Acid cherry extract for relieving acute and chronic pancreatitis and preparation method thereof

Abstract

The invention discloses a sour cherry extract for relieving acute and chronic pancreatitis and a preparation method thereof. The preparation method of the acid cherry extract comprises the following steps: performing ultrasonic extraction on acid cherry freeze-dried powder by using an ethanol solution with the volume fraction of 40% to obtain an extracting solution containing the acid cherry extract; the parameter conditions of the ultrasonic extraction are as follows: the liquid-material ratio is 10-70mL. G <-1 >, the extraction time is 30-120min, and the extraction temperature is 30-60 DEG C. According to the invention, acute and chronic pancreatitis mouse models are established by adopting intermittent injection and repeated injection of luxau, so that the relieving effect of the sour cherry extract on pancreatitis is verified. Experimental results show that the sour cherry extract has a remarkable effect of relieving acute and chronic pancreatitis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to an extract of tart cherry with the function of alleviating acute and chronic pancreatitis and a preparation method thereof. Background Art

[0002] Pancreatitis is a pancreatic disease caused by the activation of trypsin and resulting in pancreatic autophagy. It is mainly divided into two types: acute and chronic. Its pathogenic factors are complex, including alcoholism, smoking, genetics, cholelithiasis, hypertriglyceridemia, etc. Dietary habits are closely related to pancreatitis. Research shows that regular alcoholism and single excessive intake of meat rich in saturated fat and cholesterol are positively correlated with the risk of pancreatitis. While when we consume more fruits rich in high fiber and high polyphenols, the risk of acute and chronic pancreatitis can be reduced. This may be because the high content of antioxidants in fruits can prevent the onset of pancreatitis by reducing the level of oxidative stress. Therefore, searching for natural dietary supplements with pancreatic protection potential and implementing intervention strategies from the dietary perspective is a beneficial way to alleviate the symptoms of pancreatitis.

[0003] Tart cherry is rich in polyphenolic compounds and has significant health benefits such as anti - inflammation, uric acid reduction, and prevention of cardiovascular diseases. However, due to its sour and astringent taste, it cannot be eaten fresh and can only be processed into primary products with low economic value, resulting in low utilization rate. At the same time, whether tart cherry has a protective effect on the pancreas has not been disclosed in the prior art.

[0004] Therefore, developing an extract of tart cherry with the function of alleviating pancreatitis and realizing the high - value utilization of tart cherry is of great significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an extract of tart cherry with optimized high polyphenol yield and anthocyanin yield as a dietary intervention for alleviating acute and chronic pancreatitis. It aims to provide a preparation method of an extract of tart cherry that is efficient, safe, fast, and suitable for large - scale production, and to verify the efficacy of the extract of tart cherry in alleviating pancreatitis through animal experiments, laying a foundation for the high - value utilization of tart cherry and the development of dietary foods.

[0006] To solve the above - mentioned technical problems, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides a preparation method of an extract of tart cherry with high polyphenol yield and anthocyanin yield.

[0008] The preparation method of the extract of tart cherry provided by the present invention includes the following steps: ultrasonically extracting freeze - dried powder of tart cherry with an ethanol solution having a volume fraction of 40% to obtain an extractive solution containing the extract of tart cherry;

[0009] The parameter conditions of the ultrasonic extraction are as follows: liquid - to - material ratio is 10 - 70 mL·g-1 、The extraction time is 30 - 120 min, and the extraction temperature is 30 - 60 °C.

[0010] Preferably, the parameters of the ultrasonic extraction are as follows: the liquid - to - solid ratio is 45 mL·g -1 、The extraction time is 30 min, and the extraction temperature is 40 °C.

[0011] Furthermore, the extraction solution of the sour cherry extract is subjected to vacuum evaporation to remove water, obtaining the sour cherry extract.

[0012] Even further, the vacuum evaporation can be carried out by rotary evaporation under reduced pressure at 40 °C.

[0013] In the sour cherry extract prepared by the above - mentioned optimal extraction process, the polyphenol yield is 23.40 ± 0.24 mg GAE / g DW (dry weight of the extract), and the anthocyanin yield can reach 173.15 ± 2.04 mg Cyd - 3 - glu / 100g DW (dry weight of the extract).

[0014] In a second aspect, the present invention provides the sour cherry extract prepared in the first aspect.

[0015] In a third aspect, the present invention provides the application of the sour cherry extract described in the second aspect.

