CN115400160B

CN115400160B - Application of gleditsia sinensis endosperm extract in preparation of medicine for treating ulcerative colitis

Info

Publication number: CN115400160B
Application number: CN202211033883.7A
Authority: CN
Inventors: 王祥培; 吴红梅; 徐锋; 丁芹; 张梅; 秦苹; 方镕泽
Original assignee: Guizhou Minzu University
Current assignee: Guizhou Minzu University
Priority date: 2022-04-27
Filing date: 2022-08-26
Publication date: 2023-06-20
Anticipated expiration: 2042-08-26
Also published as: CN115400160A

Abstract

The invention provides an application of a Chinese honeylocust endosperm extract in preparing medicaments for treating ulcerative colitis, which comprises the following steps of (1) taking Chinese honeylocust endosperm, pulverizing into powder, sieving, slowly adding into boiling water, stirring while adding, uniformly mixing, boiling with small fire, and concentrating to a certain concentration to obtain Chinese honeylocust endosperm liquid medicine. (2) Pulverizing endosperm of Gleditsia sinensis, sieving, adding into water, and heating to obtain product A; pulverizing fructus Gleditsiae Abnormalis endosperm, sieving, adding above product A, stirring, mixing, decocting with small fire, and concentrating to obtain fructus Gleditsiae Abnormalis endosperm liquid medicine. The prepared gleditsia sinensis endosperm extract has the effects of moistening dryness, relaxing bowels, dispelling wind, relieving swelling, strengthening spleen, supplementing qi, eliminating dampness and the like, thereby playing a role in treating ulcerative colitis. It also has certain improving effect on lung, liver and kidney injury.

Description

Application of gleditsia sinensis endosperm extract in preparation of medicine for treating ulcerative colitis

Technical Field

The invention relates to a new application of a Chinese honeylocust endosperm extract, in particular to an application of the Chinese honeylocust endosperm extract in preparing a medicament for treating ulcerative colitis.

Background

The gleditsia sinensis endosperm is gleditsia sinensis lamGleditsia sinensisLam.) or fructus Gleditsiae AbnormalisGleditsia japonica var. delavayi(Franch.) A processed dry product of the endosperm of L.C.Li) seed is obtained by seedBoiling in water, peeling off seed coat, collecting endosperm, and drying; or collecting fresh seeds, peeling off seed coat, collecting endosperm, and drying. Belongs to high-energy, high-carbohydrate, low-protein and low-fat foods. And contains vegetable dietary fiber. The book of materia medica book of changes: for white meat in the nucleus, it is used as a lung-treating drug. The 'white meat in the core' is endosperm of the seeds of Chinese honeylocust. At present, the research on the chemical composition and pharmacological activity of the gleditsia sinensis endosperm is less, and particularly, the research on ulcerative colitis is not reported in the related data.

Ulcerative Colitis (UC) is a chronic, nonspecific inflammatory disease of the colon and rectum of which the etiology is not yet clear, and the lesions are limited to the mucosa and submucosa of the large intestine. Lesions are mostly located in the sigmoid colon and rectum, but can also extend to the descending colon, even the entire colon. The disease course is long and the attack is repeated frequently. The disease is seen at any age, but is most seen between 20 and 30 years old. The etiology of ulcerative colitis has remained unknown to date. The genetic factors may be in a certain position. Psychological factors play an important role in disease progression, with the originally present pathological mental states such as depression or social distance being significantly improved after colectomy. Ulcerative colitis is considered an autoimmune disease.

The onset of inflammatory bowel disease is currently thought to be the result of foreign substances causing interactions of host response, genes and immune effects. The existing treatments are generally classified into drug treatment and surgical treatment. The medicine treatment is mainly western medicines, the sulfasalazine salicylic acid preparation is mainly therapeutic medicines, such as Ai Disha, mesalamine and the like, and the corticosteroid is prednisone or dexamethasone usually, but long-term hormone maintenance is not considered to prevent relapse at present. Hydrocortisone or dexamethasone can be used for intravenous drip in the acute attack stage, but whether hormone should be continuously used in the chronic stage is still different, and long-term use is not claimed in most cases because of certain side effects. The traditional Chinese medicine for treating diarrhea type ulcerative colitis can be used for treating the diarrhea type ulcerative colitis by using traditional Chinese medicine, has ideal effect, small side effect and high safety.

