Application of wheat germ extract in preparing a composition for improving functions related to memory and cognition

Abstract

The present invention discloses the use of wheat germ extract in the preparation of a composition for improving functions related to memory and cognition. The test results show that the wheat germ extract can enhance the vitality of nerve cells, reduce neuroinflammation, enhance the plasticity of synapses, and reduce brain amyloid plaques, and has the function of improving functions related to memory and cognition. Compared with other chemical drugs, wheat germ extract has the characteristics of non-toxicity, safety, and nutrition. Therefore, it is of certain practical significance to develop wheat germ extract into a product for improving functions related to memory and cognition.

Description

Technical Field

[0001] The present invention relates to a novel application of a wheat germ extract, and in particular to an application of the wheat germ extract in preparing a composition for improving functions related to memory and cognition. Background Art

[0002] Wheat germ accounts for 2.5-3% of wheat grains and is the "source of life" of wheat grains. In addition to high-quality protein, wheat germ also contains fats and dietary fiber, minerals, vitamins and other ingredients that are beneficial to human health. It is a rare natural healthy food resource. Therefore, it is particularly necessary to deeply develop and utilize wheat germ resources.

[0003] At present, the utilization of wheat germ is mainly concentrated in three aspects: first, most of the wheat germ is mixed with bran and used as feed, which causes a great waste of resources; second, it is directly added to food, such as wheat germ baked food, soup, snack food, etc.; third, wheat germ oil is extracted, and the defatted wheat germ is used as feed and sold cheaply. The main reason why wheat germ is not reasonably utilized is that the research and development of wheat germ is still at the level of nutritional source, and the research on its health function is not in-depth enough.

[0004] Wheat germ has been proven to have antioxidant, anti-aging, anti-fatigue and other activities. Currently, there is no literature report on the use of wheat germ extract in the preparation of a composition for improving functions related to memory and cognition. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an application of wheat germ extract in the preparation of a composition for improving functions related to memory and cognition. Studies have found that the wheat germ extract prepared by the present invention can enhance the vitality of nerve cells, reduce neuroinflammation, enhance the plasticity of synapses, and reduce brain amyloid plaques. It has the effect of improving functions related to memory and cognition, and provides a new choice for the preparation of a composition for improving functions related to memory and cognition.

[0006] Preferably, the wheat germ extract contains spermidine and / or spermine.

[0007] Preferably, the spermidine content in the wheat germ extract is greater than 0.18%.

[0008] The improvement of memory and cognition-related functions of the present invention is to improve the degeneration or damage of memory, cognition, and action-related functions caused by Alzheimer's disease, Parkinson's disease, inflammatory neurological diseases, and aging.

[0009] The present invention has found through research that wheat germ extract improves functions related to memory and cognition by enhancing the activity of nerve cells, reducing neuroinflammation, enhancing the plasticity of synapses, and reducing amyloid plaques in the brain.

[0010] The method for preparing the wheat germ extract of the present invention comprises the following steps:

[0011] Step S1, soaking wheat germ powder in a 60-80% ethanol solution containing 0.6-1% NaOH, and filtering and collecting the extracted supernatant as a soaking solution after soaking;

[0012] Step S2, combining the soaking liquid as an extracting liquid, adjusting the pH of the extracting liquid to 5-7, stirring, and standing to obtain a solid-liquid mixture;

[0013] Step S3, centrifuging the solid-liquid mixture to collect a solid phase rich in polyamines;

[0014] Step S4, drying the polyamine-rich solid phase to obtain a polyamine-rich wheat germ extract.

[0015] Preferably, in step S1, the volume ratio of wheat germ powder to ethanol solution is 1:5-10; the soaking temperature is 25°C to 40°C, and the soaking times are 1-3 times.

[0016] The above method uses an alkaline alcohol solution for extraction. On the one hand, the alkaline solution can make the wheat germ raw material tissue swell, and at the same time can avoid the dissolution of a large amount of starch and protein, thereby facilitating the free release of polyamine substances, and playing a more effective role in dissolving and extracting polyamine components. Through the treatment of step S1, the polyamines are released into the soaking liquid; after the acidification of step S2, the polyamines are precipitated from the soaking liquid in the form of salts, and then through solid-liquid separation and drying treatment, a wheat germ extract rich in polyamines can be obtained.

