CN114558026B

CN114558026B - Application of barbed skullcap herb polysaccharide in preparing medicine for treating inflammatory intestinal diseases

Info

Publication number: CN114558026B
Application number: CN202210067780.6A
Authority: CN
Inventors: 吴霞; 胡兴江; 张乔; 徐娜娜; 吴明兰; 叶子奇; 李卫芬; 刘剑; 李济忱; 喻松霞; 赵青威; 刘健; 古佳艳; 张天芳; 胡希; 杨希; 李鑫; 李小冬
Original assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Current assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2023-04-21
Anticipated expiration: 2042-01-20
Also published as: CN114558026A

Abstract

The invention relates to application of barbed skullcap herb polysaccharide in preparing medicine for treating inflammatory bowel disease. The barbed skullcap herb polysaccharide is obtained by separating and purifying barbed skullcap herb of Labiatae, has the advantages of wide source, convenient material taking and the like, and is convenient for industrialized mass preparation; the PSB is extracted and prepared by an alcohol precipitation water-soluble method, and the technology simplifies the preparation flow of the PSB, and has the advantages of simple operation, low cost and high purity; the PSB prepared by the method has potential IBD resisting effect, and the action mechanism of the PSB is possibly related to anti-inflammatory and intestinal flora regulation, so that a research basis and a theoretical basis are provided for the application of the PSB in IBD resisting.

Description

Application of barbed skullcap herb polysaccharide in preparing medicine for treating inflammatory intestinal diseases

Technical Field

The invention relates to application of barbed skullcap herb polysaccharide in preparing medicine for treating inflammatory bowel disease.

Background

Inflammatory bowel disease (Inflammatory bowel disease, IBD) including ulcerative colitis (Ulcerative Colitis, UC) and Crohn's Disease (CD) is a group of chronic intestinal inflammatory diseases of unknown cause, which have become common digestive system diseases in our country due to increasing incidence, and are also listed by WHO as one of the modern refractory diseases. The current main clinical treatment comprises aminosalicylate, corticosteroid, immunosuppressant, biological agent and the like, but has the problems of limited curative effect, high recurrence rate, long treatment period, easy drug resistance generation and the like, and is often accompanied by serious side effects. Thus, there is still significant clinical value in finding cheaper and less toxic, highly effective, orally administrable natural drugs. The traditional Chinese medicine has the advantages of small toxic and side effects, low cost, multiple targets, multiple ways and the like, and has great clinical application prospect, so that the traditional Chinese medicine with high efficiency and low toxicity and the effective components thereof are further explored in the UC treatment field, and the action mechanism of the traditional Chinese medicine is fully clarified, so that the traditional Chinese medicine has important social and economic significance.

Herba Scutellariae Barbatae of LabiataeScutellaria barbataD.don.) dried whole herb, pungent tasteBitter, cold-natured, and has the effects of clearing heat, removing toxicity, removing blood stasis and promoting urination. Is used as an anti-inflammatory, antioxidant, antitumor and diuretic agent in China, and is used for treating cancers, snake bites and inflammatory diseases clinically. Among them, the barbed skullcap herb Polysaccharide (PSB) may have important functions in antioxidation, anticancer, immunity regulation and the like, but no report on the activity related to the PSB anti-IBD and the action mechanism thereof is yet seen. In the same way, related patents (application publication numbers CN107216400A and CN 201611209220.0) in the prior art adopt ultrasonic extraction methods for the extraction and separation of PSB, and relate to organic solvents such as acetone, so that the method has high toxicity, high cost and difficulty in realizing industrial production.

Disclosure of Invention

The invention aims to provide an application of barbed skullcap herb polysaccharide in preparing a medicament for treating inflammatory bowel disease, and solves the problem of lack of related medicaments in the current treatment of IBD.

The technical scheme adopted by the invention is as follows:

the application of herba Scutellariae Barbatae polysaccharide in preparing medicine for treating inflammatory bowel disease is provided. The barbed skullcap herb polysaccharide is extracted from barbed skullcap herb, and the effective components include arabinose, galacturonic acid, galactose, glucose, glucuronic acid, etc.

The inflammatory bowel disease includes ulcerative colitis or Crohn's disease. Ulcerative colitis (ulcerative colitis) is a chronic nonspecific inflammatory disease of the colon and rectum of which the etiology is not yet clear, and lesions are limited to the mucosa and submucosa of the large intestine. Crohn's disease is an unexplained inflammatory disease of the intestinal tract that occurs anywhere in the gastrointestinal tract but is commonly found in the terminal ileum and right half colon.

