A composition for improving cognitive impairment and its preparation method

Abstract

This invention relates to the field of pharmaceutical technology, and discloses a composition for improving cognitive impairment and its preparation method. The preparation method of the composition for improving cognitive impairment includes the following steps: mixing oats, sea buckthorn, okra, green tea, and purple sweet potato, followed by crushing, freeze-drying, treatment with a dielectric barrier discharge plasma device, sonication, centrifugation, and filtration to obtain a mixed plant extract; using liquid chromatography to perform gradient elution of the mixed plant extract to separate and purify it to obtain a mixed plant extract. Mixing the mixed plant extract with soybean extract yields the composition for improving cognitive impairment. This invention utilizes multiple plants with natural antioxidant and anti-inflammatory substances in combination with soybean, enhancing the effect of soybean in improving cognitive impairment. The dielectric barrier discharge plasma device treats the plant materials, which can destroy the cell walls of the plants, thereby improving extraction efficiency.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a composition for improving cognitive impairment and a method for preparing the same. Background Technology

[0002] Age-related cognitive impairment primarily manifests as memory loss, decreased learning ability, impaired executive function, and slowed information processing speed, commonly seen in Alzheimer's disease (AD) and vascular dementia (VD). Its pathological features include neuronal loss, synaptic dysfunction, β-amyloid deposition, and neurofibrillary tangles, accompanied by oxidative stress, mitochondrial dysfunction, and exacerbated neuroinflammation. These impairments not only reduce patients' quality of life but also increase the burden of care on families and social healthcare costs; in severe cases, it can lead to complete loss of independent living ability. With the increasing global aging population, cognitive impairment has become a significant challenge in the field of public health.

[0003] Strategies for preventing and treating age-related cognitive impairment encompass lifestyle interventions (such as the Mediterranean diet, regular exercise, and cognitive training), pharmacological interventions (such as cholinesterase inhibitors and NMDA receptor antagonists), and the development of natural medicines. Natural medicines have attracted significant attention due to their multi-target nature and low toxicity; for example, curcumin protects neurons through anti-inflammatory and antioxidant effects, ginkgo biloba extract improves cerebral blood flow and neuroplasticity, and ginsenosides regulate neurotransmitter metabolism. These components offer new directions for preventing and treating cognitive decline.

[0004] Soybeans are rich in isoflavones (such as daidzein and genistein), lecithin, and polyphenols, possessing antioxidant, anti-inflammatory, and estrogen-like effects. Studies have shown that soy isoflavones can alleviate oxidative stress by activating the Nrf2 pathway and relieve neuroinflammation by inhibiting NF-κB signaling. Clinical studies also suggest a positive correlation between soybean intake and cognitive function in the elderly, making soybeans a safe and accessible dietary supplement. However, the effect of using soybean extracts alone on improving age-related cognitive impairment is limited. Furthermore, soybeans contain a wide variety of bioactive substances, and how to extract, enrich, and improve the efficacy of these compounds in improving age-related cognitive impairment is a problem that urgently needs to be solved by those skilled in the art. In addition, soybean cell walls contain cellulose (35%), hemicellulose (15%), and lignin (2%), and their dense structure makes cell wall disruption difficult during extraction. Simple methods such as crushing and sonication have low extraction efficiency for bioactive substances within the cells. On the other hand, traditional methods require the use of large amounts of organic solvents during extraction, which may leave residues and harm the user's health. Therefore, a safe and efficient method for extracting bioactive substances from soybeans is urgently needed. Summary of the Invention

[0005] In view of this, the present invention proposes a composition for improving cognitive impairment and a method for preparing the same, aiming to solve the problems of limited efficacy of soybean extract in improving age-related cognitive impairment and the difficulty in extracting relevant active substances from soybeans in the current technology.

[0006] This invention provides a method for preparing a composition for improving cognitive impairment, comprising the following steps: 1) Mix oats, sea buckthorn, okra, green tea and purple sweet potato, crush and freeze dry to obtain precursor powder; 2) The precursor powder was mixed with sodium hydroxide solution and then processed using a dielectric barrier discharge plasma device; 3) Mix the processed precursor powder from step 2) with water and adjust the pH to 6.5-7. Then, perform ultrasonication, centrifugation, and filtration to obtain a mixed plant extract. 4) The mixed plant extracts described in step 3) were subjected to gradient elution by liquid chromatography to separate and purify the mixed plant extracts. 5) Soybeans are subjected to cyclic freeze-thaw treatment and microwave extraction to obtain soybean extract A, a gaseous condensate, and microwave-treated soybeans; the microwave-treated soybeans are crushed and mixed with water for microwave extraction again; after microwave extraction, the mixture is filtered and the filtrate is freeze-dried to obtain soybean extract B; soybean extract A and soybean extract B are mixed to obtain soybean extract. 6) Mix the mixed plant extracts obtained in step 4) with the soybean extract obtained in step 5) to obtain a composition for improving cognitive impairment.

[0007] Preferably, the mass ratio of oats, sea buckthorn, okra, green tea and purple sweet potato in step 1) is 2~4:1~2:1~2:2~5:1~3; The precursor powder has a particle size of 40-80 mesh.

