Preparation method and application of Bombyx Batryticatus extract

Abstract

The invention relates to the technical field of silkworm extract, and specifically discloses the use of silkworm extract in the preparation of drugs for improving and / or preventing and treating cerebral small vessel disease and cerebral small vessel disease-related diseases. The silkworm extract includes extraction treatment, alcohol precipitation treatment and dialysis treatment, wherein the extraction treatment is to soak the white silkworm in water at 2-5°C, and then heat and extract to obtain the extract after the soaking is completed; the alcohol precipitation treatment is to add anhydrous ethanol to the extract so that the ethanol concentration in the extract is 65-75%, and the alcohol precipitation is carried out at 2-5°C for 8-14 hours, and then heat and evaporate to remove the ethanol after the alcohol precipitation is completed; the dialysis treatment is to put the extract after the ethanol is removed into a dialysis bag for dialysis treatment, and then concentrate and sterilize after the dialysis is completed. The present invention can prevent and treat CSVD and related diseases, and experiments have shown that it is better than heparin. The preparation method of the silkworm extract of the present invention is suitable for batch production and has a high product yield.

Description

Technical Field

[0001] The invention relates to the technical field of silkworm extracts, and in particular to a preparation method and application of a silkworm extract. Background Art

[0002] Cerebral small vessel disease (CSVD) is a clinical syndrome caused by various pathogenic factors that lead to pathological microcirculation in the brain, which can progress to ischemic stroke (IS). Its onset is insidious, and clinical diagnosis mainly relies on imaging examinations. The treatment of CSVD is based on the principle of improving blood circulation in the ischemic area of ​​the brain as soon as possible and promoting neurological recovery. However, thrombolysis or anticoagulation intervention is often performed only when the patient progresses to IS. Thrombolysis mainly uses streptokinase and urokinase, while anticoagulation mainly uses heparin or dicoumarol to prevent thrombus extension and new thrombus formation. These drugs are not specific drugs and have a certain degree of side effects. Therefore, it is of great significance to explore more and more effective CSVD prevention and treatment drugs to achieve pre-onset intervention of IS.

[0003] Bombyx batryticatus is the dried body of the 4th to 5th instar larvae of the silkworm, Bombyx mori, which is an insect of the family Bombyx mori, that is infected or artificially inoculated with the white silkworm. Modern research shows that Bombyx batryticatus mainly contains fat, protein, amino acids and trace elements. The commonly used method is to use it as a decoction of Bombyx batryticatus, either by decoction or oral administration, or by oral administration in the form of pills or powders, which has the effects of calming wind and relieving convulsions, dispelling wind and relieving pain, and resolving blood stasis and dispersing nodules. However, there is currently no record of using Bombyx batryticatus for the prevention and treatment of CSVD. Summary of the invention

[0004] One of the technical problems solved by the present invention is to provide an application of a Bombyx batryticatus extract in the preparation of a drug for improving and / or preventing and treating cerebral small vessel disease and cerebral small vessel disease-related diseases.

[0005] The second technical problem solved by the present invention is to provide a method for preparing the Bombyx batryticatus extract, so that the product yield is high and the product is suitable for preparing drugs for preventing and treating cerebral small vessel disease and cerebral small vessel disease-related diseases.

[0006] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0007] Application of Bombyx Batryticatus extract, application of Bombyx Batryticatus extract in the preparation of drugs for improving and / or preventing and treating cerebral small vessel disease and cerebral small vessel disease-related diseases.

[0008] Furthermore, the silkworm extract is used as a medicinal ingredient alone or in combination with a pharmaceutically acceptable excipient to prepare a pharmaceutical preparation for intramuscular injection or intravenous administration.

[0009] Furthermore, the cerebral small vessel disease-related diseases include ischemic stroke.

[0010] A method for preparing a Bombyx batryticatus extract, comprising:

[0011] Extraction treatment: soak the white silkworm in water at 2-5°C, and heat and extract to obtain an extract after soaking;

[0012] Alcohol precipitation: add anhydrous ethanol to the extract to make the ethanol concentration in the extract 65-75%, and precipitate at 2-5℃ for 8-14h. After the precipitation, heat and evaporate to remove the ethanol. The control of the alcohol precipitation conditions is critical. Improper handling can easily lead to excessive impurity proteins and the risk of allergies.

[0013] Dialysis treatment: The extract from which ethanol has been removed is dialyzed, and after the dialysis is completed, the permeate is concentrated and sterilized.

