CN113662979B

CN113662979B - Purification method and application of acyl-rich collard flavone with colitis relieving effect

Info

Publication number: CN113662979B
Application number: CN202111069836.3A
Authority: CN
Inventors: 聂少平; 王辉; 陈晓敏; 黄晓君; 殷军艺
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2021-09-13
Filing date: 2021-09-13
Publication date: 2022-09-16
Anticipated expiration: 2041-09-13
Also published as: CN113662979A

Abstract

The invention discloses a method for purifying collard flavone rich in acyl group and having the function of relieving colitis and application thereof. The method comprises the steps of obtaining a collard flavone extract by an alcohol extraction method, separating and purifying by NKA-9 macroporous resin to obtain collard total flavone, purifying by thermoplastic resin containing repeated amide groups, and separating to obtain an acyl flavone component which can directionally reach the colon. The purity of the collard total flavonoids obtained by macroporous resin separation and purification reaches 31.16 percent, the recovery rate reaches 85.64 percent, and the adsorption capacity can reach 15.93mg per gram of resin; the purity of the kale flavone purified by the thermoplastic resin containing the repeated amide groups is up to 37.02 percent, and the acyl flavone with the capability of directionally reaching colon is 5.02 times of that of the flavone separated and purified by the macroporous resin. The collard total flavone rich in acyl provided by the invention can effectively relieve ulcerative colitis.

Description

Purification method and application of acyl-rich collard flavone with colitis relieving effect

Technical Field

The invention belongs to the technical field of separation and purification of flavone, and particularly relates to a purification method and application of acyl-rich collard flavone with a colitis relieving effect.

Background

Kale (a)Brassica oleracea var. sabellica) Is a biennial plant belonging to the family Brassicaceae, and is widely cultivated in central Europe, northern Europe, North America, etc. Each kale can be harvested 3-5 leaves each time, immature young leaves are left to continue growing, and harvesting is carried out successively. In early spring or late autumn, the leaves of kale are crisp and tender in texture and good in flavor. In summer, the leaves are harder, more fiber and poor in flavor at high temperature. Old leaves which are not harvested in time in summer are abandoned, and great waste of resources is caused. Researches show that the kale is rich in flavonoids and has a plurality of varieties, and the varieties of the flavonoids in some varieties are even more than 70. Flavonoids are secondary metabolites widely distributed in plants.The flavonoid compound has strong antioxidation and can remove free radicals in organisms; it is also effective in resisting aging, cancer, virus, allergy and diabetes, but impurities and low purity limit the exertion of the functional activity of flavonoids.

Ulcerative colitis is a chronic and recurrent inflammatory disease of the intestinal tract with a high risk of developing colon cancer, with lesions affecting the entire colon and terminal ileum, manifested clinically as diarrhea, abdominal pain and hematochezia. Patients with ulcerative colitis experience severe intestinal inflammation and restricted diet, which leads to weight loss and a severe impact on the quality of life of the patients. Statistically, millions of people in the world suffer from ulcerative colitis, especially in northern europe and america, which poses a great threat to human health. The etiology of ulcerative colitis is related to genetic, immunological, and environmental factors. In addition, the site of the onset of ulcerative colitis is the colon, and many conventional oral medications have limited efficacy due to direct absorption in the small intestine which results in too low an effective concentration at the focal site. Therefore, the colon-targeted drug delivery can be directly achieved, and the treatment effect on the ulcerative colitis can be greatly improved.

The macroporous resin method is a widely used purification method, and compared with the macroporous resin method, the solvent extraction method needs to use a large amount of harmful organic solvent, so that the problems of environmental protection and health exist. Microwave, ultrasonic, enzyme extraction, etc. cannot improve the purity of flavone. Compared with macroporous resin method, it has the advantages of large capacity, high yield, high purity, simple operation, no toxicity and no pollution. However, the types of flavonoids from different sources are very different, and different flavonoids need to be matched with different macroporous resins, especially plants with the same types as collard and very different chemical properties, so that the purification of the flavonoids in the plants is difficult. Therefore, there is a need to develop a method for effectively separating and purifying acylated collard flavone and an application for alleviating ulcerative colitis.