[0008] Preferably, the concentration of the sodium hydroxide solution in step 2) is 0.2~1 mol / L; The water content of the system after mixing the precursor powder with the sodium hydroxide solution is 10-20%.

[0009] Preferably, the parameters of the dielectric barrier discharge plasma device in step 2) are set as follows: The discharge voltage is 12~22kV; The discharge frequency is 6~9kHz; The processing time is 0.5~5 minutes; The discharge gap is 1~3mm; The processing temperature is 5~15℃; The treatment atmosphere is a mixture of ozone and inert gas, wherein the volume ratio of ozone to inert gas is 1~2:8~10, and the gas flow rate is 3~8L / min.

[0010] Preferably, the mixing ratio of the processed precursor powder to water in step 3) is 1g: 3~5mL; The ultrasound frequency is 20~50kHz, the duration is 10~20min, and the temperature is 0~10℃.

[0011] Preferably, the parameters for the liquid chromatography in step 4) are set as follows: The length of the chromatographic column is 50~100cm, and the inner diameter is 2.5~5cm; The chromatographic column packing material is cross-linked dextran with a particle size of 30~50μm; The eluent is a mixture of ethanol and water; The flow rate of the eluent is 1-3 mL / min.

[0012] Preferably, the gradient elution procedure in step 4) is as follows: Ethanol:water = 0:100, 10~15 min; Ethanol:water = 10:90, 5~10 min; Ethanol:water = 25:75, 5~10 min; Ethanol:water = 50:50, 8~12 min; Ethanol:water = 70:30, 5~10 min; Ethanol:water = 90:10, 3~8 min; Ethanol:water = 100:0 10~20min.

[0013] Preferably, the mixed plant extract is the effluent from a gradient elution process where the ratio of ethanol to water is between 25:75 and 90:10.

[0014] Preferably, the mass ratio of the mixed plant extract to the soybean extract is 1:1 to 3.

[0015] The present invention also provides a composition for improving cognitive impairment prepared by the above preparation method.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The composition for improving cognitive impairment described in this invention is a natural plant extract with high safety. It can be well applied to drugs and foods for the prevention and treatment of age-related cognitive impairment, providing a new, safe and effective natural medicine for the prevention and treatment of age-related cognitive impairment.

[0017] This invention utilizes a variety of plants with natural antioxidant and anti-inflammatory substances, such as oats, sea buckthorn, okra, green tea, and purple sweet potato, in combination with soybeans to effectively enhance the effect of soybeans in improving cognitive impairment.

[0018] This invention first uses freeze-drying to process natural plants, which not only preserves the activity of active substances but also allows ice crystals generated during the freezing process to pierce cell membranes and cell walls, thereby improving the extraction efficiency of active substances. Then, a dielectric barrier discharge plasma device is used to treat the precursor powder containing a certain amount of moisture, which causes the dissociation of macromolecules within the plant cell walls, thus improving the extraction efficiency of subsequent ultrasonic extraction. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention provides a method for preparing a composition for improving cognitive impairment, comprising the following steps: 1) Mix oats, sea buckthorn, okra, green tea and purple sweet potato, crush and freeze dry to obtain precursor powder; 2) The precursor powder was mixed with sodium hydroxide solution and then processed using a dielectric barrier discharge plasma device; 3) Mix the processed precursor powder from step 2) with water and adjust the pH to 6.5-7. Then, perform ultrasonication, centrifugation, and filtration to obtain a mixed plant extract. 4) The mixed plant extracts in step 3) were subjected to gradient elution by liquid chromatography to separate and purify the mixed plant extracts. 5) Soybeans are subjected to cyclic freeze-thaw treatment and microwave extraction to obtain soybean extract A, a gaseous condensate, and microwave-treated soybeans; the microwave-treated soybeans are crushed and mixed with water for microwave extraction again; after microwave extraction, the mixture is filtered and the filtrate is freeze-dried to obtain soybean extract B; soybean extract A and soybean extract B are mixed to obtain soybean extract. 6) Mix the mixed plant extracts obtained in step 4) with the soybean extract obtained in step 5) to obtain a composition for improving cognitive impairment.

[0025] In this invention, the mass ratio of oats, sea buckthorn, okra, green tea, and purple sweet potato in step 1) is 2~4:1~2:1~2:2~5:1~3, preferably 2.5~3.5:1.2~1.8:1.2~1.8:2.5~3.5:1.5~2.5, more preferably 2.8~3.2:1.4~1.6:1.4~1.6:2.8~3.3:1.8~2.2, and even more preferably 3:1.5:1.5:3:2. Oats are rich in β-glucan, sea buckthorn contains a large amount of vitamins and flavonoids, okra is rich in polysaccharides, green tea contains tea polyphenols, and purple sweet potato contains anthocyanins. These components all have antioxidant and anti-inflammatory properties, and their synergistic effect can improve cognitive impairment from multiple targets, significantly enhancing the effect of a single component (such as soybean).

