Sea cucumber viscera extract as well as preparation method and application thereof

Abstract

The invention discloses a sea cucumber viscera extract as well as a preparation method and application thereof, and belongs to the technical field of marine biological medicines. The sea cucumber viscera extract is obtained by heating, refluxing and extracting sea cucumber viscera medicinal materials with 70% ethanol aqueous solution or water, filtering, concentrating and drying. The prepared sea cucumber viscera extract has a remarkable anti-colitis effect, can be used for preparing anti-colitis medicines and auxiliary medicines or used as a healthy food additive, and has a relatively high application value and a relatively high market prospect. According to the invention, the waste sea cucumber viscera in the sea cucumber industry is subjected to resource utilization, an active extract or additive raw material is provided for the anti-colitis medicine and food industry, and the preparation method is simple and easy to implement, low in cost, easy to realize industrialization and suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of marine biomedicine, and in particular relates to a sea cucumber viscera extract and a preparation method and application thereof. Background Art

[0003] The Chinese medicinal material Holothurian viscera (HV) is the viscera of animals such as Apostichopus japonicus (Selenka), Stichopus variegatus Semper, and Stichopus chloronotus Brandt, which belong to the Stichopodidae family, Holothuroidea, Aspidochirota, Echinodermata. The main tissues and organs include sea cucumber intestines, gonads, respiratory trees, etc. It is commonly known as "sea cucumber flowers" and contains ingredients such as protein, polysaccharides, fatty acids, and saponins. Both the "Chinese Materia Medica Dictionary" and the "Chinese Marine Materia Medica" record that sea cucumber viscera can be used to treat gastrointestinal ulcers, etc. Sea cucumber viscera was included for the first time in the 2022 edition of the "Shandong Province Traditional Chinese Medicine Standards", and has both medicinal and edible value. Summary of the invention

[0004] The purpose of the present invention is to provide an anti-colitis extract prepared based on sea cucumber viscera, and to provide a preparation method and specific application of the extract, so as to make up for the deficiencies of the prior art.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions:

[0006] The invention discloses a sea cucumber viscera extract. The extract takes the sea cucumber viscera as raw material and is extracted by using ethanol or water to obtain a sea cucumber viscera ethanol extract (HVE) or a sea cucumber viscera water extract (HVW).

[0007] The preparation method of the sea cucumber viscera extract comprises the following steps:

[0008] (1) grinding the sea cucumber viscera medicinal material and passing it through a 40-mesh sieve; adding 60-80% ethanol aqueous solution at a weight / volume (g / mL) ratio of 1:8-10, stirring evenly and soaking for 0.5 hours, heating and refluxing for 2-4 times, each time for 0.5 hours, filtering, combining the filtrate, recovering ethanol and drying to obtain the sea cucumber viscera ethanol extract (HVE);

[0009] (2) The sea cucumber viscera medicinal material was crushed, passed through a 40-mesh sieve, and pure water was added at a weight / volume (g / mL) ratio of 1:8-10. After stirring evenly, it was soaked for 0.5 hours, heated under reflux for extraction 2-4 times, each time for 0.5 hours, filtered, the filtrate was combined, ethanol was recovered and dried to obtain the sea cucumber viscera water extract (HVW).

[0010] Furthermore, the concentration of the ethanol aqueous solution is 70%.

[0011] Furthermore, the drying in the preparation method is freeze drying, vacuum reduced pressure drying or oven drying.

[0012] The use of the sea cucumber viscera extract (sea cucumber viscera ethanol extract (HVE) or sea cucumber viscera water extract (HVW)) in the preparation of anti-colitis products; the products are drugs, auxiliary drugs, health products, nutritional supplements, food additives, etc. for preventing or treating colitis.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The method for preparing the sea cucumber viscera extract provided by the present invention is low in cost, simple and easy to implement, and is easy to realize industrial and large-scale production. The sea cucumber viscera discarded from sea cucumber processing are used for extraction, and the obtained extract has significant anti-colitis activity and can be used to prepare drugs, auxiliary drugs, health products, nutritional supplements, food additives, etc. for preventing or treating colitis. The present invention makes full resource utilization of the sea cucumber viscera, which is the waste of the sea cucumber industry, and reduces resource waste, and has high application value and market prospects.

