A delicious boletus extract, its preparation method and application

Abstract

The application discloses a delicious boletus extract, a preparation method and application thereof, and provides application of the delicious boletus extract in preparation of medicines for preventing and / or treating senile intestinal dysfunction caused by colon atrophy and degeneration, wherein the preparation method of the delicious boletus extract is as follows: the delicious boletus is crushed, extracted by water or an alcohol solvent, filtered to obtain an extract, the extract is concentrated and dried to obtain the delicious boletus extract. The delicious boletus extract provided by the application can up-regulate expression of tight junction protein ZO-1 and mucin MUC-2 in an aging organism colon, promote growth of colon length and crypt depth, and further delay colon atrophy and degeneration and enhance intestinal digestion and absorption and immune functions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a Boletus edulis extract, a preparation method and application thereof, more particularly to the Boletus edulis extract, a preparation method and application thereof in preparing a drug for preventing and / or treating senile intestinal dysfunction caused by colon atrophy. BACKGROUND

[0002] With the intensification of global population aging, age-related digestive system function decline has become an important problem affecting the quality of life of the elderly. In terms of colon, aging is usually accompanied by atrophic changes in colon tissue, manifested as shortening of colon villi and shallowing of crypt depth, leading to decreased absorption efficiency of nutrients. More importantly, the intestinal barrier function is impaired, characterized by down-regulation of expression of tight junction proteins (such as ZO-1) and mucins (such as MUC2). This can lead to increased intestinal permeability ("leaky gut"), triggering chronic low-grade inflammation, and weakening the intestinal immune defense, which is closely related to the occurrence and development of many senile diseases.

[0003] Currently, there are limited interventions for senile intestinal function decline, mainly through the supplementation of probiotics, prebiotics or dietary fiber. However, the effectiveness of these methods varies from person to person, and has limited effect on fundamentally improving intestinal structure and barrier function. Therefore, it is of great clinical significance and market value to develop a new type of natural product that can effectively delay colon degeneration and strengthen the physical and immune barrier of the intestine.

[0004] Boletus edulis is a precious edible fungus rich in polysaccharides, terpenes, phenols and other bioactive ingredients. Existing researches mainly focus on its antioxidant, immune regulation or anti-tumor activity, but there is no report on using its specific extract to specifically improve the morphological and functional decline of the colon related to aging. SUMMARY

[0005] The present application aims to provide a Boletus edulis extract that enhances the intestinal immunity of an aging body, a preparation method thereof and its application in preparing a drug for preventing and / or treating senile intestinal dysfunction caused by colon atrophy.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The present application provides the application of a Boletus edulis extract in preparing a drug for preventing and / or treating senile intestinal dysfunction caused by colon atrophy, wherein the preparation method of the Boletus edulis extract comprises the following steps: crushing Boletus edulis, extracting with water or an alcohol solvent, filtering to obtain an extract, concentrating and drying the extract to obtain the Boletus edulis extract.

[0008] Preferably, the medicine delays atrophy of the colon, enhances digestive absorption and immune function of the intestinal tract.

[0009] Preferably, the medicine promotes growth of the length of the colon and the depth of the crypt.

[0010] Preferably, the medicine up-regulates expression of tight junction protein ZO-1 and mucin MUC-2 in the colon of the aging organism.

[0011] Preferably, the water or alcohol solvent extraction is carried out by adding 10-30 L of water or alcohol solvent per kg of Boletus edulis, and the alcohol solvent is 70%-80% ethanol aqueous solvent.

[0012] Boletus edulis (Bull.: Fr.) Fr. Boletus edulis , commonly known as white bolete, is one of the most recognized safe top-grade edible fungi in the world, and is also one of the few wild edible fungi that can be eaten raw (but it is still recommended to be cooked).

[0013] Preferably, the Boletus edulis is further subjected to freeze-drying treatment before being crushed.

[0014] The Boletus edulis is crushed to 100-200 mesh.

[0015] Preferably, the extraction method can be selected from immersion extraction, hot reflux extraction, ultrasonic extraction, or any combination of two thereof.

[0016] Preferably, the hot reflux extraction time is 1-5 h, and the extraction times is 1-4 times, more preferably, the hot reflux extraction time is 2-3 h, and the extraction times is 2-3 times.

[0017] Preferably, the preparation method of the Boletus edulis extract further comprises the following purification steps: dissolving the alcohol extract with water, extracting with ethyl acetate, collecting the ethyl acetate extract, concentrating, and drying.

