Pulsatilla chinensis vesicle, preparation method thereof and application of pulsatilla chinensis vesicle in intestinal inflammation

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to pulsatilla chinensis vesicles, a preparation method thereof and application of the pulsatilla chinensis vesicles in intestinal inflammation. The preparation method comprises the following steps: adding water into fresh pulsatilla chinensis, juicing, filtering, and collecting filtrate; centrifuging the filtrate at a low speed, and collecting supernate; and finally centrifuging the supernate, collecting the precipitate, suspending the precipitate with PBS, and drying to obtain the product. The prepared pulsatilla chinensis vesicles are small in particle size and are nanoscale carriers; rich active substances such as lipid, protein and the like are carried, and through the synergistic effect of multiple components, the natural targeting property is achieved on the diseased region; the safety is high, the immunogenicity is low, and ethical dispute does not exist.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a Pulsatilla scaly, a preparation method thereof, and an application thereof in intestinal inflammation. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific intestinal inflammatory disease. Clinically, IBD presents a variety of clinical symptoms, including abdominal discomfort, diarrhea, bloody stools, fever, fatigue and weight loss, etc. In addition to the above frequently reported symptoms, IBD patients often exhibit extraintestinal complications, which may involve skin diseases (erythema nodosum and pyoderma gangrenosum), eye diseases (conjunctivitis, uveitis), rheumatic diseases (ankylosing spondylitis and peripheral arthritis), hepatobiliary diseases (primary sclerosing cholangitis) and urinary system diseases (kidney stones), etc.

[0003] IBD mainly includes ulcerative colitis (UC) and Crohn's disease (CD), which have the same clinical course, and the difference is limited to the location and nature of the inflammatory lesions. Among them, UC is characterized by diffuse inflammation of the colonic mucosa, and the course of the disease is repeated remission. There is an urgent need to develop better UC treatment drugs, especially drugs that can act locally in the inflamed colon without systemic exposure and related side effects, which is of great significance for the treatment of UC colitis.

[0004] Extracellular vesicles are a general term for tiny membrane-structured vesicles that are actively secreted by cells. They are mainly divided into three categories: exosomes, microvesicles, and apoptotic bodies according to their size, biological characteristics, and formation process. Extracellular vesicles have a typical lipid bilayer structure, carrying proteins, lipids, RNA, and other substances, and play an important role in cell communication, cell migration, and the occurrence and development of cancer.

[0005] Pulsatilla Pulsatilla chinensis (Bunge) Regel) has a significant inhibitory effect on the secretion of IL-6 by macrophages stimulated by Pulsatilla striata of bacterial lipopolysaccharide, and the effect increases over time, thereby reducing excessive inflammatory response and systemic damage; Pulsatilla striata and its complexes inhibit skin fungi, yeasts, cones, and Candida albicans; fresh juice and explosive of Pulsatilla striata are most sensitive to Staphylococcus aureus and Pseudomonas aeruginosa; it also has a good inhibitory effect on dysentery and typhoid bacteria.

[0006] There is no research on Pulsatilla-like extracellular vesicles at present, therefore, it is very necessary to develop Pulsatilla-like extracellular vesicles for the treatment of inflammatory bowel disease. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a Pulsatilla scaly vesicle and a preparation method thereof and application thereof in intestinal inflammation.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A method for preparing Pulsatilla scaly vesicles comprises the following steps: (1) First, add water to fresh Pulsatilla chinensis to squeeze out the juice, filter it, and collect the filtrate; (2) Centrifuge the filtrate and collect the supernatant; (3) Finally, the supernatant was centrifuged, the precipitate was collected, the precipitate was suspended with PBS, and dried to obtain. Wherein, the centrifugation in step (2) is 150 × g-250 × g for 10 min-20 min, 3500 × g-4500 × g for 15 min-25 min, The centrifugation in step (3) is 10000 × g-14000 × g for 35 min-45 min, and 80000 × g-120000 × g for 65 min-75 min.

[0009] Preferably, the mass volume ratio of the fresh Pulsatilla chinensis product and water is 150 g-240 g:800 mL-1500 mL.

[0010] Preferably, the number of times of squeezing the juice in step (1) is 2 to 4 times.

[0011] Preferably, the centrifugation temperature in steps (2) and (3) is 3.5°C-4.5°C.

[0012] Preferably, the mass volume ratio of the precipitate to PBS in step (3) is 0.3 g-0.5 g:8 mL-12 mL.

