CN113499351B

CN113499351B - Extraction method and application of fomes fomentarius extract for treating UC

Info

Publication number: CN113499351B
Application number: CN202110769683.7A
Authority: CN
Inventors: 成晓霞; 张旭维; 姬一凡
Original assignee: Xian University
Current assignee: Xian University
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2022-09-06
Anticipated expiration: 2041-07-07
Also published as: CN113499351A

Abstract

The invention discloses an extraction method of fomes fomentarius extract for treating UC, which comprises the following steps: step 1, cleaning, drying, stripping and crushing fruit bodies of Fomitopsis pinicola to obtain flocculent Fomitopsis pinicola powder; step 2, uniformly mixing the product obtained in the step 1 with an organic solvent to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract; step 3, adding deionized water into the product obtained in the step 2, and concentrating under reduced pressure to obtain an extract; and 4, mixing and dissolving the extract with ethanol, then carrying out silica gel column chromatography on the ethanol solution of the fomes fomentarius extract, eluting with chloroform as an eluent, concentrating and drying to obtain the fomes fomentarius total terpene. The fomes pinicola extract extracted by the invention can relieve ulcerative colitis symptoms induced by dextran sodium sulfate.

Description

Extraction method and application of fomes fomentarius extract for treating UC

Technical Field

The invention belongs to the technical field of research of inflammatory bowel diseases, and relates to an extraction method of a fomes fomentarius extract for treating UC.

Background

Inflammatory Bowel Disease (IBD) is a general term for a series of intestinal diseases, mainly including Ulcerative Colitis (UC) and Crohn Disease (CD), which have different pathogenesis but similar clinical manifestations. Ulcerative colitis is a kind of intestinal tract disease mainly caused by nonspecific inflammation of the submucosa of rectum and colon, mainly involving rectum and sigmoid colon, and also invading the whole colon and terminal ileum, and clinically manifested as abdominal pain, diarrhea, mucopurulent bloody stool, etc. The disease has repeated attack and prolonged duration, and complications including toxic megacolon, intestinal perforation, lower gastrointestinal tract hemorrhage, etc. can be accompanied by anemia, angular stomatitis, intestinal perforation, intestinal obstruction, and cancer risk, and can bring great mental anxiety to patients and possibly induce depression. Epidemiological investigation shows that UC has no sex difference but has regional difference, and the higher the latitude of the residential area is, the higher the incidence rate is; the incidence reason of the UC is probably related to factors such as heredity, diet, immunity and the like, the incidence rate of the UC in western developed countries is considered to be high in the past, the incidence rate of the UC in Asian countries is low, but with the development of economy, the dietary structure and the life style are changed greatly, the incidence rate of the UC in China rises year by year, and the incidence of the UC in 1999-2003 is 8 times that of the UC in 1989-1993. Because the pathogenesis of UC is unknown and specific drugs are lacked, the prior clinical treatment mostly adopts salicylic acid inhibitor, corticosteroid and immunosuppressant, and although the onset of action is quick, the difficult problems of hormone dependence, hormone resistance, toxic and side effects, drug resistance and the like still need to be solved urgently. The expert scholars in China try to relieve the clinical symptoms of patients to a certain extent by adopting traditional Chinese medicine oral administration, acupuncture and moxibustion, application and enema. Therefore, the traditional Chinese medicine brings first-line dawn for treating 'modern refractory diseases' listed in the world health organization as the top list of UC.

Fomitopsis pinicola (Swartz.: Fr.) Karst, also called Fomitopsis pinicola, Russula sanguinea and the like, belonging to Basid-fungi of Basidiomycetes, Aphyllophorales, Polyporaceae and Fomitopsis, is a wood rotting fungus with semicircular or horseshoe-shaped pileus, the color of the pileus changes from white to reddish brown to black brown during growth, the edge changes from milky white to light yellow or reddish brown, and the pileus is widely distributed in China and is a common medicinal fungus. Fomitopsis pinicola is slightly bitter in taste and neutral in nature, has the effects of tonifying lung and kidney, harmonizing stomach and strengthening spleen, tranquilizing mind, dispelling wind and removing dampness and the like, has a certain curative effect on nephritis, bronchitis, cancer, heart disease, diabetes and the like, and is commonly used for treating cold leg diseases of miners in folk. Recent pharmacological studies also prove that the phellinus igniarius sporocarp and the mycelium extract cultured in vitro have the physiological activities of resisting tumors, inhibiting bacteria, resisting inflammation, reducing blood sugar, regulating immunity, resisting oxidation and the like.