[0018] Preferably, in the ethyl acetate extraction, the volume ratio of the water phase to ethyl acetate is 1:1-5, and the extraction is carried out 2-4 times.

[0019] Preferably, the medicine further comprises one or more pharmaceutically acceptable carriers or excipients.

[0020] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of sustained-release agents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, lubricants, and the like, and other optional combinations, which will not be repeated here.

[0021] Preferably, the dosage form of the drug is a tablet, injection, capsule, granule, pill, decoction, syrup or mixture.

[0022] The beneficial effects of the present application are:

[0023] The present application first found that Boletus edulis extract can specifically improve the aging-induced decline of colon morphology and function, and the mechanism is clear. The experimental results prove that Boletus edulis extract can significantly promote the growth of colon length and crypt depth, reverse colon atrophy from the anatomical structure, and lay the foundation for improving the digestive and absorptive function. Boletus edulis extract can efficiently up-regulate the expression of tight junction protein ZO-1, reinforce the connection between intestinal epithelial cells, and reduce intestinal permeability, thereby reducing endotoxin translocation and systemic inflammation. Boletus edulis extract can promote the high expression of gel-forming mucin MUC2, enhance the thickness and integrity of the intestinal mucus layer, and play a role in lubricating the intestinal tract, isolating pathogenic bacteria, and strengthening the first line of immune defense. Boletus edulis is a edible fungus, which is highly safe and easy to be accepted, and is suitable for developing into a long-term taken drug. It provides a new and effective natural solution for preventing and treating senile intestinal dysfunction caused by colon atrophy and degeneration. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 HE staining and PASS staining results of colon tissue;

[0025] Figure 2 ZO-1 and MUC2 protein expression level detection results of intestinal mucin. DETAILED DESCRIPTION

[0026] The present application will be further described in detail below in conjunction with the accompanying drawings and examples, but in no way limits the present application, any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0027] The process, conditions, reagents, experimental methods, etc. for implementing the present application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the present application has no special limitation. The experimental methods not specified in the specific conditions in each example are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0028] Unless otherwise specified, the meanings of all professional terms and scientific terms used in the present specification are the same as those generally understood by the person skilled in the art to which the present application belongs. However, if there is a conflict, the present specification including the definition shall prevail.

[0029] The following are the sources of some materials in the examples:

[0030] Boletus edulis (Vill. Boletus edulisFresh product, purchased from Yunnan Lijiang Zhongyuan Green Food Co., Ltd.

[0031] 95% ethanol, purchased from Tianjin Fenshen Chemical Reagent Co., Ltd., purity ≥ 99.5%

[0032] Petroleum ether, purchased from Tianjin Fenshen Chemical Reagent Co., Ltd., purity ≥ 99.5%;

[0033] Ethyl acetate, purchased from Tianjin Fenshen Chemical Reagent Co., Ltd., purity ≥ 99.5%;

[0034] Dexamethasone, purchased from Beijing Solabio Biotech Co., Ltd., purity ≥ 99.5%;

[0035] Methotrexate, purchased from Beijing Solabio Biotech Co., Ltd., purity ≥ 99.5%;

[0036] C57BL / 6 mice, SPF level, purchased from Yunnan University Animal Experiment Management Center.

[0037] Example 1

[0038] Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and ground to 200 mesh to prepare Boletus edulis powder. Take an appropriate amount of powder, add 75% ethanol solution according to the solid-liquid ratio of 1:20 (g / mL), heat reflux extraction at 75°C for 3 times (2 h each time), combine the extract, and concentrate under reduced pressure to obtain Boletus edulis extract.

[0039] Example 2

[0040] Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and ground to 200 mesh to prepare Boletus edulis powder. Take an appropriate amount of powder, add water according to the solid-liquid ratio of 1:20 (g / mL), heat reflux extraction for 3 times (2 h each time), combine the extract, and concentrate under reduced pressure to obtain Boletus edulis extract.

[0041] Example 3

[0042] Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and ground to 200 mesh to prepare Boletus edulis powder. Take an appropriate amount of powder, add 70% ethanol solution according to the solid-liquid ratio of 1:10 (g / mL), heat reflux extraction for 2 times (3 h each time), combine the extract, and concentrate under reduced pressure to obtain Boletus edulis extract.

