阅读说明：本技术 一种具有防治结肠炎功效的羊肚菌提取物及其应用 (Morchella extract with effect of preventing and treating colitis and application thereof ) 是由 许瀛引 张志远 唐杰 王勇 彭卫红 周洁 刘天海 于 2021-08-02 设计创作，主要内容包括：本发明涉及一种具有防治结肠炎功效的羊肚菌提取物的制备方法,包括以下步骤：(1)取羊肚菌子实体,粉碎,按照羊肚菌子实体粉与乙醇溶液的质量体积比为1:10-1:50(g/mL)的料液比投加乙醇溶液,在60℃～80℃温度下,加热回流提取1-10h,过滤,收集并合并滤液,浓缩、离心,得上清液；(2)将上清液送入离子交换树脂柱中进行分离,再用洗脱溶剂对离子交换树脂柱进行洗脱,收集洗脱液；(3)将洗脱液浓缩,干燥,得到羊肚菌提取物。本发明制得的羊肚菌提取物,黄酮含量高,可用于缓解结肠炎。(The invention relates to a preparation method of a morchella extract with a colitis prevention and treatment effect, which comprises the following steps: (1) taking morchella sporocarp, crushing, adding an ethanol solution according to the mass-to-volume ratio of morchella sporocarp powder to the ethanol solution of 1:10-1:50(g/mL), heating and refluxing for extraction for 1-10h at the temperature of 60-80 ℃, filtering, collecting and combining filtrate, concentrating and centrifuging to obtain supernatant; (2) sending the supernatant into an ion exchange resin column for separation, eluting the ion exchange resin column by using an elution solvent, and collecting the eluent; (3) concentrating the eluate, and drying to obtain Morchella esculenta extract. The morchella extract prepared by the invention has high flavone content and can be used for relieving colitis.)

1. A preparation method of a morchella extract with a colitis prevention and treatment effect is characterized by comprising the following steps:

(1) taking morchella sporocarp, crushing, adding an ethanol solution according to the mass-to-volume ratio of morchella sporocarp powder to the ethanol solution of 1:10-1:50(g/mL), heating and refluxing for extraction for 1-10h at the temperature of 60-80 ℃, filtering, collecting and combining filtrate, concentrating and centrifuging to obtain supernatant;

(2) sending the supernatant into an ion exchange resin column for separation, eluting the ion exchange resin column by using an elution solvent, and collecting the eluent;

(3) concentrating the eluate, and drying to obtain Morchella esculenta extract.

2. The method of claim 1, wherein the pulverizing is that the pulverized morchella sporocarp powder is sieved by a 10-50 mesh sieve.

3. The method according to any one of claims 1 to 2, wherein the ethanol solution is added at the step (1) at a concentration of 60 to 90% by mass.

4. The method of any one of claims 1 to 3, wherein the extraction temperature is from 65 ℃ to 70 ℃.

5. The method according to any one of claims 1 to 4, wherein the concentration in step (1) is 20 to 50% by volume of the total filtrate.

6. The method according to any one of claims 1 to 5, wherein the ion exchange resin column is a macroporous resin column, preferably any one of a D101 resin column, a NKA resin column, an X-5 resin column or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the elution solvent is a 20% to 80% strength ethanol solution and is used in an amount of 3 to 5 times the volume of the resin column.

8. The method according to any one of claims 1 to 7, wherein the extract has a flavonoid content of not less than 10% (w/w), preferably not less than 50% (w/w), more preferably not less than 60% (w/w).

9. The morchella vulgaris extract having the effect of preventing and treating colitis, prepared by the method according to any one of claims 1-8, wherein the flavonoid content in the extract is not less than 10% (w/w), preferably not less than (w/w) 50%, more preferably not less than 60% (w/w).

10. Use of the morchella vulgaris extract with the effect of preventing and treating colitis, which is prepared according to any one of claims 1-8, in preparing a product for preventing and treating colitis.

Technical Field

The invention relates to the field of medicines, and particularly relates to a morchella extract with a colitis prevention and treatment effect, and a preparation method and application thereof.