[0014] Furthermore, in the extraction step, the silkworms are taken out before the heating extraction, and the soaking liquid is divided into 2 to 4 portions. Before each heating extraction, one portion of the soaking liquid is mixed with the silkworms, and after boiling, it is simmered on low heat to obtain an extract, and the extracts obtained by multiple heating extractions are combined.

[0015] Furthermore, before the alcohol precipitation treatment, the extract is concentrated, and the liquid-to-mass ratio of the concentrated extract to the Bombyx batryticatus is in the range of 2 to 4:1.

[0016] Furthermore, in the alcohol precipitation step, the evaporation is carried out in a water bath at 55-65° C. Too high a temperature will lead to the destruction and deterioration of the active ingredients, directly affecting the efficacy of the product, and too high a temperature may ignite the alcohol, causing the drug to burn and deteriorate.

[0017] Furthermore, the dialysis treatment is carried out in a dialysis bag, and the dialysis includes three dialysis sessions, wherein the first dialysis session lasts 1 to 3 hours, the second dialysis session lasts 5 to 7 hours, and the third dialysis session lasts 10 to 14 hours.

[0018] Furthermore, the cut-off volume of the dialysis bag is 5000Da. The product obtained by this cut-off volume can retain the functional components to the greatest extent, and has low impurities, can meet the requirements of preparing injections, and fully exerts its efficacy.

[0019] Furthermore, the yield of the extract after the extraction treatment is higher than 44%, the yield after the alcohol precipitation treatment is higher than 20%, and the yield after the dialysis treatment is higher than 15%.

[0020] Beneficial effects: The medicine prepared from the silkworm extract of the present invention can prevent and treat CSVD by combining anticoagulation and immune regulation. It can effectively improve the brain inflammation of rats with cerebral small vessel ischemia-reperfusion model and thus play a neuroprotective role, and can activate coagulation factor ⅩⅡ (FXⅡ) to mediate cerebral angiogenesis in rats with cerebral small vessel ischemia-reperfusion model.

[0021] The silkworm extract of the present invention can be used for the preparation of IS prevention and treatment drugs, and has better effect than heparin.

[0022] The method for preparing the silkworm extract of the present invention is suitable for batch production, the production nodes are easy to control, the product quality is stable, the product yield is high, and the prepared silkworm extract has a good effect in improving and / or preventing and treating cerebral small vessel disease and cerebral small vessel disease-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of Bombyx batryticatus extract on the morphology of the cerebral cortex of I / R rats (HE staining, ×100). (control: blank group; Model control: model group; Positive control heparin group; L-DG: low-dose Bombyx batryticatus extract group; M-DG: medium-dose Bombyx batryticatus extract group; H-DG: high-dose Bombyx batryticatus extract group.)

[0024] Figure 2 The experimental results of the effect of Bombyx batryticatus extract on the kinin releasing system and angiogenesis in rat brain tissue after ischemia-reperfusion ( n=3). (Part A represents the overall results, FXII and KK belong to the kinin release system, VEGF is an endothelial cell growth factor that promotes angiogenesis, CD31+ and Brdu+ / vWF+ are surface markers of new blood vessels, Part B is the immunofluorescence test results of KK, Part C is the immunofluorescence test results of CD31, and Figure D is the immunofluorescence test results of Brdu+ / vWF+. * indicates P<0.05 compared with the blank group; ** indicates P<0.01 compared with the blank group; Δ indicates P<0.05 compared with the model group). (control: blank group; Model control: model group; Positive control heparin group; L-DG: low-dose group of Bombyx mori extract; M-DG: medium-dose group of Bombyx mori extract; H-DG: high-dose group of Bombyx mori extract.)

[0025] Figure 3 The morphology of brain microvascular endothelial cells in oxygen-glucose deprivation model rats (n=3).

[0026] Figure 4 The proliferation of brain microvascular endothelial cells in the oxygen-glucose deprivation model rats ( n=3) (A) Proliferation of microvascular endothelial cells. (B) VEGF expression of microvascular endothelial cells. control: blank group; plasma: blank plasma group; BB plasma: Bombyx mori extract combined with plasma group; BB-medicated plasma: Bombyx mori extract-medicated plasma group; Bs: Bismuth subgallate positive drug group; Positive: heparin group; Serum: serum group; BB: Bombyx mori extract group; IG: BKR2 inhibitor group. * indicates P<0.05 compared with the blank group; ** indicates P<0.01 compared with the blank group; Δ indicates P<0.05 compared with the model group. DETAILED DESCRIPTION

[0027] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0028] Example 1

[0029] The preparation method of silkworm pupa liquid comprises the following steps:

[0030] Extraction treatment: weigh 100.0g of white silkworm, add 1L of water and soak at 4℃ for 10h; take out the silkworm before heating, divide the soaking liquid into 3 parts, mix one part of the soaking liquid with the silkworm before each heating extraction, boil it and then turn to low heat to decoct to obtain the extract, and combine the extracts after three decoctions.