[0043] Example 4

[0044] Fresh Boletus edulis was washed with deionized water, sliced, freeze-dried to constant weight, and ground to 200 mesh to obtain Boletus edulis powder. An appropriate amount of the powder was added to 80% ethanol solution at a solid-liquid ratio of 1:30 (g / mL), and hot reflux extraction was performed twice (2 h each time). The combined extract was concentrated under reduced pressure to obtain Boletus edulis extract.

[0045] Example 5

[0046] The Boletus edulis extract in Example 1 was dissolved in water, and 3 volumes of ethyl acetate were added for extraction. The extraction was performed 3 times, and the combined extract was concentrated under reduced pressure using a rotary evaporator and dried to obtain the Boletus edulis ethyl acetate extract.

[0047] Example 6

[0048] The Boletus edulis extract in Example 1 was dissolved in water, and 5 volumes of ethyl acetate were added for extraction. The extraction was performed 2 times, and the combined extract was concentrated under reduced pressure using a rotary evaporator and dried to obtain the Boletus edulis ethyl acetate extract.

[0049] Example 7

[0050] The Boletus edulis extract in Example 1 was dissolved in water, and 1 volume of ethyl acetate was added for extraction. The extraction was performed 4 times, and the combined extract was concentrated under reduced pressure using a rotary evaporator and dried to obtain the Boletus edulis ethyl acetate extract.

[0051] Test Example 1: Effect of Aging Intestinal Immunity Experiment

[0052] 1. Test sample

[0053] The extract samples were prepared according to Examples 1-7.

[0054] 2. Test animals

[0055] C57BL / 6 mice, 6-8 weeks old, SPF level.

[0056] 3. Test method

[0057] 3.1 Modeling

[0058] After the C57BL / 6 mice were adaptively fed for 1 week, 10 mice were randomly selected as the blank control group, and 0.5 mg / kg·bw of normal saline was injected once a day. The remaining C57BL / 6 mice were injected intraperitoneally with 25 mg / kg·bw of dexamethasone injection once a day to establish an aging intestinal mouse model. The diet and body weight changes of the C57BL / 6 mice were measured every two days from the 1st to the 21st day, and the pull force physical fitness changes of the mice were monitored.

[0059] 3.2 Test sample intervention

[0060] After the molding is finished, the experimental group is randomly divided into 9 groups, 10 C57BL / 6 mice in each group, wherein groups 1-7 are respectively given the extracts of embodiments 1-7 of the application by gavage (the dosage is 60 mg / kg·bw, the extract is dissolved into a 12 mg / ml solution with water and then given by gavage), once a day, group 8 is a model control group, once a day, and group 9 is a positive control group, once a day, and the like. The weight of the test sample is intervened for 21 days,

[0061] The colon length is calculated, and the colon length and crypt depth are detected.

[0062] 3.3 Colon HE staining (hematoxylin-eosin staining) experiment for colon detection

[0063] The colon tissues of the blank control group, the model group, the embodiment 1 group and the embodiment 5 group are paraffin sectioned, and the paraffin section is distilled with xylene I and II for 10-20 minutes, anhydrous ethanol I and II for 10 minutes, 95%, 90%, 80% and 70% ethanol for 5 minutes, and then washed with distilled water. Then the section is fixed with 4% paraformaldehyde for 24 hours. The section is immersed in hematoxylin staining solution for 3 minutes. Washed with tap water for 3 minutes. Differentiate with 1% hydrochloric acid ethanol for 10 seconds, and then washed with running water for 1 minute. Immersed in eosin staining solution for 2 minutes, and then washed with tap water for 1 minute. Finally, gradient dehydration is performed: 80%, 95%, anhydrous ethanol for 2-5 minutes, xylene transparency for 2 times, 5 minutes each time, neutral balsam mounting, and microscopic examination.

[0064] 3.4 Colon sugar binding PAS staining (periodic acid-Schiff staining) experiment for colon detection

[0065] The paraffin section is deparaffinized to water (the same as the HE staining step). The section is treated with 1% high iodine acid solution for 5-10 minutes, and then washed with running water for 5 minutes. Immersed in Schiff reagent for 15 minutes, and then washed with running water for 7 minutes (to wash away the excess reagent). Mayer hematoxylin re-staining for 1 minute, running water blueing, gradient dehydration (the same as HE), xylene transparency, neutral balsam mounting, and microscopic examination.

