Pulsatilla peridermium, a preparation method thereof and application thereof in intestinal inflammation

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a Pulsatillae radix vesicle, a preparation method thereof and application thereof in intestinal inflammation. The preparation method comprises the following steps: crushing fresh Pulsatillae radix, squeezing juice, filtering, and collecting the filtrate; centrifuging the filtrate at low speed, collecting the supernatant; and finally centrifuging the supernatant, collecting the precipitate, suspending the precipitate in PBS, and drying, to obtain the Pulsatillae radix vesicle. The Pulsatillae radix vesicle prepared by the method has a small particle size, is a nanometer carrier, carries rich active substances such as lipids and proteins, has natural targeting to the pathological site through multi-component synergistic effect, has high safety, low immunogenicity, and no ethical controversy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Pulsatilla chinensis vesicle, its preparation method, and its application in intestinal inflammation. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific inflammatory bowel disease. Clinically, IBD presents with a variety of symptoms, including abdominal discomfort, diarrhea, bloody stools, fever, fatigue, and weight loss. In addition to the symptoms frequently reported above, IBD patients often exhibit extraintestinal complications, which may involve skin diseases (erythema nodosum and pyoderma gangrenosa), 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). Both have similar clinical courses, differing only in the location and nature of the inflammatory lesions. UC is characterized by diffuse inflammation of the colonic mucosa, with a course of recurrent remissions. There is an urgent need to develop better drugs for treating UC, especially those that act locally in the inflamed colon without systemic exposure and related side effects. These drugs are of great significance for the treatment of UC colitis.

[0004] Extracellular vesicles are a collective term for tiny membrane-bound vesicles actively secreted by cells. Based on their size, biological characteristics, and formation process, they are mainly classified into three categories: exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles typically possess a lipid bilayer structure and carry various substances such as proteins, lipids, and RNA, playing a crucial role in cell communication, cell migration, and the occurrence and development of cancer.

[0005] White-headed old man ( Pulsatilla chinensis (Bunge) Regel has a significant inhibitory effect on the secretion of IL-6 by macrophages stimulated by bacterial lipopolysaccharide in Pulsatilla chinensis, and this effect increases over time, thereby reducing excessive inflammatory response and systemic damage; Pulsatilla chinensis and its complexes inhibit dermatophytes, yeasts, pyramidal filaments, and Candida albicans; fresh juice and extracts of Pulsatilla chinensis show the highest sensitivity to Staphylococcus aureus and Pseudomonas aeruginosa; it also has a good inhibitory effect on dysentery and typhoid bacteria.

[0006] There is currently no research on extracellular vesicles of Pulsatilla chinensis. Therefore, developing extracellular vesicles of Pulsatilla chinensis is essential for the treatment of inflammatory bowel disease. Summary of the Invention

[0007] The present application is directed to the problems existing in the prior art, and provides a Pulsatilla chinensis bubble, a preparation method thereof and application thereof in intestinal inflammation.

[0008] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0009] A preparation method of Pulsatilla chinensis bubble comprises the following steps:

[0010] (1) First, juice the fresh Pulsatilla chinensis with water, filter, and collect the filtrate;

[0011] (2) Then centrifuge the filtrate, and collect the supernatant;

[0012] (3) Finally, centrifuge the supernatant, collect the precipitate, suspend the precipitate with PBS, and dry, to obtain,

[0013] In step (2), the centrifugation is 150 ×g-250 ×g for 10 min-20 min, and 3500 ×g-4500 ×g for 15 min-25 min, in sequence.

[0014] In step (3), the centrifugation is 10000 ×g-14000 ×g for 35 min-45 min, and 80000 ×g-120000 ×g for 65 min-75 min, in sequence.

[0015] Preferably, the mass-volume ratio of the fresh Pulsatilla chinensis and water is 150 g-240 g:800 mL-1500 mL.

[0016] Preferably, the number of times of juicing in step (1) is 2-4 times.

