CN114948977A

CN114948977A - Application of flavanone glycoside derivative in preparing medicine for preventing and treating colitis

Info

Publication number: CN114948977A
Application number: CN202110210470.0A
Authority: CN
Inventors: 胡立宏; 吕祁; 刘健; 张毅楠; 胡杨; 王平; 邢瑶
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2021-02-25
Filing date: 2021-02-25
Publication date: 2022-08-30
Anticipated expiration: 2041-02-25
Also published as: CN114948977B

Abstract

Experiments prove that the compound can obviously reduce DAI scores of mice with colitis, obviously inhibit the shortening of colon length, obviously reduce the MPO activity of colon tissues of the mice with colitis, obviously relieve pathological injury of the colon tissues of the mice induced by DSS, has obviously better effect than that of dihydroflavonoid glycoside and positive control, and has potential pharmaceutical application in resisting ulcerative colitis.

Description

Application of flavanone glycoside derivative in preparing medicine for preventing and treating colitis

Technical Field

The invention belongs to the pharmaceutical technology, and particularly relates to an application of a flavanone glycoside derivative in preparing a medicine for preventing and treating colitis.

Background

Ulcerative Colitis (UC) is a chronic nonspecific inflammatory disease with an undefined etiology and easily recurrent attacks, and is characterized by continuous inflammation of the mucosal and submucosa layers of the colon, often affecting the rectum and spreading to the whole colon and even the terminal of the ileum. Epidemiological data indicate that UC has a high incidence and prevalence throughout the world, particularly in developed countries such as europe and north america.

The clinical manifestations of UC are diarrhea, abdominal pain, mucus, bloody pus, stool, etc., and the pathological features are that lymphocytes, plasma cells, monocytes, etc. are diffused in the inherent layer of the colonic mucosa; the number of goblet cells in the active phase is reduced, a large amount of neutrophils and eosinophils infiltrate, and cryptitis and abscess can occur; chronic inflammation can lead to structural and functional impairment of the colon, increasing the risk of colorectal cancer. UC has a long course of disease and is difficult to cure radically due to repeated attack. With the development of the disease, some patients are also accompanied with certain degree of parenteral manifestations such as skin, joint, mucous membrane inflammation and the like, which causes serious disease burden to the patients. Clinically, no specific treatment method for UC exists, and the main treatment aims at relieving symptoms, preventing and treating complications and preventing canceration. The current therapeutic drugs for UC mainly comprise aminosalicylates (sulfasalazine and mesalamine), glucocorticoids (dexamethasone and budesonide), immunosuppressants (cyclosporine and methotrexate), biological agents (infliximab and adalimumab) and the like. Although the amount is large, the traditional Chinese medicine preparation has the defects of large side effect, easy relapse, easy drug resistance, high price and the like. Therefore, the development of safe and effective therapeutic drugs is urgently needed in clinic.

At present, no report is found about the application of the compound shown in the formula I or the pharmaceutical composition thereof in preventing and treating ulcerative colitis.

Disclosure of Invention

The purpose of the invention is as follows: in view of the above prior art, the present application provides the use of the dihydroflavonol glycoside derivatives of formula I in the preparation of a medicament for the prevention and treatment of ulcerative colitis.

The technical scheme is as follows: the application discloses an application of a flavanone glycoside derivative shown as a formula I in preparing a medicament for preventing and treating colitis,

wherein R is ₁ Is selected from C ₁ -C ₄ An alkoxy group; r ₂ Selected from hydrogen, C ₁ -C ₄ An alkoxy group.

As a preferred embodiment, wherein R ₁ Is methoxy.

As a preferred embodiment, R ₂ Selected from hydrogen, C ₁ -C ₃ An alkoxy group.

Preferably, the flavanone glycoside derivative of formula I is selected from the following compounds:

the compound 1 is extracted from Clinopodium polycephalum herba Clinopodii.

The application of the clinopodium polycephalum extract in preparing the medicines for preventing and treating the colitis is also within the protection scope of the application.

The application also discloses application of the plant extract containing the active ingredient of the formula I in preparing the medicines for preventing and treating the colitis.