The barbed skullcap herb polysaccharide may be added with pharmaceutically acceptable supplementary material to prepare one of the following dosage forms: capsule, tablet, granule, injection, sustained release preparation, oral liquid or dripping pill.

When the barbed skullcap herb polysaccharide is used as a medicine, the medicinal dosage of the barbed skullcap herb polysaccharide is 25-50 mg/kg.

Specifically, the barbed skullcap herb polysaccharide is prepared by the following steps: adding water with the mass of 20-25 times into the barbed skullcap, heating and refluxing for extraction for 1-3 hours, filtering and taking filter residues, adding water with the mass of 10-20 times, continuously refluxing for extraction for 1-2 hours, filtering, combining filtrates, concentrating under reduced pressure (vacuum degree-0.07 to-0.1, temperature 70 ℃), slowly adding absolute ethyl alcohol with the mass of 3-5 times into the concentrated solution, continuously stirring, standing overnight at 4 ℃, taking out, filtering, drying filter cakes, and separating and purifying to obtain barbed skullcap crude polysaccharide.

The separation and purification method comprises the following steps: dissolving crude barbed skullcap herb polysaccharide in water, adding a solution with the volume ratio of dichloromethane to n-butanol being 5:1, centrifuging, removing a lower organic layer and an intermediate layer, repeating the operation (5-8 times) until protein is removed, and obtaining an upper aqueous solution for freeze drying to obtain the barbed skullcap herb polysaccharide.

The beneficial effects of the invention are mainly as follows:

the invention provides an application of barbed skullcap herb polysaccharide in preparing a medicament for treating inflammatory bowel disease, the barbed skullcap herb polysaccharide is separated and purified from barbed skullcap herb of Labiatae, and has the advantages of wide source, convenient material taking and the like, and is convenient for industrialized mass preparation; the PSB is extracted and prepared by an alcohol precipitation water-soluble method, and the technology simplifies the preparation flow of the PSB, and has the advantages of simple operation, low cost and high purity; the PSB prepared by the method has potential IBD resisting effect, and the action mechanism of the PSB is possibly related to anti-inflammatory and intestinal flora regulation, so that a research basis and a theoretical basis are provided for the application of the PSB in IBD resisting.

Drawings

FIG. 1 is a flow chart of the PSB extraction process in example 1;

FIG. 2 shows the results of analysis of the chemical composition of PSB in example 2; (a) FT-IR spectrum of PSB; (B) HPGP profile of PSB; (C) monosaccharide composition of PSB; (D) chromatogram of monosaccharide standard.

FIG. 3 is the effect of PSB on DSS-induced UC mice in example 3; (a) a weight change profile; (B) DAI change curve. Effect of PSB on DSS-induced mice spleen factor. (C) representative pictures of spleen; (D) statistical results of spleen coefficients (n=6). In comparison with the control group, ^### P<0.001；p compared to model<0.001。

FIG. 4 is a graph showing the effect of PSB on DSS-induced inflammatory cytokine levels in colon tissue of UC mice in example 4; (A) Colon slice H&Representative pictures of E staining. (B) morphological scoring of colon slices. Each point is expressed as mean ± standard deviation (n=6). In comparison with the control group, ^### p<0.001， ^# p<0.05. compared with the model, p<0.01，***p<0.001.PSB inhibits the levels of the pro-inflammatory cytokines (C) TNF- α (D) IFN- γ (E) IL-1β (F) IL-18 and (G) IL-6 in colon tissue. In comparison with the control group, ^# p<0.05， ^### p<0.001; compared with the model, p<0.05，**p<0.01，***p<0.001。

FIG. 5 shows the anti-inflammatory effect of PSB by inhibiting the activation of NF- κB and STAT3 pathways in example 5; (A) PSB inhibits activation of DSS mice p-NF- κ B, p-IκBα; (B) PSB inhibits activation of DSS mouse STAT 3; (C) relative protein expression rate of p-NF- κB/NF- κB; (D) relative protein expression rate of p-iκbα/iκbα; (E) Relative protein expression rate of p-STAT3/STAT3 (n=6). In comparison with the control group, ^### p<0.001; in contrast to the set of models, ^*** p<0.001。

FIG. 6 is a graph showing the results of differential microbial community diversity analysis following PSB group activation in example 6; (A) Wen diagram (Venn diagram), (B) strain richness index (Chao 1), (C) observed species index (observed species index), (D) Shannon diversity index (Shannon's diversity index), (E) Simpson diversity index (Simpson index).