Background

Colitis refers to inflammatory lesions of the colon caused by various causes. The clinical manifestations of colitis are diarrhea, abdominal pain, mucous stool, bloody purulent stool, tenesmus, even constipation, and inability to get rid of the stool within several days; it is often accompanied by emaciation, hypodynamia, etc., and is frequently recurrent. More and more studies have shown that the onset of colitis is associated with changes in the intestinal flora and chronic inflammatory reactions caused by immune dysfunction of the intestinal mucosa.

Morchella is a rare edible and medicinal fungus, is known as high-quality protein food, contains 7 kinds of amino acids necessary for human body, and contains amino acids with total amount of about 40%, fat content of less than 1%, and rich cellulose and mineral elements. The compendium of materia medica records that the medicine has sweet and cold taste, mild nature, no toxicity, can benefit intestines and stomach, and can resolve phlegm and regulate qi. Modern medical research proves that the morchella can improve the immunity of the organism, has killing capacity on tumor cells of liver cancer, lung cancer, colon cancer, breast cancer and the like, and has the effects of resisting fatigue, reducing blood fat and relieving toxic and side effects caused by radiotherapy and chemotherapy. Therefore, the medicinal development of morchella and the extract thereof becomes a research hotspot in the field. However, no research related to the use of morchella extract for preventing and treating colitis exists in the prior art.

Disclosure of Invention

A preparation method of Morchella esculenta extract with colitis prevention and treatment effects comprises the following steps:

(1) taking morchella sporocarp, crushing, adding an ethanol solution according to the mass-to-volume ratio of morchella sporocarp powder to the ethanol solution of 1:10-1:50(g/mL), heating and refluxing for extraction for 1-10h at the temperature of 60-80 ℃, filtering, collecting and combining filtrate, concentrating and centrifuging to obtain supernatant;

(2) sending the supernatant into an ion exchange resin column for separation, eluting the ion exchange resin column by using an elution solvent, and collecting the eluent;

(3) concentrating the eluate, and drying to obtain Morchella esculenta extract.

In the preferable technical scheme of the invention, the crushing is that the crushed morchella sporocarp powder is sieved by a sieve with 10-50 meshes.

In the preferable technical scheme of the invention, in the step (1), the mass concentration of the added ethanol solution is 60-90%.

In the preferred technical scheme of the invention, the extraction times are 2-5 times.

In the preferable technical scheme of the invention, the feed-liquid ratio is 1:20-1:30 (g/mL).

In the preferred technical scheme of the invention, the extraction time is 3-6 h.

In the preferred technical scheme of the invention, the extraction temperature is 65-70 ℃.

In a preferred technical scheme of the invention, the filtration is any one of gauze filtration and centrifugal filtration.

In a preferred technical scheme of the invention, in the step (1), the concentration is performed until the total volume of the filtrate is 20-50%.

In a preferred embodiment of the present invention, the concentration is selected from any one of vacuum concentration, membrane concentration and atmospheric concentration.

In the preferable technical scheme of the invention, the concentration temperature is 65-70 ℃.

In the preferable technical scheme of the invention, the centrifugation is carried out for 10-20min at the temperature of 4-10 ℃ and at the rotating speed of 6000-8000 rpm/min.

In a preferred technical scheme of the invention, the ion exchange resin column is a macroporous resin column, preferably any one of a D101 resin column, an NKA resin column and an X-5 resin column or a combination thereof.

According to a preferred embodiment of the present invention, the height/diameter ratio of the resin column is (3-10):1, preferably (6-8): 1.

According to the preferable technical scheme, the working temperature of the resin column is 20-25 ℃.

In the preferred technical scheme of the invention, the flow rate of the supernatant liquid fed into the ion exchange resin column is 0.5-3mL/min, preferably 1-2 mL/min.

In the preferred technical scheme of the invention, the elution flow rate of the elution solvent fed into the ion exchange resin column is 0.5-3mL/min, and the preferred elution flow rate is 1-2 mL/min.

In the preferred technical scheme of the invention, the elution solvent is 20-85% ethanol solution, preferably 50-80% ethanol solution, and the dosage of the elution solvent is 3-5 times of the volume of the resin column.