The application also discloses a pharmaceutical composition which comprises one or more of the flavanone glycoside derivatives shown in the formula I as an active ingredient, and pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and/or diluents, and is used for preventing and treating ulcerative colitis.

Further, the application also discloses application of the pharmaceutical composition in preparing a medicine for preventing and treating colitis.

Has the advantages that: experiments prove that the compound can obviously reduce the DAI score of a colitis mouse, obviously inhibit the shortening of the colon length, obviously reduce the MPO activity of colon tissues of the colitis mouse, obviously relieve the pathological injury of the colon tissues of the mouse induced by DSS, has obviously better effect than the dihydroflavonoid glycoside and a positive control, and has potential pharmaceutical application of anti-ulcerative colitis.

Detailed Description

The present application will be described in detail with reference to specific examples.

Example 1: process for producing compounds 1 to 5

Preparation of compound 1:

taking 5kg of herba Clinopodii (the content of the compound 1 is 0.5-0.6%), adding 10 times of 70% ethanol, soaking for 30min, heating and refluxing for 120min, extracting with petroleum ether, removing chlorophyll, standing overnight, concentrating the filtrate under reduced pressure, and evaporating to dryness; adopting D101 type macroporous resin, wherein the dosage ratio of the resin to a sample loading solution is 1: 3, the concentration of the sample loaded on a column is 0.70mg/mL, washing off tannin by using 0.5mol/L NaOH solution during elution until the effluent NaOH solution is light yellow, washing by using distilled water until the effluent NaOH solution is neutral, then washing by using 3BV 30% ethanol to remove impurities, and finally eluting by using 4BV 70% ethanol at an adsorption flow rate of 0.5mg/mL, wherein the elution flow rate is 1.0 mg/mL. The fractions were purified by silica gel column to obtain white needle crystals 1. ESI-MS (M/z) [ M + Na ]] ⁺ ＝617.2(calcd:617.2)。 ¹ H NMR(500MHz,DMSO-d ₆ ):δ12.04(s,1H),7.48(d,J＝8.7Hz,2H),6.99(d,J＝8.7Hz,2H),6.14(d,J＝2.0Hz,2H),5.59(dd,J＝12.6,2.9Hz,1H),5.42(d,J＝4.7Hz,1H),5.20(s,2H),4.99(d,J＝7.5Hz,1H),4.71(s,1H),4.61(s,1H),4.53(s,2H),3.81(s,1H),3.78(s,3H),3.63(s,1H),3.57-3.53(m,1H),3.44-3.36(m,4H),3.27-3.22(m,2H),3.17-3.10(m,2H),2.79(dd,J＝17.1,3.0Hz,1H),1.09(d,J＝6.2Hz,3H).

General procedure for the Synthesis of Compounds 2-5:

hesperidin was purchased from shanghai shidan standard technical service ltd.

Under the protection of nitrogen and under the condition of ice bath, dropping iodoalkane (RI, 0.48mmol) into a mixed solution of hesperidin (100mg,0.16mmol), potassium carbonate (67.9mg, 0.48mmol) and N, N-dimethyl formyl (5mL), removing the ice bath after dropping, and stirring at normal temperature until TLC is finishedAnd (5) detecting the completion of the reaction. The reaction mixture was poured into an ice-water bath, extracted with ethyl acetate (10 mL. times.3), and the organic layer was successively washed with 1M hydrochloric acid solution (20mL), saturated NaHCO ₃ (10mL), washed with saturated brine (10mL), and the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give a pure product.