FIG. 7 shows the results of bacterial community diversity analysis for intestinal contents of different populations in example 6.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1: preparation of Scutellaria barbata Polysaccharide (PSB)

1.1 extraction:

putting 1kg of barbed skullcap herb into a 40L decoction pot, adding 20-25 times of water, extracting under reflux for 1-3 hours, and filtering with 150 meshes; and adding 10-20 times of water into the filter residues, continuously extracting under reflux for 1-2 hours, filtering with 150 meshes, and combining the filtrates. And (3) taking filtrate, concentrating under reduced pressure (the vacuum degree is-0.07 to-0.1, the temperature is 70 ℃) to 1-2 times of the feeding amount, slowly adding 3-5 times of absolute ethyl alcohol, continuously stirring, standing overnight at the temperature of 4 ℃, taking out, filtering, and drying a filter cake to obtain the crude barbed skullcap herb polysaccharide.

1.2 purification:

dissolving crude calyx seu fructus Physalis polysaccharide obtained in step 1.1 in water, adding 1/2 volume of dichloromethane and n-butanol (5:1) solution, centrifuging for 10 min, removing lower organic layer and middle layer, and repeating the operation until protein is removed (repeating the operation for 8 times) to obtain upper aqueous solution. The upper aqueous solution was freeze-dried to give the barbed skullcap polysaccharide (76.2 g) (PSB extraction process flow diagram see FIG. 1).

Example 2: structural identification of barbed skullcap herb polysaccharide

The FT-IR spectrum is shown in FIG. 2A, and Table 1 summarizes the properties of the peaks. FT-IR spectra showed PSB at 3407.60, 2933.20 and 1614.13 cm ^-1 The compositions exhibit characteristic absorption of polysaccharides for O-H, C-H and C-O stretching vibrations, respectively. 1000-1200 cm ^-1 The absorption at this point is due to the C-O-C and C-O-H in the pyranose ring. HPGPC spectra showed that PSB had a single symmetrical peak, meaning that PSB was a homogeneous polysaccharide (fig. 2B) with a weight average molecular weight (Mw) of 1.25×106Da (Mw/mn=1.86). Further, the number average molecular weight (Mn) thereof was 0.67×106da, and the peak molecular weight (Mp) thereof was 0.9×106da. As shown in fig. 2C, D and table 2, the monosaccharide composition of PSB consisted mainly of arabinose, galacturonic acid, galactose, glucose and glucuronic acid, in a molar ratio of 1.00:2.09:4.52:4.73:4.90.

Table 1: properties of peaks in FT-IR absorption Spectrum

Wave number/cm-1	Attribution of peak
		3407.60	Stretching vibrations of O-H
2933.20	Asymmetric stretching vibration of CH ₂
		1614.13	Asymmetric stretching vibration of C=O
1411.64	Shear vibration of CH ₂
		1261.22	Bending vibrations of C-H
1064.51	Pyranoside

Table 2: monosaccharide composition of PSB

Number	Retention time （min）	Monosaccharides	Composition （%）
				1	17.337	Mannose	3.81
2	21.582	Aminoglucose	0.66
				3	22.613	Ribose	1.36
4	24.316	Rhamnose	2.70
				5	25.910	Glucuronic acid	25.24
6	29.607	Galacturonic acid	10.76
				7	36.167	Galactosamine	/
8	41.173	Glucose	24.35
				9	43.996	Galactose	23.28
10	45.596	Xylose	1.54
				11	48.242	Arabinose	5.15
12	53.597	Fucose	0.89

Example 3: effects of PSB on DSS-induced weight changes, disease Activity Index (DAI) score and spleen factor in UC mice

The experimental method comprises the following steps: 30 (20.+ -. 2 g) male C57BL/6 SPF mice were randomly divided into 5 groups (6 per group): control, model, SASP, PSB (25 mg/kg) and PSB (50 mg/kg). All mice were free to drink 3% dss solution for 7 days to induce UC, except for the control group. Simultaneously, the corresponding medicine is infused into the stomach 2 times a day according to the dosage of 0.1 mL/10g, and the medicine is continuously administrated for 7 days. Mice body weight, stool, and other abnormalities were recorded daily and blindly scored for Disease Activity Index (DAI). Mice were sacrificed on day 8 for cervical dislocation anatomic sampling. Spleen was isolated, photographed and weighed. Meanwhile, spleen factor was calculated as organ weight/body weight (unit: g).