Disclosure of Invention

Aiming at the problems mentioned in the background technology, the invention provides a purification method of collard flavone rich in acyl group and having the function of relieving colitis and application thereof, which is realized by the following technical method:

a method for purifying acyl-rich collard flavone with colitis relieving effect comprises the following steps:

1) preparing a collard flavone extract:

stirring and heating collard powder with ethanol water solution for extraction, centrifuging at 4800 rpm/min for 10 min, collecting supernatant, vacuum rotary evaporating for concentration until liposoluble components precipitate, centrifuging at 4800 rpm/min for 10 min to remove liposoluble components, collecting supernatant, and further concentrating to obtain extract;

2) purifying collard total flavonoids:

diluting the extract obtained in the step 1) with distilled water to obtain a flavone solution, adjusting the pH value of the solution, pouring the solution into a container filled with macroporous resin, stirring and adsorbing, collecting the macroporous resin, eluting the macroporous resin with distilled water until the macroporous resin is colorless, then stirring and eluting with an ethanol water solution as an eluent, collecting the eluent, performing vacuum rotary evaporation and concentration, performing freeze-drying to obtain purified kale total flavone, and detecting and calculating the adsorption rate, desorption rate, recovery rate and desorption amount of the flavone;

3) purification of the fraction of acyl kale flavonoids that can be directed to the colon:

dissolving the total flavonoids of the purified kale obtained in the step 2) by using low-carbon alcohol, adding an adsorption filler, uniformly stirring, volatilizing, loading into a chromatographic column, eluting the chromatographic column by using an eluent with gradually increased concentration, respectively collecting the eluates, performing vacuum rotary evaporation and concentration, freeze-drying to obtain an acyl kale flavonoid component which can directionally reach the colon, detecting and calculating the content of the flavonoids, and identifying the flavonoid component.

Further, the volume fraction of the ethanol aqueous solution in the step 1) is 60-80%, and the material-liquid ratio of the ethanol aqueous solution to the kale powder is 1: 10-30 (g/mL); the heating temperature is 45-60 ℃.

Further, the macroporous resin in the step 2) is D101, NKA-9, HPD-400, HPD-500, AB-8, HPD-450 or XDA-1.

Further, the concentration of the flavone solution obtained by diluting in the step 2) is 0.1-2 mg/mL, and the pH value of the flavone solution is 2-8 after the pH value is adjusted.

Further, the volume fraction of the ethanol water solution in the step 2) is 30-95%.

Further, rutin is used as a standard substance in the detection of the flavone content in the steps 2) and 3), and the aluminum trichloride colorimetric method is adopted for quantification.

Further, step 2) calculating the concentration of flavone in the measurement solution, and the adsorption rate (%) = (total flavone amount in solution before adsorption-total flavone amount in solution after adsorption)/total flavone amount in solution before adsorption × 100%; desorption rate (%) = total flavone amount in eluate after elution/(total flavone amount in solution before adsorption-total flavone amount in solution after adsorption) × 100%; and recovery (%) = adsorption (%) × desorption (%) × 100%; the desorption amount is the total amount of flavone in the eluent after elution.

Further, the adsorption filler in step 3) is thermoplastic resin containing repeated amide groups, silica gel or diatomite.

Further, the flow rate of the elution in the step 3) is 0.5-2 BV/h.

Further, the eluents with increasing concentrations in the step 3) are sequentially: the gradient 1 eluent is 10 to 25 percent of low-carbon alcohol aqueous solution, and the dosage is 2 to 5 BV; the eluent of the gradient 2 is 30 to 45 percent of low-carbon alcohol aqueous solution, and the dosage is 2 to 5 BV; the gradient 3 eluent is 50-65% of low-carbon alcohol aqueous solution, and the dosage is 2-5 BV; the gradient 4 eluent is 70-90% of low-carbon alcohol aqueous solution, and the dosage is 2-5 BV; the lower alcohol is ethanol and/or methanol.

Further, the flavone identification method in the step 3) adopts ultra-high performance liquid phase-ion trap-tandem mass spectrometry (AB SCIEX Q-Ttap 4500) to carry out negative ion detection, and takes Quercetin-3-O-D-glucoside and Kaempferol-3-O-D-glucoside as standard substances to carry out relative quantification.

Further, the content of acylated flavone in the acyl kale flavone component which can be oriented to reach the colon and is obtained in the step 3) is 5.02 times of that of the purified kale total flavone obtained in the step 2).