The research results of Zhao Xiuhong et al suggest that methylene chloride and petroleum ether extracts of Phellinus pinicola fruiting bodies can remarkably inhibit mouse ear swelling caused by xylene, increase the superoxide dismutase level in mouse plasma and right ear tissue fluid, remarkably reduce malondialdehyde and prostaglandin E2 levels of mouse serum and right ear tissue fluid, and show anti-inflammatory, analgesic and antipyretic effects. Until now, reports related to treatment of the phellinus rimosus on the ulcerative colitis have not been seen.

Disclosure of Invention

The invention aims to provide an extraction method of a fomes fomentarius extract for treating UC, and the fomes fomentarius extract extracted by the method can relieve ulcerative colitis symptoms induced by sodium dextran sulfate.

The technical scheme adopted by the invention is that the extraction method of the fomes fomentarius extract for treating UC specifically comprises the following steps:

step 1, cleaning, drying, stripping and crushing fruit bodies of Fomitopsis pinicola to obtain flocculent Fomitopsis pinicola powder;

and 2, mixing the product obtained in the step 1 with an organic solvent according to the weight ratio of 1 g: uniformly mixing the materials in a ratio of 20-40 mL to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract;

step 3, adding deionized water which is 10-20 times of the weight of the fomes fomentarius extract obtained in the step 2 into the fomes fomentarius extract, stirring at 45 ℃ until the fomes fomentarius extract is completely dissolved, then adding a sodium bicarbonate solution with the concentration of 5% into the solution, adjusting the pH of the system to be 7-8, stirring until the fomes fomentarius extract is completely dissolved, and concentrating under reduced pressure to obtain a fomes fomentarius extract;

step 4, mixing the fomes fomentarius extract obtained in the step 3 with ethanol according to the ratio of 1 g: mixing and dissolving 10-20 mL of the extract in a ratio to obtain an ethanol solution of the fomes fomentarius extract, performing silica gel column chromatography on the ethanol solution of the fomes fomentarius extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the fomes fomentarius total terpene.

The invention is also characterized in that:

the drying temperature in the step 1 is 45-60 ℃.

The organic solvent in the step 2 is ethanol.

The volume fraction of the ethanol in the step 2 is 70-90%.

In the step 2, the volume of ethanol added into 1g of powder is 20-40 mL.

The ultrasonic power of ultrasonic treatment in the step 2 is 280-360 w; the ultrasonic time is 45-75 minutes.

The invention has the beneficial effects that: according to the invention, the mouse model of DSS-induced ulcerative colitis is treated by gavage FPKc (Fomitopsis pinicola total terpenoids), and the discovery shows that the Fomitopsis pinicola terpenoids can obviously improve the mental state of the mouse, relieve the decrease of hematochezia and body weight, gradually recover the colon length and crypt structure of the UC mouse, and have exact curative effect on UC. Further experimental results suggest that the action mechanism of the polypeptide is probably closely related to the activation of immune regulation and the down-regulation of the expression of inflammatory factors IL-6, IL-1 beta and TNF-alpha in serum. After the drug action, the content ratio of the glutamic-pyruvic transaminase to the glutamic-pyruvic transaminase in the blood serum of the UC mouse tends to a normal control group, which indicates that FPKc can relieve liver injury caused by UC.

Drawings

FIG. 1 is a graph of weight change during 14 days of drug intervention over the course of an experiment involving a method of extraction of Fomitopsis pinicola extract for the treatment of UC in accordance with the present invention;

FIG. 2 is an OB kit for testing occult blood in stool of an extraction method of fomes erythrorhizoides extract for treating UC according to the present invention;

FIG. 3 is a graph of the histopathological changes of the colon during the course of the experiment of the extraction method of Fomitopsis pinicola extract for the treatment of UC according to the invention;

FIG. 4 shows the colon length of mice obtained from different experimental groups of the extraction method of Fomitopsis pinicola extract for the treatment of UC according to the present invention;

FIGS. 5(a) - (c) are bar charts of IL-6, IL-1 beta, and TNF-alpha contents in mouse serum during experiment of the extraction method of Fomitopsis pinicola extract for treating UC according to the present invention;

FIG. 6 is a histogram of the ratio of contents of glutamic acid and glutamic-pyruvic transaminase (DRR) in mouse serum during the experiment of the extraction method of Fomitopsis pinicola extract for treating UC of the present invention.