[0016] The application of the sour cherry extract provided by the present invention is its application in the preparation of products for preventing, alleviating and / or treating pancreatitis.

[0017] In a fourth aspect, the present invention provides a product for preventing, alleviating and / or treating pancreatitis.

[0018] The product for preventing, alleviating and / or treating pancreatitis provided by the present invention contains the sour cherry extract described in the second aspect of the present invention.

[0019] Furthermore, the product may further include other ingredients having the efficacy of preventing, alleviating and / or treating pancreatitis and / or pharmaceutically acceptable carriers. The carrier is an inactive ingredient that conforms to the route of administration or dosage form and is non - toxic to the human body. The carrier can be a solid or liquid excipient. Solid excipients, for example, include microcrystalline cellulose, mannitol, lactose, pre - gelatinized starch, low - substituted hydroxypropyl cellulose, cross - linked polyvinylpyrrolidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium dodecyl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, polyvinylpyrrolidone, starch, magnesium stearate, sodium carboxymethyl starch and talc powder; liquid excipients, for example, include water, ethanol, syrup and glycerol.

[0020] Furthermore, the product can be a drug, a health product and / or a functional food.

[0021] In the present invention, the dosage form of the drug is not limited, as long as it can effectively deliver the active ingredient to the body, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, buccal tablets, granules, instant granules, pills, powders, elixirs, suspensions, powders, solutions, injections, etc.

[0022] In the present invention, the pancreatitis mentioned includes acute and chronic pancreatitis and / or chronic pancreatitis.

[0023] In the present invention, the prevention, alleviation and / or treatment of pancreatitis are specifically reflected in at least one of the following aspects:

[0024] 1) Improving pancreatic injury caused by acute pancreatitis;

[0025] 2) Improving hyperfunction of exocrine function caused by acute pancreatitis;

[0026] 3) Improving infiltration of inflammatory cells in pancreatic tissue caused by acute pancreatitis;

[0027] 4) Inhibiting the increase in the levels of pro-inflammatory factors in serum and pancreas caused by acute pancreatitis;

[0028] 5) Alleviating the process of pancreatic atrophy caused by chronic pancreatitis;

[0029] 6) Improving pancreatic fibrosis caused by chronic pancreatitis;

[0030] 7) Improving exocrine insufficiency caused by chronic pancreatitis.

[0031] Furthermore, the improvement of pancreatic injury caused by acute pancreatitis described in 1) above is specifically manifested as: reducing pancreatic interstitial dilation caused by acute pancreatitis and inhibiting the increase in pancreatic pathological scores caused by acute pancreatitis.

[0032] Furthermore, the improvement of hyperfunction of exocrine function caused by acute pancreatitis described in 2) above is specifically manifested as: inhibiting the increase in urinary amylase, serum amylase and serum lipase caused by acute pancreatitis.

[0033] Furthermore, the improvement of infiltration of inflammatory cells in pancreatic tissue caused by acute pancreatitis described in 3) above is specifically manifested as: inhibiting the increase in the number of infiltrating inflammatory cells in pancreatic tissue caused by acute pancreatitis.

[0034] Furthermore, the pro-inflammatory cytokines described in 4) above include IL-6, IL-1β and TNF-α.

[0035] Furthermore, the improvement of exocrine insufficiency caused by chronic pancreatitis described in 7) above is specifically manifested as: inhibiting the decrease in the content of lipase in serum caused by chronic pancreatitis.

[0036] The present invention uses intermittent injection and repeated injection of cerulein to establish acute and chronic pancreatitis mouse models to verify the alleviating effect of tart cherry polyphenols on pancreatitis. The experimental results show that the tart cherry extract has a significant effect on alleviating acute and chronic pancreatitis.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention prepares a tart cherry extract with a high polyphenol yield and a high anthocyanin yield by a single-factor optimization experimental method. The experimental results show that the tart cherry extract prepared by the present invention has a good effect on alleviating acute and chronic pancreatitis. By inducing acute pancreatitis in C57BL / 6J mice by intermittent intraperitoneal injection of cerulein, it was found through dietary intervention with tart cherry polyphenols that the degree of pancreatic tissue edema in mice was improved, the levels of hematuramylase and serum lipase were reduced, the positive expression of MPO in pancreatic tissue was decreased, and the levels of TNF-α, IL-6 and IL-1β in serum and pancreas were decreased. By inducing chronic pancreatitis in C57BL / 6J mice by repeated intraperitoneal injection of cerulein, it was found that dietary intervention with tart cherry polyphenols could improve pancreatic atrophy, reduce the degree of pancreatic fibrosis, and significantly increase the serum lipase level. Description of the Drawings