[0015] The invention makes full use of sea cucumber viscera to prepare the extract of sea cucumber viscera, and experimental verification shows that the sea cucumber viscera extract prepared by the invention has a significant anti-ulcerative colitis effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 1 is a graph showing the experimental results of mice in an embodiment of the present invention; wherein a is a graph showing the weight changes of mice in each group; and b is a graph showing the comparison of DAI scores of mice in each group.

[0017] Figure 2 These are the H&E staining results of the mouse colon section samples of the embodiments of the present invention, wherein a: control group; b: DSS group; c: 5-ASA group; d: LE group; e: ME group; f: HE group; g: LW group; h: MW group; i: HW group. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below in conjunction with embodiments and drawings.

[0019] Example 1 Preparation method of sea cucumber viscera ethanol extract (HVE)

[0020] The sea cucumber viscera medicinal material was crushed and passed through a 40-mesh sieve. 40.0 g of sea cucumber viscera medicinal material powder was weighed and evenly stirred with 400 mL of 70% ethanol aqueous solution, and then soaked for 0.5 hours. The mixture was heated under reflux at 75°C and extracted twice, each time for 0.5 hours. The filtrate was combined and concentrated under reduced pressure to an extract using a rotary evaporator. The mixture was dried in an oven at 45°C to constant weight to obtain 12.7 g of sea cucumber viscera ethanol extract (HVE).

[0021] Example 2 Preparation method of sea cucumber viscera water extract (HVW)

[0022] The sea cucumber viscera medicinal material was crushed and passed through a 40-mesh sieve. 40.0 g of sea cucumber viscera medicinal material powder was weighed and evenly stirred with 400 mL of pure water. After soaking for 0.5 hours, it was heated to reflux at 75 ° C for extraction twice, each time for 0.5 hours, filtered, and the filtrate was combined and concentrated under reduced pressure using a rotary evaporator to an extract state. The extract was dried in an oven at 45 ° C to constant weight to obtain 9.78 g of sea cucumber viscera water extract (HVW).

[0023] Example 3: Alleviation and improvement of sea cucumber viscera extract on DSS-induced colitis

[0024] Animal experiments were conducted using the sea cucumber viscera extracts prepared in Example 1 and Example 2 at different doses. The experimental method is as follows:

[0025] 1. Animal Grouping and Dosing

[0026] Mice were randomly divided into 9 groups, including normal control group (Control group), model control group (DSS group), and 7 treatment groups: HVE high-dose group (HE group), HVE medium-dose group (ME group), HVE low-dose group (LE group), HVW high-dose group (HW group), HVW medium-dose group (MW group), HVW low-dose group (LW group), positive control group (5-ASA group), 6 mice in each group, with free access to food and water. On the 1st to 5th day of the experiment, the drinking water of mice in the Control group was pure water, and the drinking water of mice in other groups was 2% (w / v) DSS aqueous solution. From the 6th to the 12th day, purified water was used as drinking water for mice in each group. At the same time, drugs were administered by gavage for 7 consecutive days, once a day, with a volume of 10 mL / kg. The Control group and the DSS group were administered with a solvent of 0.5% CMC-Na aqueous solution. The high-dose group, medium-dose group, and low-dose group of the HVE and HVW treatment groups were administered with raw medicinal material concentrations of 2400 mg / kg, 1600 mg / kg, and 800 mg / kg, respectively. The 5-ASA group was administered with mesalazine (5-ASA) (200 mg / kg) by gavage.

[0027] 2. Experimental Methods

[0028] The mice were weighed every day, the stool viscosity and blood in the stool were recorded, and the disease activity index (DAI) was scored according to Table 1.

[0029] Table 1 DAI scoring criteria

[0030]

[0031]

[0032] Note: Narrow and hard stool is normal, wet and large stool is soft, residue in the anus is diarrhea, and blood stains attached to the anus are bloody stools.

