CN115212225B - Application and preparation of RS3 resistant starch in drugs for preventing and treating ischemic stroke - Google Patents

Abstract

The present invention discloses the application of RS3 resistant starch in drugs for preventing and treating ischemic stroke and its preparation. The present invention finds the application of RS3 resistant starch in the preparation of drugs for preventing or treating ischemic stroke, and drugs for alleviating or improving complications of ischemic stroke. RS3 resistant starch can help restore weight gain after ischemic stroke, improve neurological deficits after ischemic stroke, improve abnormal blood lipids and blood rheology caused by ischemic stroke, and repair brain tissue and intestinal mucosal damage after ischemic stroke. RS3 resistant starch improves the disordered intestinal microecology after stroke and can be used as a dietary supplement for stroke patients. The Pueraria RS3 resistant starch of the present invention is extracted and gelatinized by water decoction, the protein is removed by improved Sevag method, the small molecular active components and impurities in the Pueraria starch are removed by dialysis method, and the ethanol precipitation is followed by freeze drying to obtain the obtained product, which is simpler to operate.

Description

Application and preparation of RS3 resistant starch in drugs for preventing and treating ischemic stroke

Technical Field

The invention belongs to the field of pharmaceutical technology, relates to the development and utilization of Pueraria root, and specifically relates to the application of RS3 resistant starch in drugs for preventing and treating ischemic stroke and the preparation thereof.

Background technique

Cerebrovascular disease refers to ischemic or hemorrhagic diseases of the brain caused by hyperlipidemia, hypertension, atherosclerosis, etc. Take stroke as an example. Stroke is also called "cerebral infarction", which seriously threatens the life of patients. Stroke can be divided into two categories: hemorrhagic (cerebral hemorrhage) and ischemic (cerebral infarction). Among them, ischemic stroke accounts for 87% of the total number of strokes. Research data show that stroke ranks first in the list of causes of death among Chinese residents. The disease has a high incidence, high mortality rate, and high disability rate, which brings great pain and heavy economic burden to patients, families and society. Since the occurrence and development of cerebrovascular disease is a gradual process, early prevention and early treatment have become effective ways to reduce the harm of cerebrovascular disease.

At present, the standard treatment for acute ischemic stroke in clinical practice is mainly thrombolysis, anticoagulation and neuroprotection. According to the "Guidelines for the Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases in China", for patients with acute ischemic stroke, intravenous injection of 0.9 mg/kg of recombinant tissue plasminogen activator (r-tPA, Human Tissue Plasminogen Activator) is recommended for thrombolysis to quickly restore blood perfusion in the ischemic area. However, there is a big problem in the use of r-tPA to treat ischemic stroke, that is, the time window for r-tPA to treat ischemic stroke is within 3.0 to 4.5 hours. Therefore, most patients in my country cannot be effectively treated within the time window. Moreover, after the application of r-tPA thrombolysis, accompanied by blood reperfusion, it can induce brain edema, neuronal metabolic disorders, inflammatory response, and cell apoptosis. In addition, the use of r-tPA can activate the systemic fibrinolytic system and induce complications such as secondary intracranial hemorrhage. These shortcomings limit the clinical application of r-tPA. Therefore, it is particularly urgent to find and develop safe and effective drugs that can treat or improve stroke.

Summary of the invention

Pueraria root is the dried root of Pueraria lobata (Willd.) Ohwi, a leguminous plant. It has the effects of relieving muscle and fever, raising yang and stopping diarrhea, and promoting blood circulation. It is often used to treat exogenous fever and headache, stiff neck and back pain, measles, heat dysentery, and diarrhea. Modern pharmacological studies have found that puerarin, a hallmark ingredient of pueraria root, has the effects of dilating cardiovascular and cerebrovascular vessels, improving myocardial contractile function, and promoting blood circulation. Puerarin was approved as a national Class IV new drug in 1993 for the treatment of ischemic cardiovascular and cerebrovascular diseases. However, the oral bioavailability of puerarin is only 3.95%, and oral absorption is poor, which contradicts the good pharmacological activity of pueraria root. The traditional way of administering Chinese medicine is to take it orally after decocting it in water. The pueraria decoction after decocting in water contains not only small molecule active ingredients of flavonoids such as puerarin and daidzein, but also contains a large amount of macromolecular components such as starch, soluble polysaccharides and protein. Currently, the research and development on pueraria is mostly focused on small molecule flavonoid compounds that can be absorbed into the blood, while there is very little research and development on macromolecular components that cannot be absorbed into the blood. This has greatly limited the in-depth development and utilization of pueraria resources.

