Application of Nereidia serrata in preparing medicine for treating constipation

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the use of nereids in the preparation of drugs for treating constipation. The present invention provides for the first time the use of nereid polypeptides in the preparation of products for improving intestinal function, products for improving immunity, and / or products for treating constipation. The present invention found that after nereids intervened in constipated mice, the mice gained weight, defecation time was shortened, feces moisture content increased, and stool shape was uniform, indicating that nereids have the effect of improving the intestinal function of constipated mice. In addition, after nereid intervention, the spleen and thymus indexes of mice showed significant increases, and the thymus index and spleen index were significantly improved. It can be considered that nereids can improve the body's immune capacity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of lugworms in preparing medicines for treating constipation. Background Art

[0002] Constipation is one of the common gastrointestinal diseases. According to statistics, the global prevalence of constipation in adults ranges from 2.5% to 79%, and the prevalence in middle-aged and elderly people is significantly higher than that in adolescents. Constipation not only increases the economic pressure on patients, but also increases the psychological burden on patients and reduces their quality of life. The occurrence of constipation is related to intestinal flora disorders. Figures show that 40% of adults and 70% of children use antibiotics at least once a year, and one in ten of them will have side effects of varying degrees after use, including constipation. The use of antibiotics can cause intestinal flora disorders, weakened intestinal motility, and lead to constipation. The current treatment effect of constipation is not ideal. According to the Rome Criteria Asian Working Group, 46.1% of constipation patients are dissatisfied with the current treatment effects. Finding a treatment method that is effective and highly satisfactory to constipation patients is a difficult problem that needs to be solved urgently.

[0003] Studies have found that changing the dietary structure has a regulatory and intervention effect on constipation. The double-toothed perinecrosis is a nutritious and delicious seafood. The "Sea Medicine Materia Medica" records that "sea silkworms" can replenish the body and make people shiny, light and long-lived after long-term consumption, while the "Chinese Marine Drug Dictionary" records that "sea silkworms" have the effects of tonifying the spleen and stomach, nourishing blood, promoting diuresis and reducing swelling. They are mainly used to treat symptoms such as yin deficiency, spleen and stomach weakness, anemia, and limb swelling. However, it is not clear whether the sandworm can relieve the symptoms of constipation, and there are no reports on the use of sandworms in the preparation of products for treating constipation. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide the application of sandworms.

[0005] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides any one of the uses of neriworm polypeptides in a1) to a3):

[0007] a1) Preparation of products that improve intestinal function;

[0008] a2) Preparation of products for improving immunity;

[0009] a3) Preparation of products for treating constipation.

[0010] Preferably, the sandworms include at least one of the dicynoglossiella, the Japanese spiny sandworm, the multi-spined sandworm, and the warty-mouthed sandworm;

[0011] Preferably, the sandworm includes the bilaterian sandworm.

[0012] Preferably, the lureworm polypeptide includes at least one of a crude lureworm product and a lureworm polypeptide.

[0013] Preferably, the crude processed products of lugworms include at least one of living lugworms, lugworm homogenate, dried lugworms and lugworm powder.

[0014] Preferably, the crude processed product of lugworms includes lugworm powder.

[0015] Preferably, the molecular weight of the neriworm polypeptide includes at least one of <5 kDa, 5-8 kDa, and >8 kDa.

[0016] Preferably, the molecular weight of the neriworm polypeptide is comprised between 5 and 8 kDa.

[0017] Preferably, the method for preparing the lugworm polypeptide comprises the following steps: enzymatic hydrolysis of lugworm and filtering.

[0018] Preferably, the enzyme for enzymolysis includes at least one of alkaline protease, pepsin, trypsin and plant protease.

[0019] Preferably, the ratio of the enzyme to lugworm is 2000-4000 U / g.

[0020] Preferably, the enzymatic hydrolysis time is 3 to 12 hours.