Compound 2, yield 62.8%. ESI-MS (M/z) [ M + Na ]] ⁺ ＝647.2(calcd:647.2)。 ¹ H NMR(500MHz,DMSO-d ₆ ):δ12.05(s,1H),7.15(d,J＝9.8Hz,1H),7.07(d,J＝10.1Hz,1H),6.99(d,J＝8.4Hz,1H),6.15(s,2H),5.58(dd,J＝9.3,2.9Hz,1H),5.41(d,J＝5.0Hz,1H),5.20(dd,J＝7.1,5.3Hz,2H),4.99(d,J＝7.5Hz,1H),4.70(d,J＝5.5Hz,1H),4.61(d,J＝4.4Hz,1H),4.53(s,1H),4.48(d,J＝6.1Hz,1H),3.81(s,1H),3.79(s,3H),3.77(s,3H),3.63(s,1H),3.57-3.52(m,1H),3.46-3.37(m,4H),3.29-3.21(m,2H),3.17-3.12(m,2H),2.78(dd,J＝17.1,2.9Hz,1H),1.09(d,J＝6.2Hz,3H).

Compound 3, yield 47%. ESI-MS (M/z) [ M + Na ]] ⁺ ＝661.2(calcd:661.3)。 ¹ H NMR(500MHz,DMSO-d ₆ ):δ12.03(s,1H),7.13(d,J＝9.8Hz,1H),7.06(d,J＝10.1Hz,1H),6.99(d,J＝8.4Hz,1H),6.15(d,J＝4.2Hz,2H),5.55(dd,J＝9.3,2.9Hz,1H),5.41(d,J＝5.0Hz,1H),5.20(dd,J＝7.1,5.3Hz,2H),4.99(t,J＝7.5Hz,1H),4.70(s,1H),4.61(dd,J＝4.4,4.3Hz,1H),4.53(s,1H),4.49(d,J＝6.1Hz,1H),4.04-4.01(m,2H),3.81(s,1H),3.77(s,3H),3.63(d,J＝4.5Hz,1H),3.57-3.52(m,1H),3.46-3.37(m,4H),3.29-3.21(m,2H),3.17-3.12(m,2H),2.78(dd,J＝17.1,2.9Hz,1H),1.33(t,J＝7.0Hz,3H),1.09(dd,J＝2.5,6.2Hz,3H).

Compound 4, in 43% yield. ESI-MS (M/z) [ M-H ]] ^- ＝651.2(calcd:651.2)。 ¹ H NMR(500MHz,DMSO-d ₆ ):δ12.05(s,1H),7.16(d,J＝8.8Hz,1H),7.08(d,J＝9.0Hz,1H),7.00(d,J＝9.2Hz,1H),6.16(s,2H),5.58(dd,J＝9.4,3.0Hz,1H),5.40(d,J＝5.0Hz,1H),5.20(dd,J＝8.0,4.8Hz,2H),5.02(d,J＝7.8Hz,1H),4.66(d,J＝5.8Hz,1H),4.60(d,J＝5.2Hz,1H),4.52(s,1H),4.46(d,J＝5.4Hz,1H),3.99(t,J＝7.2Hz,2H),3.81(s,1H),3.79(s,3H),3.60(s,1H),3.58-3.54(m,1H),3.46-3.40(m,4H),3.28-3.23(m,2H),3.18-3.14(m,2H),2.78(dd,J＝17.1,3.0Hz,1H),1.72-1.69(m,2H),1.09(d,J＝6.1Hz,3H),0.99(t,J＝8.0Hz,3H).

Compound 5, yield 48%. ESI-MS (M/z) [ M-H ]] ^- ＝651.2(calcd:651.2)。 ¹ H NMR(500MHz,DMSO-d ₆ ):δ12.03(s,1H),7.20(d,J＝9.6Hz,1H),7.10(d,J＝8.8Hz,1H),7.01(d,J＝8.8Hz,1H),6.15(s,2H),5.62(dd,J＝9.5,3.0Hz,1H),5.40(d,J＝4.8Hz,1H),5.21(dd,J＝7.2,5.2Hz,2H),4.96(d,J＝7.8Hz,1H),4.79-4.76(m,1H),4.66(d,J＝5.8Hz,1H),4.60(d,J＝4.2Hz,1H),4.51(s,1H),4.48(d,J＝6.4Hz,1H),3.82(s,1H),3.78(s,3H),3.62(s,1H),3.57-3.53(m,1H),3.46-3.39(m,4H),3.29-3.20(m,2H),3.18-3.11(m,2H),2.78(dd,J＝17.1,2.8Hz,1H),1.32(d,J＝7.0Hz,6H),1.09(d,J＝6.1Hz,3H).

