TW202042655A - Composition for preventing stroke and improving the severity of stroke and use thereof including an effective amount of Lactobacillus reuteri GMNL-89 and Lactobacillus paracasei GMNL-133 - Google Patents

Abstract

This invention provides a composition for preventing stroke and improving the severity of stroke. The composition includes an effective amount of Lactobacillus reuteri GMNL-89 and Lactobacillus paracasei GMNL-133, wherein the Lactobacillus reuteri GMNL-89 is deposited with the serial number of BCRC910340, and the Lactobacillus paracasei GMNL-133 is deposited with the serial number of BCRC910520. The composition can be ingested daily by normal person and continuously administrated after stroke attack and has the effects of improving brain infarction, athletic ability and intestinal microflora after stroke.

Description

Composition for preventing stroke and improving severity of stroke and its use

The present invention relates to the technical field of probiotics for improving lactobacillus isolates, for preventing stroke and improving the severity of stroke.

Cerebrovascular diseases are consistently ranked among the top ten causes of death in the world. Stroke injury may affect the whole body, causing disability and death, and is a huge burden on social medical resources and families. Among cerebrovascular diseases, ischemia stroke has the highest incidence, accounting for about 80% of the total proportion of stroke patients. However, currently the main clinical treatment of ischemia stroke is the use of thrombolytic therapy. The treatment method dredges the infarcted ischemic area of ischemic stroke patients, restores blood flow, and reduces the infarcted area. However, in recent years, it has been discovered that after the ischemic tissue restores blood flow and reperfusion, it produces a large amount of free radicals or reactive oxygen species, causing more serious ischemic cell damage, leading to ischemia-reperfusion injury (ischemia-reperfusion injury), and central central deficit When blood reperfusion injury occurs, it will produce oxidative stress, cause DNA damage, lipid peroxidation and other damages. It will greatly increase cytokines and chemokines, causing a large number of white blood cells to infiltrate around the infarcted area, causing inflammation. Causes apoptosis of nerve cells and damages nerve function.

The microbial flora in the intestine will interact with the central nervous system, the autonomic nervous system and the immune system. It can form bacteria through the three pathways of the gut-brain axis (immunity, neuroendocrine and vagus nerve)-intestine-brain Axis (microbiome-gut-brain axis), to the central nervous system The system has a major impact, which means that if the normal intestinal microflora is maintained, the intestinal barrier function can be maintained, the intestinal immunity can be enhanced, and the occurrence of excessive inflammation can be reduced.

Intestinal bacteria are highly correlated with stroke and brain injury. Experimental results found that in the middle cerebral artery occlusion (MCAO) mouse model, the intestinal flora will be imbalanced and the intestinal barrier function will be reduced. And the increase in intestinal permeability will make the course of stroke worse. In the middle cerebral artery ligation mouse mode, some bad bacteria that can cause intestinal inflammation are increased, such as: Bacteroides , Escherichia Shigella , Haemophilus , Eubacterium nodatum group, Collinsella, Enterococcus , Proteus , Alistipes , Klebsiella , Shuttleworthia and Faecalibacterium ... etc.; in addition, some good bacteria that regulate immune response and enhance the barrier function of intestinal epithelial cells have been reduced, such as: Alloprevotella , Ruminococcaceae , Oscillospira , Lachnospiraceae NK4B4 group , Akkermansia and Megasphaera .. etc.

Pretreatment of mice with broad-acting antibiotics can reduce the infarct area after ischemic stroke surgery. Antibiotic mice can activate regulatory T cells (Treg) and reduce the increase of IL-17+γδ T cells, thereby reducing its downstream related trends The accumulation of chemical factors in the brain reduces the severity of stroke; however, antibiotics cannot be used for body maintenance or disease prevention. Excessive use can lead to many side effects. Therefore, the use of safe and non-side-effect probiotics to regulate the intestinal flora, maintain the intestinal barrier and reduce excessive inflammation is a better strategy to prevent ischemic stroke in the future.

In addition, studies have found that in the animal model of bilateral carotid artery ligation and reperfusion injury, it has been found that the administration of Clostridium butyricum (1x10 ⁹ CFU/kg) can reduce the oxidative pressure and cell apoptosis caused by cerebral ischemia-reperfusion injury. Death, achieve the effect of neuroprotection. However, bilateral common carotid artery ligation is not the main cause of clinical ischemic stroke. Most clinical ischemic strokes occur in the middle cerebral artery or its branch ischemia, but there is no research to evaluate the function The relationship between the protective effect of sexual probiotics in the occurrence of ischemic stroke and the intestinal flora.

