Extract capable of recovering colon histopathologic injury and preparation method thereof

Abstract

The invention discloses a preparation method of an extract capable of recovering colon histopathologic injury, which comprises the following steps: taking purple dendrobium, adding pure water, carrying out wet crushing, decocting after crushing, cooling, filtering, concentrating to obtain purple dendrobium primary pulp, sterilizing, adding a proper amount of xylitol and purified water, blending, canning, and carrying out secondary sterilization, thereby obtaining the extract capable of recovering colon histopathologic injury. The extract is obtained. The extract disclosed by the invention can be used for effectively recovering weight loss caused by constipation, shortening the time of first black excrement, increasing the defecation frequency and the water content of excrement, improving the propulsion rate of small intestines and recovering pathological injury of colon tissue.

Description

Technical Field

[0001] The present invention relates to the technical field of extract synthesis, in particular to an extract capable of restoring pathological damage of colon tissue and a preparation method thereof. Background Art

[0002] With changes in modern society's lifestyles and dietary patterns, the number of people experiencing slow-transmission constipation is on the rise. Constipation is a global gastrointestinal disease that is increasingly affecting more and more people worldwide. It is more common among women, the elderly, and people of lower socioeconomic status. Prolonged constipation can easily lead to adverse symptoms such as hemorrhoids, anal tears, and prolapse, or damage colon tissue. Severe cases may also induce malignant diseases such as colon cancer or rectal cancer, increasing not only the financial burden but also the psychological burden on patients, leading to a decline in their quality of life. Summary of the Invention

[0003] To this end, the present invention provides a preparation method of an extract capable of restoring pathological damage to colon tissue. The preparation method comprises the following steps: taking purple dendrobium, adding pure water, crushing by wet method, decocting after crushing, filtering after cooling, and then concentrating to obtain purple dendrobium pulp, sterilizing, adding appropriate amounts of xylitol and purified water for preparation, canning and then sterilizing again to obtain the extract.

[0004] Furthermore, the decoction temperature is 96±4° C., and the decoction time is 2 to 3 hours.

[0005] Further, the mixture was cooled to below 50°C and filtered.

[0006] Furthermore, the added amount of xylitol is 2% to 4% of the weight of the purple dendrobium pulp, and the added amount of purified water is 1% to 3% of the weight of the purple dendrobium pulp.

[0007] The invention also discloses application of the extract, which is used for restoring colon tissue pathological damage.

[0008] The invention also discloses another application of the extract, which is used for treating constipation.

[0009] The beneficial effects of the present invention are that the extract of the present invention can effectively restore weight loss caused by constipation, shorten the time to first black stool, increase defecation frequency and stool water content, improve small intestinal propulsion rate and restore colon tissue pathological damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The effect of each test group on the apparent defecation indexes of constipated mice;

[0011] Figure 2The effects of each test group on the colon tissue morphology of constipated mice (a is the normal group; b is the constipation group; c is the mosapride group; d is the group in Example 1). DETAILED DESCRIPTION

[0012] Example 1

[0013] A method for preparing an extract capable of restoring pathological damage to colonic tissue comprises the following steps: taking inspected raw material purple dendrobium, adding purified water (material:liquid mass ratio = 1:5), pulverizing by wet method, boiling at 96°C for 2 hours, filtering after the temperature drops below 50°C, and then concentrating to obtain purple dendrobium pulp, sterilizing at 100°C for 30 minutes, and adding xylitol and purified water to the pulp, wherein the amount of xylitol added is 3% by weight of the purple dendrobium pulp and the amount of purified water added is 2% by weight of the purple dendrobium pulp. After canning, the extract is sterilized again to obtain the extract capable of restoring pathological damage to colonic tissue.

[0014] Test Method

[0015] 1. Establishment of experimental mouse model and drug administration

[0016] Mice were housed under SPF conditions at (24±1)°C, relative humidity 50%-55%, and a 12-hour light and dark cycle. They were acclimated for 7 days, with body weight and food intake recorded daily. After 1 week of acclimation, the mice were randomly divided into 6 groups of 12 animals each, namely:

[0017] Normal group (CK): daily infusion of normal saline, normal diet: 0.25mL normal saline + 0.25mL normal saline

[0018] Constipation group (LOP): Loperamide hydrochloride: 0.25 mL of loperamide + 0.25 mL of normal saline

[0019] Mosapride group (MOS): (loperamide hydrochloride + mosapride citrate tablets 2.5 mg / kg: 0.25 mL loperamide + 0.25 mL drug.