Starch is the most abundant macromolecular component in Pueraria root. According to the different rates and degrees of enzymatic digestion of starch in vitro, it can be divided into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). Resistant starch, also known as resistant starch or indigestible starch, cannot be enzymatically hydrolyzed in the small intestine and can only enter the colon to be fermented by intestinal flora and converted into beneficial metabolites such as short-chain fatty acids. Therefore, resistant starch can improve the intestinal environment and promote the colonization of beneficial intestinal microorganisms. It has important biological functions such as regulating blood sugar balance and improving metabolism.

RS3 resistant starch is a physically modified starch. During the processing, the starch granules are heated and expanded in a large amount of water and eventually disintegrate. During the cooling process, the starch chains re-approach, entangle and fold. Due to the retrogradation during the period, the starch crystal structure is arranged in a directional manner and is not easy to bind to amylase. It can be seen that the preparation of RS3 resistant starch has the advantages of safe production process, easy control and good thermal stability. Moreover, its content can be increased by physical methods to achieve industrial production. Therefore, RS3 resistant starch is a type of resistant starch with the most industrial production potential and broad application prospects. At present, there is no research on the prevention and treatment of ischemic stroke with Pueraria RS3 resistant starch.

In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide the application of RS3 resistant starch in drugs for preventing and treating ischemic stroke and its preparation. The specific technical scheme is as follows:

The first aspect of the present invention provides the use of RS3 resistant starch in the preparation of a drug for preventing or treating ischemic stroke, or a drug for alleviating or improving complications of ischemic stroke.

The second aspect of the present invention provides the use of RS3 resistant starch in the preparation of a dietary supplement or prebiotic for improving the intestinal microecology of patients with ischemic stroke, as well as its use as a colon-targeted adjuvant or in the preparation of a health food for improving dyslipidemia caused by ischemic stroke.

In the above application, the RS3 resistant starch is prepared from Pueraria root.

The third aspect of the present invention provides a pharmaceutical composition for preventing or treating ischemic stroke, wherein the active ingredient of the pharmaceutical composition includes RS3 resistant starch;

Preferably, the RS3 resistant starch is prepared from Pueraria lobata.

Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient;

Preferably, the excipient is selected from acceptable pharmaceutical excipients for oral preparations;

Preferably, the pharmaceutical composition is an oral dosage form;

Preferably, the oral dosage form is a powder, granule, capsule, tablet, pill, sustained-release preparation, controlled-release preparation or liquid preparation;

Preferably, the liquid preparation is an oral solution, suspension, emulsion or syrup.

A fourth aspect of the present invention provides a pharmaceutical composition for alleviating or improving complications of ischemic stroke, wherein the active ingredient of the pharmaceutical composition includes RS3 resistant starch;

Preferably, the RS3 resistant starch is prepared from Pueraria root;

Preferably, the stroke is ischemic stroke.

Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient;

Preferably, the excipient is selected from acceptable pharmaceutical excipients for oral preparations;

Preferably, the pharmaceutical composition is an oral dosage form;

Preferably, the oral dosage form is a powder, granule, capsule, tablet, pill, sustained-release preparation, controlled-release preparation or liquid preparation;

Preferably, the liquid preparation is an oral solution, suspension, emulsion or syrup.

A fifth aspect of the present invention provides a dietary supplement, prebiotic or health food for stroke patients, wherein the dietary supplement, prebiotic or health food comprises RS3 resistant starch;

Preferably, the RS3 resistant starch is prepared from Pueraria root;

Preferably, the stroke is ischemic stroke.

A sixth aspect of the present invention provides a method for preparing RS3 resistant starch, comprising:

(1) decocting the kudzu root medicinal material, collecting the filtrate, further decocting the residue, combining the filtrate, and concentrating the filtrate;

(2) adding Sevag reagent to the concentrated filtrate, stirring evenly, centrifuging, and collecting the top layer of solution;

(3) The top layer solution is dialyzed, and ethanol solution is added to the dialyzed solution. The solution is allowed to stand at 4°C, and the white flocculent precipitate is collected by centrifugation and freeze-dried to obtain the product.

Furthermore, during the water decoction treatment in step (1), the volume ratio of the Pueraria root medicinal material to water is 1:(10-20), and the decoction is performed 1-4 times, each decoction lasting 0.5-2 hours;

Preferably, the filtrate after concentration in step (1) is placed at 4° C. for 24 to 48 hours;

Preferably, the amount of Sevag reagent added in step (2) is 1/5 to 1/3 of the volume of the concentrated filtrate;

Preferably, the amount of Sevag reagent added in step (2) is 1/4 of the volume of the concentrated filtrate;

Preferably, the volume ratio of n-butanol to chloroform in the Sevag reagent in step (2) is 1:4;

Preferably, the dialysis treatment time in step (3) is 8-24 hours;

Preferably, the volume fraction of the ethanol solution in step (3) is 70-90%;

Preferably, in step (3), the mixture is allowed to stand at 4° C. for 12-24 hours.