The present invention uses the middle cerebral artery ligation (MCAO) mouse model to simulate the occurrence of ischemic stroke, to evaluate whether functional probiotics have preventive and protective effects and their correlation with intestinal bacteria. As the current research on probiotics for preventing stroke and improving symptoms after stroke is not extensive, it is urgent to find effective probiotics for improvement.

The object of the present invention is to provide a composition for preventing stroke and improving the severity of stroke, which comprises an effective amount of Lactobacillus reuteri GMNL-89 and Lactobacillus paracasei GMNL-133; wherein The deposit number of Lactobacillus reuteri GMNL-89 is BCRC910340, and the deposit number of Lactobacillus paracasei GMNL-133 is BCRC910520.

In order to achieve the aforementioned purpose of the invention, it is taken by normal people on a daily basis or after a stroke.

Another object of the present invention is to provide a probiotic for the preparation of a stroke prevention and stroke severity improvement composition, wherein the probiotic is selected from Lactobacillus reuteri ( Lactobacillus reuteri ) GMNL-89, side cheese milk The group consisting of Lactobacillus paracasei GMNL-133; wherein the deposit number of Lactobacillus reuteri GMNL-89 is BCRC910340, and the deposit number of Lactobacillus paracasei GMNL-133 is BCRC910520.

In order to achieve the aforementioned object of the invention, the improvement of stroke is improvement of ischemic stroke.

In order to achieve the aforementioned object of the invention, the improvement of stroke severity is to reduce stroke Infarcted area of the brain.

In order to achieve the aforementioned object of the invention, the improvement of the severity of stroke is to improve the recovery status of the patient after the stroke.

In order to achieve the aforementioned object of the invention, the recovery condition of the patient is exercise capacity.

In order to achieve the purpose of the foregoing invention, the dosage of the probiotic bacteria is 1×10 ⁷ to 1×10 ¹⁰ live bacteria per day.

Another object of the present invention is to provide a use of probiotics for preparing a composition for improving intestinal flora after stroke, wherein the probiotics are selected from Lactobacillus reuteri GMNL-89, side cheese milk The group consisting of Lactobacillus paracasei GMNL-133; wherein the deposit number of Lactobacillus reuteri GMNL-89 is BCRC910340, and the deposit number of Lactobacillus paracasei GMNL-133 is BCRC910520.

In order to achieve the aforementioned object of the invention, the improvement of the intestinal flora after stroke is an increase in the richness of good bacteria.

In order to achieve the purpose of the aforementioned invention, the good bacteria are Ruminococcaceae or Oscillospira.

In order to achieve the purpose of the foregoing invention, the dosage of the probiotic bacteria is 1×10 ⁷ to 1×10 ¹⁰ live bacteria per day.

In summary, the probiotic composition of the present invention can reduce the cerebral infarction area of the individual after a stroke, restore exercise capacity, and increase the intestinal microflora, thereby achieving the effects of preventing stroke and improving the severity of stroke.

S01~S06‧‧‧Experimental process

Figure 1A is the experimental process.

Figure 1B is a diagram of the experimental process and operation mode.

Figure 2 shows the effects of different probiotics on the cerebral infarct area caused by ischemic stroke.

Figure 3 shows the effect of different probiotics on motor dysfunction caused by ischemic stroke-Grip test.

Figure 4 is the analysis of the microbial richness of the ischemic stroke mice in different probiotic groups (phyla level).

All technical and scientific terms mentioned in this specification, unless otherwise defined, have meanings commonly understood by those skilled in the art.

The composition described herein may include, but is not limited to: food, beverage, health food, animal drinking water additives, animal feed additives, medical compositions for animals and humans, food additives, beverage additives, etc. applicable to the present invention The application form.

The present invention is illustrated by the following examples, but the present invention is not limited by the following examples. The drugs and biological materials used in the present invention are all commercially available and easy to obtain. The following are only examples of available channels.

The present invention is illustrated by the following examples, but the present invention is not limited by the following examples.

The Lactobacillus reuteri GMNL-89 used in the present invention is deposited in the Biological Resources Preservation and Research Center of the Hsinchu Food Industry Development Research Institute, the deposit date is November 14, 2006, and the deposit number is BCRC910340; and is deposited in the China Type Culture Collection Center, the deposit date is November 19, 2007, and the deposit number is CCTCC M207154.