Disclosure of Invention

In order to solve the technical problems, the invention provides application of the gleditsia sinensis endosperm extract in preparing medicaments for treating ulcerative colitis, and solves the problem of how to extract the gleditsia sinensis endosperm extract to treat ulcerative colitis. In order to achieve the above purpose, the invention is realized by the following technical scheme:

use of fructus Gleditsiae Abnormalis endosperm extract in preparing medicine for treating ulcerative colitis is provided.

The application of the Chinese honeylocust endosperm extract in preparing the medicament for treating ulcerative colitis comprises the following steps of: pulverizing fructus Gleditsiae Abnormalis endosperm, sieving, slowly adding into boiling water under stirring, decocting with small fire, and concentrating to obtain extract or soft extract.

In particular to application of the saponin endosperm extract in preparing medicaments for treating ulcerative colitis, and the preparation method of the saponin endosperm extract comprises the following steps: pulverizing fructus Gleditsiae Abnormalis endosperm, sieving with 8-500 mesh sieve, slowly adding into 2-12 times of boiling water, stirring, mixing, decocting with small fire for 10-60min, and concentrating to obtain fluid extract or soft extract.

The application of the Chinese honeylocust endosperm extract in preparing the medicament for treating ulcerative colitis comprises the following steps of:

a) Pulverizing endosperm of Gleditsia sinensis, sieving, adding into water, and heating to obtain product A;

b) Pulverizing fructus Gleditsiae Abnormalis endosperm, sieving, adding above product A, stirring, decocting with small fire, and concentrating to obtain fluid extract or soft extract.

5. Use of the endosperm extract of gleditsia of claim 4 in the manufacture of a medicament for the treatment of ulcerative colitis, wherein: the preparation method of the gleditsia sinensis endosperm extract comprises the following steps:

a) Pulverizing fructus Gleditsiae Abnormalis endosperm, sieving with 8-500 mesh sieve, adding into 3-12 times of water, and heating for 2-10 min to obtain product A;

b) Pulverizing fructus Gleditsiae Abnormalis endosperm, sieving with 8-500 mesh sieve, adding into above product A under stirring, decocting with small fire for 10-60min, and concentrating to obtain fluid extract or soft extract.

Use of gleditsia endosperm in the preparation of a medicament for the treatment of ulcerative colitis is provided.

A medicament for treating ulcerative colitis, which comprises a saponaria endosperm extract or saponaria endosperm.

A medicine for treating colitis ulcerosa comprises fructus Gleditsiae Abnormalis endosperm extract or fructus Gleditsiae Abnormalis endosperm as main active ingredient.

The application uses the saponaria endosperm to treat ulcerative colitis, and the saponaria endosperm can inhibit the excessive activation of TLR4/NF- κB/NLRP3 signal paths, down regulate the expression of TLR4, NF- κB-p65, NLRP3 and Caspase-1 proteins and genes, reduce the release of IL-1 beta, IL-6, IL-17 and TNF-alpha pro-inflammatory factors, and play a role in treating ulcerative colitis.

The invention has the beneficial effects that:

the gleditsia sinensis endosperm can improve the symptoms of weight reduction, loose stool, hematochezia and the like of patients with ulcerative colitis, has the effects of protecting and repairing colon pathological tissue injury of patients with ulcerative colitis, and inhibiting the release of relevant inflammatory factors, and has the anti-inflammatory effect, thereby inhibiting the occurrence and development of ulcerative colitis. Can also be used for improving lung injury.