Compared with the prior art, the invention has the beneficial effects that:

1. the method adopts NKA-9 macroporous resin to purify the kale flavone, the purity of the obtained kale total flavone reaches 31.16 percent, the recovery rate reaches 85.64 percent, the adsorption capacity can reach 15.93mg per gram of resin, and the purity and the recovery rate of the kale flavone in the selected macroporous resin are optimal. Compared with the purity of 3.8 percent of the collard flavone extract flavone before purification, the purity is improved by 8.2 times; the purity of the collard flavone is up to 37.02% after the subsequent purification by the thermoplastic resin containing the repeated amide groups, and the collard flavone has the characteristics of directional arrival of colon and potential targeted medicinal value.

2. The method for separating and purifying the acyl-rich collard flavone is simple to operate, high in recovery rate and purity, improves the economic value of the collard, and is suitable for enterprises and large-scale production.

3. The collard flavone rich in acyl obtained by the separation and purification method can effectively relieve ulcerative colitis.

Drawings

FIG. 1 is a Q-trap mass spectrum of collard flavones and collard total flavones which may be directed to the colon.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The D101, NKA-9, HPD-400, HPD-500, AB-8, HPD-450, and XDA-1 macroporous resins used in the examples of the present invention were obtained from Shanghai Moshi scientific instruments, Inc., the thermoplastic resins containing a repeating amide group were obtained from China national drug group, Inc., silica gel was obtained from American Merck, and diatomaceous earth was obtained from Tianjin three factories.

Purification of total flavonoids from kale

Example 1

Step 1, preparation of collard flavone extract

Accurately weighing a certain amount of dried collard powder in a 1L beaker, and adding the mixture into the beaker according to a material-liquid ratio of 1: extracting with 20 (g/mL) 80% ethanol at 50 deg.C under heating and stirring for 60 min, centrifuging at 4800 rpm/min for 10 min to obtain supernatant, vacuum rotary evaporating for concentrating until liposoluble components are separated out, centrifuging at 4800 rpm/min for 10 min to remove liposoluble components, collecting supernatant, and further concentrating to obtain extract;

step 2, purification of collard total flavonoids

Diluting collard flavone extract with distilled water to 0.15 mg/mL flavone water solution, adjusting pH of the flavone solution to 4, pouring 10mL flavone solution into a beaker filled with 1 g pretreated NKA-9 macroporous resin, stirring and adsorbing for 12 h; collecting the macroporous resin, and leaching the macroporous resin with distilled water until the macroporous resin is colorless; collecting macroporous resin, taking 25 mL of 95% ethanol water solution as an eluent, and stirring and eluting for 12 h; collecting the eluent, concentrating by vacuum rotary evaporation, and freeze-drying to obtain brown yellow powder. Detecting and calculating the adsorption rate, desorption rate and recovery rate of the flavone.

Example 2

Step 1, preparation of collard flavone extract

step 2, purification of kale total flavonoids

Diluting collard flavone extract with distilled water to obtain 2 mg/mL flavone water solution, adjusting pH of the flavone solution to 4, pouring 10mL flavone solution into a beaker filled with 1 g pretreated NKA-9 macroporous resin, and stirring for adsorption for 12 h; collecting the macroporous resin, and leaching the macroporous resin with distilled water until the macroporous resin is colorless; collecting macroporous resin, taking 25 mL of 80% ethanol water solution as eluent, and stirring and eluting for 12 h; collecting the eluent, concentrating by vacuum rotary evaporation, and freeze-drying to obtain brown yellow powder. And detecting and calculating the adsorption rate and desorption amount of the flavone.

Comparative example 1

Comparative example 1 differs from example 1 in the type of macroporous resin, as shown in table 1, and the measured adsorption and desorption properties are shown in table 1.

Comparative example 2

Comparative example 2 is different from example 1 in that the diluted flavone solution has a pH of 2 to 8, respectively, and the measured adsorption properties are shown in Table 2.

Comparative example 3

Comparative example 3 differs from example 2 in that the eluents have different ethanol concentrations of 30% to 90%, respectively, and the desorption properties measured are shown in table 3.

Adsorption rate (%) = (total flavone amount in solution before adsorption-total flavone amount in solution after adsorption)/total flavone amount in solution before adsorption × 100%; desorption rate (%) = total flavone amount in eluate after elution/(total flavone amount in solution before adsorption-total flavone amount in solution after adsorption) × 100%; and recovery (%) = adsorption (%) × desorption (%) × 100%; the desorption amount is the total amount of flavone in the eluent after elution.