[0013] Preferably, the drying temperature in step (3) is -80°C to -90°C, and the drying time is 10 h to 14 h.

[0014] The present invention also provides the Pulsatilla scaly vesicles prepared by the above preparation method.

[0015] Preferably, the particle size of the Pulsatilla scaly vesicle is 180 nm-190 nm, and the molecular weight of the Pulsatilla scaly vesicle is 52KDa-105K Da.

[0016] The present invention also provides the use of the Pulsatilla scaly vesicle in preparing a drug for treating intestinal inflammation.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0018] Preferably, the dosage form of the drug is any one or more of injection, capsule, tablet, oral liquid and granule.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The Pulsatilla scaly vesicles prepared by the present invention have a small particle size and are nanoscale carriers; they carry abundant active substances such as lipids and proteins, and have natural targeting to the lesion site through the synergistic effect of multiple components.

[0020] (2) The Pulsatilla scaly vesicles prepared by the present invention are of natural origin, have high safety, low immunogenicity, and no ethical controversy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the TEM image of the Pulsatilla scaly vesicles prepared in Example.

[0022] Figure 2 This is the NTA image of the Pulsatilla scaly vesicles prepared in Example.

[0023] Figure 3 This is the electrophoretic distribution diagram of the Pulsatilla scaly vesicles prepared in Example.

[0024] Figure 4 This is a graph of weight change rate.

[0025] Figure 5 This is the disease activity index DAI score chart.

[0026] Figure 6 HE staining of the cross section of the colon.

[0027] Figure 7 This is a histopathological scoring diagram.

[0028] Figure 8 A diagram of the mouse colon.

[0029] Fig. 9 Figure 2 is a graph of colon length.

[0030] Figure 4 , Figure 5 , Figure 7 and Fig. 9 Compared with the normal group, **** Indicates p<0.0001; compared with the model group, ## indicates p<0.01, #### Indicates p<0.0001; compared with the positive drug group, ^^^ indicates p<0.001, ^^^^ Indicates p<0.0001; compared with the low-dose group of Pulsatilla truncatula, && indicates p<0.01, &&&&means p<0.0001; ns means no significant difference.

[0031] Figure 6 and Figure 8 In the figure, from left to right, they are the normal group, model group, positive drug group, low-dose Pulsatilla scutellaria vesicle group, and high-dose Pulsatilla scutellaria vesicle group. DETAILED DESCRIPTION

[0032] It is worth noting that the raw materials used in the present invention are all common commercially available products. Fresh Pulsatilla chinensis was purchased from Chengde Shenshan Medicinal Materials Co., Ltd. of China National Pharmaceutical Group.

[0033] Example A method for preparing Pulsatilla scaly vesicles, comprising the following steps: (1) First, wash 200 g of fresh Pulsatilla chinensis thoroughly with sterile water, chop it into small pieces, add 1000 mL of pure water, and squeeze the juice three times in a juicer (voltage / power: 220 V / 1 KW), filter, and collect the filtrate; (2) Centrifuge the filtrate at 200 × g for 10 min and 4000 × g for 20 min at 4°C, and collect the supernatant. (3) Finally, the supernatant was centrifuged at 4°C, 12,000 × g for 40 min and 100,000 × g for 70 min. The precipitate was collected and resuspended in PBS at a mass-to-volume ratio of 10 mL:0.4 g. The precipitate was then freeze-dried in a -85.6°C freeze dryer for 12 h to obtain Pulsatilla striata vesicles.

[0034] The prepared Pulsatilla scaly vesicles were characterized by morphology, particle size and molecular weight. The TEM image is shown in Figure 1 and the NTA image is shown in Figure 2. Figure 2 As shown in Figure 2, it can be seen that the particle size of the Pulsatilla sphaerocarpa vesicle is 187.5 nm; the electrophoresis distribution diagram is shown in Figure 2 Figure 3 As shown, it can be seen that the molecular weight of Pulsatilla stoloniferum vesicles is 52K Da-105K Da.

[0035] Test case: The therapeutic effect of Pulsatilla scaly on ulcerative colitis 1. Experimental Animals C57 mice, 6-8 weeks old, male, were purchased from Beijing Weitonglihua Co., Ltd., animal license: SYXK (Beijing) 2021-0017, and were housed in a specific pathogen-free (SPF) environment with a light / dark cycle of 12 h. Food and water were freely available, and all animal care and experimental procedures were in accordance with the guidelines approved by the Ethics Committee of the Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences.

