Application of purple rice seed coat extract in preparing medicine for preventing and treating acute pancreatitis

Abstract

The invention discloses a new application of a purple rice seed coat extract in the preparation of a drug for preventing and treating acute pancreatitis, and belongs to the field of biomedicine technology. The purple rice seed coat extract of the invention is used to prepare a drug for preventing and treating acute pancreatitis, which broadens the application field of the purple rice seed coat and provides a new choice and idea for preventing and treating acute pancreatitis; the animal model of acute pancreatitis induced by caerulein is used to confirm the effect of the purple rice seed coat extract in reducing serum amylase, lipase and the expression of MPO and SOD in plasma, alleviating pancreatic tissue damage, acinar cell vacuoles and inflammatory cell infiltration, reducing the expression of pancreatic tissue inflammation and oxidative stress-related factors, and reducing pancreatitis-related intestinal permeability.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of a purple rice seed coat extract in preparing a medicine for preventing and treating acute pancreatitis. Background Art

[0002] Acute pancreatitis (AP) is one of the most common acute abdominal diseases in clinical practice. It is an inflammatory disease caused by damage to exocrine acinar cells. Its pathological characteristics are self-digestion of the pancreas by pancreatic enzymes, accompanied by excessive infiltration of inflammatory cells and release of pro-inflammatory factors. Severe AP can cause systemic inflammatory response syndrome and multiple organ dysfunction and failure. The pathogenesis of AP is not fully understood, but studies have reported that the intestine is involved in the development of severe organ failure in AP. 60% of AP patients have intestinal barrier dysfunction, and intestinal mucosal damage in the intestinal barrier causes increased intestinal permeability and bacterial translocation, which is an important link leading to pancreatic infection and necrosis. Therefore, it is particularly important to explore the pathogenesis of AP and develop efficient methods to prevent and treat AP, in order to prevent the occurrence of AP and block the spread of inflammation, avoid the development of acute pancreatitis into severe acute pancreatitis or even pancreatic cancer, and reduce the mortality and complication rate of pancreatitis.

[0003] Purple rice (Oryza sativa L.indica) is a kind of glutinous rice, also known as purple glutinous rice. It is a relatively precious rice variety and is only cultivated in small quantities in Yunnan, Hunan, Sichuan and other places. Purple rice is sweet and warm in nature. It is used as a folk tonic. It has the effects of replenishing qi and blood, warming the stomach and spleen, nourishing the liver and kidneys, reducing urination, and relieving cough and asthma. It has certain therapeutic effects on stomach cold pain, thirst, frequent urination at night, etc., so it is also known as the "Medicine Valley". Studies have shown that purple rice has antioxidant, anti-inflammatory, anti-atherosclerotic and anti-tumor activities. At present, the protective effect of purple rice on AP is still unclear. Therefore, the present invention studies the protective effect of purple rice seed coat ethanol extract on AP and related tissue damage. Summary of the invention

[0004] The main purpose of the present invention is to provide a new use of a purple rice seed coat extract, which is applied to the preparation of a drug for preventing, treating and / or relieving acute pancreatitis.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Application of purple rice seed coat extract in preparing medicine for preventing and treating acute pancreatitis.

[0007] Purple rice seed coat extract can prevent and treat acute pancreatitis, including at least one of the following effects:

[0008] (1) Reduce serum lipase, amylase, and plasma MPO and SOD levels;

[0009] (2) Alleviate pancreatic tissue damage, acinar cell vacuoles, and inflammatory cell infiltration;

[0010] (3) reduce the expression levels of inflammation and oxidative stress markers in pancreatic tissue;

[0011] (4) reduce pancreatitis-related intestinal permeability;

[0012] (5) Reduce the risk of acute pancreatitis.

[0013] The purple rice seed coat extract is obtained by extracting purple rice seed coat powder with 70-80% acidified ethanol.