[0066] 3.5 Detection of ZO-1 and MUC2 protein expression levels in intestinal mucosa

[0067] (1) Extraction of colon protein tissue of blank control group, model group, Example 1 group and Example 5 group of mice: about 20 mg of mouse colon tissue sample was weighed and placed in a 2 ml round-bottom EP tube, 2 steel balls were added, 1 mL of 1% PMSF cell lysis solution was added to each tube, and the tissue was broken in a tissue crusher at 30 s / time; after complete breaking, the sample was placed on ice for 30 min, and centrifuged at 4°C and 12000 rpm for 15 min; the supernatant was transferred to a new 1.5 mL centrifuge tube, and the centrifugation operation was repeated until the supernatant was clear and transparent, and then stored at -80°C for standby.

[0068] (2) Quantitative sample preparation: the concentration of protein in the sample was detected by BCA kit, first a standard curve was prepared, 1 mg / ml standard protein sample was diluted by equal volume (0.5, 0.25, 0.125, 0.0625, 0.03125) in a 96-well dilution plate, and the blank well was added with filtered PBS as 0, 2 μL of the protein to be tested was diluted 40 times in 38 μL of filtered PBS, and then mixed by gun. Take 20 μL of mixed standard protein and test protein to the enzyme-labeled plate, three parallel samples for each sample. Add 200 μL of BCA working solution (A liquid: B liquid = 50:1) to each well, incubate in a 37°C oven for 30 min. Measure the absorbance of the enzyme-labeled plate at 590 nm and 630 nm, calculate the standard curve, and substitute the absorbance of the sample protein into the standard curve to calculate the sample protein concentration. After calculation, add reducing loading liquid (3-5 μL of 5×reducing loading liquid to 30 μg of total protein) to the sample protein, then cook the sample in a 95°C sample cooker for 5-10 min to denature the protein, and cool naturally at room temperature. Membrane protein is cooked at 37°C for 30 min.

[0069] (3) Sample loading: according to the molecular weight of the desired protein, prepare 1× electrophoresis solution, 8% concentration separation gel and prepared protein sample. Fill the electrophoresis tank with electrophoresis solution, slowly pull out the gel plate comb in the electrophoresis tank, clean the residual gel particles in the lane, and then prepare for sample loading. 20 μL of protein sample was taken by a 20 μL pipette and slowly and evenly added to the sample well (to ensure that the sample protein is in the middle well as much as possible), and the remaining wells were supplemented with 5× reducing loading liquid.

[0070] (4) Electrophoresis: set the electrophoresis program, a first program constant voltage 50 V, time 30 min; b second program constant voltage 120 V, time 90 min, according to the size of the required protein, observe the pre-dye maker termination electrophoresis of the sample.

[0071] (5) Transfer: PVDF membrane is labeled and activated in methanol for 30 seconds, and then transferred to cold 1x transfer buffer prepared in advance to wash the methanol; after the electrophoresis program is completed, the upper concentrated gel is removed, and a sandwich structure is prepared in the order of sponge, 4 layers of filter paper, PVDF membrane, separation gel, 4 layers of filter paper and sponge (air bubbles are avoided between the separation gel and the PVDF membrane); the sandwich structure is clamped using a clamp, and the positive and negative electrodes are determined and placed in the transfer tank. At the same time, the positive and negative directions on the electrode cover are noted, and the transfer program is started: constant current 200 mA, time 60 minutes; the transfer tank is placed in an ice water bath.

[0072] (6) Blocking: after the transfer is completed, the PVDF membrane is taken out using tweezers, the surface transfer buffer is washed in 1x TBST, and the PVDF membrane is placed in 5% skimmed milk powder blocking buffer prepared with 1x TBST, and is slowly shaken at room temperature for at least 1 hour to avoid specific bands in the subsequent process.

[0073] (7) Primary antibody incubation: after the blocking is completed, the blocking solution is recovered, the residual skimmed milk powder on the PVDF membrane is washed away using 1x TBST, the membrane is washed for 5-10 minutes after being washed three times, and the operation is repeated three times. After washing, the PVDF membrane is placed in an incubation box, the primary antibody is completely immersed in the PVDF membrane, and the incubation is performed overnight at 4°C in a refrigerator shaking bed.

[0074] (8) Secondary antibody incubation: the primary antibody is recovered, the membrane washing operation is repeated, and the residual primary antibody on the membrane is washed away; then the secondary antibody corresponding to the primary antibody is incubated at room temperature for 1 hour; the membrane washing operation is repeated, and the residual secondary antibody on the membrane is washed away.