[0031] Alcohol precipitation treatment: Concentrate the combined extract to 300 ml, add anhydrous ethanol to the concentrated extract to make the ethanol concentration in the extract 70%, then precipitate at 4°C for 10 hours, and after the precipitation, evaporate in a water bath at 60°C to remove the ethanol;

[0032] Dialysis treatment: the extract from which ethanol has been removed is placed in a dialysis bag with a cutoff of 5000Da for dialysis treatment. The dialysis treatment includes three dialysis treatments in sequence, and the duration of the three dialysis treatments is 2h, 6h, and 12h, respectively. After the dialysis is completed, the extract is concentrated to 100ml, sterilized under high pressure, and configured into a product with a concentration of 1g / ml. The product is sealed and stored at 4°C for future use.

[0033] In this embodiment, the yield of the extract after the extraction treatment is 44.2%, the yield after the alcohol precipitation treatment is 20.9%, and the yield after the dialysis treatment is 15.1%.

[0034] Example 2

[0035] The use of the Bombyx Batryticatus extract obtained in Example 1 in the preparation of a drug for preventing and treating cerebral small vessel disease.

[0036] By converting the human dosage and mouse dosage, rat dosage = human dosage × conversion factor = 10g·70kg -1 ·d -1 ×6.3≈0.9mg / g -1 ·d -1 The high, medium and low doses of Bombyx batryticatus extract were 1.8 mg / g -1 ·d -1 , 0.9mg / g -1 ·d -1 , 0.45mg / g -1 ·d -1 .

[0037] 1. Materials and methods

[0038] 1.1 Research subjects: 60 SPF male SD rats weighing 200-220 g were purchased from Hunan Slake Jingda Co., Ltd. After 24 hours of adaptive feeding, they were randomly divided into: sham operation group (0.9% NaCl), model group (0.9% NaCl), positive drug (heparin) group (150 IU), low-dose Bombyx batryticatus extract group (0.45 mg / g -1 ·d -1 ), medium dose group of Bombyx batryticatus extract (0.9 mg / g -1 ·d -1 ), high-dose group of Bombyx batryticatus extract (1.8 mg / g -1 ·d -1 ), the corresponding drugs were injected intraperitoneally 4 days before surgery and 3 days after surgery, once a day for a total of 7 days.

[0039] Cerebral small vessel disease model - bilateral common carotid artery repeated clamping and recanalization rat I / R model. 25% urethane and 10% chloral hydrate 1:1 mixture was intraperitoneally injected for anesthesia, and the bilateral common carotid arteries were isolated. Under the small animal speckle imager, the artery was clamped for 1.5 minutes and then released for 1 minute. After 5 consecutive operations, a significant decrease in microvascular blood flow of 30% was observed in the imager, and the skin was sutured. In the sham operation group, only the bilateral common carotid arteries were isolated and the skin was sutured, and no clamping and recanalization surgery was performed.

[0040] 1.2 Rat behavioral testing

[0041] BBT is a classic behavioral model for studying reduced motor ability or cerebral cortical damage in animals. During the test, rats were placed on a 1.5 cm wide wooden bar with one end suspended in the air and the other end fixed in the center of a 40 cm × 40 cm flat plate. The balance ability of the rats was recorded within a 2-minute test period. The scoring criteria were: 1 point for standing firmly on the bar without shaking for 2 minutes; 2 points for standing firmly on the bar, shaking left and right without sliding down for 2 minutes; 3 points for standing on the bar, sliding to one side without falling down for 2 minutes; 4 points for falling off the bar within 2 minutes of standing on the bar; 5 points for trying to stand firmly on the bar but falling down within a few seconds; 6 points for no standing ability at all.