Detailed Description

The invention is described in detail below with reference to the drawings and the detailed description.

The invention relates to an extraction method of a fomes fomentarius extract for treating UC, which comprises the following steps:

step 1, cleaning, drying, stripping and crushing fruit bodies of Fomitopsis pinicola to obtain flocculent Fomitopsis pinicola powder; the drying temperature is 45-60 ℃.

And 2, mixing the product obtained in the step 1 with an organic solvent according to the weight ratio of 1 g: uniformly mixing the materials in a ratio of 20-40 mL to obtain a mixture; performing ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, and performing suction filtration and concentration on the ultrasonic treatment mixture to obtain a fomes fomentarius extract; the organic solvent is ethanol; the volume fraction of the ethanol is 70-90 percent; the ultrasonic power of ultrasonic treatment is 280-360 w; the ultrasonic time is 45-75 minutes.

step 4, mixing the fomes pinicola extract obtained in the step 3 with ethanol according to a ratio of 1 g: mixing and dissolving 10-20 mL of the extract in a ratio to obtain an ethanol solution of the Fomitopsis pinicola extract, performing silica gel column chromatography on the ethanol solution of the Fomitopsis pinicola extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the Fomitopsis pinicola total terpene.

Example 1

The extraction method of the fomes fomentarius extract for treating UC (UC is short for ulcerative colitis) specifically comprises the following steps:

step 1, cleaning, drying, stripping and crushing fruit bodies of Fomitopsis pinicola to obtain flocculent Fomitopsis pinicola powder; the drying temperature is 45 ℃.

And 2, mixing the product obtained in the step 1 with ethanol according to a ratio of 1 g: uniformly mixing the materials in a ratio of 20mL to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract; the volume fraction of ethanol is 80%; the ultrasonic power of ultrasonic treatment is 320 w; the sonication time was 60 minutes.

Step 3, adding deionized water which is 10 times of the weight of the fomes pinicola red edge extract into the fomes pinicola red edge extract obtained in the step 2, stirring at 45 ℃ until the fomes pinicola red edge extract is completely dissolved, then adding a sodium bicarbonate solution with the concentration of 5% into the solution, adjusting the pH of the system to be 7, stirring until the solution is completely dissolved, and concentrating under reduced pressure to obtain the fomes pinicola red edge extract;

step 4, mixing the fomes fomentarius extract obtained in the step 3 with ethanol according to the ratio of 1 g: mixing and dissolving in a proportion of 10mL to obtain an ethanol solution of the Fomitopsis pinicola extract, performing silica gel column chromatography on the ethanol solution of the Fomitopsis pinicola extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the Fomitopsis pinicola total terpene.

Example 2

step 1, cleaning, drying, stripping and crushing fruit bodies of Fomitopsis pinicola to obtain flocculent Fomitopsis pinicola powder; the drying temperature is 50 ℃.

And 2, mixing the product obtained in the step 1 with ethanol according to a ratio of 1 g: uniformly mixing the components in a ratio of 30mL to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract; the volume fraction of ethanol is 70%; the ultrasonic power of ultrasonic treatment is 360 w; the sonication time was 60 minutes.

Step 3, adding deionized water which is 15 times of the weight of the fomes fomentarius extract obtained in the step 2 into the fomes fomentarius extract, stirring at 45 ℃ until the fomes fomentarius extract is completely dissolved, then adding a sodium bicarbonate solution with the concentration of 5% into the solution, adjusting the pH of the system to 7.5, stirring until the solution is completely dissolved, and concentrating under reduced pressure to obtain the fomes fomentarius extract;

step 4, mixing the fomes pinicola extract obtained in the step 3 with ethanol according to a ratio of 1 g: mixing and dissolving in a proportion of 15mL to obtain an ethanol solution of the fomes fomentarius extract, performing silica gel column chromatography on the ethanol solution of the fomes fomentarius extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the fomes fomentarius total terpene.