The Lactobacillus paracasei GMNL-133 used in the present invention is deposited at the Biological Resources Preservation and Research Center of Hsinchu Food Industry Development Institute, the deposit date is July 5, 2010, and the deposit number is BCRC910520; and the deposit is deposited in the China Type Culture Collection The date is September 26, 2011, and the deposit number is CCTCC M2011331.

experimental method

Stroke experimental animal model: The experimental process is shown in Figure 1A and Figure 1B (top). The experiment used male C57BL/6 mice (8-12 weeks; 22-28g), and the experimental animals were purchased from Lesco Biotech. Co., Ltd., the environment for raising animals is controlled at room temperature (24±1℃), humidity is 55±5%, and the light cycle of day and night is maintained for each twelve hours. Experimental animals are allowed to eat and drink freely.

Mice were given the antibiotics sulfamethoxazole (0.8mg/ml) and trimethoprim (0.16mg/ml) for 10 days before the middle cerebral artery ligation operation, and then drank pure water for 2 days. After that, all the experimental animals were divided into different groups: Vehicle group: tube-fed water; GMNL-662: tube-fed 8.2x10 ⁷ CFU (low dose) or 8.2x10 ⁸ CFU (high dose) per kilogram of mouse; GMNL-89: tube-fed 8.2x10 ⁷ CFU (per kg mouse) Low dose) or 8.2x10 ⁸ CFU (high dose); GMNL-133: 8.2x10 ⁷ CFU (low dose) or 8.2x10 ⁸ CFU (high dose) per kilogram of mice; GMNL-89+GMNL-133(1 : 1): 8.2x10 ⁷ CFU per kilogram of mice (half each of GMNL-89 and GMNL-133, low dose) or 8.2x10 ⁸ CFU (half each of GMNL-89 and GMNL-133, high dose).

The preparation of lactic acid bacteria is to dissolve the freeze-dried powder of the strain in sterile water, mix it evenly, and administer it by tube feeding. It is fed once a day. It is fed for 4 consecutive days. On the 5th day, the middle cerebral artery is ligated after the preparation is fed. The mice were fed with probiotics for 6 days, and the mice were evaluated for 7 days after the middle cerebral artery ligation operation. The animals were fed with water or lactic acid bacteria solution for 11 consecutive days.

Ligation of the right middle cerebral artery (MCA) caused ischemia of the right cerebral cortex. The experimental procedure is shown in the schematic diagram of Figure 1B (bottom). First, anesthetize with 2% isoflurane, and then shave the neck and head hair . Cut away from the midline of the neck, and find the left and right common carotid arteries (CCA) on both sides of the trachea. After the CCA is isolated, the C57BL/6 mouse is placed sideways, from the right corner of the eye (canthux) Make an incision between the pinna and separate the muscles. Use a bone drill to drill a small hole at the junction of the zygoma bone and the squamosal bone to form a bone window with a diameter of about 3mm. Under the operating microscope, use fine forceps to remove the dura, and you can see the right middle cerebral artery. Use a bipolar electric knife to burn the MCA to block the blood flow. After the electric burn, use the CCA to block the blood flow for 20 minutes. The mouse was injured by local central ischemia, then the vascular clamp was loosened, blood was reperfused, and the wound was sutured with a 4/0 suture. During the operation, the mouse was placed on an electric blanket to maintain a constant body temperature. Afterwards, the mice were also placed on the electric blanket to wait for the mice to wake up to prevent the mice from losing temperature.

Assessment of cerebral infarction injury area: On the 7th day after the operation, the animal was decapitated and sacrificed after anesthesia. The brain was taken out and washed with PBS. The appearance of the brain was checked for hemorrhage or infection. When the experimental animal had abnormal cerebrovascular sites or middle cerebral arteries The anatomical position was abnormal and the experimental animal was excluded. Then put the brain tissue of the mouse in the cold physiological solution for 10 minutes, then place it in the brain tissue section mold and cut into brain slices with a thickness of 1 mm. In a dark environment, infiltrate at 37°C, 2% After staining in 2,3,5 triphenyltetrazolium chloride (TTC) for 30 minutes, the brain tissue was fixed with 10% formalehyde for 24 hours. After scanning it, it was scanned using an image processing system (Image J software, National Institutes of Health (NIH) ),USA) Calculate the area of cerebral infarction injury. Milky white area or necrotic liquefaction area (defect area) belong to cerebral infarction injury area.

Animal behavior evaluation: The time points of the behavior evaluation test are set on the day before the stroke and the third and sixth days after the stroke. The Grip Test can test the grasping reflex and sense of balance of the mouse using its front paws to grasp the metal strip and maintain the suspension. The test mode is to pick up the tail of the mouse first, and hang the mouse’s front paw in the middle of a 50cm-long metal strip 20cm above the table. The test time is 30 seconds. Table 1 shows the scoring standard of the Grip Test.