[0039] Figure 1 Effect of solid-liquid ratio on the extraction yield of tart cherries in Example 1: A, polyphenols; B, anthocyanins; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05;

[0040] Figure 2 Effect of ethanol volume fraction on the extraction yield of tart cherries in Example 1: A, polyphenols; B, anthocyanins; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05;

[0041] Figure 3 Effect of extraction time on the extraction yield of tart cherries in Example 1: A, polyphenols; B, anthocyanins; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05;

[0042] Figure 4 Effect of extraction temperature on the extraction yield of tart cherries in Example 1: A, polyphenols; B, anthocyanins; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05;

[0043] Figure 5 Effect of tart cherry extract on the pancreas of mice with acute pancreatitis in Example 2: A, hematoxylin-eosin staining results, 1 bar = 100 μm; B, pathological tissue score; C, pancreatic index; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05.

[0044] Figure 6Effect of tart cherry extract on the exocrine function of mice with acute pancreatitis in Example 2: A, urine amylase; B, serum amylase; C, serum lipase; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05.

[0045] Figure 7 Effect of tart cherry extract on the MPO expression of mice with acute pancreatitis in Example 2, 1 bar = 100 μm.

[0046] Figure 8 Effect of tart cherry extract on the inflammation of mice with acute pancreatitis in Example 2: A, content of serum TNF-a; B, TNF-a mRNA expression in pancreas; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05.

[0047] Figure 9 Effect of tart cherry extract on the inflammation of mice with acute pancreatitis in Example 2: A, content of serum IL-6; B, IL-6 mRNA expression in pancreas; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05.

[0048] Figure 10 Effect of tart cherry extract on the inflammation of mice with acute pancreatitis in Example 2: A, content of serum IL-1β; B, IL-1β mRNA expression in pancreas; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05.

[0049] Figure 11 Effect of tart cherry extract on the pancreas of mice with chronic pancreatitis in Example 3: A, results of hematoxylin-eosin staining, 1 bar = 100 μm; B, pathological tissue score; C, pancreas index; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05.

[0050] Figure 12 Effect of tart cherry extract on pancreatic fibrosis of mice with chronic pancreatitis in Example 3: A, results of Masson staining, 1 bar = 100 μm; B, volume fraction of collagen fibers; C, hydroxyproline content; D, expression of pancreatic COL-1 mRNA; E, expression of pancreatic a-SMA mRNA; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05.

[0051] Figure 13 Effect of tart cherry extract on the endocrine and exocrine functions of mice with chronic pancreatitis in Example 3: A, fasting blood glucose; B, serum amylase; C, serum lipase; Note: Different lowercase letters indicate significant differences between different groups, p < 0.05. Detailed implementation manners

[0052] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0053] In the experimental methods of the following embodiments, unless otherwise specified, they are all conventional methods and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0054] Example 1. Preparation of Sour Cherry Extract

[0055] The extraction process of sour cherry extract was optimized. Through single-factor experiments, the optimal process parameters for ultrasonic-assisted extraction of sour cherry extract were determined as follows: liquid-solid ratio of 45 mL·g -1 , ethanol volume fraction of 40%, extraction time of 30 min, extraction temperature of 40 °C. The polyphenol yield was 23.40 ± 0.24 mg GAE / g DW, and the anthocyanin yield could reach 173.15 ± 2.04 mg Cyd-3-glu / 100 g DW.

[0056] The optimization of the above-mentioned sour cherry polyphenol extraction process was obtained through the following method:

[0057] The sour cherries were washed, dried, and pitted, then frozen at -20 °C for 24 h and placed in a freeze dryer for freeze-drying to obtain freeze-dried sour cherries. Under liquid nitrogen protection, the freeze-dried sour cherries were ground into powder using a mortar to obtain freeze-dried sour cherry powder.

[0058] Single-factor experiment on liquid-solid ratio: Weigh 1.0 g of freeze-dried sour cherry powder into a conical flask, fix the extraction time at 30 min, the extraction temperature at 30 °C, and the extraction solvent as an ethanol solution with a volume fraction of 70%. Respectively measure the ethanol solution with a volume fraction of 70% according to the liquid-solid ratios of 15, 25, 35, 45, 65 mL·g -1 and pour it into the conical flask for ultrasonic extraction to explore the influence of the liquid-solid ratio on the extraction yields of polyphenols and anthocyanins in sour cherries.