[0021] Preferably, the temperature of the enzymatic hydrolysis is 40-60°C.

[0022] Preferably, the product of the first aspect of the present invention further comprises a pharmaceutically acceptable excipient.

[0023] Preferably, the pharmaceutically acceptable excipients include at least one of a filler, a lubricant, a flavoring agent, a stabilizer, a desiccant, an antioxidant, a disintegrant, a colorant, a dispersant, a pH adjuster, and a coating material.

[0024] Preferably, the product may further include any one or more second active ingredients.

[0025] Preferably, the product is a pharmaceutical product.

[0026] Preferably, the drug is an antioxidant drug.

[0027] The beneficial effects of the present invention are:

[0028] The present invention provides for the first time the use of lugworms in the preparation of products for improving intestinal function, products for improving immunity, and / or products for treating constipation. The present invention found that after lugworms intervened in constipated mice, the mice gained weight, defecation time was shortened, feces moisture content increased, and the stool shape was uniform, indicating that lugworm polypeptides have the effect of improving the intestinal function of constipated mice. In addition, after the intervention of lugworms, the spleen and thymus indexes of mice showed significant increases, and the thymus index and spleen index were significantly improved. It can be considered that lugworms can improve the body's immune capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the fecal morphology of mice in the blank group and model group after antibiotic treatment.

[0030] Figure 2 This is the small intestinal propulsion rate of mice in the blank group and model group after antibiotic treatment.

[0031] Figure 3 This is the effect of antibiotic treatment on the discharge time of the first black stool in the small intestine of mice in the blank group and the model group.

[0032] Figure 4 The figure shows the effects of different treatment groups on the body weight of mice at different times.

[0033] Figure 5 This is the effect of different treatment groups on the intestinal propulsion rate of mice.

[0034] Figure 6 This is the effect of different treatment groups on the time of discharge of the first black stool in mice.

[0035] Figure 7 This is the effect of different treatment groups on the water content of mouse feces.

[0036] Figure 8 The effects of different treatment groups on the spleen of mice.

[0037] Fig. 9 This is the effect of different treatment groups on the mouse thymus. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0039] The 5kDa ultrafiltration membrane used in the present invention is the product number C6JR77589-020 of Millipore Company, and the 8kDa ultrafiltration membrane is the product number C6MR12468-029 of Millipore Company. Other ultrafiltration membranes with the same molecular retention capacity on the market can also achieve the technical effect of this invention.

[0040] Example 1 A Nereid polypeptide

[0041] This example prepares a nereid polypeptide with a molecular weight of <5 kDa, and the specific process is as follows:

[0042] Weigh 500g of lugworm (Periglomerulus bipunctatus), homogenize and add alkaline protease (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10154) at a ratio of 3000 U / g, with a material-water ratio of 1:5, adjust pH to 8.5, place in a 50℃ shaker for 7h, and after the enzymatic hydrolysis is completed, place in 100℃ boiling water for 10 minutes to inactivate the enzyme. After the enzyme inactivation is completed, cool to room temperature at room temperature, centrifuge at 8000 r / min to obtain the supernatant.

[0043] The supernatant is filtered through a 5 kDa ultrafiltration membrane, and the permeate is the neriworm polypeptide of less than 5 kDa, which is freeze-dried into dry powder and dissolved in ultrapure water when used.

[0044] Example 2 A Nereid polypeptide

[0045] This example prepares a nereid polypeptide with a molecular weight of 5 to 8 kDa, and the specific process is as follows:

[0046] Weigh 10g of sandworms, homogenize and add alkaline protease (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10154) at a ratio of 3000 U / g, with a material-water ratio of 1:5, adjust pH=8.5, place in a 50℃ shaker for 7 hours, and after the enzymatic hydrolysis is completed, place in 100℃ boiling water for 10 minutes to inactivate the enzyme. After the enzyme inactivation is completed, cool to room temperature at room temperature, centrifuge at 8000 r / min to obtain the supernatant.