[0036] 2. Experimental Methods 2.1 Animal modeling: The mice were given an aqueous solution containing 2% dextran sulfate sodium (DSS) by weight for 1 week without any other water source. The ulcerative colitis model was established.

[0037] 2.2 Grouping and drug administration: 50 C57 mice were divided into 5 groups, namely, normal group, model group, low-dose Pulsatilla vesicle group (1.5 mg / mouse), high-dose Pulsatilla vesicle group (3 mg / mouse) and positive drug group (5-aminosalicylic acid, i.e. 5-ASA). Except for the normal group, the mice in the other groups were modeled with ulcerative colitis.

[0038] 2.3 Index detection: (1) Body weight change rate: After inducing colitis, the mice were weighed daily to observe dynamic changes.

[0039] Weight change rate (%) = (current weight − baseline weight) ÷ baseline weight × 100% The baseline body weight refers to the initial body weight of the mice.

[0040] (2) Disease activity index (DAI): During the modeling period, the general condition of the mice (activity status, coat color, weight changes), stool characteristics, and fecal occult blood (or bloody stool) were observed, and the disease activity index (DAI) of the mice was evaluated according to the criteria established by McCarthy (reference: Kobayashi T, Siegmund B, Le Berre C, et al. Ulcerative colitis: Recent advances inpathophysiology and targeted therapies. Int J Mol Sci. 2023;24(23):16877.DOI:10.3390 / ijms242316877.).

[0041] DAI score = (weight loss + stool characteristics + fecal occult blood) ÷ 3.

[0042] (3) HE staining and histopathological scoring of colon cross section HE staining of colon cross section, the steps are as follows: A. Dewaxing and hydration: Place the paraffin sections in environmentally friendly dewaxing solution I for 20 min → environmentally friendly dewaxing solution II for 20 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 75% alcohol for 5 min, and wash with tap water.

[0043] B. Frozen Section and Rewarming Fixation: Take out the sections from the -20°C refrigerator and let them return to room temperature. After fixing with 4% paraformaldehyde for 15 min, rinse with running water, and then pre-treat the sections in hematoxylin-eosin (H&E) staining solution for 1 min.

[0044] C. Hematoxylin Staining: Immerse the sections in hematoxylin staining solution for 3 - 5 min and wash with tap water.

[0045] D. Differentiation and Blueing: Differentiate with hematoxylin differentiation solution, wash with tap water, blue with hematoxylin blueing solution, and rinse with running water.

[0046] E. Eosin Staining: Dehydrate the sections in 95% ethanol for 1 min, and then stain in eosin staining solution for 15 s.

[0047] F. Dehydration and Clearing: Immerse the sections successively in absolute ethanol I for 2 min → absolute ethanol II for 2 min → absolute ethanol III for 2 min → n-butanol I for 2 min → n-butanol II for 2 min → xylene I for 2 min → xylene II for 2 min until clear, and mount with neutral balsam.

[0048] G. Finally, examine under a microscope and collect and analyze the images.

[0049] Histopathological Scoring: Score according to the scoring criteria (References: Kobayashi T, Siegmund B, Le Berre C, et al. Ulcerative colitis: Recent advances in pathophysiology and targeted therapies[J]. Int J Mol Sci. 2023;24(23):16877. DOI:10.3390 / ijms242316877.).

[0050] (4)Colon Length: After the administration ended, take out the colons of mice in each group and measure the colon length of mice in each group with a steel ruler.

[0051] 3. Experimental Results (1)Body Weight Change: The experimental results are as Figure 4 shown. Compared with the normal group, the body weight of the model group showed a significant downward trend (p < 0.0001). Compared with the model group, the body weights of the positive drug group, the low-dose Pulsatilla vesicle group, and the high-dose Pulsatilla vesicle group showed an upward trend, but there were no statistical differences compared with the model group. It indicates that the body weight of mice with ulcerative colitis shows a significant downward trend, which is in line with the basic characteristics of the ulcerative colitis model mice.