Drawings

FIG. 1 mice weight change profile;

fig. 2 mouse DAI index (compared to normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01, representP<0.05; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; dose group in the endosperm of Z gleditsia; g high endosperm dose group of honeylocust fruits);

FIG. 3 is a graph of the colon of a mouse;

fig. 4 colon length statistics (compared to normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01, representP<0.05; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; dose group in the endosperm of Z gleditsia; g-honeylocust endosperm high dose group）；

Fig. 5 mice spleen index change (compared to normal control group: ^△ representation ofP<0.05; comparison to model set: ^** representation ofP<0.01， ^* Representation ofP<0.05; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; dose group in the endosperm of Z gleditsia; g high endosperm dose group of honeylocust fruits);

fig. 6 effect of colon tissue score in mice (compared to normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01， ^* Representation ofP<0.05; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; dose group in the endosperm of Z gleditsia; g high endosperm dose group of honeylocust fruits);

FIG. 7 colon histopathological section (x 100) of mice (C normal group; M model group; Y positive drug mesalamine group; D low dose group; Z medium dose group; G high dose group of saponaria endosperm);

fig. 8 effect of saponaria endosperm on inflammatory factor content in colon tissue of mice (compared to normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01， ^* Representation ofP<0.05; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; dose group in the endosperm of Z gleditsia; g high endosperm dose group of honeylocust fruits);

FIG. 9 is a graph of bands for TLR4, NF-. Kappa.B-p 65, NLRP3, caspase-1 protein and internal reference protein in the colon of mice for each group (C normal group; M model group; Y positive drug mesalamine group; D low dose group; Z medium dose group; G high dose group of saporin endosperm);

FIG. 10 histogram of TLR4, NF-. Kappa.B-p 65, NLRP3 and Caspase-1 protein gray scale expression in mouse colon tissue (compare to normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; z soapDose group in horny endosperm; g high endosperm dose group of honeylocust fruits).

FIG. 11 histogram of TLR4, NF-. Kappa.B-p 65, NLRP3 and Caspase-1 mRNA expression in mouse colon tissue (compared to normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; dose group in the endosperm of Z gleditsia; g high endosperm dose group of honeylocust fruits);

FIG. 12A pulmonary histopathological section (x 100) of mice (C normal group; M model group; Y positive drug mesalamine group; D low dose group; Z medium dose group; G high dose group)

Fig. 13 is a bar graph of ALT, AST, CREA, UREA content in mouse serum (compared to normal control group: ^△△ representation ofP<0.01， ^△ Representation ofP<0.05; comparison to model set: ^** representation ofP<0.01， ^* Representation ofP<0.05; c, normal group; m model group; mesalamine group as a Y positive drug; d low dose group of honeylocust endosperm; dose group in the endosperm of Z gleditsia; g Chinese honeylocust endosperm high dose group

In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention.

Detailed Description

Example 1:

the process comprises the following steps:

pulverizing 100g of fructus Gleditsiae Abnormalis endosperm, sieving with 8-500 mesh sieve, slowly adding into 2-12 times of boiling water, stirring under stirring, mixing, decocting with small fire for 5-60min, and concentrating to obtain fructus Gleditsiae Abnormalis endosperm liquid medicine.

Mixing fructus Gleditsiae Abnormalis endosperm liquid 100g with 80% pregelatinized starch, granulating, sieving, and making into capsule of 1000 pieces.

Efficacy: moistening dryness, relaxing bowels, dispelling pathogenic wind, relieving swelling, invigorating spleen, invigorating qi, and eliminating dampness.

The using method comprises the following steps: is orally taken.

The usage amount is as follows: orally taken 2 granules at a time and 3 times a day.

Example 2:

the process comprises the following steps:

a) Pulverizing 50g of fructus Gleditsiae Abnormalis endosperm, sieving with 8-500 mesh sieve, adding into 3-12 times of water, and heating for 2-10 min to obtain product A;

b) Pulverizing 50g of fructus Gleditsiae Abnormalis endosperm, sieving with 8-500 mesh sieve, adding into above product A under stirring, decocting with small fire for 5-60min, and concentrating to obtain fructus Gleditsiae Abnormalis endosperm liquid medicine.

The using method comprises the following steps: is orally taken.