The beneficial effects of the present invention are:

1. The present invention finds the use of RS3 resistant starch in the preparation of drugs for preventing or treating stroke, and drugs for alleviating or improving stroke complications. RS3 resistant starch can help restore weight gain after ischemic stroke, improve neurological deficits after ischemic stroke, improve abnormal blood lipids and blood rheology caused by ischemic stroke, reshape the disordered intestinal flora during ischemic stroke, repair brain tissue and colon mucosal damage after ischemic stroke, treat stroke from multiple links, alleviate and improve stroke complications, and has broad application prospects in the treatment of cerebrovascular diseases.

As a new type of prebiotic made from Pueraria root, Pueraria RS3 resistant starch can effectively increase the number and abundance of beneficial bacteria in the intestine (such as Prevotella_9, Lactococcus, Butyricimonas, Anaeroplasma, Akkermansia, Bifidobacterium, Romboutsia, Ruminiclostridium, etc.), reduce the abundance of pathogenic bacteria (such as Alistipes, Klebsiella), and improve the disordered intestinal microecology after stroke. It can be added to traditional foods as an additive to develop into a special dietary supplement for the rehabilitation of stroke patients, which is conducive to the secondary development of Pueraria root resources and obtain greater economic value and social benefits.

2. The method for preparing kudzu root RS3 type resistant starch of the present invention is different from the prior art in that kudzu root starch is first extracted and then the resistant starch is prepared by pressure heat method, enzymolysis method, acid hydrolysis method, etc. Instead, kudzu root starch is extracted and gelatinized by water decoction method, protein is removed by improved Sevag method, and small molecular active components and impurities in kudzu root starch are removed by dialysis method. The extraction and preparation of RS3 type resistant starch (PLR-RS3) from kudzu root is simpler to operate, less chemical reagents are introduced during the treatment process, there is no hidden danger of chemical residues, it is easy to realize industrial production, and the production efficiency is improved. In addition, the kudzu root starch content obtained by the method for preparing kudzu root RS3 type resistant starch of the present invention is as high as 79%, the resistant starch (RS3) content is not less than 52%, the purity of kudzu root resistant starch is high, and its effect is significantly better than the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an infrared spectrum and an X-ray diffraction diagram of PLR-RS3 in Example 1.

FIG. 2 shows the daily weight gain and survival rate of rats in Example 2 (n=10).

Figure 3 is a comparison of neurological function scores and cerebral infarction rates in each group of Example 3. (A) Neurological function scores for a total of 14 days and (B) daily (n=10); (C) TTC staining images of the brain (n=6); (D) quantification of cerebral infarction rate (n=6).

FIG. 4 is a HE-stained micrograph of brain tissue and colon tissue of Example 4.

Figure 5 shows the blood lipid levels of each group in Example 5 (n=6). (A) Serum total cholesterol level; (B) Serum triglyceride level; (C) Serum high-density lipoprotein level; (D) Serum low-density lipoprotein level.

Figure 6 shows the blood rheology characteristics of each group in Example 5 (n=6). (A) Blood viscosity (200 mPa·s shear rate); (B) Blood viscosity (5 mPa·s shear rate); (C) Fibrinogen; (D) Platelet aggregation rate.

Figure 7 is an analysis of key bacterial genera in Example 6 (n=10). (A) Relative abundance of bacterial communities at the genus level; (B) Cladogram.

FIG8 is the LEfSe analysis in Example 6 (n=10).

Figure 9 shows the expression of tight junction proteins in the brain and colon and the brain-gut correlation in Example 7 (n=6). Concentrations of (A) occludin, (B) claudin-5, and (C) ZO-1 in the brain; concentrations of (D) occludin, (E) claudin-5, and (F) ZO-1 in the colon; brain-gut correlation of (G) occludin, (H) claudin-5, and (I) ZO-1

Figure 10 shows the intestinal flora translocation in Example 7 (n=6). (A) FD-4 concentration in plasma; (B) diamine oxidase activity level in serum; (C) LPS activity in serum; (D) D-lactate concentration in serum.

FIG. 11 shows the daily relative body weight gain of rats in Example 8 (Control, Saline, PLR-RS groups n=10, PLR-NS group n=3).

FIG. 12 shows the neurological function scores of Example 8 every day (Control, Saline, PLR-RS groups n=10, PLR-NS group n=3).