Example 2: anti-ulcerative colitis Activity assay for Compounds 1-5

UC is a chronic inflammatory disease with the main pathological change characteristic of massive inflammatory cell infiltration at the colon mucosa part, and clinically, patients have symptoms of abdominal pain, diarrhea, mucus, bloody pus, stool, tenesmus and the like. At present, UC model animals are prepared by a plurality of methods which mainly comprise chemical induction and adoptive transfer

CD4 ⁺ T lymphocytes, gene knockouts, transgenics, and the like. Among them, the chemical induction method is most widely used because of its advantages such as simple operation and low cost. Commonly used chemical inducers include DSS, acetic acid, 2,4,6-trinitrobenzenesulfonic acid solution (TNBS), oxazolone (oxazonone), and the like. Compared with other inducers, the symptoms and colon pathological characteristics of a colitis model mouse established after DSS induction are most similar to those of human UC. In addition, the model also has the advantages of high success rate, good repeatability, even lesion of the distal colon and the like. DSS is an artificially synthesized heparin polysaccharide, has anticoagulant property, has obvious cytotoxicity to intestinal epithelial cells at the basal crypt position, destroys the integrity of the intestinal mucosal barrier, further causes intestinal contents (such as bacteria and metabolites thereof) to enter and penetrate the internal mucus layer, activates immune cells and inflammation of the intestinal solid layerSex cells. The activated immune cells and inflammatory cells cause inflammatory reactions in the intestinal tract by releasing a large amount of pro-inflammatory factors, chemokines, adhesion molecules, reactive oxygen species, and the like. In conclusion, the application adopts a DSS-induced mouse colitis model to investigate the inhibition effect of the compounds 1 and 2 on mouse colitis.

Experimental Material

Experimental reagent

The drug was prepared as described in example 1 above, code number 1-5; DSS (MW 36,000-50,000) was purchased from MP Biomedicals, Inc., USA; 5-Aminosalicylic acid (5-ASA) available from pharmacia, Eyre, France; a Myeloperoxidase (MPO) kit is purchased from Nanjing to build a bioengineering research institute; o-toluidine was purchased from Shanghai Crystal purifications science and technology Ltd; hydrogen peroxide (H) ₂ O ₂ ) And glacial acetic acid was purchased from Nanjing chemical reagents, Inc.

Laboratory animal

Female C57BL/6 mice, 6-8 weeks old, 18-20g in weight, purchased from Qinglong mountain animal farm in Jiangning district, Nanjing City [ production license number: SCXK (su) 2017-. Animals are fed with water and food at room temperature of 22 + -2 deg.C and humidity of 45 + -10%. After 3 days of adaptive feeding, the obtained mixture was used for subsequent experiments.

Experimental methods

Establishment and group administration of mouse colitis model

Female C57BL/6 mice, 6-8 weeks old, weighing 18-20g, were randomly divided by body weight into normal, model, Compound 1(1,2,4mg/kg), Compound 2(1,2,4mg/kg), hesperidin (10,20,40mg/kg) and Positive control 5-ASA (200 mg/kg). The mice in the other groups, except the normal group, were given 2.5% DSS solution for 7 consecutive days, followed by 3 days of free drinking with single distilled water. Recording day when modeling is started as day 1, and performing intragastric administration of the compound 1, the compound 2, hesperidin and 5-ASA for 1 time every day for 10 days continuously, wherein the administration volume is 0.1mL/10 g; mice in the normal and DSS groups were given an equal volume of vehicle (0.5% CMC-Na solution).

Disease Activity index score

Mice were observed and recorded daily for weight, diarrhea and occult blood, and were scored for Disease Activity Index (DAI), which is (weight score + stool trait score + stool bleeding score/3). The specific scoring criteria are as follows:

disease Activity index Scoring criteria

And (3) fecal occult blood condition determination: collecting the mouse feces, placing the mouse feces in a 24-hole culture plate, adding equal volume of o-toluidine glacial acetic acid and 3% hydrogen peroxide solution, and judging the mouse feces to be positive if the mouse feces are blue brown within 2 min.