In the preferable technical scheme of the invention, the elution step is to elute by using an ethanol solution with the volume of 3-5 times of the resin column and the mass concentration of 20 percent or 50 percent or 80 percent and collect the eluent.

In the preferable technical scheme of the invention, the elution step comprises the steps of firstly eluting by using 20 mass percent ethanol solution with 3-5 times of the volume of the resin column, discarding the eluent, then eluting by using 50 mass percent ethanol solution with 3-5 times of the volume of the resin column, and collecting the eluent.

In the preferable technical scheme of the invention, in the step (3), the concentration is 1/10-1/50 of the volume of the original eluent after decompression concentration in water bath at 40-60 ℃.

In a preferred embodiment of the present invention, the separation is selected from any one of filtration, centrifugation, and membrane treatment.

In a preferred embodiment of the present invention, the drying is selected from any one of vacuum drying, atmospheric drying, spray drying, fluidized drying and pneumatic drying.

In the preferable technical scheme of the invention, the drying temperature is not lower than 80 ℃, and preferably is 100-110 ℃.

In the preferable technical scheme of the invention, the content of the flavone in the extract is not less than 10 percent

(w/w), preferably not less than 50% (w/w), more preferably not less than 60% (w/w).

The invention aims to provide a morchella extract with the effect of preventing and treating colitis, wherein the content of flavone in the extract is not less than 10% (w/w), preferably not less than 50% (w/w), and more preferably not less than 60% (w/w).

In a preferred technical scheme of the invention, the preparation method of the extract comprises the following steps:

(1) taking morchella sporocarp, crushing, adding an ethanol solution according to the mass-to-volume ratio of morchella sporocarp powder to the ethanol solution of 1:10-1:50(g/mL), heating and refluxing for extraction for 1-10h at the temperature of 60-80 ℃, filtering, collecting and combining filtrate, concentrating and centrifuging to obtain supernatant;

(2) sending the supernatant into an ion exchange resin column for separation, eluting the ion exchange resin column by using an elution solvent, and collecting the eluent;

(3) concentrating the eluate, and drying to obtain Morchella esculenta extract.

In the preferable technical scheme of the invention, the crushing is that the crushed morchella sporocarp powder is sieved by a sieve with 10-50 meshes.

In the preferable technical scheme of the invention, in the step (1), the mass concentration of the added ethanol solution is 60-90%.

In the preferred technical scheme of the invention, the extraction times are 2-5 times.

In the preferable technical scheme of the invention, the feed-liquid ratio is 1:20-1:30 (g/mL).

In the preferred technical scheme of the invention, the extraction time is 3-6 h.

In the preferred technical scheme of the invention, the extraction temperature is 65-70 ℃.

In a preferred technical scheme of the invention, the filtration is any one of gauze filtration and centrifugal filtration.

In a preferred technical scheme of the invention, in the step (1), the concentration is performed until the total volume of the filtrate is 20-50%.

In a preferred embodiment of the present invention, the concentration is selected from any one of vacuum concentration, membrane concentration and atmospheric concentration.

In the preferable technical scheme of the invention, the concentration temperature is 65-70 ℃.

In the preferable technical scheme of the invention, the centrifugation is carried out for 10-20min at the temperature of 4-10 ℃ and at the rotating speed of 6000-8000 rpm/min.

In a preferred technical scheme of the invention, the ion exchange resin column is a macroporous resin column, preferably any one of a D101 resin column, an NKA resin column and an X-5 resin column or a combination thereof.

According to a preferred embodiment of the present invention, the height/diameter ratio of the resin column is (3-10):1, preferably (6-8): 1.

According to the preferable technical scheme, the working temperature of the resin column is 20-25 ℃.

In the preferred technical scheme of the invention, the flow rate of the supernatant liquid fed into the ion exchange resin column is 0.5-3mL/min, preferably 1-2 mL/min.

In the preferred technical scheme of the invention, the elution flow rate of the elution solvent fed into the ion exchange resin column is 0.5-3mL/min, and the preferred elution flow rate is 1-2 mL/min.