13 is the neurological function scores of Example 8 for a total of 14 days (Control, Saline, PLR-RS groups n=10, PLR-NS group n=3).

FIG. 14 is a picture of TTC staining of the brains of Example 8 (Control, Saline, PLR-RS groups n=6, PLR-NS group n=3).

FIG. 15 is a quantification of the cerebral infarction rate in Example 8 (Control, Saline, PLR-RS groups n=6, PLR-NS group n=3).

Detailed ways

In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

1 Experimental Animals

Male Sprague-Dawley rats (220 ± 20 g, SPF grade) were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China, Animal Qualification Certificate No. SCXK (Yue) 2016-0041). Rats were housed under standard light and dark cycle (12 h:12 h) conditions with free access to food and water. All animal studies were performed in accordance with the protocols and guidelines approved by the Animal Ethics Committee of the Experimental Animal Center of Southern Medical University.

2 Experimental reagents

3. Solution preparation

2% TTC (2,3,5-Triphenyltetrazolium chloride) solution: weigh 2 g of TTC powder and dilute to 100 mL with normal saline.

4% formaldehyde solution: weigh 2.901 g of sodium dihydrogen phosphate (Na ₂ HPO ₄ ·12H ₂ O), 0.296 g of sodium dihydrogen phosphate (NaH ₂ PO ₄ ·2H ₂ O), formaldehyde solution (40%, 10 mL), and distilled water to make up to 100 mL.

10% chloral hydrate solution: weigh 4 g of chloral hydrate powder and dilute to 40 mL with distilled water.

4 Establishment and evaluation of ischemic stroke model

4.1 Establishment of ischemic stroke model

The rat right middle cerebral artery occlusion model (MCAO) was prepared. The rats were anesthetized by intraperitoneal injection of chloral hydrate solution (10%, 3mL/kg). The rats were fixed on the operating table in a supine position. An incision was made in the middle of the neck. The common carotid artery was separated after the muscle tissue was separated with forceps. Then the external carotid artery and the internal carotid artery were separated upward along the common carotid artery and distributed in a "Y-shaped" manner. After the blood vessels were separated, the proximal end of the common carotid artery and the distal end of the external carotid artery were ligated with surgical threads of different thicknesses. An incision was made at the external carotid artery with ophthalmic scissors. The thread plug was slowly inserted from the incision into the external carotid artery to the middle cerebral artery. After the thread plug was inserted, the ligature of the common carotid artery was fixed. After 2 hours of ischemia, the rats were anesthetized, and the thread plug was pulled out to loosen the ligature of the common carotid artery to form reperfusion. Finally, the wound was sutured and disinfected with iodine tincture, and then the rats were returned to the cage and given feed and water.

4.2 Evaluation of ischemic stroke model

4.2.1 Neurological function score

The Longa 5-point system was used to score neurological function, and the scoring criteria were as follows: 0 means that the rat can stretch both upper limbs to the ground without neurological deficit symptoms; 1 means that the left forelimb cannot be fully extended; 2 means that the rat turns to the left when walking; 3 means that the rat falls to the left when moving autonomously; 4 means that the rat cannot walk spontaneously and is accompanied by impaired consciousness. A cumulative score of 1 or above indicates a successful model.

4.2.2 Determination of cerebral infarction rate

The cerebral infarction rate of rats in each group was determined by TTC staining. After the rats were killed, the brain was quickly removed (maintaining its integrity), frozen at -20°C for 20 minutes, and placed in the brain trough. Each brain tissue was evenly cut into 6 slices of 2 mm thick with a blade, and the slices were quickly placed in 2% TTC staining solution and stained at 37°C in the dark for 10 minutes. During the period, the slices were turned over to make them stain evenly. After staining, the brain slices were fixed with 4% formaldehyde solution. After fixation, the brain slices were taken out and arranged in order, photographed, and the cerebral infarction rate was calculated using Image J software. The infarction area = the area of the non-ischemic hemisphere - the area of the non-infarcted area of the ischemic hemisphere. The sum of the infarction areas of each brain slice multiplied by the thickness (2 mm) was the total infarction volume, and the ratio of the volume of the cerebral infarction focus to the total brain volume was the percentage of the cerebral infarction volume.

