Application of ginseng natto complex ferment in preparation of alzheimer's disease drugs

Abstract

The application of ginseng natto compound ferment in preparation of Alzheimer's disease medicine relates to the technical field of medicine, and solves the technical problems of safety risk and the like of the existing Alzheimer's disease prevention and treatment medicine.The ginseng natto compound ferment comprises natto kinase, soybean glycoside, soybean glycoside, genistin, daidzein, genistein, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rc.The soybean is soaked in ginseng extract solution until the soybean absorbs the ginseng extract solution completely;the soaked soybean is sterilized as a fermentation substrate;the bacillus subtilis is inoculated, shaken and continuously cultured;and the product after the culture is dried, crushed and sieved to obtain the ginseng natto compound ferment.The application provides a new solution for upgrading of functional food to neuroprotective agent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to an application of a ginseng-natto compound fermentation product in preparation of an Alzheimer's disease drug. BACKGROUND

[0002] With the aggravation of population aging, the prevention of Alzheimer's disease (AD) is increasingly urgent, but the existing prevention and treatment drugs are mainly chemical synthetic drugs or biological agents, which have significant limitations. For example, chemical synthetic drugs are single-targeted, only temporarily alleviate cognitive symptoms, and cannot inhibit the pathological process; biological agents have safety risks, such as brain edema in some patients using monoclonal antibody drugs, and high annual treatment costs.

[0003] Natto is a traditional fermented soybean food prepared by Bacillus subtilis fermentation of soybeans, which has rich nutritional value. Studies have shown that natto is rich in a fibrinolytic enzyme, nattokinase, which can prevent and assist in the treatment of cardiovascular and cerebrovascular diseases and has no toxic side effects.

[0004] Ginseng is the dried root and rhizome of Panax ginseng of the Araliaceae family, which is a precious traditional Chinese medicine. Ginsenosides are one of the main active components of ginseng, which have antioxidant, hypoglycemic, and memory-improving effects. As a representative species of the "food-medicine homology" strategy in China, ginseng has been listed as a new resource food since 2012. Its health care functions of tonifying qi and blood, promoting blood circulation and unblocking meridians have been deeply rooted in people's hearts. Its application scenarios have expanded from traditional Chinese medicine decoction pieces to functional food field, and a series of functional health foods have been derived. Although studies have confirmed that ginsenoside Rg1 can reduce the toxicity of Aβ1-42 by inhibiting the GSK-3β pathway, it is difficult to achieve effective brain delivery alone due to its low blood-brain barrier penetration rate.

[0005] Although biotransformation technology has been successfully applied to active ingredient enhancement, there is still a lack of research on ginseng-natto synergistic fermentation system, and there is no related product application in Alzheimer's disease. Currently, the industry generally uses physical compounding technology, such as mechanically mixing ginseng ethanol extract and natto freeze-dried powder, but this method does not utilize microbial metabolism to modify the structure of the components, and does not involve the prevention of Alzheimer's disease. Therefore, the use of integrated fermentation technology based on microbial co-metabolism is crucial to fill the gap in the application of natural medicines in Alzheimer's disease drugs and to improve the safety of Alzheimer's disease prevention and treatment drugs. SUMMARY

[0006] In order to solve the technical problems of safety risks of existing Alzheimer's disease prevention and treatment drugs, the present application provides an application of a ginseng-natto compound fermentation product in preparation of an Alzheimer's disease drug.

[0007] The technical scheme of the present application is as follows:

[0008] Application of a ginseng-natto compound fermentation product in preparation of an Alzheimer's disease drug, wherein the ginseng-natto compound fermentation product comprises the following components:

[0009] natto kinase, soybean glycoside, daidzein, genistin, daidzein, genistein, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Rc.

[0010] The natto kinase activity is 6645.3673±109.6755 IU / g; the contents of the soybean glycoside, daidzein, genistin, daidzein, genistein, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Rc are 0.3261±0.0077 mg / g, 0.9571±0.0070 mg / g, 0.1160±0.0055 mg / g, 0.9716±0.0166 mg / g, 0.0332±0.0015 mg / g, 0.0433±0.0019 mg / g, 0.2729±0.0044 mg / g, and 0.0942±0.0022 mg / g, respectively.