[0059] Single-factor experiment on ethanol volume fraction: Weigh 1.0 g of freeze-dried sour cherry powder into a conical flask, fix the extraction time at 30 min, the extraction temperature at 30 °C, and prepare ethanol solutions with volume fractions of 20, 30, 40, 50, 60, 70, 80% respectively. Measure the ethanol solution according to the liquid-solid ratio of 45 mL·g -1 and pour it into the conical flask for ultrasonic extraction to explore the influence of the ethanol volume fraction on the extraction yields of polyphenols and anthocyanins in sour cherries.

[0060] Single-factor experiment on extraction time: Weigh 1.0 g of freeze-dried tart cherry powder into a conical flask, fix the solid-liquid ratio at 45 mL·g -1 , the extraction solvent is ethanol solution with a volume fraction of 40%, the extraction temperature is 30 °C, and ultrasonic extraction is carried out for 10, 20, 30, 40, and 50 min respectively to explore the effect of extraction time on the extraction yields of polyphenols and anthocyanins from tart cherries.

[0061] Single-factor experiment on extraction temperature: Weigh 1.0 g of freeze-dried tart cherry powder into a conical flask, fix the solid-liquid ratio at 45 mL·g-1, the ethanol volume fraction at 40%, and the extraction time at 30 min. Set the extraction temperatures at 30, 40, 50, 60, and 70 °C respectively to explore the effect of extraction temperature on the extraction yields of polyphenols and anthocyanins from tart cherries.

[0062] The total phenols in the tart cherry extract were determined by the Folin-Ciocalteu method. Pipette 1 mL of appropriately diluted tart cherry extract or gallic acid standard solutions with different concentrations into a 10 mL test tube, add 0.5 mL of Folin-Ciocalteu reagent, mix well and let stand for 8 min, then add 1.5 mL of 20% sodium carbonate solution, and make up the volume to 10 mL with distilled water. After mixing well and reacting in the dark for 2 h, measure the absorbance at 765 nm, and calculate the total phenol content of the tart cherry extract in terms of gallic acid (GAE) equivalent.

[0063] The anthocyanins in the tart cherry extract were determined by the pH differential method. Take 1 mL of the extract into a 10 mL test tube, add 9 mL of pH 1.0 and pH 4.5 buffer solutions respectively, and measure the absorbance at 520 nm and 700 nm within 50 min. Calculate the anthocyanin concentration according to the following formula 2-1, and the anthocyanin content is calculated in terms of cyanidin-3-glucoside (Cyd-3-glu).

[0064] Anthocyanin content (mg / L) = (A × Mr) / (ε × 1) × D f × 1000 (Formula 2-1)

[0065] In the formula: A = (A 520nm pH1.0 — A 700nm pH1.0 ) — (A 520nm pH4.5 — A 700nm pH4.5 )

[0066] Mr is the relative molecular mass of cyanidin-3-glucoside (449.2 g / mol); ε is the molar extinction coefficient of cyanidin-3-glucoside (26900 L / mol); D f is the dilution factor.

[0067] The screening results of the above single-factor experiments are shown in Figures 1 - 4 .

[0068] Example 2: Effects of Intervention with Sour Cherry Extract on a Mouse Model of Acute Pancreatitis Induced by Intermittent Intraperitoneal Injection of Cerulein

[0069] Preparation of sour cherry extract: According to the optimal extraction process parameters screened in Example 1, prepare the sour cherry extract solution, and then rotary evaporate under reduced pressure at 40 °C until the water is completely evaporated, and re-dissolve with water to make intragastric suspension solutions with concentrations of 10 mg / mL, 20 mg / mL, 40 mg / mL, and 80 mg / mL.

[0070] Preparation of sour cherry concentrated juice: Using eight-ripe fresh sour cherries as raw materials, after cleaning and removing impurities, crush them at a speed of 700 r / min, beat them into a pulp and heat to 90 °C, quickly cool to 60 °C after 45 s, extract for 30 min, then add pectinase to adjust the concentration to 150 - 200 ppm, enzymatically hydrolyze for 45 min, press with a juicer, filter, concentrate under negative pressure at 30 - 45 °C, and then fill after pasteurization. The total phenol content of the sour cherry concentrated juice is: 26.22 ± 0.52 mg / g concentrated juice, and the total anthocyanin content is 1.01 ± 0.05 mg / g concentrated juice.