The inventors conducted a large number of experiments, and the following are studies of the extraction method of the present invention:

1. reagent preparation and Experimental grouping

1. Preparation of medicinal liquid

1.1 one of the preparation of the liquid soap endosperm is: pulverizing fructus Gleditsiae Abnormalis endosperm, sieving (8-500 mesh) to obtain powder, slowly adding into 2-12 times of boiling water, stirring, mixing, decocting with small fire for 5-60min, and concentrating to obtain fructus Gleditsiae Abnormalis endosperm liquid medicine.

1.2 preparation of a liquid soap endosperm formulation two: heating 3-12 times of water of the powder of the Chinese honeylocust endosperm for 2-10 min, taking the powder of the Chinese honeylocust endosperm, stirring while adding, mixing, decocting with small fire for 5-60min, and concentrating to obtain Chinese honeylocust endosperm liquid medicine.

Reagent of the rest

Mesalamine (Losan Pharma GmbH); dextran sodium sulfate (MP company) was formulated as a 2.5% solution prior to use.

Animal for experiment

SPF grade Kunming mice, 4 weeks old, 18-22 g, male, s Bei Fu (Beijing) biotechnology limited, provided [ license number: SCXK (Beijing) 2019-0010, and is fed in a cage to a pharmacological laboratory of the university of Chinese medicine in Guizhou.

Method for molding

Dextran Sodium Sulfate (DSS) powder is prepared into 2.5% concentration DSS solution by using baby haha purified water, and is used for daily free drinking of a mouse model group, a positive medicine group and a high-medium-low dosage group of gleditsia sinensis endosperm, the freshly prepared DSS solution is replaced every two days, and molding lasts for 8 days.

5. Experimental grouping

SPF-grade male Kunming mice were randomly divided into 6 groups of 12. The compositions are respectively a blank group, a model group, a positive medicine mesalamine control group (the administration dosage is 0.52 g/kg), a low-dosage group of gleditsia sinensis endosperm (the administration dosage is 0.3379 g/kg), a medium-dosage group of gleditsia sinensis endosperm (the administration dosage is 0.6758 g/kg) and a high-dosage group of gleditsia sinensis endosperm (the administration dosage is 1.3515 g/kg). The amount of the stomach is 0.2ml/10g, and the blank group is subjected to the same amount of distilled water for the stomach, twice a day and continuously for 10d.

After the last administration, the eyeballs of the mice were taken out for blood collection, the blood of the mice was collected and centrifuged at 12000 r at 4℃for 10 min, and the separated serum was stored in a-80℃refrigerator. Rapidly dissecting abdomen along the center line of abdomen, cutting colon specimen (from anus upwards to cecum), taking out, placing on ice, immediately measuring and recording length thereof with graduated scale, then washing intestinal cavity with physiological saline for 3 times, sucking dry with filter paper, breaking colon along longitudinal axis, observing and recording histological change; then, part of colon is taken and placed in 4% paraformaldehyde for fixation for HE detection; the remaining colon was stored in a-80℃freezer for further use.

2. Medicinal effect of gleditsia sinensis endosperm for preventing and treating ulcerative colitis

1. Effect of Gleditsia sinensis endosperm on the disease Activity index of mice

Following administration of DSS solution, mice were recorded daily for general status and scored for Disease Activity Index (DAI). Dai= (weight loss fraction+stool trait fraction+hematochezia fraction)/3. Specific scoring criteria are shown in table 1, and the results are shown in fig. 1 and 2.

TABLE 1 DAI index scoring criteria

Weight loss (%)	Stool characteristics	Fecal occult blood	Scoring of
				0	Normal state	Normal state	0
1-5	Soft stool for shaping	Blood stool (+)	1
				5-10	Thin stool	++	2
10-20	Thin stool	+++	3
				>20	Serious loose stool	>+++	4

Compared with the normal group, the DAI index of the mice in the model group is extremely obviously increased [ ]P< 0.01). Compared with a model group, the positive medicine mesalamine can reduce the DAI index of UC mice, but has no obvious differenceP0.05), the low dosage of the gleditsia sinensis endosperm can extremely obviously reduce the DAI index of UC micePLess than 0.01), the DAI index of UC mice can be obviously reduced by high dosage in the endosperm of Chinese honeylocustP＜0.05）。

Effect of Gleditsia sinensis endosperm on colon length in mice

After the last administration, the mice were taken out of the eyes and were quickly dissected along the midline of the abdomen, the colon specimen was taken out (from anus up to cecum), and the specimens were taken out and placed on ice, and the length was measured and recorded immediately with a graduated scale, and the results are shown in table 2 and fig. 3 and 4.