In the preferred technical scheme of the invention, the elution solvent is 20-85% ethanol solution, preferably 50-80% ethanol solution, and the dosage of the elution solvent is 3-5 times of the volume of the resin column.

In the preferable technical scheme of the invention, the elution step comprises the steps of firstly eluting by using 20 mass percent ethanol solution with 3-5 times of the volume of the resin column, discarding the eluent, then eluting by using 50 mass percent ethanol solution with 3-5 times of the volume of the resin column, and collecting the eluent.

In the preferable technical scheme of the invention, in the step (3), the concentration is 1/10-1/50 of the volume of the original eluent after decompression concentration in water bath at 40-60 ℃.

In a preferred embodiment of the present invention, the separation is selected from any one of filtration, centrifugation, and membrane treatment.

In a preferred embodiment of the present invention, the drying is selected from any one of vacuum drying, atmospheric drying, spray drying, fluidized drying and pneumatic drying.

In the preferable technical scheme of the invention, the drying temperature is not lower than 80 ℃, and preferably is 100-110 ℃.

In a preferred embodiment of the present invention, the content of flavone in the extract is not less than 10% (w/w), preferably not less than 50% (w/w), more preferably not less than 60% (w/w).

The invention also aims to provide application of the morchella extract with the effect of preventing and treating colitis in preparing a product for preventing and treating colitis.

Unless otherwise indicated, when the present invention relates to percentages between liquids, said percentages are volume/volume percentages; the invention relates to the percentage between liquid and solid, said percentage being volume/weight percentage; the invention relates to the percentages between solid and liquid, said percentages being weight/volume percentages; the balance being weight/weight percent.

Unless otherwise indicated, the following methods are used in the present invention:

1. and (3) flavone content: AlCl₃Colorimetric method (reference document: Bengecap et al, 2018. Pholiota nameko flavone alcohol extraction process and antioxidant activity research. agricultural product processing (first half month) (4):26-30.)

Shannon index and Simpson index: high throughput sequencing method (reference: Li, Bing, et al. sequencing of tetracyclic bacterial communication in saline activated sludge using substrate digestion with high-through put sequencing and analysis. Water research 47.13(2013): 4207. 4216.)

Compared with the prior art, the beneficial technical effects of the invention comprise:

1. the morchella extract prepared by the invention has high flavone content, is used for treating colitis, can increase the weight of a colitis mouse, recover the length of a contracted colon, obviously increase the colonic-villi hiding ratio, reduce proinflammatory factors IFN, IL-1 beta, IL-6 and TNF-alpha, increase an inflammation inhibiting factor IL-10, and obviously reduce indexes (DAO and D-Lactate) for indicating the degree of colon inflammation.

2. The morchella extract extracted by the method can affect intestinal flora and flora metabolites of the intestinal flora of colitis mice, improve the biological diversity of the intestinal flora, increase dominant flora, reduce harmful flora and achieve the effect of relieving colitis.

3. The invention has simple process and convenient preparation and can be used for industrial production.

Drawings

FIG. 1: colon histopathological changes in mice in normal, adult and experimental groups

Detailed Description

The present invention is described below with reference to examples. The invention is not limited to the examples.

Example 1

(1) Pulverizing Morchella esculenta sporophore, sieving with 10-50 mesh sieve, collecting sieved Morchella esculenta sporophore powder 200g, adding 70% ethanol solution at a material-to-liquid ratio of 1:20(g/mL), heating and reflux extracting at 60 deg.C for 2 times, extracting for 6 hr each time, filtering the extractive solution with three layers of gauze for 2 times, mixing filtrates, concentrating under reduced pressure in 40 deg.C water bath condition to 25% of the total volume of the filtrate, centrifuging at 4 deg.C at 6000rpm for 15min, and collecting supernatant.