[0011] Preferably, the preparation of the ginseng-natto compound fermentation product comprises the following steps:

[0012] S1. Soybeans and ginseng are weighed according to a mass ratio of 6:1;

[0013] S2. A ginseng extract is prepared, the ginseng extract is concentrated to 1.2 times the mass of the soybeans, and the soybeans are added to the ginseng extract for soaking until the soybeans completely absorb the ginseng extract;

[0014] S3. The soaked soybeans are sterilized as a fermentation substrate;

[0015] S4. Bacillus subtilis is inoculated, shaken, and continuously cultured, and the product after the culture is dried, crushed, and sieved to obtain the ginseng-natto compound fermentation product.

[0016] Preferably, the preparation of the ginseng extract is as follows: the ginseng is decocted in distilled water twice, each time for 1 h, and the extract is combined to obtain the ginseng extract.

[0017] Preferably, the sterilization is performed at 121℃ and 0.1 MPa for 30 min.

[0018] Preferably, the inoculation amount of the Bacillus subtilis is 6.2%.

[0019] Preferably, the fermentation seed age of the Bacillus subtilis is 5 h.

[0020] Preferably, the culture temperature is 34 DEG C, and the culture time is 37.2 h.

[0021] Preferably, the sieving is sieving through a No. 3 sieve.

[0022] Compared with the prior art, the present application has the following specific advantages:

[0023] The present application innovatively proposes to apply the product of co-fermentation of Bacillus subtilis, ginseng and soybean to prevention and treatment of Alzheimer's disease. Experimental data show that the fermentation product can significantly improve the learning and memory ability of AD model mice, and regulate the cholinergic system, oxidative stress and inflammatory response, and no similar fermentation process has been reported for AD prevention.

[0024] The special fermentation process of the present application converts isoflavone glycosides (such as daidzin and genistin) in soybean into highly active aglycone forms, and the bioavailability is significantly improved. The fermentation product synergistically inhibits the cholinergic system, oxidative stress and inflammatory response, forming a multi-dimensional intervention on AD pathology, which is not possessed by the traditional compounding process.

[0025] The product provided by the present application can be applied to prevention and treatment of Alzheimer's disease, and provides a new solution for upgrading of natural food to neuroprotective agent. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a growth curve of Bacillus subtilis;

[0027] Figure 2 is a standard curve of urokinase;

[0028] Figure 3 is a chromatogram of mixed reference substance at a wavelength of 254 nm;

[0029] Figure 4 is a chromatogram of mixed reference substance at a wavelength of 203 nm;

[0030] Figure 5 is a chromatogram of ginseng natto compound fermentation product at a wavelength of 254 nm;

[0031] Figure 6 is a chromatogram of ginseng natto compound fermentation product at a wavelength of 203 nm;

[0032] Figure 7 is the motion trajectory result of each group of mice in the positioning navigation experiment;

[0033] Figure 8 is the latency experiment result in the positioning navigation experiment;

[0034] Figure 9For the total distance experiment results in the positioning navigation experiment;

[0035] Figure 10 For the number of times of crossing the platform experiment results in the space exploration experiment;

[0036] Figure 11 For the effective area movement time experiment results in the space exploration experiment;

[0037] Figure 12 For the ACh expression level experiment results of mice;

[0038] Figure 13 For the AChE expression level experiment results of mice;

[0039] Figure 14 For the SOD expression level experiment results of mice;

[0040] Figure 15 For the MDA expression level experiment results of mice;

[0041] Figure 16 For the IL-6 expression level experiment results of mice;

[0042] Figure 17 For the TNF-α expression level experiment results of mice;

[0043] Figure 18 For the mouse brain tissue staining section microscopic observation figure. DETAILED DESCRIPTION

[0044] In order to make the technical solutions of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings of the specification of the present application. It should be noted that the following embodiments are only used to better understand the technical solutions of the present application, and should not be understood as a limitation of the present application.

[0045] Embodiment 1.

[0046] The Bacillus subtilis stored at 4℃ was activated in the NA liquid medium, the liquid volume was 40 mL / 250 mL, and was placed in a 37℃, 180 r·min-1 shaking bed for 12 h. 5% of the bacterial liquid was taken for secondary activation, the liquid volume was 40 mL / 250 mL, and the optical density of the Bacillus subtilis liquid diluted appropriately was measured every 1 h by using a spectrophotometer. The OD 660 value was measured until the 15th hour, and the time was taken as the abscissa and the OD 660 value was taken as the ordinate to plot a growth curve of the Bacillus subtilis. The bacterial liquid in the logarithmic growth phase was inoculated.