[0014] Preferably, the extraction method of the purple rice seed coat extract is:

[0015] The purple rice is ground and then sieved to obtain purple rice seed coat powder; the purple rice powder is evenly mixed with a solvent and then extracted to obtain an extract; the extract is repeated 3 to 5 times and then the extract is combined; the combined extract is suction filtered and the filtrate is rotary evaporated to obtain a thick extract after the solvent is completely evaporated; the thick extract is vacuum freeze-dried and then ground into powder to obtain a powdery purple rice seed coat extract; the powdery purple rice seed coat extract is stored in the dark at -20 to -25°C for later use.

[0016] Preferably, the sieving is through a 40-60 mesh sieve.

[0017] Preferably, the solvent is acidified ethanol, containing 1% to 5% HCl, 70% to 80% ethanol, and the remainder water by mass.

[0018] Preferably, the solid-liquid ratio of the purple rice powder to the solvent is 1:4-6.

[0019] Preferably, the extraction is carried out at 3-4°C for 10-15 hours.

[0020] Preferably, the temperature of the rotary evaporation is 38-42°C.

[0021] Preferably, the administration dosage of the purple rice seed coat extract is 200-600 mg / day / kg.

[0022] Preferably, the drug comprises purple rice seed coat extract. The drug containing purple rice extract is taken into the gastrointestinal tract of a mammal; the mammal includes but is not limited to humans.

[0023] The new drug for preventing, treating and / or alleviating acute pancreatitis of the present invention uses purple rice seed coat extract as an active ingredient, and may also be added with one or more adjuvants acceptable to pharmaceutical preparations, or may be compounded with other active ingredients to exert a synergistic effect in treating acute pancreatitis; the drug is an oral preparation, and the oral preparation is granules, pills, tablets, capsules, powders, etc.

[0024] The beneficial effects of the present invention are:

[0025] 1. The invention provides a new use of purple rice seed coat extract in drugs for preventing and / or relieving acute pancreatitis, and proves that purple rice seed coat extract can play a protective role against acute pancreatitis in mice induced by caerulein, and can be added as an effective ingredient to drugs for preventing and treating acute pancreatitis, reducing the occurrence and development of acute pancreatitis and the degree of inflammation in clinical practice. Therefore, purple rice seed coat extract is expected to be well used in the preparation of drugs for the treatment and prevention of severe acute pancreatitis.

[0026] 2. The present invention uses purple rice seed coat extract to preventively treat the caerulein-induced mouse acute pancreatitis model, and verifies the effect of purple rice seed coat extract on alleviating pancreatic tissue damage, acinar cell vacuoles and inflammatory cell infiltration, reducing serum amylase, lipase and the expression of MPO and SOD in plasma, reducing pancreatic tissue inflammation and the expression of oxidative stress factors, and reducing pancreatitis-related intestinal permeability.

[0027] 3. The present invention broadens the application field of purple rice seed coat extract and provides new options and ideas for preventing and treating acute pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The total ion chromatogram of the negative ion mode of the UPLC-MS / MS principal component analysis of the purple rice seed coat extract of the present invention;

[0029] Figure 2 The purple rice seed coat extract of the present invention is applied to acute pancreatitis mice and the expression of serum lipase (A), amylase (B) and plasma MPO (C), SOD (D);

[0030] Figure 3 The purple rice seed coat extract of the present invention was applied to mice with acute pancreatitis and HE staining was used to observe pancreatic tissue damage, acinar cell vacuoles and inflammatory cell infiltration;

[0031] Figure 4 The expression of factors related to pancreatic tissue inflammation and oxidative stress after the rice seed coat extract of the present invention is applied to mice with acute pancreatitis;

[0032] Figure 5 The present invention is a study on pancreatitis-related intestinal permeability after the purple rice seed coat extract of the present invention is applied to mice with acute pancreatitis. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] The terms "first", "second" and "third" in this application are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined as "first", "second" and "third" can explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. All directional indications in the embodiments of the present application (such as up, down, left, right, front, back...) are only used to explain the relative position relationship, movement, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication also changes accordingly. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not limited to the listed steps or units, but optionally also includes steps or units that are not listed, or optionally also includes other steps or units inherent to these processes, methods, products or devices.