[0042] 1.3 Animal handling and sampling

[0043] After the BBT score of rats, 4 rats were randomly selected from each group and anesthetized by intraperitoneal injection of 25% urethane and 10% chloral hydrate in a ratio of 1:1. The brains were removed after cardiac perfusion with 4% paraformaldehyde and paraffin sections were used for HE staining and immunofluorescence staining. Fresh brain tissues were taken from the remaining rats in each group, and the cerebral cortex (CC) regions were separated and placed in different EP tubes and stored at -80°C for ELISA and non-target metabolomics detection.

[0044] 1.4 HE staining

[0045] The paraffin blocks were taken, brain slices were cut, and they were stained with 1% eosin and 0.2% hematoxylin dyes, respectively. After color separation with 1% hydrochloric acid ethanol, images were collected under a common microscope.

[0046] 1.5 Immunofluorescence staining

[0047] Paraffin blocks were taken, brain slices were cut, the membrane was broken with 0.5% Triton, blocked with 5% BCA, and primary antibodies against CD45+, CD11b+, and Arg-1+ (1:500 dilution) were incubated at 4°C overnight. The primary antibodies were discarded, the samples were rinsed with PBS three times, and the secondary antibodies (1:1000) were incubated at 37°C for 1 hour. The slices were sealed with DAPI, and images were collected under a fluorescence microscope.

[0048] 1.6 Detection of cytokine expression in the cerebral cortex by enzyme-linked immunosorbent assay

[0049] The rat cerebral cortex was added to RIPA, homogenized on ice, ultrasonically disrupted, and total protein was extracted. The ELISA instructions were followed to detect the absorbance of brain tissue inflammatory factors IL-1β, IL-6, IL-4, and IL-10 at a wavelength of 450 nm, and the content of each inflammatory factor in the sample was calculated according to the standard curve.

[0050] 1.7 In vitro intervention of silkworm extract in four coagulation tests of human plasma

[0051] Whole blood of healthy people (inclusion criteria: 20-65 years old, no acute or chronic diseases such as cold, hypertension, coronary heart disease, no history of taking anticoagulants such as aspirin and warfarin) was obtained from the Health Management Department of the First Affiliated Hospital of Hunan University of Chinese Medicine. Plasma was prepared by centrifugation in the Laboratory of Cardiovascular and Cerebrovascular Disease Prevention and Treatment of Integrated Traditional Chinese and Western Medicine of Hunan University of Chinese Medicine and divided into blank group (0.9% NaCl), low-dose group of Bombyx mori extract (3 mg·mL-1), medium-dose group of Bombyx mori extract (6 mg·mL-1), and high-dose group of Bombyx mori extract (9 mg·mL-1). Plasma was mixed with the intervention components of each group in a ratio of 1:1 and then tested on an automatic coagulation instrument for PT, APTT, TT, and FIB.

[0052] 1.8 Non-targeted metabolomics test of metabolite changes in Bombyx batryticatus extract

[0053] The rat plasma and cerebral cortex tissue samples of Bombyx mori extract and the sham operation group, model group, and medium-dose Bombyx mori extract group were sent to Beijing Novogene Technology Co., Ltd. for non-targeted metabolomics detection.

[0054] The drug-containing plasma of Bombyx mori was compared with the plasma of the model group to find new metabolites and those with significantly increased levels; the cerebral cortex of rats treated with Bombyx mori was compared with the cerebral cortex of rats in the model group to find new metabolites and those with significantly increased levels; the new metabolites and those with significantly increased levels in the plasma and cerebral cortex of rats in the Bombyx mori intervention group were compared to find common new metabolites and those with significantly increased levels; the common new metabolites and those with significantly increased levels in rats and cerebral cortex of Bombyx mori extract and Bombyx mori intervention group were compared.

[0055] 2. Test results

[0056] 2.1 Effects of Bombyx Batryticatus Extract on Mouse Behavior

[0057] The results showed that Bombyx batryticatus extract could significantly reduce the behavioral scores of rats with cerebral small vessel disease I / R model. As shown in Table 2, compared with the sham operation group, the BBT scores of rats in the model group increased on the 1st and 3rd days; compared with the model group, after low, medium and high doses of Bombyx batryticatus extract intervention, the BBT scores of rats with I / R on the 1st and 3rd days were significantly reduced, while there was no statistically significant difference in the positive drug (heparin) group.

[0058] Table 1 Effects of Bombyx Batryticatus Extract on Behavior of I / R Rats ( n=4)

[0059]

[0060] Note: Compared with sham operation group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01.