Table 2 colon length of mice (' x±s, n=8)

Group of	Colon length (cm)
		Normal group	10.338±0.628
Model group	8.250±0.975 ^△△
		Positive group	9.438±1.074
Low dose group	9.963±0.890 ^*
		Medium dose group	9.563±0.883
High dose group	10.238±0.571 ^**

Note that: comparison to the normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01； ^* Representation ofP<0.05。

As shown in Table 3 and figures 3 and 4, the colon length of the mice in the model group is extremely significantly shortened compared with that in the normal groupP< 0.01). Compared with the model group, the low dosage of the gleditsia sinensis endosperm obviously inhibits the colon shortening of UC micePLess than 0.05), the high dosage of the gleditsia sinensis endosperm can obviously inhibit the colon shortening of UC micePLess than 0.01), the dosage in the endosperm of the gleditsia sinensis and positive medicine mesalazine can inhibit the colon shortening of UC mice, but no obvious difference is causedP＞0.05）。

Effect of Gleditsia sinensis endosperm on mouse spleen index

After the mice were dissected, spleens were weighed, and the spleen index of the mice was calculated, and the results are shown in table 3 and fig. 5.

Table 3 mouse spleen index (' x±s, n=8)

Group of	Spleen index
		Normal group	3.698±0.746
Model group	5.743±1.112 ^△
		Positive group	5.238±1.900
Low dose group	3.571±1.129 ^*
		Medium dose group	3.510±0.614 ^**
High dose group	3.035±0.879 ^**

Note that: comparison to the normal control group: ^△ representation of P<0.05; comparison to model set: ^** representation ofP<0.01； ^* Representation ofP<0.05。

Compared with the normal group, the spleen index of the mice in the model group is obviously increasedP< 0.05). Compared with the model group, the dosage in the endosperm of the Chinese honeylocust and the high dosage extremely obviously reduce the spleen index of the UC micePLess than 0.01), the low dosage of the gleditsia sinensis endosperm obviously reduces the spleen index of UC miceP< 0.05), positive medicine mesalazine can also reduce spleen index of UC mice, but no significant difference is causedP＞0.05）。

Effect of Gleditsia sinensis endosperm on colon histopathological changes in mice

The colon tissue of the mice was observed by HE staining and subjected to histopathological scoring, and the results are shown in Table 4 and FIG. 6, and the pathological section of the colon tissue of the mice is shown in FIG. 7.

Table 4 mouse colon TDI index (' x±s, n=8)

Group of	TDI
		Normal group	0.00±0.000
Model group	1.266±0.493 ^△△
		Positive group	0.600±0.830 ^*
Low dose group	0.332±0.408 ^**
		Medium dose group	0.668±0.625
High dose group	0.000±0.000 ^**

The colon tissue structure of the normal group mice is clear and complete, the mucosal surface is coated with a single layer of columnar epithelium, the morphology of columnar epithelium is normal, the arrangement of large intestine glands in the intrinsic layer is relatively dense, the number of goblet cells is normal, the myofibrillar structure of the myolayer is complete and clear, other obvious pathological changes are not seen, and the histological score is relatively low; the structural integrity of colon tissue of model group is seriously damaged, epithelial cell is necrotized, nucleus is dissolved and inherentLayer cell necrosis, cytoplasmization, cytokinesis, nuclear shrinkage disintegration, myolayer myofiber necrosis, and inflammatory cell infiltration in necrotic areas, mainly single round deeply-stained lymphocytes and rod-shaped nuclear neutrophils, and also fibrous tissue hyperplasia including fibroblast proliferation with oblong nucleus and fibrous cell proliferation with long spindle-shaped nucleus, and the histological score is remarkably increased compared with that of normal groupP<0.01). Compared with model group, the low dose and high dose of the gleditsia sinensis endosperm can reduce the inflammatory cell infiltration degree of colon tissue of UC mice, restore the arrangement of goblet cells and glands and have lower histological scoreP< 0.01); positive medicine improves the necrosis of epithelial cells and lamina propria cells of colon tissue of UC mice, reduces the infiltration degree of inflammatory cells and has lower histological scoreP< 0.05); although the dose in the endosperm of the Chinese honeylocust has improved inflammatory cell infiltration degree, the histological score is lower without obvious differenceP＞0.05）。