Example 1: Preparation of Pueraria RS3 Resistant Starch (PLR-RS3)

Weigh an appropriate amount of Pueraria root medicinal material, soak for 1 hour, add 10-20 times the volume of distilled water, decoct for 1 hour, collect the filtrate, and continue to decoct the residue with 10-20 times the volume of distilled water for 1 hour, and collect the filtrate. Combine the two filtrates, concentrate, and place at 4°C for 24 hours. Take the concentrated filtrate and add Sevag reagent (n-butanol: chloroform (v/v) in Sevag reagent = 1:4, and the amount of Sevag reagent added is 1/4 of the volume of the concentrated solution) to remove impurities such as protein, remove the Sevag reagent by rotary evaporation, and use a dialysis bag for 24 hours to remove small molecular active ingredients. After dialysis, the solution is placed at 4°C for 8 hours with 83% (v/v) ethanol solution until a white flocculent precipitate is produced. Centrifuge at 4000rpm for 10 minutes, collect the precipitate, and freeze-dry the precipitate for 24 hours to obtain Pueraria root RS3 type resistant starch (PLR-RS3).

The structure of the obtained freeze-dried powder was confirmed by infrared spectroscopy. It can be seen from the infrared spectroscopy results (Figure 1A) that the vibration absorption observed near 3376cm ^-1 belongs to the stretching vibration of the OH group. The small band near 2925cm ^-1 is caused by CH stretching and bending vibrations. The absorption peaks observed near 1646cm ^-1 and 1413cm ^-1 are consistent with the peaks of the carboxyl and carbonyl groups, respectively, and are the characteristics of uronic acid. The peaks observed in the range of 1400-1200cm ^-1 are the characteristics of the CH bond. In addition, the peaks observed in the range of 1200-1000cm ^-1 belong to the vibration of the COH side group and the COC glycosidic bond. Among them, the absorption at 500-900cm ^-1 is similar to the skeleton characteristics of the pyranose ring. These results show that PLR-RS3 exhibits typical starch characteristic absorption peaks. X-ray diffraction analysis (Figure 1B) shows that the resistant starch is RS3 type. According to the modified Englyst method, the content of the freeze-dried powder was determined (the results are shown in Table 1). It can be seen that the total starch (TS) content of the freeze-dried powder is 78.74% ± 3.10%, while the contents of fast-digestible starch (RDS), slow-digestible starch (SDS) and resistant starch (RS) are 43.60% ± 5.62%, 4.86% ± 3.93% and 51.54% ± 5.72%, respectively. Because the preparation process is treated by dialysis, the prepared Pueraria starch does not contain any flavonoids such as puerarin, daidzein, daidzein or other small molecule active ingredients and impurities. At the same time, because fast-digestible starch and slow-digestible starch can be hydrolyzed and digested by α-amylase within 20 minutes or 20-120 minutes after entering the gastrointestinal tract, they do not exert pharmacological activity. Therefore, the subsequent pharmacodynamic experiments can exclude the influence of small molecule active ingredients, fast-digestible starch and slow-digestible starch in Pueraria, and the pharmacological activity is attributed to resistant starch.

Table 1 Composition of Pueraria starch

Example 2: Pueraria RS3 resistant starch can significantly increase the body weight of stroke rats

The change of body weight is a key factor affecting the prognosis of stroke. In order to investigate the effect of Pueraria RS3 resistant starch (PLR-RS3) on the body weight of rats with ischemic stroke, the rats were randomly divided into 5 groups in this example: control group (Control, n = 10), stroke model group (Saline, n = 15), Pueraria group (PLR, n = 12), puerarin group (Puerarin, n = 12) and Pueraria resistant starch group (PLR-RS3, n = 12). The rats in the control group did not receive modeling, and the rats in the other groups were all subjected to the preparation of the right middle cerebral artery embolism model. The rats in the Pueraria group were gavaged with Pueraria decoction (containing 50 mg/kg puerarin), the rats in the Pueraria resistant starch group were gavaged with Pueraria RS3 resistant starch (491.25 mg/kg), and the rats in the control group and the stroke model group were gavaged with an equal volume of normal saline. Once a day for 14 days.

The results are shown in Figure 2, which show that the average weight loss of rats in the stroke model group was about 5%, while the weight gain was 11.55% after 14 days of administration of Pueraria RS3 resistant starch, indicating that Pueraria resistant starch can significantly increase the weight of stroke rats.

Example 3: Pueraria RS3 resistant starch can protect the neurological function of stroke rats and reduce the area of cerebral infarction

This example uses a rat right middle cerebral artery occlusion (MCAO) model to simulate the disease state of ischemic stroke during natural onset, and investigates the effects of pueraria root, puerarin and pueraria root RS3 resistant starch on the behavior of stroke rats. The grouping, administration method and specific experimental operation are the same as in Example 2.