Specimen collection

Collecting blood from fundus venous plexus 1h after the last administration, standing at room temperature for 2h, centrifuging at 3500rpm and 4 deg.C for 15-20min, separating serum, and storing at-20 deg.C or-80 deg.C. After blood collection, the mice were sacrificed, the abdominal cavity was carefully opened, the colon was taken 1cm from the anus, the length was measured, the morphological change was observed, and a photograph was taken. And then, washing the colon by using a precooled PBS solution, measuring 0.4cm of colon tissue from the distal end, fixing the colon tissue in a 4% formaldehyde neutral buffer solution, and storing the remaining colon liquid nitrogen in ice when pathological examination is performed.

MPO Activity measurement

Myeloperoxidase MPO, one of the important markers of leukocyte invasion into tissues. 20mg of colon tissue from each group of mice was taken and 200. mu.L of precooled PBS was added to prepare a 10% homogenate. Centrifuging at 1200rpm for 5min at 4 deg.C, and sucking the supernatant. Reagents were added stepwise, exactly as described in the kit instructions, and absorbance was measured at a wavelength of 460 nm. Calculation formula of MPO vitality:

MPO activity (U/g wet weight) ═ OD of measuring tube-OD of control tube)/11.3 Xsample size (g)

Definition of enzyme activity unit: h in the reaction system at 37 ℃ per gram of wet weight ₂ O ₂ Decomposed 1 mu mol to be 1 enzyme activity unit.

Colon histopathology assay

Taking a mouse telecentric colon tissue soaked in 4% neutral formaldehyde, conventionally dehydrating, embedding with paraffin, staining with Hematoxylin-eosin (H & E), and sealing and fixing for histopathological examination. Professional pathological inspectors observe the pathological changes of the mucosa layer and the submucosa layer, the inflammatory cell infiltration degree of the mucosa lamina propria and the damage degree of the crypt under an optical microscope, and score the 3 indexes. The total score for each mouse was calculated as follows:

colon histopathology scoring

And multiplying the scores of the three indexes by the proportion (participation proportion) of the lesion area to obtain a final score.

Data statistics

All data are expressed as means ± s.e.m. and statistical differences between groups were tested using one-way ANONA and Dunnett's in the SPSS software. P values less than 0.05 are considered to be significantly different.

Results of the experiment

Effect on DAI Scoring

As shown in Table 1, the weight of mice in the DSS group was significantly reduced, severe diarrhea and hematochezia appeared, and the DAI score continued to increase on days 1-8, and slightly decreased after the DSS was discontinued. The administration of compound 1(2,4mg/kg), compound 2(2,4mg/kg), hesperidin (20,40mg/kg) and 5-ASA (200mg/kg) through gastric gavage all significantly reduced the DAI score of colitis mice, and it is noted that compound 1 and compound 2 were 10 times stronger than hesperidin and 100 times stronger than 5-ASA.

TABLE 1 DAI scores

Note:comparison with Normal group ^## P<0.01; comparison with DSS group ^* P<0.5 and ^** P<0.01.

effect on Colon Length

As shown in Table 2, the colon length was significantly shortened in the DSS group mice as compared with the normal mice, and the gastric gavage administration of Compound 1(2,4mg/kg), Compound 2(2,4mg/kg), hesperidin (20,40mg/kg) and 5-ASA (200mg/kg) significantly inhibited the shortening of the colon length. It is noteworthy that compounds 1 and 2 are 10 times more potent than hesperidin and 100 times more potent than 5-ASA.