Example 3

step 1, cleaning, drying, stripping and crushing fruit bodies of Fomitopsis pinicola to obtain flocculent Fomitopsis pinicola powder; the drying temperature is 60 ℃.

And 2, mixing the product obtained in the step 1 with ethanol according to a ratio of 1 g: uniformly mixing the components in a proportion of 40mL to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract; the volume fraction of ethanol is 80%; the ultrasonic power of ultrasonic treatment is 360 w; the sonication time was 45 minutes.

Step 3, adding deionized water which is 20 times of the weight of the fomes pinicola red edge extract into the fomes pinicola red edge extract obtained in the step 2, stirring at 45 ℃ until the fomes pinicola red edge extract is completely dissolved, then adding a sodium bicarbonate solution with the concentration of 5% into the solution, adjusting the pH of the system to be 8, stirring until the solution is completely dissolved, and concentrating under reduced pressure to obtain the fomes pinicola red edge extract;

step 4, mixing the fomes fomentarius extract obtained in the step 3 with ethanol according to the ratio of 1 g: mixing and dissolving in a ratio of 20mL to obtain an ethanol solution of the Fomitopsis pinicola extract, performing silica gel column chromatography on the ethanol solution of the Fomitopsis pinicola extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the Fomitopsis pinicola total terpene.

Example 4

step 1, washing, drying, stripping and crushing fomes fomentarius sporocarp to obtain flocculent fomes fomentarius powder; the drying temperature is 60 ℃.

And 2, mixing the product obtained in the step 1 with ethanol according to a ratio of 1 g: uniformly mixing at a ratio of 20mL to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract; the volume fraction of ethanol is 90%; the ultrasonic power of ultrasonic treatment is 360 w; the sonication time was 75 minutes.

step 4, mixing the fomes pinicola extract obtained in the step 3 with ethanol according to a ratio of 1 g: mixing and dissolving in a ratio of 20mL to obtain an ethanol solution of the Fomitopsis pinicola extract, performing silica gel column chromatography on the ethanol solution of the Fomitopsis pinicola extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the Fomitopsis pinicola total terpene.

Example 5

And 2, mixing the product obtained in the step 1 with ethanol according to the weight ratio of 1 g: uniformly mixing the components in a ratio of 30mL to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract; the volume fraction of ethanol is 80%; the ultrasonic power of ultrasonic treatment is 280 w; the sonication time was 75 minutes.

step 4, mixing the fomes pinicola extract obtained in the step 3 with ethanol according to a ratio of 1 g: mixing and dissolving in a proportion of 20mL to obtain an ethanol solution of the fomes fomentarius extract, performing silica gel column chromatography on the ethanol solution of the fomes fomentarius extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the fomes fomentarius total terpene.

Example 6

And 2, mixing the product obtained in the step 1 with ethanol according to a ratio of 1 g: uniformly mixing the materials in a ratio of 20mL to obtain a mixture; carrying out ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, carrying out suction filtration on the ultrasonic treatment mixture, and concentrating to obtain a fomes pinicola extract; the volume fraction of ethanol is 80%; the ultrasonic power of ultrasonic treatment is 360 w; the sonication time was 60 minutes.

The results of the test data of examples 1 to 6 are shown in table 1 below, and the extraction rate in table 1 is the extraction rate of total terpenes.

TABLE 1

From table 1 above, example 6 is the best extraction process;

method and steps

1. Mouse experimental grouping

50 healthy Kunming mice, half male and half female, with the weight of 18-22 g, purchased from the experimental animal center of air force military medical university [ license number: SCXK (shan) 2019-. Randomized into 5 groups: a normal control group, a model group 1, a model group 2, an FPKc (Fomitopsis pinicola total terpene) group, and a SASP (sulfasalazine) group. As shown in table 2.

TABLE 2 mouse grouping

Note: "+" indicates that the substance was ingested by the mouse, and "-" indicates that the substance was not ingested by the mouse.

2. Mouse ulcerative colitis model establishment and drug intervention

(1) Establishment of mouse ulcerative colitis model

After the mice are adaptively fed for 1 week, the other 4 groups except the normal control group are freely drunk with 4% DSS aqueous solution (DSS is dextran sodium sulfate) for 7 days, and then a mouse acute ulcerative colitis model is established.