The results are shown in Figure 3. The results show that after modeling, rats with ischemic stroke model showed behaviors of leaning to the opposite side and being unable to walk in a straight line. The Longa 5-point scoring method was used to evaluate neurological function, and it was found that the neurological function score remained at 3 points, indicating that the neurological function defects caused by acute brain injury cannot heal themselves, which is also consistent with the symptoms of clinical stroke patients. After intervention with Pueraria RS3 resistant starch, the neurological function defects of rats with ischemic stroke were greatly improved. The rats were able to stretch the forelimbs on the opposite side of the lesion normally and walk independently in a straight line. At the end of the experiment on the 14th day, the neurological function score had dropped to 0.4 points, which was significantly different from the model group (3 points). The results of whole-brain TTC staining showed that compared with normal rats, the cerebral infarction rate of rats in the stroke group increased sharply, and obvious tissue collapse and edema were seen in the cerebral infarction area, indicating that the brain tissue damage caused by cerebral infarction caused by ischemic stroke will not repair itself over time. However, after treatment with Pueraria RS3 resistant starch, the cerebral infarction rate decreased significantly, cerebral edema disappeared, and the brain morphology remained intact without any brain tissue damage or collapse. Although the puerarin group could also reduce the cerebral infarction rate, there was no significant difference compared with the stroke model group.

Example 4: Pueraria RS3 resistant starch can repair brain tissue and colon mucosal damage in stroke rats

HE staining was used to evaluate the disease state of ischemic stroke and the histological changes of brain and colon after intervention treatment. The grouping, administration method and specific experimental operation were the same as in Example 2.

The results are shown in Figure 4. Compared with normal rats, the brain cells of stroke rats were arranged irregularly and disorderly, and neurons showed obvious necrosis. After 14 days of intervention with Pueraria RS3 resistant starch, the cells in the brain tissue were arranged neatly and densely, the nuclei were large and regular, no obvious damage was found, and neuronal damage was repaired. The colon of rats in the ischemic stroke model group also showed obvious pathological changes, such as degeneration and necrosis of intestinal mucosal epithelial cells, a decrease in the number of goblet cells, and inflammatory cell infiltration. After treatment with Pueraria RS3 resistant starch, colon inflammatory cell infiltration and mucosal damage were significantly reduced, the structure of the lamina propria mucosa and muscular layer was normal, the number of intestinal glands increased, and a large number of goblet cells were closely arranged, and no obvious abnormalities were found in the tissue.

Example 5: Pueraria RS3 resistant starch can alleviate dyslipidemia caused by stroke

Blood lipid level is an independent risk factor for stroke. Given that most ischemic stroke patients have abnormal blood lipid metabolism and blood rheology, this example investigates the changes in blood lipid metabolism and blood rheology in the ischemic stroke disease state and after intervention treatment. The grouping, administration method and specific experimental operation are the same as in Example 2.

The results are shown in Figures 5 and 6. Compared with the normal group rats, the total cholesterol (T-CHO) and triglyceride (TG) levels in the serum of stroke rats increased sharply, especially in the disease state, TG increased by more than 2 times, indicating that stroke can cause abnormal increase in blood lipid and cholesterol levels, but after intervention with Pueraria lobata decoction, puerarin and Pueraria lobata resistant starch, the concentrations of T-CHO and TG decreased significantly and were close to the levels of normal group rats, and the regulation effects of Pueraria lobata decoction and Pueraria lobata resistant starch were better than Pueraria lobata. In addition, Pueraria lobata resistant starch can also regulate high-density lipoprotein (HDL) and low-density lipoprotein (LDL) to near normal levels, while significantly reducing blood viscosity and platelet aggregation rate, and improving the hemorheological characteristics of rats with ischemic stroke. Therefore, Pueraria lobata RS3 resistant starch can effectively regulate abnormal blood lipids caused by ischemic stroke.

Example 6: Pueraria RS3 resistant starch can change the key bacterial genera in the intestinal flora of rats with ischemic stroke

In order to further determine the bacterial genera that specifically changed in the intestinal flora of rats between different groups, LEfSe analysis was used in this example.

The results are shown in Figures 7 and 8. A large number of pathogenic bacteria or conditional pathogens, such as Alistipes, Klebsiella, Hungatella, etc., were enriched in the model group, indicating that the composition of the intestinal microbiota changed under ischemic stroke. However, after treatment with Pueraria lobata decoction and Pueraria lobata resistant starch, more probiotics, such as Prevotella_9, Lactococcus, Butyricimonas, Anaeroplasma, Akkermansia, Bifidobacterium, Romboutsia, Ruminiclostridium, etc., were enriched, and their relative abundance was significantly increased compared with the ischemic stroke model group. This result shows that Pueraria lobata RS3 resistant starch can improve the intestinal microbial imbalance caused by ischemic stroke by increasing the number and abundance of beneficial bacteria.