[0017] Preferably, the temperature of centrifugation in steps (2) and (3) is 3.5℃-4.5℃.

[0018] Preferably, the mass-volume ratio of the precipitate and PBS in step (3) is 0.3 g-0.5 g:8 mL-12 mL.

[0019] Preferably, the temperature of drying in step (3) is -80℃~-90℃, and the drying time is 10 h-14 h.

[0020] The present application also provides the Pulsatilla chinensis bubble prepared by the above preparation method.

[0021] Preferably, the particle size of the Pulsatilla chinensis bubble is 180 nm-190 nm, and the molecular weight of the Pulsatilla chinensis bubble is 52K Da-105K Da.

[0022] The present application also provides the application of the above Pulsatilla chinensis bubble in preparing a drug for treating intestinal inflammation.

[0023] Preferably, the medicine further comprises a pharmaceutically acceptable excipient.

[0024] Preferably, the medicine is in any one or more of an injection, a capsule, a tablet, an oral liquid, and a granule.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) The prepared whitehead vesicle has a small particle size, is a nanoscale carrier, and carries rich active substances such as lipids and proteins, and has natural targeting to the lesion site through multi-component synergistic effect.

[0027] (2) The prepared whitehead vesicle is of natural origin, has high safety, low immunogenicity, and no ethical controversy. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 TEM image of the whitehead vesicle prepared in the example.

[0029] Figure 2 NTA image of the whitehead vesicle prepared in the example.

[0030] Figure 3 Electrophoresis distribution diagram of the whitehead vesicle prepared in the example.

[0031] Figure 4 Body weight change rate diagram.

[0032] Figure 5 Disease activity index DAI score diagram.

[0033] Figure 6 Colon cross-section HE staining diagram.

[0034] Figure 7 Histopathology score diagram.

[0035] Figure 8 Mouse colon diagram.

[0036] Figure 9 Colon length diagram.

[0037] Figure 4 , Figure 5 , Figure 7 and Figure 9 Compared with the normal group, **** p<0.0001; compared with the model group, ## p<0.01, #### p<0.0001; compared with the positive drug group, ^^^ p<0.001,^^^^ p<0.0001; compared with the low-dose Pulsatilla Bubble group, && p<0.01, &&&& p<0.0001; ns represents no significant difference.

[0038] Figure 6 and Figure 8 From left to right, in order, are the normal group, the model group, the positive drug group, the low-dose Pulsatilla Bubble group and the high-dose Pulsatilla Bubble group. DETAILED DESCRIPTION

[0039] It should be noted that the raw materials used in the present application are all ordinary commercially available products. The fresh Pulsatilla product is purchased from Chengde Deep Mountain Herbal Medicine Co., Ltd. of the State Pharmaceutical Group.

[0040] EXAMPLE

[0041] A preparation method of Pulsatilla Bubble is as follows:

[0042] (1) First, 200 g of fresh Pulsatilla product is thoroughly washed with sterile water, chopped and added with 1000 mL of pure water, and then squeezed 3 times in a juicer (voltage / power: 220 V / 1 KW), filtered, and the filtrate was collected;

[0043] (2) Then, the filtrate was centrifuged at 200 x g for 10 min at 4℃, and then centrifuged at 4000 x g for 20 min, and the supernatant was collected;

[0044] (3) Finally, the supernatant was centrifuged at 12000 x g for 40 min at 4℃, and then centrifuged at 100000 x g for 70 min, and then the precipitate was collected, resuspended with PBS at a mass-volume ratio of 10 mL:0.4 g, and then freeze-dried in a freeze dryer at -85.6℃ for 12 h, to obtain Pulsatilla Bubble.

[0045] The prepared Pulsatilla Bubble was characterized in terms of morphology, particle size and molecular weight. The TEM image is shown in FIG. 1, the NTA image is shown in FIG. 2, and the electrophoretic distribution diagram is shown in FIG. 3. Figure 2 Figure 3 It can be seen from FIG. 2 that the particle size of Pulsatilla Bubble is 187.5 nm; and it can be seen from FIG. 3 that the molecular weight of Pulsatilla Bubble is 52K Da-105K Da.