[0035] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0036] The purple rice used in the following examples of the present invention was purchased from a local market in Mojiang County, Yunnan Province; the C57BL / 6J mice used were purchased from Beijing Sibeifu Experimental Animal Co., Ltd.; and caerulein was purchased from Wuhan Sewell Biotechnology Co., Ltd. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0037] The quantitative results of the following examples are expressed as mean ± standard error (Mean ± SEM); all data were statistically analyzed using Graph Pad Prism 9.0 (Graph Pad Software, San Diego, USA); differences among the groups were analyzed using one-way analysis of variance (ANOVA); all bar graphs were drawn by Graph Pad Prism 9.0; **P < 0.01, ***P < 0.001, P < 0.05 were statistically significant.

[0038] Example 1 Preparation of purple rice seed coat extract

[0039] (1) Extraction of purple rice seed coat

[0040] The purchased purple rice was ground with a high-speed grinder and passed through a 40-mesh sieve; 100 g of purple rice seed coat powder was evenly stirred with 500 mL of an acidified ethanol solvent containing 1% HCl, 70% ethanol, and 29% water, and extracted at 4°C for 12 hours, and the extraction was repeated 3 times, and the extracts were combined. The extract was filtered on filter paper, and the filtrate was evaporated on a 40°C rotary evaporator to form a thick extract; finally, the thick extract was vacuum freeze-dried, ground into powder in a mortar, and stored at -20°C in the dark for subsequent use.

[0041] (2) UPLC-MS / MS principal component qualitative analysis of purple rice seed coat extract

[0042] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS). An Agilent SB-C18 column (1.8 μm, 2.1 mm × 100 mm) was used, mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. Elution gradient: 0-9 min, 5%-95% B; 10 min, 95% B; 10-11 min, 95%-5% B. Flow rate 0.35 mL / min, injection volume 4 μL, column temperature 40 ° C; the total ion chromatogram of the negative ion mode of the UPLC-MS / MS principal component analysis of the purple rice seed coat extract is shown in the figure. Figure 1 shown.

[0043] Depend on Figure 1 It can be seen that the purple rice seed coat ethanol extract mainly contains active ingredients such as phenolic acids, flavonoids and alkaloids.

[0044] Example 2 Purple rice seed coat extract is used to prevent and treat acute pancreatitis

[0045] (1) Experimental animal grouping

[0046] The purchased mice were randomly divided into a blank control group (Control), an AP model group (Model), a purple rice extract treatment group, and a resveratrol treatment group. The purple rice seed coat extract treatment group was gavaged with 600 mg / kg and 200 mg / kg of the purple rice extract per day, respectively, and was recorded as a high-dose purple rice extract treatment group (PREH) and a low-dose purple rice extract treatment group (PREL); the resveratrol treatment group was gavaged with 100 mg / kg of the resveratrol treatment group per day, and was recorded as a positive control group (Positive). There were 10 mice in each group, and the gavage was continued for 7 consecutive days.

[0047] The blank group (Control) and the model group (Model) were gavaged with the same volume of normal saline once a day for 7 days.

[0048] (2) Establishment of mouse model of acute pancreatitis

[0049] The mice in each group after gavage for 7 days were used. Except for the mice in the blank group, the other mice were intraperitoneally injected with caerulein 50 μg / kg / h for 8 times continuously to establish the acute pancreatitis model. The mice in the blank group were injected with an equal amount of normal saline. The mice were dislocated 14 hours after the last injection, and fresh blood, pancreatic tissue and other samples were quickly collected.