[0075] (9) Exposure: the sensitizer and stabilizer A liquid and B liquid two reagents are mixed in equal volumes in a centrifugal tube (1 mL of exposure liquid is prepared for each membrane). 1 mL of the mixed reagent is taken to the exposure instrument, the PVDF membrane is fully contacted with the exposure liquid, and it is ensured that the membrane surface is uniformly exposed to the exposure liquid; the exposure machine is automatically exposed in the dark room, the picture is observed and saved, and the picture is quantitatively analyzed by using Image J analysis software.

[0076] 4. Experimental results

[0077] Table 1 is a statistical table of the colon health of C57BL / 6 mice, and the results show that the body weight of the C57BL / 6 mice in examples 1-7 of the present application is significantly increased compared with the model control (p<0.05), and under the same dose, the body weight of the example 5 group is best recovered, indicating that the ethyl acetate extract of Boletus has better effect on the recovery of body weight. In terms of colon length, the colon length of examples 1-7 is longer than that of the model group, and the colon crypt depth is deeper, indicating that the Boletus extract can delay the atrophy of the colon of the old mice and prolong the absorption of nutrients by the colon, and the example 5 has the best effect, indicating that the ethyl acetate extraction can enrich the active substances for improving intestinal dysfunction and remove most of the inactive substances.

[0078] Table 1 C57BL / 6 mice colon health

[0079]

[0080] Note: In the table, superscript # means p < 0.05 compared with the blank control example; superscript means p < 0.05 compared with the model control example; superscript a, b, c means p < 0.05 between different examples.

[0081] Figure 1 For colon tissue HE staining, PASS staining, Figure 2 The results of the detection of the expression levels of the intestinal mucin marker ZO-1 (tight junction protein) and MUC2 (gelatin protein) are shown in the table. The results show that the Example 1 group and the Example 5 group can significantly repair the aging condition in the aging colon model and promote the intestinal integrity. At the same time, it is shown by the PASS staining that the Example 1 group and the Example 5 group promote the expression of the sugar mucin in the colon and promote the lubricity of the intestinal tract. As can be seen from the pictures, the Example 5 group is superior to the Example 1 group. The comparison between the model control example and the blank control example shows that there is a significant difference in the expression of ZO-1 and MUC2 proteins (p < 0.05). Compared with the model group, the Example 1 group and the Example 5 group can significantly increase the expression levels of ZO-1 and MUC2 proteins. And the Example 5 group is more obvious than the Example 1 group. The extract of the present application can significantly increase the expression levels of ZO-1 and MUC2 proteins in the colon, increase the content of the sugar mucin in the intestinal tract, reduce the risk of intestinal diseases of the aging mice, and maintain the intestinal health, etc.

[0082] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. The use of a Boletus edulis extract in the preparation of a medicament for the prevention and / or treatment of age-related intestinal dysfunction caused by colonic atrophy and degeneration, wherein the Boletus edulis extract is prepared by pulverizing Boletus edulis, extracting with water or alcohol solvent, filtering to obtain an extract, concentrating the extract, and drying to obtain the Boletus edulis extract.

2. The application according to claim 1, characterized in that, The drug delays colonic atrophy and degeneration, and enhances intestinal digestion, absorption, and immune function.

3. The application according to claim 1, characterized in that, The drug promotes the growth of colon length and crypt depth.

4. The application according to claim 1, characterized in that, The drug upregulates the expression of tight junction protein ZO-1 and mucin MUC-2 in the colon of aging organisms.

5. The application according to claim 1, characterized in that, The water or alcohol solvent extraction is performed by adding 10-30L of water or alcohol solvent per kilogram of Boletus edulis, wherein the alcohol solvent is 70%-80% ethanol-water solvent.

6. The application according to claim 1, characterized in that, The extraction method may be immersion extraction, hot reflux extraction, ultrasonic extraction, or any combination of two thereof.

7. The application according to claim 1, characterized in that, The preparation method of the delicious Boletus extract also includes the following purification steps: dissolving the alcohol extract in water, extracting with ethyl acetate, collecting the ethyl acetate extract, concentrating, and drying.

8. The application according to claim 7, characterized in that, In the ethyl acetate extraction, the volume ratio of the aqueous phase to ethyl acetate is 1:1 to 5, and the extraction is performed 2 to 4 times.

9. The application according to any one of claims 1-8, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers or excipients.

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