[0017] The present invention also provides another method for preparing a wheat germ extract, comprising the following steps:

[0018] (1) soaking the wheat germ raw material in a dilute acid solution of pH 3-4 containing 0.8-1.2 wt% VC (ascorbic acid);

[0019] (2) percolating the soaked wheat germ raw material with a pH 3-4 dilute acid solution, and collecting the percolation liquid;

[0020] (3) filtering the percolation liquid through an organic membrane and / or an inorganic membrane, and collecting the clear liquid;

[0021] (4) Pass the clear liquid through a 2500-3500 Da1 ultrafiltration membrane and collect the retentate;

[0022] (5) The retentate is boiled, sterilized, and dried to obtain a wheat germ extract containing spermidine.

[0023] Preferably, in step (3), the organic membrane is a suction filter bag, and the inorganic membrane is a ceramic membrane.

[0024] Preferably, in step (1), the amount of the dilute acid solution used is 3-10 times the amount of the raw material used.

[0025] Preferably, in step (5), the drying is spray drying, and the air inlet temperature of the spray dryer is controlled at 185-215°C, and the air outlet temperature is controlled at 90-110°C.

[0026] The method uses an acid solution containing ascorbic acid (VC) to soak the raw material, then uses the acid solution to treat the soaked raw material, removes impurities in the obtained treatment solution and intercepts target components to prepare a wheat germ extract containing polyamines. The preparation process has a high extraction rate for polyamines.

[0027] Compared with the prior art, the present invention has the following excellent effects:

[0028] The present invention provides a new use of wheat germ extract in preparing a composition for improving functions related to memory and cognition. The test results show that the wheat germ extract can enhance the vitality of nerve cells, reduce neuroinflammation, enhance the plasticity of synapses, and reduce brain amyloid plaques, and has the function of improving functions related to memory and cognition. Compared with other chemical drugs, wheat germ extract has the characteristics of non-toxicity, safety, and nutrition. Therefore, it is of certain practical significance to develop wheat germ extract into a product for improving functions related to memory and cognition. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a preparation process diagram of wheat germ extract;

[0030] Figure 2 The effect of wheat germ extract on the survival of 3×Tg-AD neural cells;

[0031] Figure 3 The effect of wheat germ extract on the expression of inflammatory factors in nerve cells;

[0032] Figure 4 The effect of wheat germ extract on 3×Tg-AD neurons was tested for JC-1 mitochondrial membrane potential;

[0033] Figure 5 The effect of wheat germ extract on the expression of synaptic proteins in 3×Tg-AD neurons;.

[0034] Figure 6To evaluate the effect of wheat germ extract on improving memory in zebrafish in a memory impairment model;

[0035] Figure 7 The effect of wheat germ extract on the escape latency of mice in the Morris water maze experiment;

[0036] Figure 8 Western Blot was used to detect the effect of wheat germ extract on the expression of Aβ in amyloid plaques of transgenic mice with dementia model. DETAILED DESCRIPTION

[0037] The present invention is further described below by means of specific implementation methods. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0038] Raw materials: wheat germ (spermidine content 0.0339%, spermine content 0.0093%), commercially available.

[0039] The experimental data in the present invention are expressed as The data are presented as mean ± standard error of mean (SEM), and statistical analysis was performed using GraphPad Prism 7.0. After testing for homoskedasticity, one-way ANOVAs were performed to compare differences among groups, and post hoc tests were performed using the Tukey method.

[0040] Example 1: Preparation method of wheat germ extract

[0041] Step S1: passing the wheat germ powder through a 60-mesh sieve, soaking it in an 80% ethanol solution containing 1% NaOH at 40° C., wherein the volume ratio of the wheat germ powder to the ethanol solution is 1:5, filtering and collecting the extracted supernatant after soaking as a soaking solution, and the soaking times are 1-3 times;

[0042] Step S2: combining the soaking liquids as an extracting liquid, adjusting the pH of the extracting liquid to 5-7, stirring for 10 minutes, and standing for 10 minutes to obtain a solid-liquid mixture;

[0043] Step S3: centrifuging the solid-liquid mixture to collect a solid phase rich in polyamines;

[0044] Step S4: hot-air drying the polyamine-rich solid phase at 40° C. to obtain a wheat germ extract.