[0020] Example 1 Group (DP): 800 mg / kg of Dendrobium officinale puree.

[0021] This animal experiment used a simultaneous modeling and drug administration approach. All mice in the blank group, except the control group, were gavaged with a 10 mL / kg loperamide suspension dissolved in normal saline once daily for 14 consecutive days to establish an STC model. All groups, except the model group, were simultaneously gavaged with the corresponding medication.

[0022] 2. Mouse defecation experiment

[0023] During the experiment, the weight and feces of the mice were monitored every other day. Feces were collected and photographed to record the appearance of the feces. At the end of the animal experiment, fecal samples were collected for further analysis. On the 14th day of modeling and administration, the weight of each mouse was measured. The mice were fasted overnight starting at 5 pm with free access to water. After 16 hours, the mice were transferred to clean, empty cages, one per cage, with free access to food and water. Red ink was administered orally: 6 grams of phenol red was added to 100 mL of 1% carboxymethylcellulose solution, stirred continuously, and maintained at 37°C. The time when each mouse first excreted red feces was recorded.

[0024] Defecation metrics for each mouse were recorded, including the time of first onset of black stool, total weight, and number of stools. Fecal samples were divided into three equal parts for assessment of fecal water content, 16S rRNA sequencing, and short-chain fatty acids (SCFA).

[0025] Feces were collected from each mouse within 3 hours, the number of fecal pellets was recorded, and the feces were dried in a 90°C oven for 3 hours. The fecal moisture content was calculated according to formula (1):

[0026]

[0027] 3 Small intestinal motility test

[0028] After collecting the feces of the mice, 0.2 mL of blood was collected from the eyeballs. After standing for 1 hour, the blood sample was centrifuged at 4000 r / min for 15 minutes to obtain serum. Using 10% chloral hydrate, the mice were anesthetized, dislocated and killed. The serum, gastrointestinal tissues and feces were stored at -80 ° C for further study. At 3 pm on the 14th day, the mice were fasted overnight with free access to water. After 16 hours, 0.3 mL of 10% activated carbon suspension was gavaged, and the mice were killed by dislocation of the neck 30 minutes later. The mesentery was separated, and the small intestine was completely removed. The small intestine was flattened on filter paper without traction. The distance from the pylorus to the end of the carbon powder propulsion and the total length of the small intestine were measured and recorded, and the intestinal transit rate of the mice was calculated according to formula (2):

[0029]

[0030] In addition, blood samples were collected from the orbit before intestinal dissection, and the separated serum was used for gastrointestinal hormone determinations such as gastrin and endothelin.

[0031] Colon histopathological observation

[0032] 1) Hematoxylin-eosin staining

[0033] Mouse colons were fixed with 10% formalin. After 48 hours, the fixed tissue was fixed and washed in running water for 4 hours. The samples were embedded in paraffin, cut into 5 μm slices, and then unfolded in a warm water culture dish. Finally, the samples were stained with H&E. The morphological characteristics of the samples were observed using light microscopy. The tissues were observed at 400× magnification and images were captured.

[0034] 2) Immunohistochemistry (IHC) analysis

[0035] Fix the tissue and wash it in running water for 4 hours. Then embed the sample in paraffin and cut into 5 μm sections. The sections were then unfolded in a warm water dish. Immunohistochemical analysis of colonic tissue was performed using HRP-conjugated goat anti-rabbit IgG. The staining was observed under a microscope, and micrographs were taken.

[0036] 3) TUNEL staining

[0037] Colonocyte apoptosis was detected using a TUNEL apoptosis detection kit. Fix the tissue and wash it in running water for 4 hours. Dewax the sections and rinse them three times with PBS for 5 minutes each. Apply 50 μL of the TUNEL mixture to the air-dried sections, cover with a coverslip, and incubate at 37°C in the dark for 1 hour. After washing with PBS, air-dry the sections and counterstain with 50 μL of DAPI. Store at 37°C in the dark for 30 minutes. Wash again with PBS, remove the sections, and apply a fluorescence quencher to mount them on slides. Finally, observe the staining under a microscope and photograph.

[0038] Results and Analysis

[0039] 1. Effects on the defecation ability of mice

[0040] Table 1 Defecation of mice during modeling and treatment

[0041]

[0042] Note: Different lowercase letters indicate significant differences between different groups (P<0.05); different uppercase letters indicate significant differences between different periods (P<0.05).