[0061] 2.2 Effects of Bombyx Batryticatus Extract on the Morphology of Cerebral Cortex in I / R Rats

[0062] The results are as follows Figure 1 As shown in the figure, the sham operation group had neatly arranged cerebral cortical cells, rounded nuclei and plump cytoplasm; a large number of anomalous cells were seen in the model group, which were loosely and disorderly arranged, with some nuclei condensed, intercellular spaces widened, and vacuolar changes; the positive drug (heparin) group showed no obvious improvement; after intervention with Bombyx mori extract, the degree of cell transport disorder was significantly alleviated, and the number of nuclear condensed and vacuolar cells was significantly reduced. It can be seen that Bombyx mori extract can significantly improve the morphology of the cerebral cortex of rats with cerebral small vessel disease I / R model.

[0063] 2.3 Effect of Bombyx Batryticatus Extract on Hematogenous Immune Cell Infiltration in Brain Tissue of I / R Rats

[0064] The results are shown in Table 2. Compared with the sham operation group, the CD45 fluorescence intensity in the CC brain area of ​​the model group was significantly increased; compared with the model group, after the intervention of Bombyx mori extract, the CD45 fluorescence intensity in the medium and high dose groups of Bombyx mori extract decreased, and although the CD45 fluorescence intensity in the low dose group of Bombyx mori extract decreased slightly, the difference was not statistically significant. It can be seen that medium and high doses of Bombyx mori extract can significantly reduce the infiltration of blood-borne immune cells in the brain tissue of rats with cerebral small vessel disease I / R model and promote the polarization of microglia to M2 type, which has a significant positive effect on the body's immune regulation.

[0065] Table 2 Effect of Bombyx Batryticatus Extract on the Mean Fluorescence Intensity of CD45 in CC Brain Region of I / R Rats ( n=6)

[0066]

[0067] 2.4 Effect of Bombyx Batryticatus Extract on Inflammation in CC Brain Region of I / R Rats

[0068] The results are shown in Table 3. Compared with the sham operation group, the protein expression levels of proinflammatory factors IL-1β and IL-6 in the CC brain region of rats in the model group were increased, and the protein expression levels of anti-inflammatory factors IL-4 and IL-10 were decreased; compared with the model group, the proinflammatory factors IL-1β and IL-6 in the CC brain region of the low, medium and high dose groups of Bombyx mori extract were downregulated on average, and the anti-inflammatory factors IL-4 and IL-10 were upregulated on average, and there was no statistically significant difference in the positive drug (heparin) group, indicating that Bombyx mori extract significantly alleviated the inflammatory response in the brain tissue of rats with cerebral small vessel disease I / R model and played a neuroprotective role.

[0069] Table 3 Effects of Bombyx Batryticatus Extract on the Expression of IL-1β, IL-6, IL-4 and IL-10 Proteins in CC Brain Region of I / R Rats ( n=9)(pg·mL-1)

[0070]

[0071]

[0072] 2.5 Effect of Bombyx Batryticatus Extract on Anticoagulation

[0073] The results are shown in Table 4. Compared with the blank group, the Bombyx batryticatus extract intervention group significantly prolonged the APTT, PT, and TT of human plasma in a dose-dependent manner, but had no effect on FIB, with no statistically significant difference.

[0074] Table 4 Quality control of anticoagulant efficacy of Bombyx batryticatus extract ( n=4)

[0075]

[0076] Note: Compared with sham operation group 1) P<0.05, 2) P<0.01.

[0077] 2.6 Results of non-targeted metabolomics analysis of Bombyx batryticatus extract

[0078] The test showed that 809 metabolites were identified in Bombyx Batryticatus extract, 137 metabolites were identified in Bombyx Batryticatus extract intervention in I / R rat plasma, and 172 metabolites were identified in Bombyx Batryticatus extract intervention in I / R rat brain tissue. Compared with I / R rat plasma and brain tissue, 57 metabolites of Bombyx Batryticatus extract entered the blood and 45 metabolites entered the brain. After Bombyx Batryticatus extract was used, the metabolites entering the blood and brain were compared, and there were 15 common metabolites, which are shown in Table 5.

[0079] Table 5 Common metabolites of Bombyx batryticatus extract entering brain and blood ( n=3)

[0080]

[0081]

[0082] In summary, Bombyx batryticatus extract has a significant anticoagulant effect, can reduce the behavioral scores of rats with cerebral small vessel disease I / R model, improve the cerebral cortical morphology of rats with cerebral small vessel disease I / R model, reduce the infiltration of hematogenous immune cells in the brain tissue of rats with cerebral small vessel disease I / R model and promote the polarization of microglia to M2 type, and significantly alleviate the inflammatory response in the brain tissue of rats with cerebral small vessel disease I / R model to play a neuroprotective role. It can prevent and improve CSVD through combined anticoagulation and immune regulation, and can be expected to effectively improve the clinical symptoms of IS patients, which is better than heparin.