Effect of Gleditsia sinensis endosperm on expression of inflammatory factors IL-1 beta, IL-6, IL-17 and TNF-alpha in colon tissue of mice

The contents of inflammatory factors IL-1 beta, IL-6, IL-17 and TNF-alpha in colon tissues of mice were measured according to the method required by the kit, and the results are shown in Table 5 and FIG. 8.

Table 5 groups of mice colon IL-1 beta, IL-6, IL-17, TNF-alpha (' X.+ -. S, n=8)

Group of	IL-1β（pg/mL）	IL-6（pg/mL）	IL-17（pg/mL）	TNF-α（pg/mL）
					Normal group	8.298±0.610	29.910±1.386	8.895±0.624	68.485±7.373
Model group	12.507±0.677 ^△△	38.873±5.018 ^△△	13.501±1.360 ^△△	83.720±3.650 ^△△
					Positive group	10.932±0.747 ^**	28.838±2.560 ^**	10.495±0.797 ^**	73.495±4.644 ^**
Low dose group	9.810±0.871 ^**	31.979±5.076 ^**	11.015±1.497 ^**	75.223±5.314 ^*
					Medium dose group	10.802±1.422 ^**	31.517±3.902 ^**	11.385±1.873 ^**	75.701±10.292 ^*
High dose group	9.977±0.660 ^**	31.396±5.328 ^**	11.802±0.624 ^**	73.771±8.185 ^**

Note that: comparison to the normal control group: ^△△ representation ofP<0.01; comparison to model set: ^** representation ofP<0.01。

Compared with the normal group, the colon tissue of the model group mice has extremely obvious increase of IL-1 beta, IL-6, IL-17 and TNF-alpha contentP< 0.01). Compared with the model group, the low dose, the medium dose, the high dose and the positive medicine mesalazine of the gleditsia sinensis endosperm can obviously reduce the content of IL-1 beta, IL-6 and IL-17 in colon tissues of UC micePLess than 0.01), the high dosage of the gleditsia sinensis endosperm and the positive medicine mesalazine can obviously reduce the content of TNF-alpha in colon tissues of UC micePLess than 0.01), the low and medium doses of the gleditsia sinensis endosperm can obviously reduce the TNF-alpha content in colon tissues of UC miceP＜0.05）。

Investigation of mechanism of action of gleditsia sinensis endosperm in preventing and treating ulcerative colitis based on TLR4/NF- κB/NLRP3 signal path

(1) Effect of Gleditsia sinensis endosperm on TLR4, NF- κB-p65, NLRP3 and Caspase-1 protein expression in mouse colon tissue

Western blotting was performed on colon tissue of mice, and the results are shown in Table 6, FIGS. 9 and 10.