[0071] After C57BL / 6J male mice were adaptively fed for 1 week, they were randomly divided into 7 groups (6 mice in each group), including a blank control group (Control), an acute pancreatitis model group (AP), and 5 intervention groups: sour cherry concentrated juice 5 mL / kg / d group (SJ), sour cherry extract 100 mg / kg / d dose group (SCE100), sour cherry extract 200 mg / kg / d dose group (SCE200), sour cherry extract 400 mg / kg / d dose group (SCE400), and sour cherry extract 800 mg / kg / d dose group (SCE800). After the experiment started, continuous intragastric administration was carried out for two weeks. The Control group and the AP group were intragastrically administered distilled water. Modeling started on the 15th day. 12 hours before modeling, food was withheld but water was not restricted. An acute pancreatitis model was constructed by intermittent intraperitoneal injection of cerulein. One injection was given every hour, and a total of 8 injections of cerulein (100 μg / kg) were given, with an interval of 1 h between each injection. The Control group was injected with normal saline.

[0072] Six hours after the last injection for modeling, blood was collected from the orbital cavity to measure serum amylase. Urine, blood, and fresh tissues were collected 24 hours after modeling. After urine collection, it was temporarily stored in liquid nitrogen, and after the sampling was completed, it was stored at -80 °C for measuring urine amylase and serum lipase. After blood collection, it was left to stand at room temperature for 1 - 2 h. After the blood was stratified, it was centrifuged at 4 °C, 3500 rpm, for 15 min, and the supernatant was aspirated and then aliquoted and stored at -80 °C for measuring the contents of serum inflammatory factors such as IL-6, IL-1β, and TNF-α in acute pancreatitis mice. The pancreas was divided into two parts. One part was fixed in 4% paraformaldehyde; the other part was put into RNA tissue preservation solution for pancreatic tissue morphology analysis, organ index detection, and pancreatic immunohistochemical analysis.

[0073] As Figure 5 shown, compared with the Control group, the pancreatic index of mice in the AP group was significantly increased, but the intervention with tart cherry extract did not significantly reduce the pancreatic index( Figure 5 C). To further clarify whether the intervention with tart cherry extract has a relieving effect on acute pancreatitis, the changes in pancreatic tissue were observed by hematoxylin-eosin staining. The pancreatic parenchyma in the Control group was compact, arranged orderly, with a complete structure, and there was no bleeding or necrosis. In the AP group, the expansion of pancreatic interstitium and between lobules was obvious, with inflammatory cell infiltration, and the pathological score was also significantly higher than that in the Control group, indicating successful modeling. After the intervention with tart cherry extract, the expansion of pancreatic interstitium was alleviated, and the pancreatic pathological score was significantly reduced. Among them, the SJ group and the SCE800 group had the best effect on improving pancreatic injury( Figure 5 A and Figure 5 B).

[0074] Pancreatic index (mg / g) = pancreatic weight (mg) / body weight of mouse (g)

[0075] Pancreatic pathological score: The acinar lobular structure was scored 0, 1, 2, 3 according to the atrophy area of 0, less than 10%, 10% - 50%, and more than 50%; the inflammatory cell infiltration was scored 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 according to the number of inflammatory cells / high-power field of view of 0 - 1, 2 - 5, 6 - 10, 11 - 15, 16 - 20, 21 - 25, 26 - 30, more than 30, and more than 40; the pancreatic fibrosis was scored 0, 2, 4 according to normal, focal, and diffuse.

[0076] From Figure 6 it can be seen that the contents of urine amylase, serum amylase, and serum lipase in the AP group were 6416.97 ± 426.67 U / L, 12541.3 ± 874.39 U / L, and 92.0 ± 8.06 U / dL respectively, which were significantly higher than those in the Control group. After the intervention with tart cherry extract, the content of hematuria amylase decreased by 27.6% - 31.3%, the content of serum amylase decreased by 26.2 - 47.7%, and the content of serum lipase decreased by 22.1 - 32.6%. And the trend of SCE800 in reducing enzyme activity was higher than that of SCE600, SCE400, and SCE200. The above results indicate that the intervention with tart cherry extract can improve the exocrine function of mice with acute pancreatitis, thus alleviating the onset of acute pancreatitis.