TABLE 1 adsorption and desorption of total flavonoids from kale by different resins

Resin type	Adsorption Rate (%)	Desorption ratio (%)	Recovery (%)
				NKA-9	95.2±1.13	89.94±3.55	85.64±4.4
HPD-400	94.89±0.43	89.31±1.1	84.74±0.66
				HPD-500	91.61±1.77	87.96±8.09	80.5±5.85
AB-8	96.38±0.26	81.8±0.02	78.84±0.19
				HPD-450	91.43±2.66	85.58±3.11	78.29±5.12
D101	93.21±0.94	82.96±1.62	77.33±2.29
				XDA-1	97.78±0.14	74.05±3.45	72.41±3.48

TABLE 2 adsorption rate of NKA-9 resin on collard flavone solutions of different pH values

pH value	Adsorption Rate (%)
		2-3	94.41±0.56-95.46±0.45
4-5	94.93±1.09-97.11±0.7
		6-8	79.81±1.85-93.9±1.14

TABLE 3 influence of different ethanol concentrations on the desorption amount of NKA-9 resin

Concentration of ethanol	Desorption amount (mg)
		30%	4.57±0.1
40%	5.33±0.06
		50%	9.48±0.02
60%	13.76±0.18
		70%	14.65±0.09
80%	15.93±0.33
		90%	14.35±0.1

Experimental results of examples 1 and 2 and comparative examples 1 to 3:

table 1 shows that the recovery rate of NKA-9 resin is highest, which reaches 85.64% + -4.4%, so that the NKA-9 macroporous resin is preferably the resin for purifying the collard flavone.

Table 2 shows that when the pH of the collard flavone aqueous solution is 3-5, the NKA-9 resin has high adsorption capacity to flavone, and the adsorption capacity is mostly more than 95%; when the pH value reaches 6-8, the adsorption capacity of the NKA-9 resin to the flavone is reduced from 93.9 +/-1.14% to 79.81 +/-1.85%.

Table 3 shows that when the ethanol concentration is 30-80%, the desorption amount of the kale flavone is gradually increased from 4.57 mg +/-0.1 mg to 15.93mg +/-0.33 mg; when the ethanol concentration reaches 90%, the desorption amount of the collard flavone is reduced to 14.35 mg +/-0.1 mg; the result also shows that the saturated adsorption capacity of NKA-9 to the kale flavone can reach 15.93mg per gram of resin. Under the optimized conditions, the pH value of the kale flavone aqueous solution is 4-5, the ethanol concentration of the elution solution is 80%, the purity of the kale total flavone is up to 31.16%, and compared with the purity of 3.8% in the kale flavone before purification, the purity is improved by 8.2 times.

Optimization of acyl kale flavone components targetable to the colon

Example 3

500 mg of total flavonoids of kale purified by the preferred method of the present invention in example 2 were dissolved in 10mL of methanol, and 20 g of silica gel was added, stirred well, evaporated, and loaded into a column. The silica gel column was subjected to gradient elution successively with the following eluents at a flow rate of 1 BV/h: gradient I, wherein an eluent is a methanol aqueous solution with the volume fraction of 20%, and the using amount is 3 BV; gradient ②, eluent is 40 percent methanol water solution by volume fraction, and the dosage is 3 BV; gradient ③, eluent is methanol water solution with volume fraction of 60 percent, and the dosage is 3 BV; gradient, eluent is methanol water solution with volume fraction of 80%, and the dosage is 3 BV. Collecting eluates, vacuum rotary evaporating, concentrating, and lyophilizing. Ultra-high performance liquid phase-ion trap-tandem mass spectrometry (AB SCIEX Q-Tcap 4500) is adopted for negative ion detection, and Quercetin-3-O-D-glucoside and Kaempferol-3-O-D-glucoside are used as standard substances for relative quantification.