[0083] Example 3

[0084] The Bombyx Batryticatus extract obtained in Example 1 is used in the preparation of a drug for preventing and treating cerebral small vessel disease and in improving cerebral ischemia-reperfusion injury.

[0085] Through the conversion of human dosage and mouse dosage, rat dosage = human dosage × conversion coefficient = 10g·70kg-1·d-1×6.3≈0.9mg / g-1·d-1, and the high, medium and low doses of Bombyx batryticatus extract were 1.8mg / g respectively. -1 ·d -1 , 0.9mg / g -1 ·d -1 , 0.45mg / g -1 ·d -1 .

[0086] 1. Test methods

[0087] 1.1 Plasma preparation

[0088] 1.1.1 Preparation of human plasma

[0089] The whole blood of healthy people in this study (inclusion criteria: 20-65 years old, no acute or chronic diseases such as cold, hypertension, coronary heart disease, no history of taking anticoagulants such as aspirin and warfarin) was obtained from the Health Management Department of the First Affiliated Hospital of Hunan University of Chinese Medicine. The whole blood was centrifuged and prepared into plasma in the Laboratory of Cardiovascular and Cerebrovascular Disease Prevention and Treatment of Hunan University of Chinese Medicine, and stored at -80℃ after aliquoting (within 6 months).

[0090] 1.1.2 Preparation of blank serum and plasma from animals

[0091] After 12 hours of adaptive feeding, 4-week-old male SD rats were given 1 mL of normal saline ip, once a day, for 7 consecutive days. 6 hours after the last treatment, the rats were anesthetized with 3% sodium pentobarbital (1 mL / kg) ip, and blood was collected from the abdominal aorta. Part of the whole blood was placed in 5 mL blank tubes, and part of the whole blood was placed in 2 mL sodium citrate blood collection tubes, which were numbered and centrifuged at 3500 r / min (serum was left to stand at 4°C overnight) for 10 min to obtain blank serum and plasma of rats, which were stored at -80°C after aliquoting.

[0092] 1.1.3 Preparation of animal drug-containing plasma

[0093] After 12 hours of adaptive feeding, 4-week-old male SD rats were given 1 mL of Bombyx batryticatus extract (105 mg / L) ip, once a day for 7 consecutive days. 6 hours after the last administration, 25% urethane (4 mL / kg) was given ip for anesthesia, and blood was collected from the abdominal aorta. The whole blood was placed in 2 mL sodium citrate blood collection tubes, numbered, and centrifuged at 3500 r / min for 10 min to obtain rat drug-containing plasma, which was stored at -80°C after aliquoting.

[0094] 1.2 Preparation of HEK-293 cells overexpressing FXII protein

[0095] HEK-293 cells were transfected with FXII plasmid according to the instructions of the nuclear transfer kit and cultured for 24 hours at 37°C and 5% CO2. The transfection rate reached 95%. HEK-293 cells that stably expressed FXII protein were screened with G418. The cell supernatant was collected and loaded onto a protein purification column at 10 μL / s. FXII purified protein was obtained by elution with 0.25 nmol / L imidazole.

[0096] 1.3FXII activation experiment

[0097] Add 40 μL of 1 μg / μL Bombyx batryticatus extract to the cell suspension containing FXII protein (the blank group used medical saline, and the agonist used: 3.94*10 -6 μg / μL Bismuth subgallate), incubated at 37℃ for 5min, added 2×SDS loading buffer and 2.5%β-ME, after hot water bath for 5min, loaded with 10% acrylamide gel, 12μL / well, 100V running, 120V wet transfer for 2h, blocked in skim milk for 1h, added FXII antibody, incubated at 4℃ overnight, washed with TBST, added secondary antibody, incubated at 37℃ for 1h, and developed by ECL luminescence method on Bio-Rad ChemiDocTM imaging system.

[0098] 1.4KK generation experiment

[0099] Take 20 μL of human plasma, treat the sample according to the FXII activation experimental method, load it on 8% acrylamide gel, 30 μL / well, run the gel at 100V, wet transfer at 120V for 2h, block in skim milk for 1h, add KK antibody, incubate at 4℃ overnight, wash the membrane with TBST, add secondary antibody, incubate at 37℃ for 1h, and develop it with ECL luminescence method on Bio-Rad ChemiDocTM imaging system.