[0047] The supernatant was filtered successively with 8kDa and 5kDa ultrafiltration membranes to obtain nereid polypeptides with a molecular weight of 5-8kDa, which were freeze-dried into dry powder and dissolved in ultrapure water when used.

[0048] Example 3 A Nereid polypeptide

[0049] This example prepares a nereid polypeptide with a molecular weight of >8 kDa, and the specific process is as follows:

[0050] Weigh 10g of lugworm (Periglomerulus bipunctatus), homogenize and add alkaline protease (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10154) at a ratio of 3000 U / g, with a material-water ratio of 1:5, adjust pH=8.5, place in a 50℃ shaker for 7h, and after the enzymatic hydrolysis is completed, place in 100℃ boiling water for 10 minutes to inactivate the enzyme. After the enzyme inactivation is completed, cool to room temperature at room temperature, centrifuge at 8000 r / min to obtain the supernatant.

[0051] The supernatant was filtered through an 8 kDa ultrafiltration membrane, and the retained portion was the neriworm polypeptide of >8 kDa, which was freeze-dried into dry powder and dissolved in ultrapure water when used.

[0052] Effect Example 1 Construction of Mouse Constipation Model

[0053] 1. Experimental animals, materials and feeding process

[0054] Experimental materials: SpF grade 6-week-old male C57BL / 6J mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.); gum arabic, rubber gloves, masks, cephalexin (purchased from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd.); amoxicillin (purchased from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd.); clarithromycin (purchased from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd.); ear tags, corn cobs, and wood shavings (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.).

[0055] Experimental instruments: analytical balance (purchased from Sartorius, Germany), constant temperature drying oven (purchased from Shanghai Boxun), ultrasonic water bath (Kunshan Ultrasonic Instrument Co., Ltd.).

[0056] The experimental animals were kept in the animal room, fed with Co-60 irradiated sterilized complete feed (nutrient composition in accordance with GB14924.3-2001) and sterilized ultrapure water. They were kept in strict accordance with the operating specifications of the Experimental Animal Center of Guangdong Ocean University (SYXK2014-0053).

[0057] 2. Reagent preparation

[0058] Antibiotic mixed suspension: Clarithromycin, cephalexin, and amoxicillin were prepared into high-concentration antibiotic mixed suspension (125 mg / mL) and low-concentration antibiotic mixed suspension (62.5 mg / mL) in a mass ratio of 2:2:1. Preparation method: Take clarithromycin, cephalexin, and amoxicillin in a mortar, grind them finely, and pour them into a clean and dry beaker. Pour ultrapure water according to the concentration calculation, seal with plastic wrap, and ultrasonicate for 20 minutes until the drugs are completely dissolved. Preparation principle: Prepare with ultrapure water and use it immediately after preparation.

[0059] Ink: Weigh 100 g of gum arabic, add 800 mL of ultrapure water, boil until the solution is transparent, weigh 100 g of activated carbon powder, add it to the above solution and boil it repeatedly three times. After the solution cools to room temperature, add ultrapure water to make up to 1000 mL for use, and shake well before use.

[0060] 3. Experimental methods

[0061] 91 6-week-old male C57BL / 6J mice were pre-raised in the quarantine room for 5-7 days to observe whether their physiological state was normal. After ensuring that the mice were physiologically normal, they were grouped. The mice were randomly divided into a blank group and a modeling group. There were 24 mice in the blank group and 67 mice in the modeling group, with 4-5 mice per cage. The modeling group was gavaged with a low-concentration antibiotic mixed suspension for 1-4 days and a high-concentration mixed suspension for 5-7 days. All mice received the same gavage dose (volume): 0.02 mL / g·bw. The blank group was gavaged with the same dose of sterilized ultrapure water. The mice were weighed and recorded before gavage every day, and the amount of feces was collected and recorded. After the gavage on the 7th day, ten mice were randomly selected from the blank group and the modeling group to confirm whether the constipation mouse modeling was successfully established. The weight and feces of each mouse were recorded, and the feces of the mice were collected and sealed in a sterile PE tube and stored in a refrigerator at -80 ℃. Then, the mice were gavaged with ink (0.02 mL / g·bw) according to their body weight. Five mice in the blank group were separated into cages to measure the time of the first black stool, and the remaining five mice were separated into cages to wait for 30 min before dissection and recording the small intestinal propulsion rate. Ten mice were selected from the modeling group as the model group, and their treatment was the same as that of the blank group.