[0043] As shown in Table 1, before modeling, there was no significant difference in the number of defecations of mice in each group (P>0.05); in the first week of modeling, except for the normal group, the average number of feces of mice in each group decreased; in the second week of modeling, except for the normal group, the average number of feces of mice in each group decreased significantly compared with the previous week; in the first week of treatment, except for the normal group and the constipation group, the number of feces of mice in each treatment group increased significantly compared with the previous week, and the number of feces of mice in the constipation group continued to decrease; in the second week of treatment, except for the normal group and the constipation group, the number of feces of mice in the mosapride group and the Example 1 group increased significantly, and the number of feces was comparable to that of the normal group. This shows that the mosapride group and the Example 1 group can effectively improve the degree of constipation in mice.

[0044] 2. Effects on the apparent defecation indicators of constipated mice

[0045] Depend on Figure 1 A shows that the time of first black stool in the constipation group mice was significantly longer than that in the blank group mice (P<0.05), indicating that loperamide hydrochloride inhibited gastrointestinal motility, thus leading to constipation. After intervention with Dendrobium officinale puree, the time of first black stool was shortened compared with the constipation group, which can effectively promote intestinal motility. Figure 1 B shows that the number of stool particles within 5 hours is also consistent with the first black stool. Figure 1 C shows that compared with the blank group, the water content of the feces of the mice in the constipation group was significantly reduced (P<0.01). After oral administration of Dendrobium officinale puree, the water content of the feces of the mice increased significantly (P<0.01). Figure 1 As shown in D, the small intestinal propulsion rate of constipated mice was significantly lower than that of the normal group, indicating that the small intestinal propulsion capacity of constipated mice is significantly reduced. This reduced small intestinal propulsion capacity leads to increased retention time of contents in the intestine, increased water content absorbed by the intestine, decreased fecal water content, dry and hard feces that are difficult to pass, and more severe constipation. After the constipated mice were treated with Dendrobium candidum puree, the small intestinal propulsion rate of the mice was significantly improved.

[0046] 3. Effects of morphology of colon tissue in constipated mice

[0047] like Figure 2 As shown, the normal group showed numerous, neatly arranged microvilli, with tight intercellular spaces, no significant epithelial cell shedding, and normal cell morphology and clear structure. In the constipation group, the small intestinal epithelial cells were severely ruptured, atrophied, and fractured, with reduced size, incomplete goblet cells, and truncated, mostly absent villi. Compared with the constipation group, the mosapride group and the Example 1 group showed improved villus morphology and an increase in the number of goblet cells. In particular, the Example 1 group showed intact, neatly arranged small intestinal epithelial cells with significantly reduced intercellular spaces. This suggests that Dendrobium officinale puree can alleviate the colonic histopathological damage caused by Lop-induced constipation in mice.

[0048] Table 2 Effects of each experimental group on organ index

[0049]

[0050] As shown in Table 2, the coefficients of various organs in the constipation group were lower than those in the blank group (P<0.05). Compared with the coefficients of various organs in the constipation group, the liver, kidney, and spleen in the Example 1 group all showed significant increases (P<0.05), indicating that the Dendrobium officinale puree has an improving effect on constipation.

[0051] The technical solutions provided by the present invention are described in detail above. For those skilled in the art, according to the ideas of the embodiments of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for preparing an extract capable of restoring pathological damage to colon tissue, characterized in that: The method comprises the following steps: taking purple dendrobium, adding pure water, crushing by wet method, decocting after crushing, filtering after cooling, concentrating to obtain purple dendrobium pulp, sterilizing, adding appropriate amount of xylitol and purified water for preparation, canning and sterilizing for secondary time to obtain the extract.

2. The method for preparing an extract capable of restoring pathological damage to colon tissue according to claim 1, characterized in that: The decoction temperature is 96±4° C., and the decoction time is 2 to 3 hours.

3. The method for preparing an extract capable of restoring pathological damage to colon tissue according to claim 1, characterized in that: Cool to below 50°C and filter.

4. The method for preparing an extract capable of restoring pathological damage to colon tissue according to claim 1, characterized in that: The added amount of xylitol is 2% to 4% of the weight of the purple dendrobium pulp, and the added amount of purified water is 1% to 3% of the weight of the purple dendrobium pulp.

5. The use of the extract prepared by the method according to claim 1, characterized in that The extract was used to restore colon histopathological lesions.

6. Use of the extract prepared by the method according to claim 1, characterized in that, The extract is used to treat constipation.

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