[0050] (3) Purple rice seed coat extract reduces serum lipase, amylase, and plasma MPO and SOD levels

[0051] When acute pancreatitis occurs, the levels of lipase and amylase in serum increase, while the recruitment and infiltration of immune cells cause an increase in MPO activity and a decrease in SOD activity. The serum lipase, amylase, mouse plasma myeloperoxidase (MPO) and superoxide dismutase (SOD) kits were used for determination. The expression of serum lipase (A), amylase (B) and plasma MPO (C), SOD (D) after purple rice seed coat extract was applied to mice with acute pancreatitis Figure 2 As shown, Figure 2 In the figure, Control represents the control group, Moedel represents the AP model group, PREH represents the model treatment group after intragastric administration of high-dose purple rice extract, PREL represents the model treatment group after intragastric administration of low-dose purple rice extract, and Positive represents the positive drug resveratrol treatment group. Figure 2 A is serum lipase in mice with acute pancreatitis, Figure 2 B is amylase, Figure 2 C is MPO in plasma, Figure 2 D stands for SOD.

[0052] Depend on Figure 2It can be seen that the levels of lipase, amylase and MPO in AP mice were significantly higher than those in the Control group (P<0.001); while the levels of serum lipase ( Figure 2 A), amylase ( Figure 2 B) and plasma MPO ( Figure 2 The expression levels of SOD in the plasma of AP mice were significantly decreased compared with those in the Control group (P < 0.01), while the SOD levels in the plasma of PRE and Positive mice were significantly decreased (P < 0.01). Figure 2 D) content was significantly increased (P<0.01). It can be seen that purple rice seed coat extract can reduce serum lipase, amylase and plasma MPO, SOD content.

[0053] (4) Purple rice seed coat extract alleviates pancreatic tissue damage, acinar cell vacuoles and inflammatory cell infiltration

[0054] The pancreases of the mice in the Control, Model, PREH, PREL and Positive groups were fixed, dehydrated, stained, dewaxed, transparentized and mounted by H&E staining of paraffin sections to observe the tissue integrity, acinar cell vacuoles and inflammatory cell infiltration of the pancreatic tissue.

[0055] The morphological observation of pancreatic pathological sections showed Figure 3 As shown. Figure 3 It can be seen that the pancreatic tissue structure of the Control group was dense and without obvious cracks; vacuoles and death of acinar cells were observed in the pancreas of the Model group mice, and a large number of inflammatory cells were infiltrated; and the pancreatic tissue damage in the PREH and PREL groups was alleviated compared with the Model group, and the treatment effect was equivalent to that of the Positive group. This shows that purple rice seed coat extract plays a protective role against acute pancreatitis in mice.

[0056] (5) Purple rice seed coat extract reduces the expression of factors related to inflammation and oxidative stress in pancreatic tissue

[0057] ELISA was used to measure the expression levels of oxidative stress and inflammation-related factors GSH and NO in mouse pancreatic tissue. Figure 4 shown.

[0058] Depend on Figure 4 It can be seen that: Figure 4 In A, compared with the Control group, the GSH content of the Model group mice was significantly decreased (P < 0.01), and the GSH content in the pancreatic tissue of the PREH and PREL groups was increased, which was equivalent to that of the Positive group. Figure 4In B, compared with the Control group, the NO content of the Model group mice was significantly increased (P < 0.01), and the NO content of the pancreatic tissue of the PREH, PREL and Positive groups was significantly reduced (P < 0.01). It can be seen that purple rice seed coat extract can reduce the expression of inflammation and oxidative stress-related factors in the pancreatic tissue of mice.

[0059] (6) Purple rice seed coat extract reduces pancreatitis-related intestinal permeability

[0060] Acute pancreatitis can lead to increased intestinal permeability. The content of intestinal D-lactic acid (D-Lac) is an important indicator of intestinal permeability. The content of D-Lac in the cecal tissue of mice was determined by ELISA kit. The intestinal permeability related to pancreatitis was evaluated after the purple rice seed coat extract was applied to mice with acute pancreatitis. Figure 5 shown.