[0033] Fecal samples of mice were collected on days 5, 7 and 11, and the hemoglobin content in the fecal samples was determined.

[0034] The mice were killed 24 hours after the last administration, and the whole blood samples obtained by removing the eyeballs were placed on ice for 0.5 hours and then centrifuged at 4°C to separate the serum, and the levels of NO, TNF-α, IL-1β, IL-6 and IL-10 in the serum were determined.

[0035] The entire colon of the mouse was taken and the colon length was measured; the distal part of the colon was taken, washed with cold PBS and fixed in a tissue fixative at 4°C for 24 hours, and then analyzed under an optical microscope (40×) after dehydration, paraffin embedding, sectioning (about 5 μm), dewaxing, hydration, and hematoxylin and eosin stains (H&E stains).

[0036] 3. Experimental results

[0037] 3.1 Effects of HVE and HVW on mouse body weight and DAI scores

[0038] Compared with the Control group, the DSS group ## indicates P < 0.01, ### indicates P < 0.001, #### Indicates P<0.0001; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared with the DSS group. Figure 1 As shown in the figure, the weight of mice drinking pure water increased slowly, while the weight of mice drinking DSS aqueous solution decreased significantly on the 6th day. Low, medium and high doses of HVE and HVW can alleviate the weight loss of mice and accelerate the recovery of their weight; compared with the DSS group, the DAI score of mice after administration of HVW and HVW was significantly reduced, indicating that HVE and HVW can improve the symptoms of ulcerative colitis in mice in a dose-dependent manner.

[0039] 3.2 Effects of HVE and HVW on the length and structure of mouse colon

[0040] Compared with the Control group, the colon in the DSS group was significantly shortened and a small amount of blood was attached to the distal colon, and the shape of the contents was not obvious. The results are shown in Table 2. The colons of mice in the 5-ASA and LE, ME, HE, LW, MW, and HW groups were significantly longer than those in the DSS group (P<0.05), indicating that HVE and HVW can alleviate the shortening of the colon in mice caused by DSS and accelerate the recovery of colon tissue.

[0041] Table 2 Effects of different doses of HVE and HVW on the colon length of mice

[0042]

[0043] Note: Compared with the Control group, the DSS group ## Indicates P<0.01; compared with the DSS group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in the treatment group.

[0044] Figure 2 The results of H&E staining of mouse colon section samples. There was no inflammatory cell infiltration in the colon tissue of mice in the Control group, and the crypt and goblet cell structures were intact. The colon of mice in the DSS group showed large areas of inflammatory infiltration, the crypts were severely damaged, and the colon crypts of some mice were completely destroyed, with a large area of ​​lack of surface epithelial cells. The colon damage of mice in the 5-ASA group was significantly relieved, the degree of inflammatory cell infiltration was reduced, and epithelial damage was restored. The colon damage of mice in each dose group of HVE was significantly relieved, the crypt structure was intact, the inflammatory cell infiltration was reduced, and the colon tissue structure was relatively intact. The colon damage of mice treated with HVW was relieved to a certain extent, and the colon tissue of some mice still had inflammatory cell infiltration, and the crypt gap was larger; the goblet cells in the LW group were more missing, there were no crypts in some colon tissues, the inflammatory cells had obvious infiltration, and the surface epithelial cells were more missing; the crypt gaps in the MW group were larger, and there were local small ulcers; the colon damage of mice in the HW group was relieved to a certain extent, and the colon tissue structure was relatively intact. The above results show that both HVE and HVW can relieve colon mucosal damage.

[0045] 3.3 Effects of HVE and HVW on fecal hemoglobin in mice

[0046] As shown in Table 3, on the 5th day of the experiment, the fecal hemoglobin content of mice drinking DSS aqueous solution was significantly increased compared with the Control group (P<0.0001). After 2 days of oral administration of HVE or HVW, the average fecal hemoglobin content of mice was significantly lower than that of the DSS group (P<0.0001), indicating that the intestinal bleeding of mice was improved after the treatment with HVE or HVW, and the treatment effect was dose-dependent. Compared with the 7th day of the experiment, the average fecal hemoglobin content of mice treated with HVE or HVW on the 11th day decreased, and was significantly lower than that of the DSS group (P<0.0001). The above results show that HVE and HVW can improve intestinal bleeding in mice with ulcerative colitis.