Example 4

500 mg of total flavonoids of kale purified by the preferred method of the present invention in example 2 was dissolved in 10mL of methanol, and 20 g of diatomaceous earth was added thereto, stirred well, evaporated, and loaded into a chromatography column. The column of diatomaceous earth was subjected to gradient elution with the following eluents in sequence at a flow rate of 1 BV/h: gradient I, wherein an eluent is a methanol aqueous solution with the volume fraction of 20%, and the using amount is 3 BV; gradient ②, eluent is 40 percent methanol water solution by volume fraction, and the dosage is 3 BV; gradient ③, eluent is methanol water solution with volume fraction of 60 percent, and the dosage is 3 BV; gradient, eluent is 80 percent methanol water solution by volume fraction, and the dosage is 3 BV. Collecting eluates, vacuum rotary evaporating, concentrating, and lyophilizing. Ultra-high performance liquid phase-ion trap-tandem mass spectrometry (AB SCIEX Q-Tcap 4500) is adopted for negative ion detection, and Quercetin-3-O-D-glucoside and Kaempferol-3-O-D-glucoside are used as standard substances for relative quantification.

Example 5

500 mg of total flavonoids of kale purified by the method preferred in example 2 of the present invention were dissolved in 10mL of ethanol, and 20 g of a thermoplastic resin containing a repeating amide group was added thereto, stirred well, evaporated to dryness, and packed in a column. The column of the thermoplastic resin containing recurring amide groups is subjected to gradient elution with the following eluents in sequence at a flow rate of 1 BV/h: gradient I, wherein an eluent is an ethanol water solution with the volume fraction of 20 percent, and the using amount is 3 BV; gradient ②, eluent is ethanol water solution with volume fraction of 40%, and the dosage is 3 BV; gradient ③, eluent is ethanol water solution with volume fraction of 60 percent, and the dosage is 3 BV; gradient, eluent is ethanol water solution with volume fraction of 80%, and the dosage is 3 BV. Collecting eluates, vacuum rotary evaporating, concentrating, and lyophilizing. Ultra-high performance liquid phase-ion trap-tandem mass spectrometry (AB SCIEX Q-Tcap 4500) is adopted for negative ion detection, and Quercetin-3-O-D-glucoside and Kaempferol-3-O-D-glucoside are used as standard substances for relative quantification.

Example 6

Qualitative and animal experiment of flavone

1. Experimental animals and dosing: SPF grade male C57BL/6J mice, weighing 20. + -.2 g. The kale total flavonoids obtained in example 2 were administered by gavage, and the colon contents of the mice were collected after 4 h.

2. Detection indexes are as follows: extracting the collected colon contents of the mouse by using 100mg/mL 80% methanol, centrifuging at 12000 rpm/min for 10 min, storing overnight at-20 ℃, centrifuging and passing through a 0.22-micron organic filter membrane, performing anion detection by using an ultra-high performance liquid phase-ion trap-tandem mass spectrum (AB SCIEX Q-Tmap 4500), and performing relative quantification by using Quercetin-3-O-D-glucoside and Kaempferol-3-O-D-glucoside as standard substances.

TABLE 4 identification of the flavonoid component of kale

Serial number

Flavonoid species

Molecular weight

A

B

C

D

E

1

Kaempferol-3-O-gentiobioside

610

+

2

Quercetin-3-O-sophoroside

626

+

3

Kaempferol-3-O-coumaroyl-sophoroside

756

+

4

Kaempferol-3-O-feruloyl-sophoroside

786

+

5

Quercetin-3-O-feruloyl-sophoroside

802

+

6

Kaempferol-3-O-sinapoyl-sophoroside

816

+

7

Quercetin-3-O-sinapoyl-sophoroside

832

+

8

Quercetin-3-O-hydroxyferuoyl-sophoroside

818

+

9

Kaempferol-3-O-D-glucoside

448

+

10

Quercetin-3-O-D-glucoside

464

+

11

Quercetin-3-O-D-glucoside-7-O-D-glucoside

626

+

12

Quercetin-3-O-sophoroside-7-O-D-glucoside

788

+

13

Isorhamnetin-3-O-D-glucoside

478

+

14

Kaempferol-3-O-hydroxyferuoyl-sophoroside

802

+

15

Kaempferol-3-O-D-glucoside-7-O-D-glucoside

610

+

…

+

37

Quercetin-3-O-sophoroside-7-O-sinapoyl-diglucoside

1156

+

38

Quercetin-3-O- hydroxyferuoyl-sophoroside-7-O-D-glucoside

980

+

39

Kaempferol-3-O-hydroxyferuoyl-sophoroside-7-O-diglucoside

1126

+

40

Quercetin-3-O-sinapoyl,feruloyl-triglucoside-7-O-diglucoside

1494

+

41

Quercetin-3-O-sinapoyl-triglucoside-7-O-sinapoyl-diglucoside

1524

+

Note: in the table, A-E are the flavonoid species in kale total flavonoids, kale flavonoids targetable to the colon, fractions in which kale total flavonoids are repurified by a thermoplastic resin containing a repeating amide group, fractions in which kale total flavonoids are repurified by diatomaceous earth, and fractions in which kale total flavonoids are repurified by silica gel, respectively; + indicates the detectable flavone in the sample.