Experimental results: FIG. 3A shows that PSB significantly inhibited weight loss in mice, particularly in the PSB (50 mg/kg) group; fig. 3B shows that PSB significantly improves DAI score; figures 3C-D show that PSB also significantly reduced the enlarged spleen. In conclusion, PSB can significantly improve symptoms and pathological injuries of UC mice.

Example 4: effects of PSB on DSS-induced UC mouse colon tissue inflammatory cytokine levels

The experimental method comprises the following steps: the treatment of mice was the same as in "example 3", and after dislocation of cervical vertebrae, the colon tissues of all mice were separated, measured and photographed as soon as possible, and the colon contents were removed and the colon was divided into two. One part was immediately frozen in liquid nitrogen and the other part was fixed with 4% paraformaldehyde, embedded, sectioned, stained and photographed under a microscope. Pathological changes such as edema, adhesion, ulcer, necrosis and the like are observed, and histological scores are evaluated.

The ELISA method is used for measuring inflammatory cytokines: the total protein concentration of the colon tissue was homogenized in PBS buffer on ice. The homogenate was then centrifuged (5 min, 5000 g,4 ℃) using a 5417R centrifuge to obtain a supernatant. Protein concentration was determined with BCA kit and proinflammatory cytokines were detected with ELISA kit. Absorbance values were measured at an optical density of 450 nm using Microplate Reader 680.

Experimental results: as shown in fig. 4A-B, the PSB (25 and 50 mg/kg) group significantly reduced the pathological changes in DSS-induced UC mice colon lesions and significantly improved the histopathological score of colon inflammation. As shown in FIGS. 4C-G, PSB significantly reduced TNF- α, IFN- γ, IL-1β, IL-6 and IL-18 levels.

Taken together, PSB (25 and 50 mg/kg) can alleviate DSS-induced colon injury in mice and significantly reduce inflammatory cytokine levels in the colon.

Example 5: PSB inhibits DSS-induced activation of NF- κB and STAT3 signaling pathways in UC mice

The experimental method comprises the following steps: protein expression is detected by a Western blot method: after extraction and centrifugation of the colon tissue of example 4, protein concentrations of all colon tissue supernatants were quantified and normalized to be equal. All protein samples were separated by SDS-PAGE and then electrophoretically transferred onto PVDF membrane. The membrane was then incubated with the corresponding primary and secondary antibodies, and the gray scale values of all protein bands were detected with an electrochemiluminescence instrument, with tubulin as an internal control, to normalize the gray density of the protein bands.

Experimental results: PSB significantly reversed the elevation of p-NF- κ B p65 and p-IκBα levels (FIGS. 5A, C and D) while significantly down-regulating STAT3 activation (FIGS. 5B and E). In conclusion, PSB (25 and 50 mg/kg) significantly inhibited DSS-induced activation of NF- κB and STAT3 in UC mice.

Example 6: PSB can regulate DSS-induced disturbance of intestinal microbiota of UC mice

The experimental method comprises the following steps:

(1) DNA extraction: DNA was extracted from all groups of "example 4" using the e.z.n.a. Fecal DNA kit and the total DNA was eluted using 50 μl elution buffer for PCR detection.

(2) PCR amplification and 16 srdnas sequencing: the V4 region of the 16S rRNA gene was amplified using primers 341F (5 '-CCTACGGGNGGCWGCAG-3') and 805R (5 '-GACTACHVGGGTATCTAATCC-3') (where N, W, H and V are degenerate bases, representing N: A/T/C/G; W: A/T; H: A/T/C; V: G/A/C, respectively). PCR amplification was performed using 25 ng template DNA, 12.5 μl PCR premix and 2.5 μl each primer.

(3) Data analysis: samples were sequenced on the Illumina HiSeq platform. FLASH (version 1.2.8) is used to allocate, truncate and merge pairs of end reads. Chimeric sequences were screened and 97% similarity was assigned to the same Operational Taxon (OTU) using ≡using Vsearch software (v 2.3.4). A representative sequence is selected for each OTU and classification data is assigned to each representative sequence using a ribosomal database entry (RDP) classifier. The difference in dominant species in the different populations was determined using the mafft software (V7.310).

Experimental results:

(1) Alpha-diversity analysis: as shown in FIG. 6A, PSB (50 mg/kg) can improve the diversity of intestinal flora to some extent. As shown in fig. 6B and table 3, DSS inhibited microbial abundance compared to control mice. The observed species index results indicate that there was a diversity difference between the control and model groups (fig. 6C). Meanwhile, although the Shannon and Simpson indices were similar in the four groups, the diversity was twisted to some extent after PSB dry (fig. 6D-E).