[0046] Test Example Treatment Effect of Pulsatilla Bubble on Ulcerative Colitis

[0047] 1. Experimental animals

[0048] ​C57 mice, 6-8 weeks old, male, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal license: SYXK (jing) 2021-0017, raised 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 Chinese Medicine, China Academy of Chinese Medical Sciences.

[0049] 2. Experimental method

[0050] 2.1 Animal modeling: mice drank a 2% dextran sodium sulfate (DSS) solution, and no other water source was given during this period. The ulcerative colitis model was established by oral administration for 1 week.

[0051] 2.2 Grouping and administration: 50 C57 mice were divided into 5 groups, namely normal group, model group, whitehead vesicle low-dose group (1.5 mg per mouse), whitehead vesicle high-dose group (3 mg per mouse) and positive drug group (5-aminosalicylic acid, 5-ASA). Except for the normal group, the rest of the mice were modeled for ulcerative colitis.

[0052] 2.3 Index detection:

[0053] (1) Body weight change rate: after inducing colitis, daily weighing was performed to observe the dynamic changes.

[0054] Body weight change rate (%) = (current body weight - baseline body weight) ÷ baseline body weight × 100%

[0055] Wherein, the baseline body weight is the initial body weight of the mouse.

[0056] (2) Disease activity index (Disease activity index, DAI): during modeling, the general state of mice (activity state, fur color, body weight change), fecal characteristics and fecal occult blood (or bloody stool) were observed, and the disease activity index (disease activity index, DAI) of mice was evaluated according to the standard established by McCarthy (reference: Kobayashi T, Siegmund B, Le Berre C, et al. Ulcerative colitis: Recent advances in pathophysiology and targeted therapies. Int J Mol Sci. 2023;24(23):16877. DOI: 10.3390 / ijms242316877.).

[0057] DAI score = (weight loss + stool characteristics + fecal occult blood) ÷ 3.

[0058] (3) HE staining of colon cross-section and histopathological scoring

[0059] HE staining of colon cross-section, the steps are as follows:

[0060] A. Dewaxing and hydration: sequentially 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 tap water.

[0061] B. Freezing section and rewarming fixation: take the section out of the -20℃ refrigerator to room temperature, fix it with 4% paraformaldehyde for 15 min, then rinse with running water, and then place the section in hematoxylin-eosin (H&E) staining solution for 1 min of pretreatment.

[0062] C. Hematoxylin staining: place the section in hematoxylin staining solution for 3-5 min, and then rinse with tap water.

[0063] D. Differentiation and blueing: differentiate with hematoxylin differentiation solution, rinse with tap water, blue with hematoxylin blueing solution, and rinse with running water.

[0064] E. Eosin staining: dehydrate the section in 95% alcohol for 1 min, and then stain in eosin staining solution for 15 s.

[0065] F. Dehydration and transparency: sequentially place the section in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, and xylene II for 2 min until transparent, and then seal with neutral balsam.

[0066] G. Finally, perform microscopic examination and image acquisition and analysis.

[0067] Histopathological scoring: score according to the scoring criteria (reference: 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.).

[0068] (4) Colon length: after the end of administration, remove the colon of each group of mice, and measure the colon length of each group of mice with a steel ruler.

[0069] 3. Experimental results

[0070] (1) Body weight change: The experimental results are shown in Table 1 and Figure 1. 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 weight of the positive drug group, the low-dose Paeonol VES group and the high-dose Paeonol VES group showed an upward trend, but there was no statistical difference compared with the model group. It showed that the body weight of the ulcerative colitis mice showed a significant downward trend, which was consistent with the basic characteristics of the ulcerative colitis model mice. Figure 4