[0077] When acute pancreatitis occurs, the main inflammatory cells in pancreatic tissue are neutrophils. MPO is an enzyme with a relatively high content in neutrophils and its content is relatively constant. Therefore, the infiltration of inflammatory cells in mouse pancreatic tissue can be evaluated by MPO immunohistochemical staining. When the cells are brownish yellow, it indicates positive expression of MPO. The degree of positive expression of MPO is reflected by the color depth of yellow, brownish yellow, brownish brown, and brown in the sections. From Figure 7 it can be seen that a large number of MPO-positive cells infiltrate the pancreas of mice in the AP group, while there is almost no positive labeling in the Control group. The expression of MPO in the pancreatic tissue of the SJ group and the SCE800 group is significantly reduced, while relatively high MPO positive expression is still seen in the pancreas of the SCE100, SCE200, and SCE400 groups. The above results indicate that the number of infiltrating inflammatory cells in the pancreatic tissue of mice in the SJ and SCE800 groups is reduced and pancreatic inflammation is alleviated.

[0078] The pathogenesis of acute pancreatitis is complex. The inflammatory factor theory is one of the mainstream pathogenesis. After inflammatory factors are triggered, they can exacerbate pancreatic inflammation in various non-specific ways through a cascade reaction and spread to systemic inflammation by pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α and various chemokines. Therefore, the content of serum inflammatory factors and the expression of inflammatory factors in pancreatic tissue were detected. From Figure 8 it can be seen that both the content of serum TNF-α and the expression of TNF-α mRNA in the pancreas in the AP group are significantly increased. The SJ group and the 4 SCE groups can significantly reduce the content of TNF-α in serum. SJ, SCE100, and SCE400 can significantly reduce the gene expression level of TNF-α in pancreatic tissue. From Figure 9 it can be seen that compared with the Control group, both the content of serum IL-6 and the expression of IL-6 mRNA in the pancreas in the AP group are significantly increased. The SJ group and the SCE200 and SCE400 groups can significantly reduce the content of IL-6 in serum. SCE100, SCE200, and SCE400 can significantly reduce the gene expression level of IL-6 in pancreatic tissue. From Figure 10 it can be seen that compared with the Control group, both the content of serum IL-1β and the expression of IL-1β mRNA in the pancreas in the AP group are significantly increased. The SJ group and the SCE200, SCE400, and SCE800 groups can significantly reduce the content of IL-1β in serum. SCE200 and SCE400 tend to reduce the gene expression level of IL-1β in pancreatic tissue.

[0079] The above results show that after dietary intervention with sour cherry extract, the degree of pancreatic tissue edema in mice was improved, the levels of blood and urine amylase and serum lipase were reduced, the positive expression of MPO in pancreatic tissue was reduced, and the levels of TNF-α, IL-6 and IL-1β in serum and pancreas were reduced. The infiltration of immune cells in pancreatic tissue can be reduced, and the levels of pro-inflammatory factors in serum and pancreas can be reduced, thereby alleviating the inflammation of acute pancreatitis to a certain extent.

[0080] Example 3: Effect of sour cherry extract intervention on the mouse model of chronic pancreatitis induced by repeated intraperitoneal injection of caerulein

[0081] Based on the intervention effect of sour cherry extract on mice with acute pancreatitis, SCE100 had the worst effect. The pancreatic pathological damage in this group was severe and there was no significant difference between the AP group and the group. Therefore, this group was removed in the experiment of sour cherry extract to relieve chronic pancreatitis. After one week of adaptive feeding, C57BL / 6J male mice were randomly divided into 6 groups (6 mice in each group), namely, blank control group (Control), chronic pancreatitis model group (CP), and 4 intervention groups: sour cherry concentrate juice 5mL / kg / d group (SJ), sour cherry extract 200mg / kg / d dose group (SCE200), sour cherry extract 400mg / kg / d dose group (SCE400), sour cherry extract 800mg / kg / d dose group (SCE800). After the start of the experiment, the chronic pancreatitis model was established by repeated intraperitoneal injection of caerulein, with a dosage of 50μg / kg / mouse. The modeling lasted for 4 weeks, with 6 injections per day on Mondays, Wednesdays, and Fridays (1h interval each time), and the control group was injected with normal saline. While modeling, the mice were gavaged with sour cherry concentrate juice and different doses of sour cherry extract every day, while the Control group and CP group were gavaged with distilled water.