TABLE 5 purity of Secondary purified collard flavone

	Purity (%)	Fold change in acylflavone content targeted to colon
			Total flavonoid of collard	31.16	1
Thermoplastic resin containing repeating amide groups	37.02	5.02
			Diatomite	19.00	0.36
Silica gel	24.77	0.95

Note: the table indicates total flavonoids of kale which are purified by the method preferred in example 2 of the present invention.

Experimental results for examples 3-6:

FIG. 1 is a Q-trap mass spectrum of collard flavone and collard total flavone which can be directed to the colon, and animal experiments show that the flavone with an early peak (higher polarity) in the collard total flavone can hardly reach the colon. While the flavones which can directionally reach the colon are mainly flavones with late peaks (with smaller polarity), and are mainly some special acylated quercetin or kaempferol through qualitative discovery of mass spectrum.

Tables 4 and 5 show that 41 flavonoids were identified from the isolated and purified kale total flavonoids from kale, 8 of which were able to reach the colon and 6 of which were acylated flavonoids. After the secondary purification, the thermoplastic resin containing the repeated amide groups can be used for more specifically purifying the flavonoid compounds from the kale, and the range is narrowed to 14 types of flavones, wherein 8 types of flavones are completely included. And the diatomite and the silica gel have no specificity, so that the screening performance of the collard flavone which can directionally reach the colon is poor, and the difference of the ingredients with the collard total flavone purified by the macroporous resin is not great. The re-purified flavone from the thermoplastic resin containing the repeated amide groups was 37.02% pure and highly pure, and the flavone fraction directed to the colon was 5.02 times higher than the flavone purified from the macroporous resin. While the purities of the flavones of the diatomite and the silica gel are respectively 19.00 percent and 24.77 percent, and are lower than the purities of the flavones of the macroporous resin, and are only 0.36 and 0.95 times.

Example 7

Animal experiments

1. Experimental animals: SPF grade male C57BL/6J mice weighing 20 + -2 g

Animal grouping and administration: the C57BL/6J male mice were divided into normal group, model group, experimental group (acyl-rich kale total flavonoids), each group was 10. The normal group and the model group take normal feed in the whole process, and the experimental group takes feed added with collard total flavonoids in the whole process. Each group ingested 14 days of the corresponding feed. The model group and the experimental group were given 3% DSS drinking water on days 7 to 14. Mice were weighed daily.

2. Detecting the index

1) Disease Activity Index (DAI): before the mice were sacrificed, the stool morphology (normal morphology, score 0; light stool, score 2; liquefied stool, score 4), stool blood (no stool blood, score 0; light stool blood, score 2; severe stool blood, score 4) and the percentage of weight loss (score 0, 0; 1% -5%, 1; 5% -10%, 2; 10% -15%, 3; > 15%, 4) of the mice were scored, and the DAI index was the sum of the three indices.

2) Colon length: after dissecting the mice, the length of the colon of the mice was measured using a ruler.

3) Colonic inflammatory factors TNF-alpha and IL-1 beta mRNA levels: colonic tissue RNA was extracted by Trizol method and fold change in mRNA levels of inflammatory factors TNF-alpha and IL-1 beta was determined by qPCR method.

3. Statistical method

Post analysis of one-way variance LSD analysis data was performed using IBM SPSS Statistics 23 statistical software. Results are expressed as mean ± standard deviation (mean ± SD). P <0.05, p <0.01, p <0.001 showed significance compared to the model group.

4. Results of the experiment

As shown in table 6, severe stool dilution, hematochezia and weight loss occurred in the ulcerative colitis model group. In addition, the colon length was significantly shortened in the model group compared to the normal group, and the colon inflammatory factors IL-1. beta. and TNF-. alpha.mRNA levels were also significantly increased. Compared with the model group, the experimental group obviously reduces the DAI index, relieves the colon length shortening and obviously reduces the colon IL-1 beta and TNF-alpha mRNA level.