The following indices were recorded: weighing the food intake of the mouse, namely weighing the mouse food before and after feeding, and calculating to obtain the daily food intake of the mouse; water intake, namely fresh drinking water is changed every day, a control group is distilled water, and the rest groups are all 4% DSS solution; (iii) change in body weight, i.e., the weight of the mouse was weighed regularly every day, and a weight loss fraction [ weight loss fraction (%) ═ weight/g on the day)/(initial weight/g) × 100% ] was calculated; and fourthly, observing the loose stool degree at regular time every day according to stool characters, and detecting bloody stool conditions by using an excrement Occult Blood (OB) kit from the third day. If the mice have the phenomena of reduced activity, lassitude, increased defecation frequency, loose stools, even bloody stools and the like, the successful establishment of the acute ulcerative colitis model of the mice is prompted, and each group of mice can recover normal drinking water.

(2) Pharmaceutical intervention

Mice were freely drunk purified water, and gavage was timed according to table 2 daily for 1 time for 14 days. The environment is kept at a constant temperature of 37 ℃, the air humidity is 60-70%, the animals can eat freely and light for a 12-hour period, and the animal rooms are kept clean and quiet.

3. Index detection

Drug intervention samples were collected on day 14 with fasting and no water deprivation for 24 hours prior to collection. Standing at room temperature for 20min after serum is collected, centrifuging at 3000r/min for 10min, collecting the upper layer serum, and storing at-20 deg.C; mice were dissected and thymus, spleen and colon tissues were collected.

(1) Evaluation of therapeutic effect of Phellinus pinicola total terpenes on ulcerative colitis

1) Disease Activity Index (DAI) score;

weighing and recording the weight change of the mice at fixed time every day, observing and recording the conditions of the food intake, the water intake, the mental state, the hair glossiness, the stool characteristics and the like of the mice, detecting and recording the bloody stool conditions by using an excrement Occult Blood (OB) kit, and scoring the mice by Disease Activity Index (DAI) according to the formula of DAI (weight fraction + stool characteristics score + stool occult blood score)/3, wherein the DAI scoring standard is shown in a table 3.

TABLE 3 DAI scoring criteria

Detection method of fecal Occult Blood (OB) kit: firstly, sampling, opening a cover on the front side of the test card, digging 10-15 mg of a fecal sample by using a sampling wood stick, smearing the fecal sample in a grid of a window A or a window B of the test card, and then closing the cover. Secondly, the back of the test card faces upwards, and the seal cover printed with Development Window is opened. And thirdly, respectively dripping one drop of color developing agent A on the left side and the right side in the square grid, and respectively dripping one drop of color developing agent B after the reagent is completely permeated. Fourthly, after the color developing agent B is added, the interpretation is finished within 2 min.

Interpretation of the test results: (ii) purple color is produced immediately upon addition of developer B, reported as (4 +); ② when adding color reagent B, purple color is generated within 10s, reported as (3 +); ③ when the color-developing agent B is added, purple color is generated within 1min, and the report is (2 +); fourthly, after the color developing agent B is added, purple is gradually generated within 1-2 min and is reported as (1 +); after the color reagent B is added, no purple color reaction is generated within the interpretation time, and the result is reported as negative (-).

2) Colonic mucosal injury index (CMDI) score

The outer wall of the colon, adipose tissue and mesentery were removed, the colon was dissected along the longitudinal axis of the mesentery edge, the wall and contents were washed with normal saline, the Colonic lesions were observed, and the Colonic mucosal lesion index (CMDI) was scored according to table 4.

TABLE 4 CMDI scoring criteria

3) Histopathological observation of colon

Paraffin sections were prepared from mouse colon tissue and stained with Hematoxylin-eosin (H & E), and histopathology was observed under a microscope.

4) Length of colon

Mouse colon tissue was taken and its length measured and recorded.

(2) Mechanism research on treatment of ulcerative colitis by using phellinus pinicola (L.) Aoshima total terpenes

1) Regulation of mouse immune system by fomes fomentarius total terpenes

Dissecting a mouse, respectively taking out the thymus and the spleen, observing and recording the color, the size and the shape of the viscera, weighing, respectively calculating the thymus index and the spleen index according to a formula of viscera index (%) -viscera wet weight (g)/mouse body weight (g) multiplied by 100%, respectively comparing the results with a normal group and a model control group, and judging whether the phellinus pinicola rupr total terpenes treat ulcerative colitis by activating immune regulation.