Example 7: Pueraria RS3 resistant starch can repair the gut-brain barrier structure and restore gut barrier function in rats with ischemic stroke

Considering that Pueraria RS3 resistant starch can repair histological damage in the brain and colon of rats with stroke (Example 4), this example uses ELISA to detect the concentrations of tight junction proteins Occludin, Claudin-5 and ZO-1 in rat brain and colon tissues to evaluate the effect of Pueraria RS3 resistant starch on the gut-brain barrier structure during ischemic stroke.

FD-4 was used to evaluate the intestinal permeability in rats and the effect of Pueraria RS3 resistant starch on the restoration of intestinal barrier function after ischemic stroke. A 20 mg/mL FD-4 solution was prepared with normal saline (keep away from light) and gavaged into rats at a dose of 200 mg/kg. After 4 hours, plasma was collected and its absorbance was detected at 488 nm using an ELISA reader, and the corresponding FD-4 concentration was calculated using a standard curve.

The levels of diamine oxidase (DAO), lipopolysaccharide (LPS) and D-lactate in serum were measured by ELISA to reflect the intestinal microbial translocation. The effect of Pueraria RS3 resistant starch on the recovery of intestinal barrier function in ischemic stroke was evaluated.

The results showed (Figures 9 and 10) that Pueraria RS3 resistant starch can significantly upregulate the levels of tight junction proteins (Claudin-5, ZO-1) in the brain and colon tissues of stroke rats, and the two are significantly positively correlated in the brain and intestine. In addition, it significantly reduces the concentration of fluorescein isothiocyanate-dextran (FD-4) in plasma and D-lactic acid in serum, reduces intestinal permeability, and weakens intestinal bacterial translocation caused by stroke. Therefore, it is believed that Pueraria RS3 resistant starch can treat ischemic stroke by reshaping the intestinal microecology of stroke rats and repairing the gut-brain barrier damage.

Example 8: Difference in the efficacy of kudzu root RS3 resistant starch and kudzu root common starch on ischemic stroke

1 Preparation of Pueraria RS3 Resistant Starch

Weigh 300g of Pueraria root Chinese medicinal material (Powdered Pueraria), soak it in distilled water at room temperature for 1h, then add 3L of distilled water for decoction, filter it while hot after 1h, add 3L of distilled water to the residue and continue to decoct for 1h before filtering, combine the two filtrates and concentrate to about 200mL. Add Sevage reagent (n-butanol: chloroform = 1:4) to the concentrated filtrate to remove protein impurities, and then remove organic reagents by rotary evaporation. Then dialyze the solution for 24h to remove small molecule impurities, and finally react the dialyzed solution with 83% (v/v) ethanol solution at 4℃ for 8h to produce a white flocculent precipitate. Place the precipitate in an oven to evaporate the ethanol, and freeze-dry it for 24h to obtain it.

2 Experimental plan and grouping

To compare the efficacy of pueraria resistant starch and common starch (Tianmeizhen pueraria powder) in the treatment of ischemic stroke, SD rats were randomly divided into 4 groups: control group (Control, n = 10), saline group (Saline, n = 10), pueraria resistant starch group (PLR-RS, n = 10) and pueraria common starch group (PLR-NS, n = 3). The rats in the control group did not receive any intervention. After 2 hours of ischemia, the rats in the Saline, PLR-RS and PLR-NS groups were gavaged with the same volume of saline, pueraria resistant starch (491.25 mg/kg) and pueraria common starch (491.25 mg/kg) once a day for 14 days. The rats were provided with sufficient diet and water during the experiment.

3. Data Statistical Analysis

IBM SPSS Statistics 25.0 and GraphPad Prism 8.0 software were used for data analysis and drawing. The experimental data were expressed as mean ± standard error (Mean ± S.E.M.). The neurological function score results were analyzed using the Kruskal Wallis test. One-way analysis of variance was used for comparison of differences among multiple groups. When the variances were equal, the LSD test method was used, and when the variances were unequal, the Dunnett’s T3 method was used for analysis. *p<0.05, **p<0.01 and ***p<0.001 were considered statistically significant.

4 Experimental results

4.1 Comparison of the effects of Pueraria lobata resistant starch and Pueraria lobata common starch on body weight gain in stroke rats

The body weight results for a total of 14 days showed (Figure 11) that the body weight of rats in the normal saline group decreased, while administration of Pueraria lobata resistant starch significantly increased the body weight of stroke rats. The body weight of stroke rats administered Pueraria lobata ordinary starch did not increase, and there was no significant difference compared with the normal saline group.