[0045] Optimization process of preparation technology:

[0046] 1.1 Effect of NaOH addition on extraction effect

[0047] Table 1: Effect of NaOH addition on extraction performance

[0048]

[0049]

[0050] It can be seen from the above table that when NaOH is added to the ethanol solution, the content of spermidine and spermine in the extract can be significantly increased. When only ethanol is added, the extraction rate of spermidine is about 20% of that of NaOH. However, if only NaOH is added, the soaking solution is too viscous and cannot be extracted and filtered to obtain the extract.

[0051] 1.2 Effect of adding different concentrations of NaOH on the extraction effect

[0052] The concentration of NaOH was adjusted, and the rest was the same as in Example 1. The contents of spermidine and spermine in the extract were detected by HPLC, as shown in Table 2.

[0053] Table 2 Effect of NaOH concentration on extraction effect

[0054]

[0055] It can be seen from the above table that with the increase of sodium hydroxide content in the alkaline alcohol solution, the dissolution rates of spermine and spermidine increase; however, when the sodium hydroxide content exceeds 1%, the dissolution rates of spermidine and spermine no longer increase, and even the dissolution rate of spermidine decreases. This may be due to the excessive alkalinity, which reduces the stability of spermidine and partially decomposes spermidine.

[0056] 1.3 Effect of different ethanol concentrations on extraction effect

[0057] The concentration of ethanol was adjusted, and the rest was the same as in Example 1. The contents of spermidine and spermine in the extract were detected by HPLC, as shown in Table 3.

[0058] Table 3 Effect of ethanol concentration on extraction effect

[0059]

[0060] As can be seen from the above table, the ethanol concentration in the range of 40-80% has little effect on the extraction and dissolution rate of spermidine. However, subsequent tests found that if the water content in the ethanol solution is too high, it will make the subsequent precipitation of spermidine more difficult. Therefore, choosing an ethanol concentration of 80% is a better choice.

[0061] 1.4 Effect of pH value on extraction effect

[0062] Taking extraction group 1 as an example, citric acid was added to the extract to acidify to the target pH value, and stirred for 10 minutes, and then allowed to stand for 10 minutes to obtain a solid-liquid mixture. The solid-liquid mixture was filtered to obtain a separated filtrate and a precipitate. The obtained precipitate was hot-air dried at 40°C to obtain a polyamine-rich wheat germ extract. The target pH values ​​of each embodiment are recorded in Table 4.

[0063] The contents of spermine and spermidine in the filtrate obtained by separation of the solid-liquid mixture and the precipitate after drying were detected by the method specified in GB5009.208-2016, and the yield of spermine and spermidine was calculated based on the weight of spermine and spermidine in the precipitate. The calculation formula was yield = total amount of target substance after drying / total amount of target substance before precipitation. The test results are recorded in Table 4.

[0064] Table 4 Effect of pH value on extraction effect

[0065]

[0066] As can be seen from the above table, the lower the pH value, the higher the precipitation rate of spermine and spermidine, that is, the higher the yield of the two. However, the lower the pH value, the more impurities are precipitated with spermine and spermidine, so the total amount of precipitate obtained is more, resulting in lower purity of spermine and spermidine. In summary, when acidified to a pH value between 4 and 7, spermine and spermidine can be well precipitated. The precipitation collection of extraction 2 and extraction group 3 under different acidified pH conditions was further tested, which had the same trend as the results shown in Table 4, and the higher the concentration of ethanol in the concentration range of 40% to 80%, the more conducive to the precipitation of spermine and spermidine. Therefore, considering the precipitation of impurities and the precipitation of the target product, selecting a pH value between 5-7 is more conducive to improving the purity of the product.

[0067] Example 2: Preparation method of wheat germ extract

[0068] See the process flow chart for Figure 1 :

[0069] (1) soaking the wheat germ raw material in a pH 3 dilute hydrochloric acid solution containing 1 wt % ascorbic acid (VC) in an amount 8 times the amount of the raw material for 1 hour;

[0070] (2) percolating the soaked wheat germ raw material with a pH 3 dilute hydrochloric acid solution in an amount 8 times the amount of the raw material, and collecting the percolation liquid;

[0071] (3) The percolate was first filtered through a 1 μm suction filter bag, and then the filtrate was transferred to a 0.1 μm ceramic membrane for filtration. During the filtration, water was replenished twice, each time with 1 times the volume of the percolate, and the clear liquid was collected;

[0072] (4) The clear liquid was passed through a 3500 Da1 ultrafiltration membrane and the retentate was collected;

[0073] (5) The retentate is boiled for sterilization and spray dried, the inlet air temperature of the spray dryer is controlled at 185-215° C., and the outlet air temperature is controlled at 90-110° C. to obtain a wheat germ extract.