In conclusion, PSB (50 mg/kg) significantly improved intestinal microbiologic diversity in DSS-induced UC mice.

Table 3: alpha-diversity analysis results

	Control	Model	SASP	PSB_50
					Chao1	796.39 ± 74.11	636.78 ± 44.47	703.01 ± 60.13	659.09 ± 56.10
Observed_otus	792.00 ± 74.08	634.17 ± 44.87	699.83 ± 59.56	656.33 ± 56.52
					Shannon	7.59 ± 0.15	6.38 ± 0.31	7.02 ± 0.22	6.46 ± 0.28
Simpson	0.99 ± 0.00	0.96 ± 0.02	0.98 ± 0.01	0.96 ± 0.01

(2) Beta diversity analysis: as shown in FIGS. 7A-E, the PSB (50 mg/kg) group tended to be the control group, indicating that PSB was able to regulate the intestinal flora towards normal.

(3) And (3) cluster analysis: as shown in FIGS. 7F-G and Table 4, PSB (50 mg/kg) has a significant regulatory effect on the changes in intestinal flora in mice model of ulcerative colitis.

Table 4: at the doorpAnd belongs togCluster analysis results at the level _)

	Control	Model	SASP	PSB_50
					p_Firmicutes	50.44	27.78 ^#	52.53*	35.78*
p_Bacteroidetes	24.36	27.17	12.81	22.23
					p_Proteobacteria	9.88	28.86 ^#	16.85*	23.43
p_Epsilonbacteraeota	8.51	9.07	8.95	9.42
					p_Deferribacteres	6.75	2.54 ^#	8.63*	8.00*
p_Tenericutes	0.06	3.26 ^#	0.11*	0.23*
					p_Bacteria_unclassified	0.00	1.33 ^#	0.11*	0.90*
g_Lachnospiraceae_NK4A136_group	14.16	4.37 ^#	24.20*	14.54*
					g_Bacteroides	0.82	17.38 ^#	5.93*	13.12*
g_Mucispirillum	6.75	2.54 ^#	8.63*	8.00*
					g_Muribaculaceae_unclassified	14.00	3.74 ^#	2.13	5.34*
g_Parasutterella	0.11	8.54 ^#	0.73*	2.81*
					g_Lactobacillus	6.07	0.22 ^#	1.10*	0.50*
g_Eisenbergiella	0.48	4.05 ^#	2.26*	0.01*
					g_Ruminococcus	3.94	0.05 ^#	0.16*	1.99*
g_Anaeroplasma	0.06	3.26 ^#	0.11*	0.23*
					g_Streptococcus	0.96	0.63 ^#	1.63*	1.69*

^# p < 0.05 vs. control，*p < 0.05 vs. model.

(4) Correlation of gut microbiota with inflammatory cytokines: as shown in FIG. 7H, at least IFN-gamma, IL-1β, IL-6 and IL-18 are highly associated with the intestinal microbiota.

Claims

1. The application of the barbed skullcap herb polysaccharide in preparing medicine for treating inflammatory bowel disease, which is characterized in that the inflammatory bowel disease is ulcerative colitis, and the barbed skullcap herb polysaccharide is prepared and obtained by the following steps: adding water with the mass of 20-25 times into the barbed skullcap, heating and reflux-extracting for 1-3 hours, filtering to obtain filter residues, adding water with the mass of 10-20 times, continuously reflux-extracting for 1-2 hours, filtering, merging filtrate, concentrating under reduced pressure, slowly adding absolute ethyl alcohol with the mass of 3-5 times into concentrated solution, continuously stirring, standing overnight at 4 ℃, taking out, filtering, drying filter cakes, and separating and purifying to obtain barbed skullcap crude polysaccharide; the separation and purification method comprises the following steps: dissolving crude herba Scutellariae Barbatae polysaccharide in water, adding solution of dichloromethane and n-butanol at a volume ratio of 5:1, centrifuging, removing lower organic layer and middle layer, repeating the operation until protein is removed, and lyophilizing to obtain upper aqueous solution to obtain herba Scutellariae Barbatae polysaccharide.

2. The use according to claim 1, wherein the barbed skullcap herb polysaccharide is formulated in one of the following forms by adding pharmaceutically acceptable excipients: capsule, tablet, granule, injection, sustained release preparation, oral liquid or dripping pill.

3. The use according to claim 1 or 2, characterized in that the pharmaceutical dosage of the barbed skullcap herb polysaccharide is 25-50 mg/kg.