[0062] 4. Experimental results

[0063] 1) Effect of antibiotic mixture on fecal morphology of mice

[0064] The results of the effect of the antibiotic mixture on the fecal morphology of mice are as follows Figure 1 As shown. The feces of the blank group were moderately soft and hard, and the feces particles were uniform in size and oval in shape. The color was darker than the normal color of mouse defecation. The feces of the model group were harder in texture, lighter in color, and dry on the surface. The amount of feces excreted in the blank group in 24 hours was much higher than that of the model group in 24 hours.

[0065] 2) Effect of antibiotic mixture on small intestinal propulsion rate in mice

[0066] The results are as follows Figure 2 As shown in the figure, the small intestinal propulsion rate of the blank group was much higher than that of the model group (P < 0.001). Compared with the blank group, the propulsion rate of the model group was significantly decreased. Antibiotics reduced the intestinal peristalsis ability of mice.

[0067] 3) Effect of antibiotic mixture on the time of first black stool discharge in mice

[0068] The results are as follows Figure 3 As shown in the figure, the time of first black stool discharge in mice showed that the time of first black stool discharge in the blank group was about 100 min, while that in the model group was about 260 min (P < 0.001). Compared with the time of first black stool discharge in the blank group, the time in the model group was significantly increased by 160 min. Antibiotics reduced the intestinal motility of mice.

[0069] from Figure 1 , 2 3. It can be seen that the amount of defecation in the blank group is much higher than that in the model group, and the feces in the blank group are moderately soft and hard, with normal color, uniform and complete fecal particles; while the feces in the model group are light in color, hard in texture, dry and hard on the surface, and uneven in fecal particles; in terms of the time of the first black stool discharge in mice, the time of the first black stool discharge in the blank group is about 100 min, while the time of the first black stool discharge in the model group is about 260 min, which is significantly longer than that in the blank group by 160 min (P<0.001); the small intestinal propulsion rate of blank mice is also much higher than that of the model group (P<0.001). According to the characteristics of constipated mice: dry and hard feces, hard texture, long defecation time, and low propulsion rate, it is judged that the constipation model of mice induced by the mixture of amoxicillin, cephalexin, and clarithromycin antibiotics was successfully established.

[0070] Effect Example 2 Regulatory Effect of Nereis Ultrafiltration Fraction on Antibiotic-Induced Intestinal Function in Mice

[0071] 1. Mouse Model

[0072] On the basis of Part 3 in Effect Example 1, 71 mice remained. Among them, 10 mice were selected from the blank group (14 mice) and continued to be gavaged with sterilized ultrapure water, and the other 4 mice were reserved. In the modeling group, 13 mice were used as the <5 kDa group, and according to body weight, they were gavaged with 7d <5 kDa nereid ultrafiltration component solution; 12 mice were used as the 5-8 kDa group, and according to body weight, they were gavaged with 7d 5-8 kDa nereid ultrafiltration component solution; 12 mice were used as the >8 kDa group, and according to body weight, they were gavaged with 7d >8 kDa nereid ultrafiltration component solution; 10 mice were used as the model group, and continued to be gavaged with low-concentration antibiotics for 7d; 10 mice were used as the self-healing group, and no gavage was performed to observe their physiological state. The amount of gavage samples in each group and each stage is shown in Table 1.

[0073] Table 1 Grouping of mice and treatment at each stage

[0074]

[0075] Treatment method: gavage dose of 0.02 mL / g·bw, neriworm polypeptide sample dissolved in water, concentration of 100 mg / mL, gavage for 7 consecutive days (8th to 14th day), record the weight of mice, feces quantity, calculate the feces water content.

[0076] After 7 days of gavage, the weight and feces of the mice were recorded and the feces were collected and stored in a -80 ℃ refrigerator. Then, ink (0.02 mL / g·bw) was gavaged according to the weight of the mice. Half of the mice in each group were separated into cages to measure the time of the first black stool, and the remaining half were separated into cages to wait for 30 min before dissecting and recording the small intestinal propulsion rate. After recording the time of the first black stool and the small intestinal propulsion rate of the mice, the mice were dissected, the spleen and thymus were removed and weighed.

[0077] Determination of the time to the first black stool and the small intestinal ink propulsion rate of mice: On the 7th day after intragastric administration of antibiotics, half of the mice in each group were housed in a separate cage and intragastrically administered with ink according to their body weight. The time from the start of intragastric administration to the discharge of the first black stool was recorded. The remaining half of the mice were dissected 30 minutes later, and the total length of the small intestine and the distance from the pylorus at the lower end of the stomach to the front edge of the ink movement were measured. The small intestinal propulsion rate was calculated according to the following formula:

[0078] Propulsion rate (%) = (ink propulsion distance rate / cm) ÷ (total length of small intestine / cm) × 100%.

[0079] 2. Experimental results

[0080] 1) Effects of ultrafiltration fractions of Nereis spp. on body weight of mice

[0081] Figure 4 The results of the weight changes of mice during the experiment, 1 to 7 days is the stage of gavage antibiotics to establish a mouse constipation model, of which 1 to 3 days is the stage of gavage low-concentration antibiotics, during which the weight of the blank group mice increased steadily, and the weight of the model group mice increased slowly. 4 to 7 days is the stage of gavage high-concentration antibiotics, during which the weight of the blank group mice increased, and the model group mice had a trend of weight loss during this stage. 8 to 14 days is the stage of gavage sample feeding, during which the model group continued to gavage low-dose antibiotics, and the weight decreased by 18.15%. The rest of the sample groups were fed with sandworm samples (Examples 1 to 3), and their weights increased significantly, with a trend close to the blank group.

[0082] 2) Effects of ultrafiltration fractions from Nereis spp. on small intestinal propulsion rate in mice

[0083] The results are as follows Figure 5 As shown, the nephrite ultrafiltration fractions prepared in Examples 1 to 3 can significantly improve the small intestinal propulsion rate of mice. Among them, the 5-8 KDa group had the most significant effect (P < 0.001). This shows that the small intestinal propulsion rate of constipated mice was improved after the intervention of nephrite ultrafiltration fractions, and the small intestinal motility was increased.

[0084] 3) Effect of ultrafiltration fractions of Nereis spp. on the time of first black stool discharge in mice

[0085] The results are as follows Figure 6 As shown in the figure, the ultrafiltration fractions of nephrite can significantly increase the time of the first black stool of mice. The defecation time of the model group was about 260 min, while the defecation time of the sample group was significantly reduced. Among them, the defecation time of the 5-8 KDa group was about 50 min, which was the most significant effect (P < 0.001). This shows that the defecation time of constipated mice was improved and the small intestinal motility was increased after the intervention of the ultrafiltration fractions of nephrite.

[0086] 4) Effect of ultrafiltration fractions of lugworm on the water content of mouse feces

[0087] The results are as follows Figure 7 As shown in the figure, compared with the first day, the water content of the model group mice decreased significantly. The water content of the blank group showed an upward trend from 5 to 6 days. The water content of the self-healing group showed an upward trend from 2 to 6 days, the water content of the sandworm group increased on the second day, the water content of the <5kDa group showed an upward trend from 2 to 6 days, the water content of the 5-8 kDa group increased on the third, fifth and sixth days, among which the third and fifth days increased significantly, and the water content of the >8 kDa group showed an upward trend from 2 to 5 days.

[0088] 5) Effects of ultrafiltration fractions from nereidia on spleen index in mice

[0089] The results are as follows Figure 8 As shown, compared with the mice in the model group, the spleen index of the mice treated in Examples 1 to 3 was significantly increased (P<0.001). After the mice were gavage-fed with a mixture of amoxicillin, cephalexin and clarithromycin antibiotics to induce constipation, in addition to affecting the mice's defecation ability, the mice's spleen index decreased significantly, indicating that antibiotics can cause a decrease in the mice's immunity. After gavage-fed with lugworms, the mice's spleen index increased significantly, indicating that lugworms have the effect of improving the mice's immunity.

[0090] 6) Effects of ultrafiltration fractions from nereidia on thymus index in mice

[0091] The results are as follows Fig. 9 As shown, compared with the model group, there were significant differences in the self-healing group, <5 kDa group, and 5-8 kDa group (P<0.05), the nereid group (P<0.01), and the >8 kDa group (P<0.001). After mice were gavaged with a mixture of amoxicillin, cephalexin, and clarithromycin antibiotics to induce constipation, in addition to affecting the defecation ability of mice, the thymus index of mice decreased significantly, indicating that antibiotics can cause a decrease in the immunity of mice, while the thymus index of mice was significantly increased after gavage with nereid ultrafiltration fractions, indicating that nereid has the effect of improving the immunity of mice.

[0092] In summary, after the present invention used clarithromycin, amoxicillin, and cephalexin three antibiotics mixed solution to gavage mice, the mice lost weight, and the mice had dry stools and constipation with difficulty in defecation. In addition, through analysis, the thymus index and spleen index of the mice gavage-fed with antibiotics decreased significantly. The thymus index and spleen index can reflect the strength of the functions of the two major immune organs, the thymus and spleen. The decrease in the thymus index and spleen index indicates a decrease in the body's immunity. After the intervention of constipated mice by gavage feeding with nereids, the mice gained weight, the defecation time was shortened, the feces moisture content was increased, and the stool shape was uniform, indicating that nereids have the effect of improving the intestinal function of constipated mice. On the other hand, after the intervention of nereid polypeptide, the spleen and thymus indexes of the mice showed significant improvement, and the thymus index and spleen index were significantly improved. It can be judged that nereid polypeptide can improve the body's immune ability. It shows that nereid polypeptide has the effect of improving the immunity of constipated mice. After the intervention of the present invention using nereid polypeptide, the immune function of mice increased and the constipation condition was improved. Therefore, the improvement of the intestinal function of constipated mice by the nereid ultrafiltration component may be related to the improvement of the immunity of constipated mice. The nereid ultrafiltration component improves the intestinal flora by improving the immune function of constipated mice, thereby ultimately improving the constipation symptoms of mice.

Claims

1. Application of Nereis polypeptide in the preparation of medicine for treating constipation; The preparation method of the lugworm polypeptide comprises the following steps: Enzymatic hydrolysis of Perineuri bipunctatus and filtration; The enzyme for enzymolysis is alkaline protease; The ratio of the enzyme to lugworm is 2000-4000 U / g; The enzymatic hydrolysis time is 3 to 12 hours; The temperature of the enzymatic hydrolysis is 40-60°C.

2. The use according to claim 1, characterized in that: The molecular weight of the neriworm polypeptide includes at least one of <5 kDa, 5-8 kDa, and >8 kDa.

3. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable excipients include at least one of fillers, lubricants, flavoring agents, stabilizers, desiccants, antioxidants, disintegrants, colorants, dispersants, pH regulators, and coating materials.

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