[0047] The growth curve of the Bacillus subtilis is as follows: Figure 1The growth stage of the strain can be divided into four periods: lag phase, logarithmic phase, stationary phase and decline phase. Figure 1 It can be seen that 0-2 h is the lag phase, the cell proliferation speed is slow in this period, and the concentration of the bacterial solution is low; 2-7 h is the logarithmic phase, the cell presents logarithmic growth in this period, the concentration of the bacterial solution increases rapidly, and the growth rate of the strain is the largest; 7-15 h is the stationary phase, the growth and death of the cell tend to be balanced in this period, and the cell is in a stable state. Therefore, the optimal fermentation age of Bacillus subtilis is 5 h.

[0048] Take 60 g of soybeans and 10 g of ginseng; decoct the ginseng to obtain 72 g of an extract; soak the soybeans in the ginseng extract until the soybeans completely absorb the ginseng extract; sterilize the soaked soybeans at 121 ℃ for 30 min as a fermentation substrate; inoculate the Bacillus subtilis with an inoculation amount of 6.2% and a strain age of 5 h, shake well, and continuously culture at 34 ℃ for 37.2 h; dry, crush, and pass through a No. 3 sieve to obtain a ginseng natto compound fermentation product.

[0049] Next, the agarose-fibrin plate method is used to detect the natto kinase activity in the compound fermentation product, and high performance liquid chromatography (HPLC) is used to detect the contents of soybean glycoside, soybean glycoside, genistin, daidzein, ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Rc in the compound fermentation product.

[0050] I. Natto kinase activity determination:

[0051] 1. Natto kinase extraction:

[0052] Precisely take 1 g of ginseng natto powder, add 5 mL of sterile physiological saline, mix well, extract for 2 h, and centrifuge at 8000 r·min -1 at low temperature for 15 min. The supernatant is the crude natto kinase extract.

[0053] 2. Natto kinase activity determination:

[0054] Prepare a fibrinogen plate and prepare urokinase standard solutions with enzyme activities of 100, 200, 400, 800, and 1600 IU·mL -1 respectively. Take 10 μL of each of the urokinase standard solution and the natto kinase crude extract, and spot them on the fibrinogen plate. After 10 min at room temperature, incubate in a 37℃ constant temperature incubator for 18 h. Measure the vertical diameter of the lysis circle with a vernier caliper, calculate the area, and plot a graph with the different concentrations of urokinase as the abscissa (x) and the lysis circle area as the ordinate (y) as shown in Figure 2The urokinase standard curve was shown, and the natto kinase activity was calculated according to the standard curve regression equation. The natto kinase activity was calculated to be 6645.3673±109.6755 IU / g by repeating the experiment three times.

[0055] II. Determination of eight components in ginseng-natto compound fermentation product by HPLC method:

[0056] 1. Preparation of mixed reference solution:

[0057] Take the appropriate amount of soybean glycoside, daidzein, genistin, daidzein, genistin, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rc reference, accurately weigh, dissolve in methanol and dilute to 10 mL volumetric flask, shake well, prepare the reference substance concentration of 4.0320, 0.4008, 4.0120, 0.2040, 0.3000, 2.0200, 1.0160, 0.5040 mg / mL respectively.

[0058] 2. Preparation of test solution:

[0059] Accurately weigh 4.0 g of ginseng-natto compound fermentation product prepared in this example, place it in a conical flask with a stopper, accurately add 20 mL of methanol, weigh the mass, ultrasonic (120 W, frequency 40 kHz) treatment for 1 h, cool down, add methanol to make up the lost mass, filter, take 10 mL of the filtrate, evaporate in an evaporating dish, dissolve the residue in methanol, transfer to a 2 mL volumetric flask, dilute to the mark, shake well, and pass through a 0.22 μm microporous filter to obtain the test solution.

[0060] 3. Chromatographic conditions:

[0061] Chromatographic column: Waters XBridge C18 (250 mm x 4.6 mm, 5 μm);

[0062] Mobile phase: acetonitrile (A) - 0.1% phosphoric acid solution (B);

[0063] Elution gradient (0.01-5 min, 90%B; 5-10 min, 90%B-85%B; 10-30 min, 85%B-82%B; 30-40 min, 82%B-80%B; 40-70 min, 80%B-74%B; 70-80 min, 74%B-71%B; 80-95 min, 71%B; 95-120 min, 71%B-60%B);

[0064] Volume flow rate 1.0 mL / min;

[0065] Detection wavelength 254 nm (10-50 min: daidzin, glycitin, genistin, daidzein; 65-75 min: genistein) and 203 nm (50-60 min: ginsenoside Rg1; 100-110 min: ginsenoside Rb1, ginsenoside Rc);

[0066] Column temperature 30℃;

[0067] Injection volume 5 μL.