TABLE 6 groups of mice vary in DAI index, colon length and colon IL-1 β and TNF- α mRNA levels

Detected index	Normal group	Model set	Experimental group
				DAI index	0±0***	10±1.33	5.7±1.49***
Length of colon	6.24±0.55**	5.44±0.39	5.97±0.59*
				Colonic IL-1 beta mRNA levels	1.016±0.18***	49.98±12.10	28.34±13.05*
Colonic TNF-alpha mRNA levels	1.04±0.09***	5.65±0.41	3.75±0.54***

Note: p <0.05, p <0.01, p <0.001 compared to model group

According to the method, the NKA-9 macroporous resin is preferably selected to purify the kale flavone, the purity of the total kale flavone is 31.16%, the recovery rate is 85.64%, the adsorption capacity can be 15.93mg per gram of resin, and compared with the purity of the kale flavone extract flavone before purification, the purity is improved by 8.2 times; the purity of the collard flavone rich in acyl after being purified again by the thermoplastic resin containing the repeated amide groups reaches 37.02 percent, which is 5.02 times of that of the macroporous resin purified flavone, and the collard flavone has the characteristics of directionally reaching colon and potential targeted medicinal value; the collard total flavonoids rich in acyl provided by the invention can effectively relieve ulcerative colitis.

The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made by one skilled in the art, and any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for purifying acyl-rich collard flavone with colitis relieving effect is characterized by comprising the following steps:

1) preparing a collard flavone extract:

stirring and heating collard powder by using an ethanol water solution for extraction, centrifuging, taking supernatant, carrying out vacuum rotary evaporation and concentration until fat-soluble components are separated out, centrifuging to remove the fat-soluble components, taking supernatant, and further concentrating into an extract for later use;

2) purifying collard total flavonoids:

diluting the extract obtained in the step 1) with distilled water to obtain a flavone solution, adjusting the pH value of the solution, pouring the solution into a container filled with macroporous resin, stirring and adsorbing, collecting the macroporous resin, leaching the macroporous resin with distilled water until the macroporous resin is colorless, then stirring and eluting with an ethanol water solution as an eluent, collecting the eluent, performing vacuum rotary evaporation and concentration, and freeze-drying to obtain purified kale total flavone;

dissolving the purified kale total flavonoids obtained in the step 2) by using low-carbon alcohol, adding a thermoplastic resin containing repeated amide groups, uniformly stirring, volatilizing, then loading into a chromatographic column, sequentially eluting the chromatographic column by using eluents with gradually increased concentrations, respectively collecting eluents, performing vacuum rotary evaporation and concentration, and freeze-drying to obtain acyl kale flavonoid components which can directionally reach the colon; the acyl kale flavone which can directionally reach the colon is acylated quercetin or kaempferol; the content of acylated flavonoids which can be directionally reached to colon in the obtained flavone component is 5.02 times of that of the total flavonoids of the kale obtained in the step 2).

2. The method of claim 1, wherein the macroporous resin of step 2) is D101, NKA-9, HPD-400, HPD-500, AB-8, HPD-450 or XDA-1.

3. The method for purifying acyl-rich kale flavone with colitis-relieving effect as claimed in claim 1, wherein the concentration of the diluted flavone solution of step 2) is 0.1-2 mg/mL, and the pH of the solution is adjusted to 2-8.

4. The method for purifying acyl-rich kale flavone with colitis-relieving effect according to claim 1, wherein the volume fraction of the ethanol aqueous solution in step 2) is 30% -95%.

5. The method for purifying acyl-rich kale flavone with colitis-relieving effect as claimed in claim 1, wherein the eluting agents with increasing concentration in step 3) are sequentially: the gradient 1 eluent is 10 to 25 percent of low-carbon alcohol aqueous solution, and the dosage is 2 to 5 BV; the gradient 2 eluent is 30 to 45 percent of low-carbon alcohol aqueous solution, and the dosage is 2 to 5 BV; the gradient 3 eluent is 50-65% of low-carbon alcohol aqueous solution, and the dosage is 2-5 BV; the gradient 4 eluent is 70-90% of low-carbon alcohol aqueous solution, and the dosage is 2-5 BV; the lower alcohol is ethanol and/or methanol.