2) Effect of Fomitopsis pinicola Total terpenes on mouse inflammatory factors

And detecting the contents of IL-6, IL-1 beta and TNF-alpha in the serum of the mouse, comparing the results with a normal group and a model control group respectively, and judging whether the total terpenes of the Fomitopsis pinicola pall can relieve the ulcerative colitis by inhibiting the expression of inflammatory factors.

3) Effect of Fomitopsis pinicola Total terpenoids on liver function in mice

Detecting the content of glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) in the serum of the mouse, and calculating the ratio (DRR) of the AST to the ALT; and comparing the results with a normal group and a model control group respectively to judge whether the total terpenes of the fomes fomentarius affect the metabolic function of the liver.

Second, statistical treatment

The data are analyzed by SPSS 25.0 software, the result is represented by X +/-S, the analysis of the difference of significance among groups is carried out by Duncan method, the multiple comparison result is marked by alphabets, A-Z represents that P is less than 0.01, and a-Z represents that P is less than 0.05.

Third, results and analysis

1. Disease Activity Index (DAI) score;

body weight changes during the 14 day period of drug intervention are shown in figure 1. Compared with a normal control group, the body weights of the model group 1 and the model group 2 are obviously reduced, which indicates that the establishment of the mouse ulcerative colitis model is successful. Compared with the model group 1, the FPKc group has higher body weight, and the body weight is increased and is related to drug intervention rather than the effect of sunflower seed oil. Compared with the model group 2, the weight of the SASP group is obviously increased, and the SASP is suggested to be capable of relieving the weight reduction trend. The results jointly show that the established UC model is stable, the curative effect of the medicament on ulcerative colitis can be effectively evaluated, the fomes fomentarius total terpene can effectively relieve the weight loss caused by UC, and the effect is equivalent to that of the clinical commonly used medicament SASP.

The stool occult blood test by OB kit is shown in FIG. 2. No pictures were provided for the bloodless stool condition in the normal group of mice. As can be seen from fig. 2, the degree of hematochezia of model group 1 and model group 2 already shows a higher degree of severity; compared with the model group 1, the fecal occult blood degree of the FPKc group is obviously reduced, and compared with the model group 2, the fecal occult blood condition of the SASP group is relieved; the fecal occult blood level in FPKc group was lower than that in SASP group. In conclusion, the relieving effect of the phellinus pinicola total terpene on the hematochezia is better than that of the clinical commonly used medicine SASP.

2. Colonic Mucosal Damage Index (CMDI) score

Removing impurities such as adipose tissues and mesentery on the outer wall of the colon, dissecting the colon along the longitudinal axis of the mesentery edge, and cleaning the intestinal wall by using normal saline, wherein the colon tissues of the normal mouse are compact and smooth; the colon of the mouse in the model group 1 and the mouse in the model group 2 is seriously hyperemic and edematous, and the mucous membrane surface has large-area ulcer; the FPKc and SASP groups showed a reduction in colonic edema, congestion, and mucosal ulceration. The FPKc group showed mild hyperemia and edema, and no heavy adhesion to the intestinal wall epithelium compared to the SASP group. The results of the inflammatory injury scores for each group are shown in table 5: the scores of the model group 1 and the model group 2 are obviously increased compared with the normal control group, and the model group have extremely obvious difference (P is less than 0.01), which shows that the mouse ulcerative colitis model is successfully established. Compared with the model group 1, the colon injury index of the mice in the FPKc group is suddenly reduced (P is less than 0.01), which indicates that the FPKc has extremely obvious effect on restoring the colon injury. Compared with model group 2, the inflammatory injury score of the SASP group is significantly reduced (P <0.01), and the therapeutic effect of the SASP on UC is verified. In conclusion, the relieving effect of the phellinus pinicola total terpene on damage of the colon mucosa is better than that of a clinical common medicament SASP.

Table 5 colitis mouse colon injury index (X ± S, n ═ 10)

Note: different letters represent significant differences between groups, a to Z represent P < 0.01.