[0047] Table 3 Effects of different doses of HVE and HVW on fecal hemoglobin in mice

[0048]

[0049] Note: Compared with the Control group, the DSS group #### Indicates P < 0.0001; compared with the DSS group, ns Indicates no significant difference, ****P<0.0001.

[0050] 3.4 Effects of HVE and HVW on serum inflammatory factors in mice

[0051] The levels of NO, IL-1β, IL-6 and TNF-α in the serum of mice in the DSS group were significantly higher than those in the Control group (P<0.0001), and IL-10 showed a downward trend. Compared with the DSS group, the levels of IL-10 in the serum of mice in the LE and WE groups were higher than those in the DSS group, with statistically significant differences; after intervention with medium and high doses of HVE and HVW, the levels of NO, IL-1β, IL-6, and TNF-α in the serum of mice were significantly reduced, and the levels of IL-10 were significantly increased. The above results show that HVE and HVW can dose-dependently reduce the levels of pro-inflammatory cytokines and increase the levels of anti-inflammatory factors in the serum of mice with DSS-induced ulcerative colitis model.

[0052] Table 4 Serum inflammatory factors of mice in each group

[0053]

[0054] Compared with the Control group, the DSS group ns Indicates no significant difference. #### Indicates P < 0.0001; compared with the DSS group, ns Indicates no significant difference. * P<0.05, ** P<0.01,**** P<0.0001.

[0055] In the above embodiment, dextran sulfate sodium (DSS) was used to induce ulcerative colitis mice, and the effects of sea cucumber viscera extract on mouse disease activity index (DAI), colonic tissue pathology, serum inflammatory factor levels, fecal hemoglobin levels, and intestinal microbial composition and abundance of colon contents were investigated. The experimental results show that compared with the model group, sea cucumber viscera ethanol extract (HVE) and sea cucumber viscera water extract (HVW) can significantly improve the colonic tissue pathological phenotype of ulcerative colitis, reduce intestinal bleeding, reduce serum proinflammatory factors and increase serum anti-inflammatory factor levels, and correct the imbalance of intestinal microorganisms. Therefore, the sea cucumber viscera extract prepared by the present invention can play an anti-colitis effect.

[0056] Finally, although this specification is described according to implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A sea cucumber viscera extract, characterized in that The extract is made from sea cucumber viscera and is extracted by using ethanol or water to obtain a sea cucumber viscera ethanol extract or a sea cucumber viscera water extract.

2. The method for preparing the sea cucumber viscera extract according to claim 1, characterized in that: The steps include: (1) Taking sea cucumber viscera medicinal materials, crushing them, and sieving them; adding 60-80% ethanol aqueous solution at a weight / volume (g / mL) ratio of 1:8-10, stirring evenly and then soaking, heating and refluxing extraction for multiple times, filtering, combining the filtrate, recovering ethanol and drying to obtain sea cucumber viscera ethanol extract HVE; (2) The sea cucumber viscera medicinal material is crushed, sieved, and pure water is added at a weight / volume (g / mL) ratio of 1:8-10. The mixture is stirred evenly and then soaked. The mixture is heated under reflux and extracted for multiple times. The filtrate is filtered, the filtrate is combined, the ethanol is recovered, and the mixture is dried to obtain the sea cucumber viscera water extract HVW.

3. The preparation method according to claim 2, characterized in that: The drying is freeze drying, vacuum drying or oven drying.

4. Use of the sea cucumber viscera ethanol extract HVE according to claim 1 in the preparation of anti-colitis products.

5. Use of the sea cucumber viscera aqueous extract HVW according to claim 1 in the preparation of anti-colitis products.

6. The use according to claim 4 or 5, characterized in that: The product is a medicine, auxiliary medicine, health product, nutritional supplement or food additive for preventing or treating colitis.

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