[0100] 20 μL of human whole plasma or FXII-deficient plasma was added to each well of a 96-well plate, followed by 40 μL of 1 μg / μL Bombyx batryticatus extract, 40 μL of saline as a blank control group, and 40 μL of kaolin solution as a positive control group. The plates were incubated at 37°C for 5 min, 20 μL of 5 mg / L S-2302 was added, and the plates were incubated at 37°C for 30 min on a microplate reader. The absorbance (A) value at 405 nm was measured every 5 minutes.

[0101] 1.5 Animal model preparation

[0102] After 12 hours of adaptive feeding, 30 SD rats were randomly divided into a blank group (normal saline), a model group (normal saline), a heparin group (150 IU), and a low-, medium-, and high-dose group of Bombyx batryticatus. The dosages of the low-, medium-, and high-dose groups were 0.45 mg / g / d, 0.9 mg / g / d, and 1.8 mg / g / d, respectively, according to the human dosage and rat conversion. Except for the blank group, the bilateral common carotid arteries of the remaining rats were isolated, and the artery clamps were clamped for 1.5 minutes and then released for 1 minute under the small animal speckle imaging device. After 5 consecutive operations, the imaging device showed a significant decrease in microvascular blood flow (30±5)%, and the skin was sutured.

[0103] 1.6 Detection of rat brain tissue indicators

[0104] The brain tissues of rats in each group were fixed with 4% paraformaldehyde, dehydrated with ethanol, transparentized with xylene, embedded in paraffin blocks, sliced, and stained with hematoxylin-eosin (HE) to observe the pathological changes of brain tissues; KK, CD31, and Brdu / vWF antibodies were incubated, and the brain tissues were observed and photographed under a microscope.

[0105] The rat brain tissue was obtained and homogenized on ice to extract protein. After the protein concentration was determined by BCA method, the protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membrane, incubated with FXII, KK, VEGF antibodies and secondary antibodies, and developed.

[0106] 1.7 Preparation of oxygen-glucose deprivation model

[0107] Rat brain microvascular endothelial cells were divided into blank group (10 μL normal saline), blank plasma group (10% blank plasma), Bombyx mori plasma group (10% 26.25 mg / L Bombyx mori extract + 10% blank plasma), Bombyx mori medicated plasma group (10% Bombyx mori extract medicated plasma), heparin group (10 IU heparin + 10% blank plasma), activator group (10 nmol / L Bismuthsubgallate + 10% blank plasma), blank serum group (10% blank serum), BKR2 inhibitor group (15 nmol / L Catibant Acetate + 10% Bombyx mori extract medicated plasma), and cultured in sugar-free medium under 5% CO2 and 1% O2 for 12 h. After reoxygenation for 30 min, the cells were observed under a microscope and photographed. Cell proliferation was detected by CCK-8 method, and intracellular VEGF protein expression was determined by Western blotting method.

[0108] 2. Results Analysis

[0109] 2.1 Effects of Bombyx Batryticatus Extract on the Kinin Release System in Brain Tissue of Rats with Ischemia-Reperfusion

[0110] like Figure 2As shown in Part A, compared with the blank group, the activation of FXII protein in the model group and heparin group was not obvious, and the expression of KK and VEGF proteins increased significantly (P < 0.05). Compared with the model group, the expression of FXII prototype protein in the Bombyx batryticatus extract group was significantly reduced (P < 0.05), which was dose-related, indicating that the protein was activated in large quantities; the expression of KK and VEGF proteins was further increased (P < 0.05), which was dose-related.

[0111] like Figure 2 As shown in the B, C, and D parts of the figure, compared with the blank group, KK expression increased in all groups (P < 0.05), but the Bombyx batryticatus extract group was better, and it was dose-related. In the immunofluorescence experiment, compared with the blank group, CD31 in the model group and heparin group + and Brdu + / vWF + No significant increase was observed; compared with the model group, the CD31 and Brdu + / vWF + There was significant expression (P<0.05, 0.01), and it was dose-related.