3. Histopathological observation of colon

After HE staining is carried out on the paraffin section of the colon tissue of the mouse, the colon tissue of the mouse in a normal group can be observed, and the crypt structure of the colon tissue of the mouse in a normal group is clear and visible; the colon crypt structures of the mice in the model group 1 and the model group 2 are completely destroyed and are in a sieve-leakage shape, so that the colon crypt structures become focus of ulcer; the crypt structure in the colon tissue of the mice in the FPKc group is gradually recovered, and the inflammation degree is obviously reduced compared with that of the model group 1; restored crypt structures were also observed in colon tissues of mice in the SASP group, with a greater number of crypts than in model group 2. In conclusion, the phellinus pinicola total terpenes and SASP can relieve the ulcer of the colon part of the mouse and gradually recover the normal physiological structure of the mouse.

4. Effect of Fomitopsis pinicola on Colon Length in mice with ulcerative colitis

Removing impurities such as adipose tissue and mesentery on the outer wall of colon, dissecting the colon along the longitudinal axis of mesentery edge, washing the colon wall with normal saline, and measuring the length of colon, wherein the result is shown in figure 4 (left), the result of statistical analysis on the length of colon of each group of mice is shown in figure 4 (right), the colon of normal control group mouse is 12.05 + -0.76 cm, the colon of model group mouse is significantly shortened (P <0.05) to 10.18 + -0.57 cm and 9.46 + -0.98 cm; the colon length of the FPKc group is 11.58 +/-0.62 cm, is obviously increased compared with that of the model group 1, and has no difference with that of the normal group mice; the colon length of mice in the SASP group is 10.79 +/-0.91 cm, the colon length is also obviously increased compared with that of the model 2 group, but the colon length is slightly lower than that of the normal group, and the differences are statistically significant (P < 0.05). In conclusion, the fomes fomentarius total terpene and the SASP can both relieve colonic inflammation and gradually recover the length of the colon, and the effect of the fomes fomentarius total terpene is superior to that of a clinical common medicament SASP.

5. Influence of Fomitopsis pinicola on immune organs of mice with ulcerative colitis

Compared with a normal control group, the spleen index and the thymus index of the mouse in the model group 1 are obviously increased, the difference has statistical significance (P is less than 0.05), and the organ index of the immune organ of the mouse in the model group 2 is not obviously different. Compared with the model group 1, the spleen index and the thymus index of mice in the FPKc group are obviously reduced, and the difference is statistically significant (P <0.05), but the spleen index and the thymus index of mice in the SASP group are not statistically different (P >0.05) compared with the model group 2. In conclusion, the relieving effect of the phellinus pinicola pall total terpene on inflammation is better than that of the clinical commonly used medicine SASP.

TABLE 6 visceral index of each group of mice

Note: different letters represent significant differences between groups, a to Z represent P <0.01, and a to Z represent P < 0.05.

6. Influence of Fomitopsis pinicola on inflammatory cytokines in mice with ulcerative colitis

After 14 days of drug gavage, the content of inflammatory factors in mouse serum was measured, and the results are shown in FIG. 5 (a). The content of IL-6 in the serum of a mouse in a normal group is 63.21 +/-1.49 pg/mL, the model group 1 is close to the normal group, and the sunflower seed oil is presumed to influence the expression of IL-6 in the serum to a certain extent, but the content of the inflammatory factor in the serum is reduced to 50.31 +/-1.13 pg/mL after FPKc administration, so that the significant difference (P <0.05) is realized, and the result shows that the total terpenes of Fomitopsis pinicola can significantly reduce the expression of IL-6 in the serum of the UC mouse and effectively relieve the inflammatory reaction. The IL-6 content in the serum of the model group 2 mouse is obviously increased (P <0.05), the IL-6 content in the serum is obviously reduced (P <0.05) after SASP is infused, and the effectiveness of clinical drugs is verified.

The effect of the drug on the inflammatory cytokine IL-1. beta. in serum is shown in FIG. 5 (b). The content of IL-1 beta in the serum of the mice in the normal group is 63.52 +/-2.04 pg/mL, the content of the model group 1 and the content of the model group 2 are respectively increased to 74.18 +/-2.52 pg/mL and 77.66 +/-1.82 pg/mL, which are obviously higher than that in the normal group (P <0.05), and the success of building the UC model is proved; after FPKc and SASP are respectively administrated, the content of IL-1 beta in the serum of the mouse is reduced to 69.97 +/-1.03 pg/mL and 67.04 +/-1.55 pg/mL, the difference with a model group is obvious (P is less than 0.05), and the level is restored to a normal level.