TABLE 2 Colon Length

Group of	Number of examples	Colon Length (cm)
			Normal	6	7.73±0.14
DSS	6	5.65±0.21 ^##
			1(1mg/kg)	6	5.72±0.12
1(2mg/kg)	6	6.48±0.17 ^**
			1(4mg/kg)	6	7.10±0.24 ^**
2(1mg/kg)	6	5.92±0.22
			2(2mg/kg)	6	6.55±0.11 ^**
2(4mg/kg)	6	7.13±0.18 ^**
			Hesperidin (10mg/kg)	6	6.03±0.16
Hesperidin (20mg/kg)	6	6.30±0.20 ^*
			Hesperidin (40mg/kg)	6	6.97±0.19 ^**
5-ASA(200mg/kg)	6	6.67±0.25 ^**

Note: comparison with Normal group ^## P<0.01; comparison with DSS group ^* P<0.5 and ^** P<0.01.

effect on MPO Activity in colon tissue

MPO is characteristic of neutrophils, has a fixed enzyme content per cell, accounts for about 5% of the dry weight of the cell, and has the ability to reduce hydrogen peroxide. According to this feature of MPO, the number of neutrophils in the tissue can be suggested by analyzing the enzyme activity. As shown in Table 3, compared with the normal group, the MPO activity in the colon tissue of the mice in the DSS group is obviously improved, the MPO activity of the colon tissue of the mice with colitis is obviously reduced by the intragastric administration of the compound 1(2,4mg/kg), the compound 2(2,4mg/kg), hesperidin (20,40mg/kg) and 5-ASA (200mg/kg), and the effects of the compounds 1 and 2 are 10 times stronger than that of hesperidin and 100 times stronger than that of 5-ASA.

TABLE 3 colonic tissue MPO viability

Group of	Number of examples	MPO vitality (U/g tissue)
			Normal	6	0.79±0.06
DSS	6	2.02±0.12 ^##
			1(1mg/kg)	6	1.95±0.11
1(2mg/kg)	6	1.63±0.09 ^*
			1(4mg/kg)	6	1.34±0.14 ^**
2(1mg/kg)	6	2.05±0.09
			2(2mg/kg)	6	1.55±0.14 ^**
2(4mg/kg)	6	1.29±0.09 ^**
			Hesperidin (10mg/kg)	6	1.91±0.12
Hesperidin (20mg/kg)	6	1.62±0.13 ^*
			Hesperidin (40mg/kg)	6	1.28±0.10 ^**
5-ASA(200mg/kg)	6	1.45±0.09 ^**

effect on Colon histopathological changes

As shown in Table 4, the colon mucosa of the normal group of mice is intact, the epithelial cells are arranged regularly, inflammatory cells are not infiltrated, the colon of the glandular organ in the inherent layer is clear, the arrangement is regular, and the goblet cells are abundant; the colon tissues of mice in the DSS group are seriously diseased, the mucous membrane layer and the submucosa are affected, the mucosal epithelial cells are necrotic, the surface is not covered by epithelium, the crypt is seriously damaged, and the infiltration of macrophages and neutrophils in the mucosa lamina propria is obvious. The compound 1(2,4mg/kg), the compound 2(2,4mg/kg), hesperidin (20,40mg/kg) and 5-ASA (200mg/kg) are administrated by stomach irrigation, so that the symptoms are obviously improved, and the pathological damage of colon tissues of mice induced by DSS is relieved.

TABLE 4 Colon histopathology scores

Claims

1. use of a flavanonoside derivative of formula I in the manufacture of a medicament for the prevention and treatment of colitis:

in the formula: r ₁ Is selected from C ₁ -C ₄ An alkoxy group; r is ₂ Selected from hydrogen, C ₁ -C ₄ An alkoxy group.

2. Use according to claim 1, characterized in that R ₁ Is methoxy.

3. Use according to claim 1, characterized in that R ₂ Selected from hydrogen, C1-C3 alkoxy.

4. Use according to claim 1, characterized in that the flavanonoside derivative is selected from the following compounds:

5. use of a plant extract comprising a flavanonoside derivative according to claim 1 in the manufacture of a medicament for the prevention or treatment of colitis.

6. A pharmaceutical composition comprising one or more of the flavanone glycoside derivatives of claims 1 to 4 as an active ingredient.

7. The use of the pharmaceutical composition of claim 6 for the manufacture of a medicament for the prevention and treatment of colitis.