4.2 Comparison of Pueraria lobata resistant starch and Pueraria lobata common starch on neurological function in stroke rats

After the MCAO model was successfully established, the neurological function scores of rats in the control group, saline group, kudzu root resistant starch group, and kudzu root ordinary starch group were monitored every day (Figures 12-13). The neurological function score of rats given saline after modeling remained at 3 points, indicating that the neurological damage caused by cerebral ischemia did not heal itself. However, the neurological deficit was greatly improved after the intervention of kudzu root resistant starch, which was significantly different from the saline group; although the rats given kudzu root ordinary starch were observed to be able to walk in a straight line in the later stage, they still could not extend the forelimbs on the opposite side of the lesion, indicating that kudzu root ordinary starch has limited effect on relieving neurological function.

4.3 Comparison of the effects of Pueraria lobata resistant starch and Pueraria lobata common starch on cerebral infarction rate in stroke rats

14 days after modeling, the whole brain was taken for TTC staining (Figures 14-15). Compared with normal rats, rats in the saline group showed obvious collapse and edema in the cerebral infarction area, and the infarction rate increased significantly, indicating that cerebral infarction caused by ischemic stroke will not repair itself. Treatment with Pueraria lobata resistant starch can keep the brain morphology intact and significantly reduce the cerebral infarction rate; however, the brain of the Pueraria lobata ordinary starch group showed collapse and edema, and its cerebral infarction rate was not significantly different from that of the saline group.

It can be seen from Example 8 that Pueraria lobata resistant starch has a good therapeutic effect on ischemic stroke, while Pueraria lobata ordinary starch has no effect on treating stroke.

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the invention.

Claims (10)

1. Use of RS3 resistant starch in the preparation of a drug for treating ischemic stroke, characterized in that the drug is a drug for protecting nerve function and/or reducing the area of cerebral infarction, and the RS3 resistant starch is prepared from Pueraria root; The preparation method of the RS3 resistant starch comprises: (1) decocting the kudzu root root, collecting the filtrate, further decocting the residue, combining the filtrate, and concentrating the filtrate; (2) adding Sevag reagent to the concentrated filtrate, stirring evenly and then centrifuging to collect the top layer of solution; the volume ratio of n-butanol to chloroform in the Sevag reagent is 1:4; (3) The top layer of solution is dialyzed, and ethanol solution is added to the dialyzed solution. The solution is allowed to stand at 4°C, and the white flocculent precipitate is collected by centrifugation and freeze-dried to obtain the product. 2. A method for preparing a pharmaceutical composition for treating ischemic stroke, characterized in that the active ingredient of the pharmaceutical composition includes RS3 resistant starch, and the RS3 resistant starch is prepared from Pueraria root; The preparation method of the RS3 resistant starch comprises: (1) decocting the kudzu root medicinal material with water, collecting the filtrate, further decocting the residue with water, combining the filtrate, and concentrating the filtrate, wherein the volume ratio of the kudzu root medicinal material to water is 1:(10-20), decocting 1-4 times, and each decoction is 0.5-2 h; (2) adding Sevag reagent to the concentrated filtrate, stirring evenly and then centrifuging to collect the top layer solution; the volume ratio of n-butanol to chloroform in the Sevag reagent is 1:4, and the amount of Sevag reagent added is 1/5 to 1/3 of the volume of the concentrated filtrate; (3) The top layer of solution is dialyzed, and ethanol solution is added to the dialyzed solution. The solution is allowed to stand at 4°C, and the white flocculent precipitate is collected by centrifugation and freeze-dried to obtain the product. 3. The preparation method according to claim 2, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. 4. The preparation method according to claim 3, characterized in that the excipients are selected from acceptable pharmaceutical excipients for oral preparations. 5. The preparation method according to claim 3, characterized in that the pharmaceutical composition is an oral dosage form; The oral dosage form is powder, granule, capsule, tablet, pill or liquid preparation; The liquid preparation is an oral solution. 6. The preparation method according to claim 3, characterized in that the pharmaceutical composition is a sustained-release or controlled-release preparation. 7. The preparation method according to claim 3, characterized in that the pharmaceutical composition is a suspension, emulsion or syrup. 8. The preparation method according to claim 2, characterized in that the filtrate after concentration in step (1) is placed at 4°C for 24 to 48 hours. 9. The preparation method according to claim 2, characterized in that the amount of Sevag reagent added in step (2) is 1/4 of the volume of the concentrated filtrate. 10. The preparation method according to claim 2, characterized in that the dialysis treatment time in step (3) is 8-24 hours; The volume fraction of the ethanol solution in step (3) is 70-90%; In step (3), incubate at 4°C for 12-24 h.