Bacterial phase analysis: collect fresh mouse feces at the end of the experiment. After extracting fecal DNA, use Illumina MiSeq System Sequencing (2 x 301 bp paired-end) to perform 16S rRNA V3-V4 region sequence, and compare the data to analyze The fecal flora changes in different groups (including the phylum to the level of bacteria). The four main groups for bacterial analysis are: vehicle, tube feeding GMNL-89 (8.2x10 ⁸ CFU/kg), GMNL-133 (8.2x10 ⁸ CFU/kg) and GMNL-89+GMNL- 133 (8.2x10 ⁸ CFU/kg) three high-dose groups.

Statistics: The data is expressed by mean±SEM. The improvement of the infarct zone and whether the behavioral dysfunction is improved are all based on one-way analysis of variance (ANOVA) and then analyzed by Student's t-test. Does the difference between groups of different concentrations of probiotics make sense? The statistical results are all regarded as significant differences with p<0.05 (*).

Experimental results:

The experiment proceeded as shown in Fig. 1A and Fig. 1B. The antibiotic water was given for 10 days and then rested for 2 days; then the individual probiotics and probiotic compositions of different doses were fed for 5 days, and then the stroke was induced. The probiotics were fed to the 11th day, during which the grip test (Grip test) was used to observe the exercise ability of the rats, and the rats were sacrificed on the 12th day to observe the brain infarct area. The results of cerebral infarction are calculated based on the calculation of the white and defects in the brain slice in Figure 2 that are included in the infarct area. The results are shown in Table 2. The defects are the result of liquefaction after infarction; the exercise ability is evaluated by the grip strength test, and the results are shown in Figure 3. ; The effects of different probiotic groups on the cerebral infarction area and exercise capacity caused by ischemic stroke are explained as follows.

Table 2. The effect of different probiotics on the cerebral infarct area caused by ischemic stroke

Lactobacillus (L.plantarum) GMNL-662 group: gavage high or low dose of Lactobacillus (L.plantarum) GMNL-662 mice were not significantly reduced the infarcted region (Table 2), to improve the ability of the stroke movement of the mouse The effect is poor (Figure 3).

Lactobacillus reuteri ( L.reuteri ) GMNL-89 group: tube feeding low-dose (8.2x10 ⁷ CFU/kg mice) Lactobacillus reuteri GMNL-89 could not reduce the cerebral infarct area (Table 2); while tube feeding High dose (8.2x10 ⁸ CFU/kg mice) of Lactobacillus reuteri GMNL-89 can significantly reduce the cerebral infarct area of mice (Table 2, *: P<0.05, compared with vehicle), and recovery of exercise ability is also better (image 3).

Lactobacillus paracasei ( L. paracasei ) GMNL-133 group: tube feeding low-dose (8.2x10 ⁷ CFU/kg mice), high-dose (8.2x10 ⁸ CFU/kg mice) Lactobacillus paracasei GMNL-133 can significantly reduce Infarct area of mice (Table 2); tube feeding high-dose Lactobacillus paracasei GMNL-133 has a better effect on improving the exercise ability of stroke mice (Figure 3).

Groups of Lactobacillus reuteri GMNL-89 and Lactobacillus paracasei GMNL-133: A combination of Lactobacillus reuteri GMNL-89 and Lactobacillus paracasei GMNL-133 was used for subsequent protection against stroke. The results showed that high dose (8.2x10 ⁸ CFU/kg mice) and low dose (8.2x10 ⁷ CFU/kg mice) of GMNL-89 and GMNL-133 complex strains (1:1) are effective in reducing the infarct area of mice and improving stroke mice In terms of exercise ability, compared with the same dose of a single bacteria, the protective effect is better (Table 2, Figure 3); it shows that the use of this compound strain combination has an additive effect, which can protect the brain against ischemia. The effect of perfusion injury is the best.

The effects of different probiotic groups on the intestinal flora caused by ischemic stroke are explained as follows.

In the analysis of the intestinal flora, it was found that the feces of the syndrome group were high in Bacteroidetes (richness of Bacteroidetes : 76.46%) and less in Firmicutes (richness of Firmicutes : 23.27%); Tube feeding of GMNL-89, GMNL-133 or composite strains (GMNL-89+GMNL-133) can relatively reduce the Bacteroides phylum and increase Firmicutes (Figure 4)