[0082] Fasting blood glucose was measured on the second day after modeling, and fresh blood and tissue were collected on the third day after modeling for the determination of serum amylase and serum lipase. The pancreatic tissue was divided into three parts, one of which was fixed in 4% paraformaldehyde for pancreatic tissue morphology analysis and pancreatic fibrosis analysis; one was placed in RNA tissue preservation solution for the expression of pancreatic COL-1 mRNA and pancreatic α-SMA mRNA; one was placed in a cryovial, temporarily stored in liquid nitrogen, and stored at -80°C after sampling for the detection of organ index and hydroxyproline determination.

[0083] The main characteristics of tissue morphology during chronic pancreatitis are loss of acinar cells, pancreatic parenchymal atrophy and fibrosis. The degree of pancreatic tissue atrophy in mice can be reflected by measuring pancreatic weight / body weight. Figure 11As shown, the pancreatic index of the mice in the Control group was 10.55 ± 0.52 mg / g, and that of the CP group was 4.75 ± 1.16 mg / g. Repeated injection of cerulein within 4 weeks induced a significant decrease in the pancreatic index of the mice in the CP group, indicating successful modeling. After intervention with tart cherry extract, the pancreatic index increased. Among them, SCE800 could significantly increase the pancreatic index, which was 23.83% higher than that of the CP group. From the pathological section results ( Figure 11 A and Figure 11 B), it can be seen that the internal morphology of the pancreas in the Control group was intact, the pancreatic parenchyma was compact and arranged orderly, and there was no acinar cell vacuolization or inflammatory cell infiltration. In contrast, the pancreatic interstitium and lobules in the CP group were significantly dilated, the pancreatic cells showed vacuolation, severe inflammatory cell infiltration, and the formation of tubular complexes, and the pathological score was also significantly increased. The number of vacuolated cells and tubular complexes in the pancreas of the SCE intervention group decreased, but the dilation of the pancreatic interstitium and infiltration of immune cells were not alleviated. The pancreatic structure of the mice in the SJ group was relatively intact, and the degree of edema was relatively reduced. Generally speaking, intervention with tart cherry extract can alleviate the process of pancreatic atrophy in chronic pancreatitis.

[0084] It can be Figure 12 seen that intervention with tart cherry extract significantly reduced the volume fraction of collagen fibers, but there was no significant difference among the intervention groups of tart cherry extract. The extracellular matrix is mainly composed of collagen and fibronectin. Hydroxyproline is a unique amino acid in collagen. Therefore, the collagen deposition can be reflected by measuring the hydroxyproline content. It can be Figure 12 seen from Figure 12 C that SJ could significantly reduce the hydroxyproline content in the pancreatic tissue. Different doses of SCE showed a trend of reducing the hydroxyproline content in the pancreatic tissue, but there was no statistical difference. COL-1 is the main collagen that constitutes the extracellular matrix. Therefore, the expression of its gene was detected at the transcriptional level. It can be Figure 12 seen from Figure 12The expression of α-SMA mRNA in the CP group as shown in E was significantly higher than that in the Control group, indicating that overstimulation with cerulein led to the transformation of normal pancreatic cells into fibroblasts, which to a certain extent damaged the pancreatic function, consistent with the decrease in serum lipase. After intervention with tart cherry extract, the expression level of this gene was significantly downregulated, and the downregulation of the transcription level of α-SMA by SCE showed a dose-dependent relationship. The above results showed that the pancreatic collagen volume fraction and the gene expression related to fiber production in the mice intervened with SJ or SCE were significantly lower than those in the CP group. SJ could significantly reduce the hydroxyproline content, and SCE showed a trend of reducing the hydroxyproline content. In general, the intervention with tart cherry extract could significantly improve the fibrosis of mice with chronic pancreatitis.