[0052] (2)DAI Score: The experimental results are as Figure 5As shown in the figure, compared with the normal group, the DAI score of the model group showed a significant upward trend (p<0.0001). Compared with the model group, the DAI scores of the positive drug group, the low-dose group of Pulsatilla striata vesicles and the high-dose group of Pulsatilla striata vesicles showed a significant downward trend (p<0.01 or p<0.0001), and the scores were comparable. Compared with the low-dose group of Pulsatilla striata vesicles, the DAI score of the high-dose group of Pulsatilla striata vesicles showed a significant upward trend (p<0.01). The above experimental results show that Pulsatilla striata vesicles and the positive drug group can significantly reduce the DAI scores of mice with ulcerative colitis, and the effect of the low-dose group of Pulsatilla striata vesicles is better than that of the high-dose group, and the efficacy of the low-dose group of Pulsatilla striata vesicles and the positive drug group is comparable.

[0053] (3) HE staining and histopathological scoring of colon cross section: The experimental results are as follows Figure 6-Figure 7 As shown in the figure, compared with the normal group, the histopathological score of the model group showed a significant upward trend (p<0.0001). Compared with the model group, the histopathological scores of the positive drug group, the low-dose group of Pulsatilla striata vesicles and the high-dose group of Pulsatilla striata vesicles showed a significant downward trend (p<0.0001). Compared with the positive drug group, the histopathological score of the low-dose group of Pulsatilla striata vesicles showed a significant downward trend (p<0.0001). Compared with the low-dose group of Pulsatilla striata vesicles, the histopathological score of the high-dose group of Pulsatilla striata vesicles showed a significant upward trend (p<0.0001). The above experimental results show that Pulsatilla striata vesicles can significantly reduce the colon histopathological score of mice with ulcerative colitis, and the effect of the low-dose group of Pulsatilla striata vesicles is better than that of the positive drug group and the high-dose group of Pulsatilla striata vesicles.

[0054] (4) Colon length: The experimental results are as follows Figure 8-Figure 9 As shown, compared with the normal group, the colon length of the model group showed a significant downward trend (p<0.0001). Compared with the model group, the colon length of the positive drug group, the low-dose group of Pulsatilla scaly and the high-dose group of Pulsatilla scaly were significantly increased (p<0.01 or p<0.0001). Compared with the positive drug group, the colon length of the low-dose group of Pulsatilla scaly showed a significant upward trend (p<0.001). Compared with the low-dose group of Pulsatilla scaly, the colon length of the high-dose group of Pulsatilla scaly showed a significant downward trend (p<0.0001). The above experimental results show that Pulsatilla scaly can significantly increase the colon length of mice with ulcerative colitis, and the effect of the low-dose group of Pulsatilla scaly is better than that of the high-dose group of Pulsatilla scaly and the positive drug group.

[0055] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing Pulsatilla scaly vesicles, characterized in that: The steps include: (1) First, add water to fresh Pulsatilla chinensis to squeeze out the juice, filter it, and collect the filtrate; (2) Centrifuge the filtrate and collect the supernatant; (3) Finally, the supernatant was centrifuged, the precipitate was collected, the precipitate was suspended with PBS, and dried to obtain. Wherein, the centrifugation in step (2) is 150 × g-250 × g for 10 min-20 min, 3500 × g-4500 × g for 15 min-25 min, The centrifugation in step (3) is 10000 × g-14000 × g for 35 min-45 min, and 80000 × g-120000 × g for 65 min-75 min.

2. The preparation method according to claim 1, characterized in that: The mass volume ratio of the fresh Pulsatilla chinensis product and water is 150 g-240 g:800 mL-1500 mL.

3. The preparation method according to claim 1, characterized in that: The number of times of squeezing the juice in step (1) is 2 to 4 times.

4. The preparation method according to claim 1, characterized in that: The centrifugation temperature in steps (2) and (3) is 3.5°C-4.5°C.

5. The preparation method according to claim 1, characterized in that: The mass volume ratio of the precipitate to PBS in step (3) is 0.3 g-0.5 g:8 mL-12 mL; the drying temperature is -80°C to -90°C, and the drying time is 10 h-14 h.

6. A Pulsatilla scaly vesicle prepared by the preparation method according to any one of claims 1 to 5.

7. The Pulsatilla scaly vesicle according to claim 6, characterized in that The particle size of the Pulsatilla scaly vesicle is 180 nm-190 nm, and the molecular weight of the Pulsatilla scaly vesicle is 52K Da-105K Da.

8. Use of the Pulsatilla scaly vesicle according to claim 6 or 7 in preparing a drug for treating intestinal inflammation.

9. The use according to claim 8, characterized in that: The drug also includes pharmaceutically acceptable excipients.

10. The use according to claim 8, characterized in that: The dosage form of the drug is any one of injection, capsule, tablet, oral liquid and granule.

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