Table 6 expression of Gray values of the mouse colons TLR4, NF-. Kappa.B-p 65, NLRP3 and Caspase-1 proteins of each group (' X.+ -.s, n=5)

Group of	TLR4	NF-κB-p65	NLRP3	Caspase-1
					Normal group	0.485±0.131	0.392±0.152	0.160±0.085	0.141±0.041
Model group	0.976±0.299 ^△△	0.946±0.621 ^△△	0.902±0.211 ^△△	0.727±0.395 ^△△
					Positive group	0.332±0.200 ^**	0.262±0.180 ^**	0.327±0.279 ^**	0.174±0.092 ^**
Low dose group	0.146±0.084 ^**	0.093±0.012 ^**	0.375±0.301 ^**	0.224±0.043 ^**
					Medium dose group	0.282±0.176 ^**	0.141±0.118 ^**	0.388±0.218 ^**	0.376±0.206 ^**
High dose group	0.192±0.074 ^**	0.158±0.130 ^**	0.327±0.330 ^**	0.334±0.125 ^**

Compared with the normal group, the expression of TLR4, NF-kappa B-p65, NLRP3 and Caspase-1 proteins in colon tissues of the model group mice is greatly increasedP< 0.01). Compared with the model group, the low dose group, the medium dose group, the high dose group and the positive medicine mesalazine of the gleditsia sinensis endosperm can greatly reduce TLR4, NF-kappa B-p65, NLRP3 and Caspase-1 protein expression in colon tissues of UC miceP＜0.01）。

(2) Effect of Gleditsia sinensis endosperm on TLR4, NF- κB-p65, NLRP3 and Caspase-1 mRNA expression in mouse colon tissue

The results of RT-PCR detection of colon tissue of mice are shown in Table 7 and FIG. 11.

TABLE 7 mouse colon TLR4, NF-. Kappa.B-p 65, NLRP3 and Caspase-1 mRNA expression profile (' X.+ -.s, n=5)

Group of	TLR4	NF-κB-p65	NLRP3	Caspase-1
					Normal group	0.983±0.255	1.108±0.220	1.164±0.693	1.118±0.207
Model group	2.918±0.651 ^△△	3.609±0.562 ^△△	17.197±7.938 ^△△	4.307±0.686 ^△△
					Positive group	1.391±0.419 ^**	1.321±0.235 ^**	3.394±2.824 ^**	1.153±0.107 ^**
Low dose group	2.155±0.212 ^**	2.878±0.141 ^**	9.691±3.738 ^**	3.352±0.717 ^**
					Medium dose group	1.674±0.264 ^**	2.186±0.061 ^**	6.442±3.297 ^**	2.714±0.646 ^**
High dose group	1.485±0.147 ^**	1.688±0.281 ^**	5.811±3.134 ^**	1.597±0.345 ^**

Compared with the normal group, the expression of TLR4, NF- κB-p65, NLRP3 and Caspase-1 mRNA in colon tissue of the model group is greatly increasedP< 0.01). Compared with the model group, the Chinese honeylocust endosperm, low dose, medium dose, high dose and positive medicine mesalazine can extremely obviously reduce the expression of TLR4, NF-kappa B-p65, NLRP3 and Caspase-1 mRNA in colon tissue of UC miceP＜0.01）。

Conclusion:

the saponaria endosperm can reduce the Disease Activity Index (DAI) of a UC mouse induced by DSS, inhibit the length shortening of colon, reduce the spleen index and the intestinal mucosa injury index, restore the structural integrity of colon tissues, improve the necrosis condition of epithelial cells, lamina propria cells and muscle fibers, reduce the infiltration of inflammatory cells in necrotic areas and inhibit the proliferation of the fibers, and the saponaria endosperm has the effect of resisting ulcerative colitis, and the action mechanism is related to inhibiting the injury of colon inflammation by inhibiting the expression of primary target molecules TLR4, NF-kappa B-P65, NLRP3 and Caspase-1 proteins and genes on a TLR 4/NF-kappa B/NLRP3 signal path, thereby inhibiting the release of IL-1 beta, IL-6, IL-17 and TNF-alpha inflammatory factors.

3. Effects of Gleditsia sinensis endosperm on mouse Lung histopathological changes

After dissection of the mice, the lung tissue was collected and observed by HE staining, and the results are shown in fig. 12.