[0085] In patients with chronic pancreatitis, due to exocrine insufficiency or total pancreatic duct stenosis, the secretion of pancreatic lipase, protease, amylase, etc. by pancreatic tissue is reduced. Figure 13 As can be seen, the content of serum lipase in the CP group of mice was significantly reduced. After intervention with tart cherry polyphenols, the content of serum lipase could be significantly increased. However, compared with the Control group, there was no significant change in the serum amylase of the CP group, SJ group and SCE group. In this experiment, chronic pancreatitis did not lead to a significant decrease in the serum amylase of mice, which was consistent with the research results of Tao et al. (Tao X F, Chen Q, Li N, et al. Serotonin-RhoA / ROCK axis promotes acinar-to-ductal metaplasia in caerulein-induced chronic pancreatitis[J]. Biomedicine&Pharmacotherapy, 2020, 125: 109999.). It was possible that in the mice with chronic pancreatitis induced by continuous injection of cerulein for 4 weeks, the retention rate of normal pancreatic acini was not too low. When Tao et al. continued to induce chronic pancreatitis with cerulein for 8 weeks, the serum amylase of the mice with chronic pancreatitis began to decrease significantly. As the course of chronic pancreatitis continued to develop, it might lead to damage to the pancreatic parenchyma and islet tissue, thus affecting the endocrine function, which also resulted in diabetes being one of the common complications of chronic pancreatitis. Therefore, the fasting blood glucose of the mice was detected to observe whether the endocrine function of the mice was normal. Figure 13 As can be seen from A, compared with the Control group, the fasting blood glucose of the CP group and the tart cherry polyphenol intervention group showed an upward trend, but there was no statistical difference, indicating that chronic pancreatitis in this experiment had not affected the endocrine function. Generally speaking, after intervention with tart cherry extract, the content of lipase in the serum could be significantly increased, thus improving the exocrine function of mice with chronic pancreatitis.

[0086] The above results indicate that repeated intraperitoneal injection of cerulein was used to induce chronic pancreatitis in C57BL / 6J mice, and it was found that dietary intervention with tart cherry polyphenols could improve pancreatic atrophy, reduce the degree of pancreatic fibrosis, and significantly increase the serum lipase content.

[0087] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements of the present invention, including those that depart from the scope disclosed in this application but are made with conventional techniques known in the art. The application of some basic features can be made within the scope of the following appended claims.

Claims

1. A method for preparing a tart cherry extract, comprising the following steps: subjecting the freeze-dried powder of tart cherries to ultrasonic extraction with an ethanol solution having a volume fraction of 40% to obtain an extract containing the tart cherry extract; The parameter conditions for the ultrasonic extraction are as follows: the liquid-solid ratio is 10 - 70 mL·g -1 , the extraction time is 30 - 120 min, and the extraction temperature is 30 - 60 °C.

2. The preparation method according to claim 1, wherein: The parameters for ultrasonic extraction are as follows: liquid-to-solid ratio is 45 mL·g -1 , extraction time is 30 min, and extraction temperature is 40 °C.

3. The preparation method according to claim 1 or 2, characterized in that: Performing vacuum evaporation on the extract containing the tart cherry extract to remove water and obtain the tart cherry extract.

4. An extract containing the tart cherry extract or the tart cherry extract prepared by the method according to any one of claims 1-3.

5. Use of the extract containing the tart cherry extract or the tart cherry extract according to claim 4 in the preparation of a product for preventing, alleviating and / or treating pancreatitis.

6. The application according to claim 5, wherein: The extract containing the tart cherry extract or the tart cherry extract is the extract containing the tart cherry extract or the tart cherry extract according to claim 4.

7. The application according to claim 5 or 6, characterized in that: The product is a drug, a health product and / or a functional food; and / or, the pancreatitis includes acute and chronic pancreatitis and / or chronic pancreatitis.

8. The application according to claim 7, wherein: The prevention, alleviation and / or treatment of pancreatitis is specifically manifested in at least one of the following aspects: 1) Improving pancreatic injury caused by acute pancreatitis; 2) Improving hyperfunction of exocrine function caused by acute pancreatitis; 3) Improving inflammatory cell infiltration in pancreatic tissue caused by acute pancreatitis; 4) Inhibiting the increase in the levels of pro-inflammatory factors in serum and pancreas caused by acute pancreatitis; 5) Alleviating the process of pancreatic atrophy caused by chronic pancreatitis; 6) Improving pancreatic fibrosis caused by chronic pancreatitis; 7) Improving exocrine insufficiency caused by chronic pancreatitis.

9. A product for preventing, alleviating and / or treating pancreatitis, which contains the extract containing the tart cherry extract or the tart cherry extract according to claim 4.

10. The product according to claim 9, characterized in that: The product further comprises other components having the efficacy of preventing, alleviating and / or treating pancreatitis and / or a pharmaceutically acceptable carrier; and / or, the product is a drug, a health product and / or a functional food; and / or, the pancreatitis includes acute and chronic pancreatitis and / or chronic pancreatitis.

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