[0068] The chromatogram of the mixed control at 254 nm and 203 nm wavelengths was respectively as shown in Figure 3 and Figure 4 The chromatogram of the ginseng-natto compound fermentation product at 254 nm and 203 nm wavelengths was respectively as shown in Figure 5 and Figure 6 Note: In the figures, 1 is daidzin; 2 is glycitin; 3 is genistin; 4 is daidzein; 5 is genistein; 6 is ginsenoside Rg1; 7 is ginsenoside Rb1; 8 is ginsenoside Rc.

[0069] The regression equation and correlation coefficient of the 8 components to be tested are shown in the following table, and the results show that the peak area and the concentration of each component have a good linear relationship.

[0070]

[0071] The content of each component was calculated and is shown in the following table.

[0072]

[0073] Effect example.

[0074] SPF healthy male ICR mice weighing 18-22 g were used. They were provided by Liaoning Changsheng Biotechnology Co., Ltd. with license number: SCXK(Liaoning)2020-0001. The feeding conditions were suitable, the feeding room had an independent ventilation system, the indoor temperature was 24±2℃, the relative humidity was 55±5%, and the mice were allowed to drink water and eat freely during the feeding period, and were adaptively fed for 7 days.

[0075] Preparation of ginseng-soybean compound: ginseng and soybeans were mixed in a mass ratio of 6:1, dried, crushed, and sieved.

[0076] Ginseng-natto compound fermentation product: the fermentation product prepared in Example 1.

[0077] The mice were randomly divided into 7 groups, namely control group (CON), model group (MOD), donepezil positive drug group (DON), ginseng soybean complex low-dose group (GSL), ginseng soybean complex high-dose group (GSH), ginseng natto complex ferment low-dose group (GNL), and ginseng natto complex ferment high-dose group (GNH). The CON group and the MOD group were given physiological saline by gavage every day, the DON group was given drug by gavage at a dose of 0.65 mg / kg, and the GSL, GSH, GNL, and GNH groups were given drug at a dose of 2.5 g / kg, 5 g / kg, 2.5 g / kg, and 5 g / kg per day, respectively. From the 6th week, the mice in the CON group were given physiological saline by gavage every day, and the mice in the other groups were given methylecgonine (2 mg / kg) by intraperitoneal injection to establish the model, followed by a behavior experiment on the mice.

[0078] I. Morris water maze behavior experiment on mice:

[0079] The Morris water maze behavior experiment was carried out, which included three steps: adaptive training, positioning navigation experiment, and spatial exploration experiment.

[0080] On the first day, the mice were given adaptive training after the water was added and heated to 23±2℃. The mice were put into the water with their faces facing the pool wall. The water maze was divided into four quadrants (east, south, west, and north). The platform was changed and the mouse was placed in a safe position. The platform was 1 cm higher than the water surface. Mice with too fast or too slow swimming speed or insufficient vision were excluded.

[0081] On the 2nd to 5th day, the positioning navigation experiment was carried out. Titanium dioxide powder was added to the water and stirred evenly to make the pool turbid. The platform position remained unchanged, and the platform was 1 cm below the water. The mice were placed in the water in different directions. If the mouse successfully climbed onto the platform within 60 seconds, the time used was the latency period. If the mouse did not climb onto the platform within 60 seconds, it was guided onto the platform and stayed for 10 seconds. The latency period was recorded as 60 seconds. The movement trajectory, latency period, and total distance on the 5th day of testing were recorded as observation indexes.

[0082] The results of the movement trajectory of the mice in each group in the positioning navigation experiment are shown in Table 1. Figure 7 As can be seen from Table 1, compared with the MOD group, the mice in the CON group could find the platform in a short time by crossing the pool. The movement trajectory of the mice in the MOD group was irregular and they did not actively search for the platform, but made circular movements along the periphery of the pool. After treatment with the positive drug donepezil, ginseng natto, and ginseng soybean, the movement trajectory improved to varying degrees, and the ginseng natto group was better than the ginseng soybean group. The results showed that ginseng natto and ginseng soybean could improve the movement trajectory of the mice searching for the platform and improve the memory retention ability of the Alzheimer's disease model mice to varying degrees, especially ginseng natto. The mice quickly found the platform after a short search, and the movement distance was significantly shortened.

[0083] The results of the latency period experiment are shown in Table 2.Figure 8 Compared with the normal CON group, the latency period in the MOD group was significantly prolonged ( P <0.01), requiring a relatively long time to find the platform; compared with the MOD group, the latency of the DON, GNL and GNH groups was significantly shortened ( P <0.01), the latency period in the GSH group was significantly shortened ( P <0.05), the latency period in the GSL group showed a trend of shortening but no significant difference ( P >0.05), the latency periods of the GSL and GNL groups, and the GSH and GNH groups, were significantly different. P <0.01).

[0084] The total distance test results are shown below. Figure 9 Compared with the normal CON group, the total distance traveled in the MOD group was significantly prolonged ( P <0.01), compared with the MOD group, the total distances of the DON, GNL and GNH groups were significantly shortened ( P <0.01); the total distance in the GSL and GSH groups showed a decreasing trend but no significant difference ( P >0.05), the total distance traveled between the GSL and GNL groups was significantly different. P <0.05), the total distance was significantly different between the GSH and GNH groups. P <0.01).

[0085] On day 6, in the space exploration experiment, titanium dioxide powder was added to the water and stirred until it became turbid. The platform was then removed, and the number of times the mice crossed the platform and the effective movement time within 60 seconds were recorded.

[0086] The experimental results of the number of platform crossings are shown below. Figure 10 Compared to the CON group, the MOD group had a significantly reduced number of platform crossings. P <0.01). Compared with the MOD group, the DON, GNL, and GNH groups showed a significantly increased number of platform crossings ( P <0.01), the number of platform crossings in the GSL and GSH groups showed an increasing trend, but there was no significant difference. P >0.05), there was a significant difference in the number of platform crossings between the GSL and GNL groups ( P <0.05), the number of platform crossings was significantly different between the GSH and GNH groups ( P <0.01).

[0087] The experimental results of effective area motion time are shown in Figure 11 Compared with the CON normal group, the effective area movement time in the MOD group was significantly shortened ( P<0.01), compared with the MOD group, DON, GNL and GNH groups were significantly prolonged (P<0.01) P <0.01), the GSL, GSH group effective area movement time had a tendency to extend but no significant difference (P>0.05) P <0.01), GSL and GNL groups, GSH and GNH groups were significantly different (P<0.01) P <0.01).

[0088] Results showed that compared with the CON group, the swimming trajectory of the MOD group was irregular, mostly edge type, latency, total distance, crossing platform times and effective area movement time had significant statistical difference (P<0.01) P <0.01), indicating that the learning and memory ability of the MOD group was significantly lower than that of the CON group, indicating that the Alzheimer's disease model was successfully established. Compared with the MOD group, the latency, total distance, crossing platform times and effective area movement time of the DON group were significantly improved (P<0.01) P <0.01), the trajectory was trend type, straight line type, indicating that donepezil had a significant effect on improving Alzheimer's disease. Compared with the MOD group, the above indexes of the GSL, GSH group and GNL, GNH group were improved to varying degrees, and the ginseng natto group was better than the ginseng soybean group, indicating that the ginseng natto group had a better effect on improving Alzheimer's disease induced by hyoscine, and the ginseng soybean group was second, and both groups showed that the high dose group was better than the low dose group.

[0089] Two, the detection of biochemical indicators of mouse brain tissue:

[0090] After the behavioral experiment, the mice in each group were sacrificed by cervical dislocation, and the whole brain tissue was taken out on ice, washed with 0.1M PBS (PH 7.4) physiological saline, dried with filter paper, weighed, added with a certain proportion of 0.1M PBS (PH 7.4), and homogenized with a homogenizer, 3000 r·min -1 centrifuged at 4℃ for 20 min, and the supernatant was detected. The levels of Ach, AchE, SOD, MDA, IL-6 and TNF-α in the brain tissue of mice were detected by ELISA method. All indexes were determined according to the instructions of the kit.

[0091] Experimental results

[0092] 1. Effect on cholinergic neurotransmitter of mice

[0093] The expression levels of ACh and AChE in mice were shown in Figure 12 and Figure 13 Compared with the CON group, the ACh level of the MOD group was extremely significantly reduced (P<0.01)P <0.01), AChE level was extremely significantly increased ( P <0.01); compared with MOD group, ACh level of DON, GNH group was extremely significantly increased ( P <0.01), AChE level was extremely significantly decreased ( P <0.01), ACh level of GNL group was significantly increased ( P <0.05), AChE level was extremely significantly decreased ( P >0.05), ACh level of GSL, GSH group had a trend of increase but no significant difference ( P >0.05), AChE level had a trend of decrease but no significant difference ( P >0.05); compared with GNH group, ACh level of GSH group had extremely significant difference ( P <0.01), AChE level had significant difference ( P <0.05).

[0094] 2. Effects on oxidative stress level of mice

[0095] The expression levels of SOD and MDA of mice were shown in Figure 14 and Figure 15 Compared with CON group, SOD level of MOD group was extremely significantly decreased ( P <0.01), MDA level was extremely significantly increased ( P <0.01); compared with MOD group, SOD level of DON, GNL and GNH group was extremely significantly increased ( P <0.01), SOD level of GSL, GSH group had a trend of increase but no significant difference ( P >0.05), MDA level of DON, GSH, GNL and GNH group was extremely significantly decreased ( P <0.01), MDA level of GSL group had a trend of decrease but no significant difference ( P >0.05); compared with GNL group, SOD level of GSL group had extremely significant difference ( P <0.01), MDA level had significant difference ( P <0.05), compared with GNH group, SOD, MDA level of GSH group had extremely significant difference ( P <0.01).

[0096] 3. Effects on inflammatory response of mice

[0097] The expression levels of IL-6 and TNF-α of mice were shown in Figure 16 and Figure 17 Compared with CON group, IL-6, TNF-α level of MOD group was extremely significantly increased ( P<0.01); compared with the MOD group, the levels of IL-6 and TNF-α in the DON, GNL and GNH groups were extremely significantly reduced (P<0.01) P <0.01), the levels of IL-6 and TNF-α in the GSL and GSH groups had a decreasing trend but no significant difference (P>0.05) P <0.05), the levels of IL-6 and TNF-α in the GSH and GNH groups had significant differences (P<0.05) P <0.05). P <0.05).

[0098] The experimental results show that the ginseng and natto compound fermentation product has a certain regulating effect on the indicators of cholinergic system, antioxidant system and inflammatory system in the brain tissue of mice. Among them, the level of Ach in the brain tissue of mice in the ginseng and natto compound fermentation product group is significantly increased, and the level of AchE is significantly decreased. Therefore, the ginseng and natto compound fermentation product can improve the scopolamine-induced Alzheimer's disease in mice by regulating cholinergic neurotransmitters; the ginseng and natto compound fermentation product can significantly increase the level of SOD and significantly reduce the level of MDA. Therefore, the ginseng and natto compound fermentation product can improve the Alzheimer's disease in mice by reducing oxidative stress damage; the ginseng and natto compound fermentation product can significantly reduce the expression levels of inflammatory factors IL-6 and TNF-α, and can regulate the balance of inflammatory response, and can improve Alzheimer's disease by inhibiting inflammatory response. In summary, the ginseng and natto compound fermentation product has a significant improvement effect on the scopolamine-induced Alzheimer's disease in mice.

[0099] III. Observation of tissue morphology by hematoxylin-eosin (H&E) staining section

[0100] After the mice were sacrificed, the brain tissue was quickly removed on ice and placed in a 15 mL EP tube containing 4% paraformaldehyde for fixation at room temperature, and then HE staining section was performed to observe the tissue morphology. The specific operation is as follows:

[0101] 1. Paraffin section deparaffinization to water: sequentially place the section in environmental protection type deparaffinization liquid I for 20 min, environmental protection type deparaffinization liquid II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% alcohol for 5 min, and tap water.

[0102] Freeze section rewarming fixation: take the freeze section out of the-20℃ refrigerator to room temperature, and fix with tissue fixing liquid for 15 min, then rinse with running water.

[0103] 2. Pretreatment: put the section into high-definition constant dyeing pretreatment liquid for 1 min.

[0104] 3. Hematoxylin staining: the section is dyed in hematoxylin dyeing solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, returned to blue with blue returning solution, and washed with running water.

[0105] 4. Eosin staining: the section is dehydrated in 95% alcohol for 1 min, and dyed in eosin dyeing solution for 15 s.

