastric tissue was improved to some extent compared with the model group, and the content was significantly increased (P<0.01). The GSH level of 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 scavenge superoxide anions, thereby serving as the first line of defense to protect gastric mucosal cells from damage caused by oxygen free radicals. The results show that compared with the normal group, the SOD activity of the model group was significantly decreased. Compared with the model group, the SOD activity of the cimetidine group was significantly increased (P<0.001), and the SOD level of the EGCG-MF group was significantly increased compared with the model group (P<0.05).

[0085] Biochemical results show that EGCG-MF can improve the ulcer damage to the mouse gastric tissue caused by alcohol by inhibiting oxidative stress. Figure 11

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

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

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

[0089] ​The experimental results: ELISA experimental results show that the content of TNF-α in the tissue after ethanol stimulation is significantly increased compared with the blank group (P<0.01), and the mice after treatment are significantly improved compared with the model group (P<0.01), which proves that EGCG-MF can improve gastric ulcer by inhibiting the generation of this pro-inflammatory factor. The increase of TNF-α content can stimulate the generation of IL-6 and aggravate the symptoms of inflammatory reaction. Then IL-6 stimulates neutrophils and macrophages at the inflammation site to produce various harmful substances that can cause damage to the gastric tissue. The IL-6 results show that, similar to the TNF-α results, the IL-6 content in the gastric tissue of the model group mice is significantly increased (P<0.01), and the IL-6 content in the gastric tissue of the mice in the administration group is significantly decreased compared with the model group (P<0.01). The IL-10 level of the model group mice has a significant difference with the blank group (P<0.01), and the improvement effect of the high-dose H-EGCG-MF group and the cimetidine group is significant (P<0.01), and the difference between the low-dose L-EGCG-MF group and the model group is more significant (P<0.05).

[0090] The ELISA experimental results all show that EGCG-MF can effectively improve the ulcer damage of the gastric tissue of the mice caused by alcohol by playing an anti-inflammatory role. Figure 12 )。

[0091] The above experimental results show that the persimmon tannin degradation product provided by the present application, EGCG-MF, can effectively reduce the gastric tissue damage index of the mice caused by alcohol and protect the gastric mucosal barrier. The alcohol-induced gastric ulcer is effectively improved by the antioxidant effect and the anti-inflammatory effect.

[0092] The specific raw materials in the present application are all existing substances, which can be directly purchased from the market.

[0093] The above is only a preferred embodiment of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A menthofuran derivative of epigallocatechin gallate, characterized in that, The derivative structure is shown as follows: 。 2. A process for the preparation of a derivative according to claim 1, characterized in that, It comprises the following steps: S1, dissolve persimmon tannin PT40 in hydrochloric acid ethanol solution to obtain reaction mother liquor I; S2, dissolve menthofuran in hydrochloric acid ethanol solution to obtain reaction mother liquor II; S3, mix reaction mother liquor I and reaction mother liquor II, and react under water bath condition, and after the reaction is completed, separate by using a preparative reverse-phase high-performance liquid separation column to obtain the menthofuran derivative of epigallocatechin gallate; Wherein, the persimmon tannin PT40 is prepared as follows: mix persimmon pulp and 1% hydrochloric acid-methanol mixed solution at a solid-liquid ratio of 1:3, condense and reflux at 80°C for 40 min, repeat three times, combine the extraction liquid and stand overnight; extract the extraction liquid, rotary evaporate at 35°C, concentrate 2000 mL of filtrate to 50 mL, and extract the extract with AB-8 macroporous resin; first, remove sugar and other soluble impurities with distilled water, wash low molecular weight phenolic compounds with 10% ethanol / water, and finally elute tannin with anhydrous ethanol; collect the eluate, remove the solvent with a vacuum rotary evaporator, and freeze-dry the residue; dissolve the freeze-dried residue in methanol, and equilibrate on a Toyopearl HW-50F column with 20% acetone / water mixture; finally, elute the target tannin with 40% acetone / water mixture, collect the fraction, evaporate under reduced pressure at 35°C, and freeze-dry to obtain the target tannin.

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

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

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

1.

6. The production method according to claim 5, wherein The mass ratio of menthofuran to persimmon tannin PT40 is 1:

1.

7. The preparation method according to claim 2, characterized in that, The water bath reaction condition in step S3 is: temperature 50-80°C, time 0.5-2 h; after the reaction is completed, place 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 condition in step S3 is: temperature 60°C, time 1.5 h.

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

10. The derivative of claim 1 in the preparation of a drug for resisting alcoholic gastric ulcer.

Images