(2) Enabling the supernatant to flow through a macroporous resin column D101 at the flow rate of 0.5mL/min, wherein the height-diameter ratio of the resin column is 6:1, and the working temperature is 20 ℃. Selecting an ethanol solution with the volume of 5 times of the column and the mass concentration of 20% as an elution solvent, flowing through a resin column at the flow rate of 1mL/min, and discarding the eluent; and selecting an ethanol solution with 5 times of column volume and 50% of mass concentration as an elution solvent, flowing through the resin column at the flow rate of 1mL/min, and collecting the eluate.

(3) Concentrating the eluate in 40 deg.C water bath under 0.08Mpa under reduced pressure to 1/50 volume of the original eluate, oven drying in 50 deg.C oven to constant weight to obtain Morchella esculenta extract, and detecting flavone content in Morchella esculenta extract to be 63.2%.

Example 2

(1) Pulverizing Morchella esculenta sporophore, sieving with 10-50 mesh sieve, collecting sieved Morchella esculenta sporophore powder 200g, adding 70% ethanol solution at a material-to-liquid ratio of 1:20(g/mL), heating and reflux extracting at 60 deg.C for 2 times, extracting for 6 hr each time, filtering the extractive solution with three layers of gauze for 2 times, mixing filtrates, concentrating under reduced pressure in 40 deg.C water bath condition to 25% of the total volume of the filtrate, centrifuging at 4 deg.C at 6000rpm for 15min, and collecting supernatant.

(2) Enabling the supernatant to flow through a macroporous resin column D101 at the flow rate of 0.5mL/min, wherein the height-diameter ratio of the resin column is 6:1, and the working temperature is 20 ℃. Selecting ethanol solution with 5 times column volume and 50% mass concentration as eluting solvent, flowing through resin column at flow rate of 1mL/min, and collecting eluate.

(3) Concentrating the eluate in 40 deg.C water bath under 0.08Mpa under reduced pressure to 1/50 volume of the original eluate, oven drying in 50 deg.C oven to constant weight to obtain Morchella esculenta extract, and detecting flavone content in Morchella esculenta extract to be 71.5%.

Example 3

(1) Pulverizing Morchella esculenta sporophore, sieving with 10-50 mesh sieve, collecting sieved Morchella esculenta sporophore powder 200g, adding 70% ethanol solution at a material-to-liquid ratio of 1:25(g/mL), heating and reflux extracting at 60 deg.C for 2 times, extracting for 6 hr each time, filtering the extractive solution with three layers of gauze for 2 times, mixing filtrates, concentrating under reduced pressure in 40 deg.C water bath condition to 25% of the total volume of the filtrate, centrifuging at 4 deg.C at 6000rpm for 15min, and collecting supernatant.

(2) The supernatant was passed through a resin column at a flow rate of 0.5mL/min, the height to diameter ratio of the resin column was 6:1, and the operating temperature was 40 ℃. Selecting 5 times of resin column volume and 20% ethanol solution as eluting solvent, flowing through resin column at flow rate of 0.5mL/min, and collecting eluate.

(3) Concentrating the eluate in 40 deg.C water bath under 0.08Mpa under reduced pressure to 1/50 volume of the original eluate, oven drying in 50 deg.C oven to constant weight to obtain Morchella esculenta extract, and detecting flavone content in Morchella esculenta extract to be 18.6%.

Example 4

(1) Pulverizing Morchella esculenta sporophore, sieving with 10-50 mesh sieve, collecting sieved Morchella esculenta sporophore powder 200g, adding 70% ethanol solution at a material-to-liquid ratio of 1:20(g/mL), heating and reflux extracting at 60 deg.C for 2 times, extracting for 6 hr each time, filtering the extractive solution with three layers of gauze for 2 times, mixing filtrates, concentrating under reduced pressure in 40 deg.C water bath condition to 25% of the total volume of the filtrate, centrifuging at 4 deg.C at 6000rpm for 15min, and collecting supernatant.

(2) Enabling the supernatant to flow through a macroporous resin column D101 at the flow rate of 0.5mL/min, wherein the height-diameter ratio of the resin column is 6:1, and the working temperature is 40 ℃. Selecting 5 times of resin column volume and 80% ethanol solution as eluting solvent, flowing through resin column at flow rate of 1mL/min, and collecting eluate.