[0061] Depend on Figure 5 It can be seen that compared with the Control group, the D-Lac content of the Model group mice was significantly increased (P < 0.001), and the D-Lac content of the intestinal tissue was significantly reduced after treatment with purple rice seed coat extract and resveratrol (P < 0.001). It can be seen that purple rice seed coat extract can reduce pancreatitis-related intestinal permeability.

[0062] Example 3

[0063] The purchased purple rice was ground with a high-speed grinder and passed through a 60-mesh sieve; 100 g of purple rice seed coat powder was evenly stirred with 600 mL of an acidified ethanol solvent containing 5% HCl, 80% ethanol, and 15% water, and extracted at 3°C ​​for 15 hours, and the extraction was repeated 5 times, and the extracts were combined. The extract was filtered on filter paper, and the filtrate was evaporated on a 42°C rotary evaporator to form a thick extract; finally, the thick extract was vacuum freeze-dried, ground into powder in a mortar, and stored at -25°C in the dark for subsequent use.

[0064] Example 4

[0065] The purchased purple rice was ground with a high-speed grinder and passed through a 50-mesh sieve; 100 g of purple rice seed coat powder was evenly stirred with 400 mL of an acidified ethanol solvent containing 3% HCl, 75% ethanol, and 22% water, and extracted at 4°C for 10 hours, and the extraction was repeated 4 times, and the extracts were combined. The extract was filtered on filter paper, and the filtrate was evaporated on a 38°C rotary evaporator to form a thick extract; finally, the thick extract was vacuum freeze-dried, ground into powder in a mortar, and stored at -23°C in the dark for subsequent use.

[0066] In summary, the present invention broadens the application field of purple rice seed coat extract, and preventively treats the acute pancreatitis model of mice induced by caerulein by using purple rice seed coat extract, and verifies the effect of purple rice seed coat extract on relieving pancreatic tissue damage, acinar cell vacuoles and inflammatory cell infiltration, reducing serum amylase, lipase and MPO, SOD expression in plasma, reducing pancreatic tissue inflammation and oxidative stress factor expression, and reducing pancreatitis-related intestinal permeability. It is proved that purple rice seed coat extract can play a protective role in acute pancreatitis of mice induced by caerulein, and can be added as an effective ingredient to the drug for preventing and treating acute pancreatitis, reducing the occurrence and development of acute pancreatitis and the degree of inflammation in clinical practice. Therefore, purple rice seed coat extract is expected to be well applied in the preparation of drugs for treating and preventing severe acute pancreatitis.

[0067] The specific implementation methods of the invention are described in detail above, but they are only examples, and the invention is not limited to the specific implementation methods described above. For those skilled in the art, any equivalent modification or substitution of the invention is also within the scope of the invention, therefore, the equalization, modification, improvement, etc. made without departing from the spirit and principle of the invention should be included in the scope of the invention.

Claims

1. Application of purple rice seed coat extract in the preparation of drugs for preventing and treating acute pancreatitis; The extraction method of the purple rice seed coat extract is: The purple rice is ground and then sieved to obtain purple rice seed coat powder; the purple rice seed coat powder is evenly mixed with a solvent and then extracted to obtain an extract; after repeating the extraction for 3 to 5 times, the extracts are combined; the combined extract is suction filtered, and the filtrate is rotary evaporated to obtain a thick extract after the solvent is completely evaporated; the thick extract is vacuum freeze-dried and then ground into powder to obtain a powdery purple rice seed coat extract; the powdery purple rice seed coat extract is stored in the dark at -20 to -25°C for standby use; The solvent is acidified ethanol, which contains 1% to 5% HCl, 70% to 80% ethanol, and the balance is water.

2. The use according to claim 1, characterized in that: The sieving is through a 40-60 mesh sieve.

3. The use according to claim 1, characterized in that: The solid-liquid ratio of the purple rice seed coat powder to the solvent is 1:4-6.

4. The use according to claim 1, characterized in that: The extraction is carried out at 3-4°C for 10-15 hours.

5. The use according to claim 1, characterized in that: The temperature of the rotary evaporation is 38-42°C.

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