The results of the effect of the drug on the inflammatory cytokine TNF-. alpha.in serum are shown in FIG. 5 (c). The content of TNF-alpha in the serum of the normal group mouse is 82.08 +/-2.87 pg/mL, the content of the model group 1 and the content of the model group 2 are respectively increased to 101.78 +/-1.72 pg/mL and 101.68 +/-1.28 pg/mL, which are obviously higher than that of the normal group (P <0.05), and the UC model is successfully established; after the FPKc is drenched, the content of TNF-alpha in the blood serum of the mouse is 96.34 +/-2.08 pg/mL, is remarkably reduced compared with a model group (P is less than 0.05), and tends to be a normal level, while the content of TNF-alpha in the blood serum of the mouse is hardly influenced by the SASP.

In conclusion, the down-regulation efficacy of the phellinus pinicola rupr total terpenes on inflammatory factors in UC mouse serum is superior to that of SASP which is a clinical common medicament.

7. Influence of Fomitopsis pinicola on liver metabolic function of mice with ulcerative colitis

Under normal conditions, ALT (glutamic-pyruvic transaminase) is mainly present in liver cytoplasm, overflows from cells when liver cells are slightly diseased and the permeability of cell membranes is increased, AST (glutamic-oxalacetic transaminase) is mainly present in mitochondria of the cytoplasm, when the liver cells are seriously damaged and die, the AST is released, when a human body is affected by external invasion to cause the metabolic function of the liver to be damaged, the content of the AST and ALT in serum is greatly increased, and the larger the ratio of the AST to the ALT is, the more serious the degree of the damaged liver function is. As can be seen from FIG. 6, the mice in the model group all had different degrees of liver damage compared to the normal control group, and the AST/ALT ratios in the serum of the mice both tended to decrease and gradually tended to the normal group after administration of FPKc and SASP. The result shows that the Fomitopsis pinicola total terpene and the clinical commonly used SASP can relieve the liver injury caused by the ulcerative colitis.

In conclusion, the phellinus pinicola pall total terpenes can regulate the content of inflammatory factors IL-6, IL-1 beta and TNF-alpha in serum by activating immunoregulation, repair liver injury, gradually improve clinical symptoms of ulcerative colitis, such as relieving hematochezia and weight reduction, recover the normal physiological structure of colon and enable the length of colon to be normal.

Claims

1. The extraction method of the fomes fomentarius extract for treating ulcerative colitis is characterized in that: the method specifically comprises the following steps:

the drying temperature in the step 1 is 45-60 ℃;

and 2, mixing the product obtained in the step 1 with an organic solvent according to the weight ratio of 1 g: uniformly mixing the materials in a ratio of 20-40 mL to obtain a mixture; performing ultrasonic extraction on the mixture to obtain an ultrasonic treatment mixture, and performing suction filtration and concentration on the ultrasonic treatment mixture to obtain a fomes fomentarius extract;

the organic solvent in the step 2 is ethanol; the volume fraction of the ethanol in the step 2 is 70-90%; in the step 2, the ultrasonic power of ultrasonic treatment is 280-360 w; the ultrasonic time is 45-75 minutes;

step 3, adding deionized water which is 10-20 times of the weight of the fomes fomentarius extract into the fomes fomentarius extract obtained in the step 2, stirring at 45 ℃ until the fomes fomentarius extract is completely dissolved, then adding a sodium bicarbonate solution with the concentration of 5% into the solution, adjusting the pH of the system to be 7-8, stirring until the fomes fomentarius extract is completely dissolved, and concentrating under reduced pressure to obtain the fomes fomentarius extract;

step 4, mixing the fomes fomentarius extract obtained in the step 3 with ethanol according to the ratio of 1 g: mixing and dissolving 10-20 mL of the extract in a ratio to obtain an ethanol solution of the Fomitopsis pinicola extract, performing silica gel column chromatography on the ethanol solution of the Fomitopsis pinicola extract under the condition that the column pressure is-0.3 Mpa, eluting by using chloroform as an eluent, collecting fractions in the elution process, concentrating and drying the collected fractions to obtain the Fomitopsis pinicola total terpene.