The lung tissue of the normal group mice has normal pleural structure, thin wall, no connective tissue hyperplasia and thickening, and all levels of bronchi have complete and clear structure, normal epithelial cell morphology, normal pulmonary respiratory alveolar epithelial cell morphology, and no obvious lesion. The bronchi structure of the lung tissue at each level is normal, the bronchus cilium epithelium is orderly arranged, no obvious cell shedding is seen, the alveolus epithelium is normal, the alveolus cavity is slightly or mildly bleeding, the lung interstitium does not see obvious fibrous tissue hyperplasia and inflammatory cell infiltration, and other diseases do not see obvious pathological changes. Compared with the model group, the low dosage of the gleditsia sinensis endosperm, the medium dosage of the gleditsia sinensis endosperm and the high dosage of the gleditsia sinensis endosperm and the positive medicine mesalazine can improve the intraalveolar hemorrhage phenomenon of the UC mice.

Conclusion:

the honeylocust endosperm can improve the bleeding condition of alveolar space in the lung tissue of UC mice, and has the prevention and treatment effect on lung injury.

4. Effect of Gleditsia sinensis endosperm on mouse liver function index

After the last administration, the eyeballs of the mice were taken out for blood collection, and serum was taken out for blood biochemical detection after blood centrifugation, and the results are shown in table 8 and fig. 13.

Table 8 conditions of ALT, AST, CREA, UREA in mouse serum (' x±s, n=5)

Group of	ALT(U/L)	AST(U/L)	UREA(mmol/L)	CREA(μmol/L)
					Normal group	29.020±3.817	132.780±13.942	7.518±1.113	28.800±8.566
Model group	37.580±3.544 ^△	166.500±18.120 ^△△	9.596±0.786 ^△△	33.840±12.073
					Positive group	37.860±4.245	137.300±4.287 ^*	10.818±1.195	30.660±10.078
Low dose group	31.260±7.270	131.860±11.160 ^**	9.206±0.476	29.260±11.186
					Medium dose group	32.620±5.282	130.740±28.721 ^**	8.216±1.565	28.200±5.335
High dose group	32.100±5.250	140.040±21.685 ^*	8.022±1.391 ^*	26.880±5.858

Note that: comparison to the normal control group: ^△△ representation ofP<0.01， ^△ Representation ofP<0.05; comparison to model set: ^** representation ofP<0.01； ^* Representation ofP<0.05。

ALT is significantly increased in serum of mice in model group compared with normal groupP< 0.05). Compared with the model group, each dosage of the gleditsia sinensis endosperm can reduce ALT content in serum of UC mice, but no significant difference is causedP> 0.05); AST extremely significant elevation in model group micePLess than 0.01), the low-dose group and the medium-dose group of the saponaria endosperm can remarkably reduce the AST content in serum of UC mice, and the positive medicine mesalamine and the high-dose group of the saponaria endosperm can remarkably reduce the AST content in serum of UC mice.

Compared with the normal group, the serum of the mice in the model group has extremely obvious increase of UREAP< 0.01). Compared with the model group, the high dosage of the saponaria endosperm can obviously reduce the content of UREA in serum of UC mice, and the low dosage and the medium dosage of the saponaria endosperm can reduce the content of UREA in serum of UC mice without obvious differenceP> 0.05); serum CREA content of model group mice is increased, but no significant difference is generatedP> 0.05), low-dose group of honeylocust endosperm, medium dose,High dosage and positive medicine mesalamine can reduce CREA content in serum of UC mice, but no significant difference is causedP＞0.05）。

Conclusion: the saponaria endosperm can reduce the content of AST, ALT, UREA and CREA in serum, and has a certain improvement effect on liver and kidney injury.

Claims

1. An application of a Chinese honeylocust endosperm extract in preparing a medicament for treating ulcerative colitis, wherein the preparation method of the Chinese honeylocust endosperm extract comprises the following steps: pulverizing fructus Gleditsiae Abnormalis endosperm, sieving with 8-500 mesh sieve, slowly adding into 2-12 times of boiling water, stirring, mixing, decocting with small fire for 10-60min, and concentrating to obtain fluid extract or soft extract.

2. The application of the gleditsia sinensis endosperm extract in preparing a medicament for treating ulcerative colitis is characterized in that: the preparation method of the gleditsia sinensis endosperm extract comprises the following steps: