Andrias davidianus active substance for relieving intestinal injury, freeze-dried powder and preparation method and application thereof

Abstract

The application provides andrelieve intestinal injury active substance of anderson's chelydra, a freeze-dried powder and a preparation method and application thereof, and belongs to the technical field of active substance extraction. The anderson's chelydra and its processing by-products are used as raw materials to prepare the freeze-dried powder, the active substance with the function of relieving intestinal injury is screened through a simple process, the method is simple, practical and convenient for industrialization, and the freeze-dried powder prepared from the mucus active substance can effectively relieve intestinal injury and promote the proliferation of intestinal epithelial cells, thereby providing material guarantee for the development of related nutritional dietary supplements and drugs, enhancing the market competitiveness of products, and improving economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of active ingredient extraction technology, specifically relating to an active ingredient from the giant salamander that alleviates intestinal damage, a freeze-dried powder, its preparation method, and its application. Background Technology

[0002] The gut is not only the primary site of nutrient digestion and absorption, but also the largest immune organ in animals, playing a vital barrier function to ensure the body absorbs nutrients while preventing the invasion of toxic and harmful substances. Studies have shown that gut damage is closely related to oxidative stress, inflammation, metabolic disorders, and insulin resistance, and can lead to a series of diseases such as fatty liver disease, type II diabetes, and endotoxemia. Therefore, alleviating gut damage and maintaining normal intestinal barrier function is crucial for overall health. Summary of the Invention

[0003] The purpose of this invention is to provide a giant salamander active substance, freeze-dried powder, preparation method and application for alleviating intestinal damage. The giant salamander active substance can effectively alleviate intestinal damage and promote the proliferation of intestinal epithelial cells; and it can be directly scaled up for production, and the relevant conditions can be achieved through industrial equipment.

[0004] The present invention provides a method for preparing active substances from giant salamanders that alleviate intestinal damage, comprising the following steps: (1) placing giant salamander meat, viscera and / or mucus in an ice-water mixture to homogenize and obtain a slurry;

[0005] (2) The slurry described in step (1) is mixed with neutral protease and then subjected to ultrasonic-assisted enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0006] (3) Mix the enzymatic hydrolysate described in step (2) with anhydrous ethanol, stir, centrifuge, and the supernatant contains the active substance of giant salamander that alleviates intestinal damage.

[0007] Preferably, the homogenization frequency in step (1) is 50~90Hz / s, and the homogenization time does not exceed 2min.

[0008] Preferably, the neutral protease activity in step (2) is 50,000 U / L, and the ultrasonic frequency of the ultrasonic-assisted enzymatic hydrolysis is 30~50kHz, and the time is 30min.

[0009] Preferably, in step (3), anhydrous ethanol is added until the volume percentage of ethanol in the mixture is 70%.

[0010] Stir the mixture at 10 rpm for 1 hour.

[0011] Preferably, the centrifugal force in step (3) is 1500g and the centrifugation time is 10min.

[0012] This invention provides a giant salamander active substance that alleviates intestinal damage, obtained using the above preparation method.

[0013] The present invention also provides a freeze-dried powder of active substances from giant salamanders that alleviate intestinal damage. The supernatant obtained by the above preparation method is concentrated, freeze-dried, and then crushed to obtain freeze-dried powder.

[0014] The present invention also provides the application of the above-mentioned giant salamander active substance or the above-mentioned giant salamander active substance lyophilized powder in the preparation of drugs to alleviate intestinal damage.

[0015] Beneficial Effects: This invention provides a method for preparing active substances from giant salamanders that alleviate intestinal damage. The method uses artificially farmed giant salamanders and their processing byproducts as raw materials. A simple process allows for the screening of active substances that alleviate intestinal damage. It is simple, practical, and easy to promote industrially. The method can be directly scaled up for production, and all relevant conditions can be achieved using industrial equipment. Furthermore, the product has a minimal fishy odor, as the smell is masked by a carrier. Furthermore, as verified by examples, preparing the active substances into freeze-dried powder can effectively alleviate intestinal damage and promote the proliferation of intestinal epithelial cells, thereby providing material support for drug development, enhancing product market competitiveness, and improving economic benefits. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a graph showing the weight statistics of mice in different treatment groups. Different lowercase letters in the superscript of the data in the same row indicate significant differences. P <0.05), the same below;

[0018] Figure 2 Statistical graph of organ indices in mice of different treatment groups;

[0019] Figure 3 A statistical graph showing the colon length of mice in different treatment groups;

[0020] Figure 4 This figure shows the effect of freeze-dried giant salamander mucus powder on the proliferation of intestinal epithelial cells. * indicates significant data in the same row. P <0.05), ** indicates extremely significant ( P <0.01), the same below;

[0021] Figure 5 Figure showing the effect of freeze-dried giant salamander mucus powder on oxidative damage to the intestinal epithelium. Detailed Implementation

[0022] The present invention provides a method for preparing active substances from giant salamanders that alleviate intestinal damage, comprising the following steps: (1) placing giant salamander meat, viscera and / or mucus in an ice-water mixture to homogenize and obtain a slurry;

[0023] (2) The slurry described in step (1) is mixed with neutral protease and then subjected to ultrasonic-assisted enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0024] (3) Mix the enzymatic hydrolysate described in step (2) with anhydrous ethanol, stir, centrifuge, and the supernatant contains the active substance of giant salamander that alleviates intestinal damage.

[0025] This invention involves homogenizing giant salamander meat, viscera, and / or mucus in an ice-water mixture to obtain a slurry. Preferably, the raw material is artificially raised giant salamanders and their processing byproducts; more preferably, it includes giant salamander meat, liver, and / or mucus; and most preferably, it is mucus. This invention does not specifically limit the extraction method of the mucus; conventional methods in the art can be used. In this invention, if the giant salamanders and their processing byproducts are not immediately used to prepare giant salamander active substances, they need to be stored and transported at 4°C. The homogenization process described in this invention is carried out in an ice-water mixture, with homogenization at 50-90 Hz / second for no more than 2 minutes. In this invention, low-temperature homogenization effectively prevents the degradation of active peptides.

[0026] After homogenizing, the slurry is mixed with a neutral protease and then subjected to ultrasound-assisted enzymatic hydrolysis to obtain a hydrolysate. The neutral protease used in this invention is preferably a solution containing 50,000 U / L of neutral protease. The neutral protease preferably includes a protease obtained from Bacillus subtilis through liquid deep fermentation and purification extraction, mainly an endopeptidase, containing small amounts of aminopeptidase and carboxypeptidase, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number D915910-1kg. The ultrasound frequency for the ultrasound-assisted enzymatic hydrolysis is preferably 30-50 kHz, and the time is preferably 30 min. During the ultrasound-assisted enzymatic hydrolysis, the temperature is preferably controlled below 30°C, and ultrasound-assisted enzymatic hydrolysis allows for more thorough contact between the enzyme and the substrate, improving the hydrolysis efficiency.

[0027] After obtaining the enzymatic hydrolysate, the present invention mixes the enzymatic hydrolysate with anhydrous ethanol, stirs, centrifuges, and the supernatant contains the active substance of giant salamander that alleviates intestinal damage.

[0028] This invention involves mixing the enzymatic hydrolysate with anhydrous ethanol. Preferably, the mucus is frozen before treatment, and the solid-liquid ratio is adjusted based on its volume in the frozen state. The mixing aims to achieve a solid-liquid ratio of 1:2 to 10, with a final ethanol concentration of 70% (v / v). The mixture of enzymatic hydrolysate and anhydrous ethanol is stirred at a speed preferably of 10 rpm for 1 hour. In this embodiment, the mixture is preferably stirred in a stirrer. Adding anhydrous ethanol followed by stirring improves the release rate of alcohol-soluble proteins and small peptides.

[0029] The present invention performs centrifugation after the stirring stops, wherein the centrifugal force is preferably 1500g and the centrifugation time is preferably 10min, which can cause some of the insoluble sludge to precipitate.

[0030] This invention provides a giant salamander active substance that alleviates intestinal damage, obtained using the above preparation method.

[0031] In this invention, functional evaluation and screening were conducted using a mouse colitis model induced by dextran sulfate sodium (DSS) and an intestinal epithelial cell inflammation and damage model induced by endotoxin (LPS). The results showed that the active substances prepared from giant salamander meat and liver can only play a certain protective role, while the active substances from giant salamander mucus have the best effect in relieving mouse colitis. Furthermore, 125 μg / mL of lyophilized giant salamander mucus powder can effectively relieve endotoxin (LPS)-induced inflammatory (oxidative) damage.

[0032] The present invention also provides a freeze-dried powder of active substances from giant salamanders that alleviate intestinal damage. The supernatant obtained by the above preparation method is concentrated by rotary evaporation, then freeze-dried and crushed to obtain freeze-dried powder.

[0033] The present invention preferably involves rotary evaporation concentration at 45°C, freeze-drying at -40°C, and crushing into powder through a 40-mesh sieve to obtain the corresponding freeze-dried powder of raw materials. In the examples, the above operations were performed using active substances derived from the mucus of giant salamanders to obtain freeze-dried powder of giant salamander mucus that has a clear effect on alleviating intestinal damage.

[0034] The present invention also provides the application of the above-mentioned giant salamander active substance or the above-mentioned giant salamander active substance lyophilized powder in the preparation of drugs to alleviate intestinal damage.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a giant salamander active substance for alleviating intestinal damage, a freeze-dried powder, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention. Example

[0036] The meat of artificially bred giant salamanders was collected, transported and stored at 4℃, and homogenized in an ice-water mixture at 70Hz / s for 1 minute. Then, it was ultrasonically treated at 40 kHz for 30 minutes in a solution containing 50,000 U / L neutral protease (Shanghai Maclean Biochemical Technology Co., Ltd., D915910-1kg). Then, anhydrous ethanol was added to achieve a solid-liquid ratio of 1:10 and an ethanol concentration of 70% (v / v). The mixture was stirred in a stirrer at 10 rpm for 1 hour, centrifuged at 1500×g for 10 minutes, and the supernatant was collected and concentrated by rotary evaporation at 45℃. The mixture was then freeze-dried at -40℃ and crushed into powder through a 40-mesh sieve. Example

[0037] The precipitate remaining after centrifugation of the supernatant in Example 1 was freeze-dried at -40°C, crushed, and sieved through a 40-mesh sieve to form powder. Example

[0038] The livers of artificially bred giant salamanders were collected and processed in the same way as in Example 1 to obtain freeze-dried powder. Example

[0039] Mucus from artificially bred giant salamanders was collected and processed in the same way as in Example 1 to obtain freeze-dried powder.

[0040] The following experiments were conducted on the lyophilized powders prepared in Examples 1-4:

[0041] 1. Experimental Grouping

[0042] Add 0.1% of the freeze-dried powder of Example 1 to the diet of mice and set it as the EE group. There are 6 replicates in total, with 1 mouse in each replicate. DSS drinking water induction is performed until day 7, and then normal drinking water is provided until day 15.

[0043] Add 0.1% of the freeze-dried powder of Example 2 to the diet of mice and set it as the FD group. There are 6 replicates in total, with 1 mouse in each replicate. DSS water induction is performed until day 7, and then normal drinking water is provided until day 15.

[0044] Add 0.1% of the freeze-dried powder of Example 3 to the diet of mice, set as group L, with a total of 6 replicates, one mouse per replicate. DSS drinking water induction was performed until day 7, and then replaced with normal drinking water until day 15.

[0045] Add 0.1% of the freeze-dried powder of Example 4 to the diet of mice, set as group M, with a total of 6 replicates, one mouse per replicate. DSS drinking water induction was performed until day 7, and then replaced with normal drinking water until day 15.

[0046] Negative control: Mice were fed a basal diet and were designated as the CTL group, with a total of 6 replicates, one mouse per replicate, and normal drinking water until day 15.

[0047] Positive control: Mice were fed a basal diet and designated as the DSS group, with a total of 6 replicates, one mouse per replicate. DSS was induced by drinking water until day 7, and then replaced with normal drinking water until day 15.

[0048] 2. Indicator Measurement

[0049] (1) Body weight and organ index

[0050] During the experiment, the weight and food intake of each mouse were measured and recorded every three days. During the DSS drinking water period, the weight and food intake of each mouse were measured and recorded daily. The average weight of each group of mice was calculated, and the mortality rate was recorded.

[0051] (2) Colon length

[0052] Spray 70% ethanol and carefully open the abdomen through a midline incision. Use forceps to lift and carefully pull the colon until the cecum is visible. Remove the colon with scissors, taking care not to stretch it while measuring its length and taking photos for record-keeping.

[0053] (3) Disease Activity Index Score

[0054] After starting DSS induction, the weight of each group of animals was measured daily, and fecal characteristics and bleeding were observed and recorded. The body weight, fecal characteristics, and bloody stool data for the last day were collected to calculate the disease activity index (DAI). DAI = (weight loss score + fecal characteristics score + bloody stool score) / 3.

[0055] 3. Results and Analysis

[0056] (1) Mice mortality and body weight in each example

[0057] Weight results as follows Figure 1 As shown, compared with the negative control, the positive control mice had a significant decrease in body weight on day 8 ( P <0.05), Example 4 significantly restored the weight of mice to the level of the negative control, while Examples 1, 2 and 3 showed no significant difference from the positive control.

[0058] The mortality rate statistics are shown in Table 1. The mortality rate of the positive control mice was 15%, the mortality rate of Example 2 was 33%, and there were no mouse deaths in the negative control, Example 1, Example 3 and Example 4.

[0059] (2) Organ index of mice in each embodiment

[0060] Organ index statistics results are as follows Figure 2 As shown, compared with the negative control, the liver index of the positive control mice was significantly increased ( P<0.05), the liver index of mice in Examples 1, 2, 3, and 4 showed no significant change. Compared with the negative control, the spleen index of mice in Example 2 increased significantly ( P <0.05%, with no significant changes in the other examples. Compared to the negative control, the kidney index of mice in Examples 1 and 2 increased significantly ( P <0.05), with no significant changes in the other embodiments.

[0061] Table 1. Mouse mortality rate in each example.

[0062] Grouping CTL DSS FD EE L M mortality rate 0% 15% 33% 0% 0% 0%

[0063] (3) Colon length in mice in each embodiment

[0064] Colitis causes intestinal atrophy in mice; therefore, colon length can directly reflect the severity of colitis in mice. Measurement results are as follows: Figure 3 As shown, compared with the negative control, the colon length of the positive control mice was significantly shortened ( P <0.05), the colon length of mice in Example 4 was significantly increased compared to the positive control ( P <0.05), but there was no significant difference between Examples 1, 2, and 3 and the positive control, indicating that Example 4 had the best effect in alleviating colitis in mice, while Examples 1, 2, and 3 played a protective role to some extent. Therefore, the lyophilized mucus powder of the giant salamander in Example 4 was used to evaluate the proliferation and inflammatory (oxidative) damage of intestinal epithelial cells (IPEC-J2) induced by bacterial endotoxin (LPS).

[0065] The results are as follows Figure 4 As shown, lyophilized powder of 0.0625~0.5 mg / mL giant salamander mucus can significantly promote the proliferation of intestinal epithelial cells. P <0.05), with the most significant effect observed at 0.125 mg / mL. Furthermore, the peroxidation product MDA in intestinal epithelial cells was further detected, and the results are as follows: Figure 5 As shown, 0.125 mg / mL (125 μg / mL) freeze-dried giant salamander mucus powder can significantly reduce endotoxin (LPS)-induced MDA production, indicating that 125 μg / mL freeze-dried giant salamander mucus powder can effectively alleviate endotoxin (LPS)-induced inflammatory (oxidative) damage.

[0066] The freeze-dried powder of giant salamander mucus processed using the extraction and separation process of this invention can effectively alleviate intestinal damage and promote the proliferation of intestinal epithelial cells. This invention is simple, practical, and easy to promote, thus providing material support for drug development, enhancing product market competitiveness, and improving economic benefits.

[0067] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a freeze-dried powder of an Andrias active substance for the preparation of a medicament for the relief of intestinal damage, characterized in that, The Andrias davidianus active substance freeze-dried powder is obtained by concentrating supernatant containing the Andrias davidianus active substance for relieving intestinal injury, freeze-drying and crushing; The preparation method of the Andrias davidianus active substance for relieving intestinal injury comprises the following steps: (1) homogenizing the Andrias davidianus mucus in an ice-water mixture to obtain a slurry; the homogenization frequency is 50-90 Hz / s, and the homogenization time is not more than 2 min; (2) mixing the slurry in step (1) with neutral protease and then performing ultrasonic-assisted enzymolysis to obtain an enzymolysis liquid; the enzyme activity of the neutral protease is 50,000 U / L, the ultrasonic frequency of the ultrasonic-assisted enzymolysis is 30-50 kHz, and the ultrasonic time is 30 min; (3) mixing the enzymolysis liquid in step (2) with anhydrous ethanol, stirring, centrifuging, and adding anhydrous ethanol to the mixture to obtain an ethanol volume percentage of 70%; the mixture is stirred at 10 rpm for 1 h; the centrifugal force of the centrifugation is 1,500 g, and the centrifugation time is 10 min.

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