[0074] Optimization process of preparation technology:

[0075] 2.1 Effect of VC addition on extraction effect

[0076] Table 5: Effect of VC addition on extraction effect

[0077]

[0078] HPLC detection adopts the method specified in GB5009.208-2016. As shown in Table 5, the addition of VC helps the release and stability of spermidine during the extraction process of spermidine. Compared with the case without adding VC, the relative yield is increased from 55.1% to 94.6%.

[0079] 2.2 Effect of VC addition on extraction effect

[0080] The amount of VC added was adjusted, and the other steps were the same as in Example 2. The content of spermidine in the extract was detected by HPLC, as shown in Table 6.

[0081] Table 6 Effect of different VC addition amounts on spermidine extraction

[0082]

[0083]

[0084] It can be seen from Table 6 that when the VC addition amount is below 0.8%, the improvement in spermidine extraction rate is not significant, and when it is above 1.2%, the effect is no longer improved, so 0.8%-1.2% is a more appropriate range.

[0085] 2.3 Effect of pH value on the extraction of spermidine from plants

[0086] The pH value of the feed solution was adjusted, and the other steps were the same as in Example 2. The content and yield of spermidine in the extract were detected by HPLC, as shown in Table 7.

[0087] Table 7 Effect of different pH values ​​on spermidine extraction

[0088] pH value of liquid Spermidine content in dried product % Relative yield% pH=1 0.039 79.13 pH = 2 0.052 82.41 pH = 3 0.115 96.22 pH = 4 0.113 94.00 pH = 5 0.101 83.32 pH = 6 0.082 59.83 pH = 7 0.069 19.02 pH=8 0.033 8.43 pH=9 0.024 5.67

[0089] Wherein, relative yield = (total amount of target component in final product / total amount of target component in raw material) × 100%.

[0090] As can be seen from Table 7, the pH value changes from high to low during the extraction process, and the corresponding relative yield and content of spermidine first increase and then decrease. pH values ​​of 1-6 are suitable for the extraction of spermidine. Under the conditions of pH values ​​of 3 and 4, the content of spermidine in the solid and the yield of spermidine reach the highest values. It is preferred to use a solvent with pH = 3-4 for extraction.

[0091] 2.4 Selection of ultrafiltration membrane molecular weight cut-off

[0092] The target component in the clear solution obtained in step (3) was retained using ultrafiltration membranes of different molecular weights, and the content of spermidine in the extract and the yield of spermidine were detected by HPLC. See Table 8 for details.

[0093] Table 8 Effect of ultrafiltration membranes with different molecular weight cutoffs on spermidine extraction

[0094] Sample name Spermidine content (dry content, %) Relative yield of spermidine (based on dry content, %) 100000Dal / (Not detected) 0 50000Dal / (Not detected) 0 10000Dal 0.06 13.4 5000Dal 0.118 61 3500Dal 0.121 91.1 2500Dal 0.112 95.1 1000Dal 0.073 97.4 500Dal 0.064 98.7

[0095] As shown in Table 8 above:

[0096] (1) In the process of separating spermidine, the 100000Dal, 50000Dal, and 10000Dal ultrafiltration membranes have a large pore size, so all solids and spermidine pass through, resulting in basically no purification effect.

[0097] (2) During the separation process of the 5000 Da1 ultrafiltration membrane, the spermidine content in the membrane concentrate can be increased, but spermidine permeates in the clear liquid, and the permeation ratio is above 30%, resulting in a low yield of the target product.

[0098] (3) During the separation of spermidine by 3500Dal and 2500Dal ultrafiltration membranes, very little spermidine permeates (less than 10%), and the content of the membrane concentrate is greatly increased, which has an enrichment effect. At the same time, the retentate (concentrate) is the target product, and the yield of spermidine is high, so the nanofiltration concentration step can be omitted and drying can be performed directly.

[0099] (4) In the process of separating spermidine using 1000Dal and 500Dal ultrafiltration membranes, although spermidine did not pass through the membrane, the amount of solid impurities that passed through was also relatively small, so no obvious purification and enrichment effect was achieved.

[0100] In summary, the best choice is an ultrafiltration membrane between 2500-3500 Da1, and the spermidine yield can reach more than 90%.

[0101] Example 3: Efficacy test of wheat germ extract

[0102] After testing, the amount of spermidine contained in the wheat germ extracts prepared in Examples 1 and 2 of the present invention is greater than 0.18%, which is 6-10 times higher than the spermidine content in the raw material, and also contains a large amount of wheat protein, soluble fiber, polysaccharides, trace elements and other substances. In this example, the wheat germ extract prepared in Example 1 with a spermidine content of 0.19% was selected for testing.

[0103] 3.1 Cell viability test

[0104] Culture cells: Dispense 3×Tg-AD neural cells into 96-well plates, add culture medium, and allow them to continue to grow and divide. Inoculate the cell suspension (100 μL / well) into the 96-well plate, and place the culture plate in an incubator for pre-culture for 4-8 hours. Aspirate the culture medium from each well, wash once with PBS, and replace with serum-free culture medium.

[0105] Processed samples: The cells were divided into a control group (Ctr group), a DMSO group, groups with different concentrations of wheat germ extract (15.625ug / ml, 31.25ug / ml, 62.5ug / ml, 125ug / ml, 250ug / ml), and a spermidine group (0.119ug / ml, calculated based on the spermidine content in 62.5ug / ml wheat germ extract) and added into the culture medium, with 6 replicate wells set up for each group.

[0106] Add CCK8 reagent (Biyuntian, C0038): add 10 μL of CCK-8 solution to each well.

[0107] Incubation: Place the culture dish in a constant temperature incubator and incubate for 30 minutes to allow the CCK8 reagent to react with the cells.

[0108] Measurement: After incubation, use an enzyme-labeled instrument to determine the absorbance at 450nm, and measure the change in absorbance in the culture dish. If the absorbance of each group does not reach 1, put it in the incubator and incubate it again for 30 minutes. Repeat the above steps until the absorbance of the duplicate wells in a group reaches 1. Stop the measurement and calculate the cell survival rate.

[0109] Statistical analysis:

[0110] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%;

[0111] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and sample solution);

[0112] Ac: absorbance of control well (containing cells, culture medium, CCK-8 solution, but not sample);

[0113] Ab: absorbance of blank well (containing culture medium and CCK-8 solution, but not cells or samples).

[0114] See also Figure 2 The results show that compared with the control group, the wheat germ extract at a concentration of 31.25ug / ml can have a significant effect on cell viability, and the effect on cell survival rate is greater with the increase of concentration. The use of spermidine alone can also promote cell viability, but compared with the wheat germ extract containing the same concentration of spermidine, there is also a significant difference between the two, indicating that the substances in the wheat germ extract of the present invention, in addition to spermidine, have other substances that work together to further enhance its efficacy.

[0115] 3.2 Detection of the effect of wheat germ extract on neuroinflammatory factors

[0116] 3.2.1 Test reagents

[0117] Reagents: RNA extraction kit (catalog number: RE-03011), reverse transcription reagent (catalog number: AG11706), qRT-PCR kit (catalog number: AG11711); human microglial cells HMC3 were from Shanghai Meiyan Biotechnology Co., Ltd. (catalog number CC-Y1754).

[0118] 3.2.2 Experimental methods

[0119] Cell culture and treatment: The human microglial cells HMC3 to be tested were seeded in a six-well plate at a cell density of 5×10 5 Pieces / cm 3 , divided into a control group, a DMSO solvent control group, an LPS group, a spermidine group, a wheat germ extract lowest concentration drug group (62.5ug / ml) obtained in Example 1, an intermediate concentration drug group (125ug / ml), and a highest concentration drug group (250ug / ml), with six replicates in each group. After 4-6 hours, the cells were replaced with serum-free medium for 24 hours, and different concentrations of the test samples were added according to different groups for 24 hours. 1μg / ml LPS was added to the LPS group and the drug group for 24 hours. When the cells were collected, they were gently washed with PBS 2-3 times to remove the interference of the culture medium on subsequent experiments.

[0120] RNA extraction: Follow the instructions of the RNA extraction kit.

[0121] cDNA synthesis: In an eight-tube concatenation, set up six replicate wells for each tube of RNA. The specific steps are as follows: 1. Removal of genomic DNA: Prepare the reaction solution according to the contents of the table below to carry out the genomic DNA removal reaction: gDNA Clean Reagent (1μl) 5X gDNAClean Buffer (2μl) Total RNA (measured RNA concentration / 1000) RNase free water (up to 10μ) Cover the lid, centrifuge briefly to mix (be careful to eliminate bubbles), put into the PCR machine, reaction conditions: 42℃2min, 4℃forever.

[0122] Reverse transcription reaction: Step (1) Reaction solution (10 μl) Evo M-MLV RTase Enzyme Mix (1 μl) RT Primer Mix (1 μl) 5X RTase Reaction Buffer Mix I (4 μl) RNase free water (4 μl).

[0123] qRT-PCR reaction: Design and synthesize primers, then use cDNA as a template and specific primers of cellular inflammatory factors (IL-1β, IL-10) for qRT-PCR amplification reaction. The specific operation is as follows: dilute each tube of cDNA with 90μl ultrapure water, reaction system (20μl) 2X Pro Taq HS Probe Premix*1X (10μl) Template (2ul) Primer F (10μM) (0.4μl) Primer R (10μM) (0.4μl) RNase free water (Up to 20μl) qPCR reaction conditions (two-step PCR reaction procedure) Step 1 95℃ (30sec cycle number: 1) Step 2 95℃ (5sec cycle number: 45) 60℃ (30sec cycle number: 45), after the reaction is completed, confirm the amplification curve and melting curve, and analyze the standard curve.

[0124] 3.2.3 Statistical analysis

[0125] The fluorescence signal was detected using a qRT-PCR instrument, and the Ct value was derived. β-actin was used as the internal reference gene, and the 2-△△CT method was used to calculate the expression level of cellular inflammatory factors.

[0126] The experimental results are as follows Figure 3The results showed that compared with the LPS-treated model group, wheat germ extract could increase the expression of the anti-inflammatory factor IL-10 and inhibit the expression of the inflammatory factor IL-1β, especially at low and medium concentrations. At the same time, wheat germ extract had an inhibitory effect on neuronal inflammation and the effect was significantly better than spermidine, thereby protecting neurons.

[0127] 3.3 Effect of wheat germ extract on mitochondrial membrane potential of JC-1 neurons:

[0128] 3.3.1 Experimental reagents

[0129] JC-1 mitochondrial membrane potential detection kit (Biyuntian, C2006).

[0130] 3.3.2 Experimental methods

[0131] 1. Cell Culture and:

[0132] The 24-well plate was pre-coated with poly-lysine, and 3×Tg-AD neural cells were added to each well for cell climbing;

[0133] 2. Preparation of JC-1 staining working solution: 0.5 ml of JC-1 staining working solution is required for every 500,000-1 million cells in the cell suspension. Take an appropriate amount of JC-1 (200X) and dilute JC-1 at a ratio of 8 ml of ultrapure water per 50 μl of JC-1 (200X). Vortex vigorously to fully dissolve and mix JC-1. Then add 2 ml of JC-1 staining buffer (5X) and mix to obtain JC-1 staining working solution.

[0134] 3. For cells after climbing, remove the culture medium from one well of the 24-well plate. If necessary, wash the cells once with PBS or other appropriate solutions according to the specific experiment. Add 0.4 ml of cell culture medium to different groups and culture for 24 hours. The cell culture medium contains serum, phenol red, wheat germ extract or spermidine.

[0135] 4. Add 0.4 ml of JC-1 staining working solution and mix thoroughly. Incubate in a cell culture incubator at 37°C for 20 minutes.

[0136] 5. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4 ml of distilled water to every 1 ml of JC-1 staining buffer (5X) and place it in an ice bath.

[0137] After incubation at 6.37°C, remove the supernatant and wash twice with JC-1 staining buffer (1X).

[0138] 7. Add 0.8 ml of cell culture medium, which may contain serum and phenol red.

[0139] 8. Observe under fluorescence microscope or laser confocal microscope.

[0140] 3.3.3 Statistical analysis

[0141] When detecting JC-1 monomer, the excitation light can be set to 488nm and the emission light can be set to 530nm; when detecting JC-1 polymer, the excitation light can be set to 525nm and the emission light can be set to 590nm.

[0142] The experimental results are as follows Figure 4 As shown, the results showed that compared with the 3×Tg-AD neural cell group without drug treatment ( Figure 4 Compared with the AD group), the addition of wheat germ extract (62.5ug / ml) Figure 4 After treatment with Tg-AD+wheat germ extract group), the red / green fluorescence intensity ratio in the chromosomes of nerve cells increased, the cell mitochondria were healthier, the mitochondrial function was improved, and the apoptosis of 3×Tg-AD nerve cells was inhibited.

[0143] 3.4 Effects of wheat germ extract on synaptic protein expression in primary neurons:

[0144] Primary neural cells (wt) were inoculated into six-well plates, and the drug (3×Tg-AD, wheat germ extract of Example 1, spermidine) was administered when the second half-fluid was changed. After 24 hours, the cells were lysed again, and 200 μL of lysate was added to each well of the six-well plate. After adding the lysate, the cells were scraped off with a cell scraper and transferred to a 1.5 mL EP tube for protein extraction and protein quantification. The extracted protein was used to detect the expression of neuronal synaptic proteins by western blot.

[0145] The results are as follows Figure 5 As shown, compared with the 3×Tg-AD treated nerve cell group, wheat germ extract can increase the expression level of synaptic proteins and is significantly better than spermidine, suggesting that wheat germ extract may improve memory by improving synaptic function.

[0146] Example 4: Animal experiment on wheat germ extract in improving memory and cognitive related functions

[0147] 4.1 Zebrafish testing

[0148] Zebrafish were kept in water at 28°C and bred by Huante Fish Farming Center. The experimental animal use license number is SYXK (Zhejiang) 2022-0004. The breeding and management are in compliance with the requirements of the international AAALAC certification (certification number: 001458). Wild-type AB strain zebrafish were bred in natural pairs. Zebrafish aged 4 days post-fertilization (4dpf) were used for the determination of the maximum detection concentration (MTC) of the sample-assisted memory improvement effect and its efficacy evaluation.

[0149] 4dpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, with 15 in each well (experimental group). Water-soluble samples (wheat germ extract in Example 1) were given respectively, and a normal control group and a model control group were set up at the same time, with a capacity of 3mL per well. Except for the normal control group, the remaining experimental groups were given aluminum chloride hexahydrate in water to establish a zebrafish memory impairment model. After 24 hours of treatment at 28°C, (1) the MTC of the sample on the model zebrafish was determined; (2) a group with 3 or fewer deaths and a similar state to the model control group was selected, and 8 zebrafish were randomly selected from each experimental group. The average movement speed per minute of the zebrafish under light and dark stimulation was measured using a behavior analyzer, and the statistical analysis results of this indicator were used to evaluate the sample's ability to assist in improving memory. The statistical analysis results were expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0150] Table 9 Drug concentration exploration results

[0151]

[0152] It can be seen from the results in Table 9 that the wheat germ extract prepared by the present invention is safe and effective without any toxicity.

[0153] For further information, see Figure 6 At three concentrations of 250 μg / m, 500 μg / m, and 1000 μg / m, wheat germ extract has a good effect on the recovery of zebrafish movement, especially when 1000 ug / ml of wheat germ extract is used, the movement speed is close to or even exceeds the normal control, and there is a significant difference compared with the model control. In addition, the present invention also tested the wheat germ extract prepared in Example 2, and its effect was similar.

[0154] 4.2 Transgenic mouse testing

[0155] Triple transgenic AD mice (3×Tg-AD) were purchased from Jax Laboratories in the United States, and the corresponding wild-type (B6129SF2 / J-WT) male mice were purchased from Nanjing Institute of Biomedicine, Nanjing University. All mice were raised under standard clean laboratory conditions, with a 12-h day / dark cycle in the animal room, a room temperature of 22±2°C, and free access to water and feed.

[0156] The test substance (wheat germ extract 1 obtained in Example 1, wheat germ extract 2 obtained in Example 2) and the placebo were mixed into the SPF-grade feed of mice, and the mice were administered with free access. The wild type was used as the control group, and the triple transgenic AD (3×Tg-AD) mice were used as the test subjects of the model group and the drug administration group. After the mice in each group were fed with a normal diet until 4 months of age, they were continued to be fed with the corresponding feed containing the test substance or placebo until 8 months of age.

[0157] 4.2.1 Behavioral testing:

[0158] Morris water maze experiment: divided into two parts: positioning navigation and space exploration. The positioning navigation lasts for 6 days and the space exploration for 1 day. The first day of positioning navigation is the training period: before the experiment, the mouse is placed on the platform to adapt for 10 seconds, and then the mouse is placed in the pool facing the wall from the corresponding position of different quadrants. The recording is terminated 2 seconds after the mouse climbs onto the platform. The longest recording time is 60 seconds. If the mouse cannot climb onto the platform within 60 seconds, it is manually guided to climb onto the platform to adapt for 10 seconds. Finally, the mouse is wiped dry and placed in the mouse cage. In this way, the mouse is placed in the pool and enters the water from four quadrants, each time with an interval of 1 hour, for positioning navigation training. The second to fifth days of positioning navigation are the detection period: enter the water in a certain quadrant order every day, four rounds a day, and record relevant data. Spatial exploration is 24 and 72 hours of memory: the timing starts from the end of positioning navigation, the platform is removed, and the mouse stays in the quadrant where the original platform is located and the number of times it crosses the original platform is recorded.

[0159] Morris water maze experimental data parameters and collation: positioning navigation escape latency, movement trajectory, average speed; spatial exploration 24, 72 hours memory stay time in the quadrant where the original platform is located and the number of times the original platform is crossed. The data were obtained through the water maze tracking system, and the Graphpad Prism 7 software was used for data collation and graphics analysis. The escape latency was analyzed by one-way repeated variance analysis, and the t-test was used to analyze the differences between the groups. p < 0.05 was statistically significant. The results are as follows Figure 7 As shown, the escape latency of mice in the model group was significantly increased compared with that in the control group, while the escape latency of mice in the wheat germ extract group was significantly decreased compared with that in the model group (**p<0.01, ***p<0.001), and the effect was better than that of using spermidine.

[0160] 4.2.2 Western blot detection

[0161] Mice were killed after the behavioral experiment. The mouse brain was dissected and the hippocampal tissue was isolated from the right hemisphere. The tissue was then homogenized in RIPA at a ratio of 1:9, a protease inhibitor cocktail and a phosphatase inhibitor were added, and centrifuged at 12,000×g for 10 min at 4°C. The supernatant was collected in a 1.5mL tube, and the protein concentration of each sample was determined using a BCA kit. All protein samples were quantified to the same concentration (3μg / μl). Then SDS-PAGE test was performed and photos were taken. The gray value of the pictures was analyzed using Image J optical density analysis software, and a t-test between groups was performed. p<0.05 was statistically significant.

[0162] like Figure 8 As shown, the results of pathological analysis showed that the expression of amyloid plaque Aβ in the wheat germ extract group mice was significantly reduced compared with that in the model group mice (*p<0.05), and the inhibitory effect was better than that using spermidine.

[0163] The above results show that wheat germ extract can improve the memory ability of transgenic mice.

Claims

1. Use of wheat germ extract in preparing a composition for improving functions related to memory and cognition, characterized in that: The preparation method of the wheat germ extract comprises the following steps: Step S1, soaking the wheat germ powder in a 60-80% ethanol solution containing 0.6-1% NaOH, and filtering and collecting the extracted supernatant as a soaking solution after soaking; Step S2, combining the soaking liquid as an extracting liquid, adjusting the pH of the extracting liquid to 5-7, stirring, and standing to obtain a solid-liquid mixture; Step S3, centrifuging the solid-liquid mixture to collect a solid phase rich in polyamines; Step S4, drying the polyamine-rich solid phase to obtain a polyamine-rich wheat germ extract.

2. The use according to claim 1, characterized in that: The spermidine content in the wheat germ extract is greater than 0.18%.

3. The use according to claim 1, characterized in that: The improvement of functions related to memory and cognition is to improve the degeneration or damage of memory, cognition and action-related functions caused by Alzheimer's disease, Parkinson's disease, inflammatory neurological diseases and aging.

4. The use according to claim 1, characterized in that: The improvement of memory and cognition-related functions is achieved by increasing the activity of nerve cells, reducing neuroinflammation, enhancing the plasticity of synapses, and reducing amyloid plaques in the brain.

5. The use according to claim 1, characterized in that: In step S1, the volume ratio of wheat germ powder to ethanol solution is 1:5-10; the soaking temperature is 25° C.-40° C., and the soaking times are 1-3 times.

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