[0112] 2.2 Effects of Bombyx Batryticatus Extract on Morphological Changes of Brain Microvascular Endothelial Cells in Oxygen-Glucose Deprivation Model Rats

[0113] like Figure 3 As shown in the figure, after modeling at the same time, the cells in each group changed, with cell shrinkage and reduced light transmittance. The blank plasma group and FXII activator group had obvious fibrin production, and obvious fibrin clumps and fibrin filaments were observed under the microscope. Although no obvious fibrin clots were observed in the blank group, heparin group, drug-containing serum group, and BKR2 inhibitor group, the number of adherent cells was significantly reduced, the shrinkage was obvious, the light transmittance was poor, and the cell proliferation was poor. Under the intervention of Bombyx mori extract and rat plasma, no obvious fibrin clumps were observed, and the number of cells was significantly higher than that in the plasma group, but some cell death and shrinkage were observed. The cell state of the Bombyx mori extract-containing plasma group was significantly better than that of the other treatment groups, with strong cell light transmittance, and no obvious cell death and morphological changes.

[0114] 2.3 Effect of Bombyx Batryticatus Extract on Proliferation of Brain Microvascular Endothelial Cells in Oxygen-Glucose Deprivation Model Rats

[0115] like Figure 4 As shown in the figure, compared with the plasma group, the heparin group, the Bombyx mori extract and plasma co-intervention group, and the Bombyx mori extract-containing plasma group had significant endothelial cell proliferation, among which the Bombyx mori extract-containing plasma group had the most obvious effect, with a statistically significant difference (P < 0.01). However, compared with the blank group and the plasma group, only the Bombyx mori extract combined with plasma group and the Bombyx mori medicated plasma group had a significant increase in VEGF protein expression (P < 0.05).

[0116] In conclusion, Bombyx batryticatus extract can effectively improve brain inflammation in rats with cerebral small vessel ischemia-reperfusion model and thus play a neuroprotective role. It can also activate coagulation factor ⅩⅡ (FXⅡ) to mediate cerebral angiogenesis in rats with cerebral small vessel ischemia-reperfusion model.

[0117] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. The application of Bombyx Batryticatus extract is characterized in that: Application of Bombyx Batryticatus extract in the preparation of medicine for improving and / or treating cerebral small vessel disease; The silkworm extract is used as a pharmaceutical ingredient alone or in combination with a pharmaceutically acceptable excipient to prepare a pharmaceutical preparation for intramuscular injection or intravenous administration; The drug exerts a neuroprotective effect by improving the intracerebral inflammation caused by cerebral small vessel ischemia-reperfusion, and activates angiogenesis of cerebral small vessel disease caused by cerebral small vessel ischemia-reperfusion mediated by intracerebral coagulation factor XII (FXII); The preparation method of the silkworm extract comprises: Extraction treatment: soak the white silkworm in water at 2-5°C, and heat and extract to obtain the extract after soaking; Alcohol precipitation treatment: add anhydrous ethanol to the extract to make the ethanol concentration in the extract 65-75%, and precipitate at 2-5℃ for 8-14h. After the precipitation, heat and evaporate to remove the ethanol; Dialysis treatment: The extract from which ethanol has been removed is dialyzed, and the cutoff volume of the dialysis bag is 5000Da; after the dialysis is completed, the permeate is concentrated and sterilized.

2. The use of the Bombyx Batryticatus extract according to claim 1, characterized in that: In the extraction process, the silkworms are taken out before the heating extraction, and the soaking liquid is divided into 2 to 4 portions. Before each heating extraction, one portion of the soaking liquid is mixed with the silkworms, and then boiled and then simmered on low heat to obtain an extract. The extracts obtained by multiple heating extractions can be combined.

3. The use of the Bombyx Batryticatus extract according to claim 1, characterized in that: Before the alcohol precipitation treatment, the extract was concentrated, and the liquid-to-mass ratio of the concentrated extract to Bombyx batryticatus was in the range of 2-4:

1.

4. The use of the silkworm extract according to claim 1 is characterized in that: In the alcohol precipitation step, evaporation is performed in a water bath at 55-65°C.

5. The use of the silkworm extract according to claim 1, characterized in that: The dialysis treatment is carried out in a dialysis bag. The dialysis includes three dialysis sessions. The first dialysis session lasts 1 to 3 hours, the second dialysis session lasts 5 to 7 hours, and the third dialysis session lasts 10 to 14 hours.

6. The use of the silkworm extract according to claim 1, characterized in that: The yield of the extract after extraction treatment is higher than 44%, the yield after alcohol precipitation treatment is higher than 20%, and the yield after dialysis treatment is higher than 15%.

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