[0071] (2) DAI score: The experimental results are shown in Table 2 and Figure 2. 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 score of the positive drug group, the low-dose Paeonol VES group and the high-dose Paeonol VES group showed a significant downward trend (p<0.01 or p<0.0001), and the scores were equivalent. Compared with the low-dose Paeonol VES group, the DAI score of the high-dose Paeonol VES group showed a significant upward trend (p<0.01). The above experimental results showed that Paeonol VES and the positive drug group could significantly reduce the DAI score of the ulcerative colitis mice, and the low-dose Paeonol VES group was better than the high-dose Paeonol VES group, and the low-dose Paeonol VES group and the positive drug group were equivalent in efficacy. Figure 5

[0072] (3) HE staining of colon cross-section and histopathological score: The experimental results are shown in Table 3 and Figure 3. 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 score of the positive drug group, the low-dose Paeonol VES group and the high-dose Paeonol VES group showed a significant downward trend (p<0.0001). Compared with the positive drug group, the histopathological score of the low-dose Paeonol VES group showed a significant downward trend (p<0.0001). Compared with the low-dose Paeonol VES group, the histopathological score of the high-dose Paeonol VES group showed a significant upward trend (p<0.0001). The above experimental results showed that Paeonol VES could significantly reduce the histopathological score of the colon tissue of the ulcerative colitis mice, and the low-dose Paeonol VES group was better than the positive drug group and the high-dose Paeonol VES group. Figure 6-7

[0073] (4) Colon length: The experimental results are shown in Table 4 and Figure 4. 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 Paeonol VES group and the high-dose Paeonol VES group showed a significant upward trend (p<0.0001). Compared with the low-dose Paeonol VES group, the colon length of the high-dose Paeonol VES group showed a significant upward trend (p<0.0001). The above experimental results showed that Paeonol VES and the positive drug group could significantly increase the colon length of the ulcerative colitis mice, and the low-dose Paeonol VES group was better than the high-dose Paeonol VES group, and the low-dose Paeonol VES group and the positive drug group were equivalent in efficacy. Figure 8-9 ​​​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 Paeonol VES group and the high-dose Paeonol VES group all showed a significant upward trend (p<0.01 or p<0.0001). Compared with the positive drug group, the colon length of the low-dose Paeonol VES group showed a significant upward trend (p<0.001). Compared with the low-dose Paeonol VES group, the colon length of the high-dose Paeonol VES group showed a significant downward trend (p<0.0001). The above experimental results show that Paeonol VES can significantly increase the colon length of ulcerative colitis mice, and the low-dose Paeonol VES group is better than the high-dose Paeonol VES group and the positive drug group.

[0074] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for preparing dried powder of Pulsatilla chinensis vesicles, characterized in that, Includes the following steps: (1) First, add water to the fresh Pulsatilla chinensis, juice it with a juicer, filter it, and collect the filtrate; (2) Centrifuge the filtrate again and collect the supernatant; (3) Finally, centrifuge the supernatant, collect the precipitate, suspend the precipitate in PBS, and freeze-dry to obtain the final product. In step (2), the centrifugation is performed sequentially as follows: 150 ×g-250 ×g for 10 min-20 min, and 3500 ×g-4500 ×g for 15 min-25 min. The centrifugation in step (3) is as follows: 10000 ×g-14000 ×g for 35 min-45 min, 80000 ×g-120000 ×g for 65 min-75 min. The centrifugation temperature in steps (2) and (3) is 3.5℃-4.5℃. In step (3), the mass-to-volume ratio of the precipitate to PBS is 0.3 g-0.5 g: 8 mL-12 mL; the freeze-drying temperature is -80℃ to -90℃, and the freeze-drying time is 10 h-14 h.

2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of fresh Pulsatilla chinensis to 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 the juice is extracted in step (1) is 2 to 4.

4. The application of the dried powder of Pulsatilla chinensis vesicles prepared by the preparation method according to any one of claims 1-3 in the preparation of drugs for treating intestinal inflammation.

5. The application according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The application according to claim 4, characterized in that, The dosage form of the drug is any one of injection, capsule, tablet, oral liquid, and granule.

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