From the results of bacterial phyla analysis, it was found that the GMNL-89 group, GMNL-133 group and the composite strain (GMNL-89+GMNL-133) did not completely adjust the intestinal flora (Figure 4); if the analysis of Lactobacillus ( Lactobacillus ) The abundance was found to be higher in the GMNL-89 group (0.26%), and lower in the GMNL-133 group (0.18%), but there was a significant increase in the postoperative syndrome group (0.06%) (Table 3); past research It was found that the abundance of related good bacteria was reduced during the occurrence of ischemic stroke. From the results of this experimental flora analysis, it was found that the two good bacteria of Ruminococcaceae and Oscillospira , the abundance in the syndrome group was Significantly lower than the three groups of probiotics (Table 3), indicating that tube feeding GMNL-89, GMNL-133 or GMNL-89+GMNL-133 may achieve the effect of protecting stroke injury through these two good bacteria; and analyzing the related bad The bacteria did not change much, which may be related to the drinking of antibiotic water before the experiment; these results infer that Lactobacillus reuteri GMNL-89 and Lactobacillus paracasei GMNL-133 increase by regulating the increase of good intestinal bacteria and stabilize the intestinal immune response , Inhibit the production of excessive inflammatory response factors, and achieve the effect of protecting the symptoms of ischemic stroke.

In summary, the probiotic composition of the present invention can prevent stroke and improve individual health after stroke, including: 1) reducing cerebral infarction area, 2) recovery of exercise ability (such as grip strength), and 3) improvement of intestinal flora , Including good bacteria Ruminococcaceae ( Ruminococcaceae ), Oscillospira ( Oscillospira ) increase; and achieve the effect of preventing stroke and improving the severity of stroke.

The above-mentioned multiple functions are indeed in full compliance with the statutory requirements for invention patents for novelty and advancement. You file an application in accordance with the law, and I implore your bureau to approve this invention patent application to encourage invention.

【Biological Material Deposit】

Domestic deposit information [please note in order of deposit institution, date and number]

1. Lactobacillus reuteri GMNL-89: Bio-resource conservation and conservation of Hsinchu Food Industry Development Institute Research center, deposit date is November 14, 2006, deposit number is BCRC910340.

2. Lactobacillus paracasei GMNL-133: Biological Resources Conservation and Research Center of Hsinchu Food Industry Development Institute, the deposit date is July 5, 2010, and the deposit number is BCRC910520.

Foreign hosting information [please note in the order of hosting country, institution, date and number]

1. Lactobacillus reuteri GMNL-89: China Type Culture Collection, the deposit date is November 19, 2007, and the deposit number is CCTCC M207154.

2. Lactobacillus paracasei GMNL-133: China Type Culture Collection, the deposit date is September 26, 2011, and the deposit number is CCTCC M2011331.

Claims (12)

A composition for preventing stroke and improving the severity of stroke, which comprises effective amounts of Lactobacillus reuteri GMNL-89 and Lactobacillus paracasei GMNL-133; wherein the Lactobacillus reuteri GMNL-133 The deposit number of GMNL-89 is BCRC910340, and the deposit number of Lactobacillus paracasei GMNL-133 is BCRC910520. The composition as described in item 1 of the scope of the patent application, which is ingested by normal people on weekdays or continuously administered after the onset of stroke. A probiotic is used for preparing a composition for preventing stroke and improving the severity of stroke, wherein the probiotic is selected from Lactobacillus reuteri GMNL-89, Lactobacillus paracasei GMNL-133 The deposit number of Lactobacillus reuteri GMNL-89 is BCRC910340, and the deposit number of Lactobacillus paracasei GMNL-133 is BCRC910520. The use described in item 3 of the scope of patent application, wherein the improvement of stroke is improvement of ischemic stroke. The use described in item 3 of the scope of patent application, wherein the improvement of stroke is improvement of cerebral infarction caused by stroke. For the purposes described in item 3 of the scope of patent application, the improvement of stroke is to improve the recovery of patients after stroke. For the purposes described in item 3 of the scope of patent application, the patient’s recovery status is exercise capacity. For the purposes described in item 3 of the scope of patent application, the dosage of the probiotics is 1×10 ⁷ to 1×10 ¹⁰ live bacteria per day. The use of a probiotic for preparing a composition for improving the intestinal flora after stroke, wherein the probiotic is selected from Lactobacillus reuteri GMNL-89, Lactobacillus paracasei GMNL-133 The deposit number of Lactobacillus reuteri GMNL-89 is BCRC910340, and the deposit number of Lactobacillus paracasei GMNL-133 is BCRC910520. The use as described in item 9 of the scope of patent application, wherein the improvement of the intestinal flora after stroke is the increase of the richness of good bacteria. The use described in item 9 of the scope of patent application, wherein the good bacteria are Ruminococcaceae or Oscillospira. For the purposes described in item 9 of the scope of patent application, the dosage of the probiotics is 1×10 ⁷ to 1×0 ¹⁰ live bacteria per day.

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