[0106] 5. Dehydration and mounting: the section is sequentially placed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, xylene II for 2 min, and transparent, and mounted with neutral balsam.

[0107] 6. Microscope examination, image collection and analysis. The results are shown in Figure 18 .

[0108] The black arrows in the figure are neuron shrinkage, the blue arrows are neuron edema, and the yellow arrows are round vacuoles. It can be seen that the number of neurons in the CON group is rich, the morphology is regular, the arrangement is dense and neat, the nucleus is large and round, the chromatin is less, and the nucleolus is obvious. In the MOD group, neuron shrinkage can be seen, the cell volume is reduced, the shape is irregular, the nucleus and cytoplasm are not clear, and the staining is deepened; a small amount of neurons are edematous, the cells are swollen, and the cytoplasm is loose and light staining; there are a small amount of round vacuoles. In the DON group, the number of neurons increases, the structure is basically clear, the arrangement is neat, and the neuron cell shrinkage phenomenon is obviously improved. In the GSL group, a large number of neurons are seen to shrink, the number of cells is reduced, the shape is irregular, and the staining is deepened. In the GSH group, a small amount of neurons are seen to shrink, the structure is loose, and the cell volume is reduced. In the GNL group, a small amount of neurons are seen to shrink, most of the neuron cells are arranged tightly, and the morphology is healthy and complete. In the GNH group, the neuron cell morphology is regular, the nucleus and cytoplasm are relatively clear, the nucleus is large, the nucleolus is obvious, and the neuron shrinkage phenomenon is reduced.

[0109] The results show that after administration of ginseng-soybean complex and ginseng-natto complex fermentation product, the degree of neuron apoptosis in the brain of Alzheimer's disease model mice is improved to a certain extent, and the improvement effect of ginseng-natto complex fermentation product is better than that of ginseng-soybean complex.

[0110] Obviously, the above examples are only examples for clearly illustrating, but not limiting the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments cannot be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Application of a ginseng natto composite ferment in the preparation of an Alzheimer's disease drug, characterized in that, The ginseng natto compound fermentation product comprises the following components: natto kinase, daidzin, glycitinn, genistin, daidzein, genistein, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rc; The natto kinase activity is 6645.3673±109.6755IU / g; the contents of daidzin, glycitinn, genistin, daidzein, genistein, ginsenoside Rg1, ginsenoside Rb1 and ginsenoside Rc are 0.3261±0.0077mg / g, 0.9571±0.0070mg / g, 0.1160±0.0055mg / g, 0.9716±0.0166mg / g, 0.0332±0.0015mg / g, 0.0433±0.0019mg / g, 0.2729±0.0044mg / g and 0.0942±0.0022mg / g respectively; The preparation of the ginseng natto compound fermentation product comprises the following steps: S1, soybeans and ginseng are weighed according to a mass ratio of 6:1; S2, a ginseng extract is prepared, the ginseng extract is concentrated to 1.2 times the mass of the soybeans, the soybeans are added to the ginseng extract for soaking until the soybeans completely absorb the ginseng extract; S3, the soaked soybeans are sterilized as a fermentation substrate; S4, Bacillus subtilis is inoculated, shaken and continuously cultured, and the product after culture is dried, crushed and sieved to obtain the ginseng natto compound fermentation product.

2. Use of the ginseng natto composite ferment according to claim 1 in the preparation of an Alzheimer's disease drug, characterized in that, The preparation of the ginseng extract is specifically as follows: the ginseng is decocted in distilled water for 2 times, 1h each time, and the extract is combined to obtain the ginseng extract.

3. Use of the ginseng natto composite ferment according to claim 1 in the preparation of an Alzheimer's disease drug, characterized in that, The sterilization is performed at 121℃, 0.1MPa for 30min.

4. Use of the ginseng natto composite ferment according to claim 1 in the preparation of an Alzheimer's disease drug, characterized in that, The inoculation amount of Bacillus subtilis is 6.2%.

5. Use of the ginseng natto composite ferment according to claim 1 in the preparation of an Alzheimer's disease drug, characterized in that, The fermentation seed age of Bacillus subtilis is 5h.

6. Use of the ginseng natto composite ferment according to claim 1 in the preparation of an Alzheimer's disease drug, characterized in that, The culture temperature is 34℃, and the culture time is 37.2h.

7. Use of the ginseng natto composite ferment according to claim 1 in the preparation of an Alzheimer's disease drug, characterized in that, The sieving is through a No.3 sieve.

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