(3) Concentrating the eluate in 40 deg.C water bath under 0.08Mpa under reduced pressure to obtain 1/50 volume of the original eluate, oven drying in 50 deg.C oven to constant weight to obtain Morchella esculenta extract, and detecting flavone content in Morchella esculenta extract to be 44.2%.

Test example 1

After 30 purchased C57BL/6 male 6-7 week-old mice were acclimatized and fed for one week, the mice were fed normally to freely take food and drink water, and then divided into a normal group, a model building group and a test group, each group containing 10 mice.

Normal group: each mouse was gavaged once daily with reverse osmosis water (200. mu.L) for 14 days. The mice normally take food and drink water in 14 days. And (5) carrying out drunken sacrifice on the mice by using the rhizoma gastrodiae.

Building a module: each mouse was gavaged once daily with reverse osmosis water (200. mu.L) for 14 days. On day 1-7 mice normally took food and water. Mice fed normally on days 8-14, and water supplemented with 3.5% sodium thiosulfate (DSS, v/w) was consumed. And (5) carrying out drunken sacrifice on the mice by using the rhizoma gastrodiae.

Test groups: each mouse was gavaged once a day with a morchella extract solution (200. mu.L, containing the morchella extract of example 1 at a concentration of 25mg/mL) for 14 days. On day 1-7 mice normally took food and water. Mice fed normally on days 8-14, and water supplemented with 3.5% sodium thiosulfate (DSS, v/w) was consumed. And (5) carrying out drunken sacrifice on the mice by using the rhizoma gastrodiae.

Evaluation index results (table 1) show that after 14 days of continuous gavage of the morchella extract solution of example 1, the weight of a colitis mouse is increased, the length of a contracted colon is recovered, the colonic villi hiding ratio is obviously increased, proinflammatory factors IFN, IL-1 beta, IL-6 and TNF-alpha are reduced, inflammation-inhibiting factors IL-10 are increased, and indexes (DAO and D-Lactate) indicating the degree of colonic inflammation are obviously reduced.

TABLE 1 colitis-related evaluation index of mice

	Normal group	Molding set	Test group
				Colon villus height (mum)	389.26±19.66	344.95±25.76**	401.99±18.94##
DAO,U/L	10.15±3.33	15.86±4.92*	11.12±2.98#
				D-Lactate,pg/mL	14.91±3.71	24.49±5.28**	11.72±2.47##
IL-1β,pg/mL	8.77±1.09	15.02±2.50**	10.01±1.34##
				IL-6,pg/Ml	11.30±1.77	19.53±4.64**	13.45±2.41#
IL-10,pg/mL	55.74±5.24	38.70±3.74**	53.79±4.72##
				TNF-α,pg/mL	83.07±5.20	117.79±7.30**	107.51±6.86#
IFN-γ,pg/mL	49.61±5.33	77.37±9.97**	65.15±7.65#
				IL-10,pg/mL	55.74±5.24	38.70±3.74**	53.79±4.72##

Note: normal group compared to the modeling group: p < 0.05 label, p < 0.01 label; the test group is compared with the modeling group: p is less than 0.05 mark #, and p is less than 0.01 mark #.

Histopathological section results show that the intestinal villi of the mice in the test group are more complete and the inflammation level is lower (see figure 1), and in conclusion, the morchella extract in the example 1 achieves the best effect of intervening in the colitis of the mice.

The Shannon index and Simpson index are used to reflect community diversity, including species abundance and species uniformity, with a larger index indicating a higher species diversity. The results of the intestinal flora show (table 2) that the diversity of the intestinal flora of the model-making group is reduced, the structure is unbalanced compared with that of the normal group, and the biological diversity of the intestinal flora of the test group is obviously improved. The result shows that the morchella extract in example 1 can achieve the effect of relieving colitis by regulating the diversity of intestinal flora.

TABLE 2 Effect of Colon bacterial community diversity in mice

Note: normal group compared to the modeling group: p < 0.05 label, p < 0.01 label; the test group is compared with the modeling group: p is less than 0.05 mark #, and p is less than 0.01 mark #.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined in the appended claims.