[0026] In this invention, rapidly freezing crushed oats, sea buckthorn, okra, green tea, and purple sweet potato causes the water within the plant tissues to form ice crystals. These ice crystals physically pierce the cell walls and cell membranes of the plants. This process not only maximizes the preservation of heat-sensitive active substances (such as vitamins and polyphenols) in the plants but also creates favorable conditions for subsequent extraction steps, greatly improving the release efficiency and extraction rate of active ingredients.

[0027] In this invention, the particle size of the precursor powder is 40-80 mesh, preferably 50-70 mesh, more preferably 55-65 mesh, and even more preferably 58-62 mesh.

[0028] In this invention, the concentration of the sodium hydroxide solution in step 2) is 0.2~1 mol / L, preferably 0.3~0.8 mol / L, more preferably 0.4~0.6 mol / L, and even more preferably 0.5 mol / L.

[0029] The water content of the system after mixing the precursor powder with sodium hydroxide solution is 10-20%, preferably 12-18%, more preferably 14-16%, and even more preferably 15%. This water content will affect the effect of subsequent dielectric barrier discharge plasma treatment. Excessive water content will inhibit the discharge efficiency. Appropriate water content will generate hydroxyl radicals during discharge treatment, which will destroy the structure of soybean cell walls and thus improve the extraction rate of active substances in cells.

[0030] In this invention, the parameter settings of the dielectric barrier discharge plasma device in step 2) are as follows: The discharge voltage is 12~22kV, preferably 14~20kV, more preferably 16~18kV, and even more preferably 17kV; The discharge frequency is 6~9kHz, preferably 6.5~8.5kHz, more preferably 7~8kHz, and even more preferably 7.5kHz; The processing time is 0.5~5 min, preferably 1~4.5 min, more preferably 1.5~4 min, and even more preferably 2~3.5 min; The discharge gap is 1~3mm, preferably 1.2~2.8mm, more preferably 1.4~2.6mm, and even more preferably 1.5~2.5mm; The processing temperature is 5~15℃, preferably 7~13℃, more preferably 8~11℃, and even more preferably 10℃; The treatment atmosphere is a mixture of ozone and inert gas, with a volume ratio of ozone to inert gas of 1~2:8~10, preferably 1.2~1.8:8.3~9.8, more preferably 1.4~1.6:8.5~9.5, and more preferably 1.5:8.8~9.2; the gas flow rate is 3~8 L / min, preferably 4~7 L / min, more preferably 5~6 L / min, and more preferably 5 L / min.

[0031] In dielectric barrier discharge plasma treatment, ozone, as the core active substance, requires strict control of its introduction rate. Excessive introduction will damage the plant cell wall and the active components inside the plant, while insufficient introduction will lead to incomplete decomposition of the plant cell wall.

[0032] Dielectric barrier discharge plasma generates a large amount of reactive oxygen species (such as hydroxyl radicals ·OH and ozone O3) and high-energy electrons during the discharge process. These reactive substances can penetrate plant cell walls, breaking the chemical bonds of macromolecules such as cellulose and hemicellulose, decomposing them into smaller molecules, thereby disrupting the dense structure of the cell wall and making it easier for the active ingredients within the cell to be extracted by subsequent solvents. Compared with traditional chemical or physical methods of cell wall disruption, plasma treatment has the advantages of high efficiency, speed, and no need to add large amounts of chemical reagents, avoiding the risk of chemical reagent residues and conforming to the development trend of green chemistry.

[0033] In this invention, the mixing ratio of the processed precursor powder and water in step 3) is 1g:3~5mL, preferably 1g:3.3~4.7mL, more preferably 1g:3.5~4.5mL, and even more preferably 1g:3.8~4.2mL.

[0034] In this invention, the frequency of ultrasound is 20-50 kHz, preferably 25-45 kHz, more preferably 30-40 kHz, and even more preferably 33-35 kHz; the duration is 10-20 min, preferably 12-18 min, more preferably 14-16 min, and even more preferably 15 min; the temperature is 0-10℃, preferably 0-8℃, more preferably 2-6℃, and even more preferably 4-5℃. When ultrasound propagates in a liquid, it generates a large number of tiny bubbles. These bubbles rapidly expand and burst instantly under sound pressure, producing a localized high temperature and high pressure "cavitation effect." This effect can further break down plant cells, accelerate the penetration of solvent (water) into the cells, and promote the diffusion of intracellular active ingredients into the solvent, thereby significantly improving extraction efficiency and yield. Furthermore, ultrasound extraction at a low temperature of 0-10℃ can effectively prevent the inactivation of heat-sensitive active ingredients (such as polyphenols and vitamins) due to high temperatures, ensuring the bioactivity of the extract.

[0035] In this invention, the parameters for liquid chromatography in step 4) are set as follows: The length of the chromatographic column is 50-100 cm, preferably 55-90 cm, more preferably 60-80 cm, and even more preferably 65-75 cm; the inner diameter is 2.5-5 cm, preferably 3-4.5 cm, more preferably 3.5-4 cm, and even more preferably 3.8 cm. The chromatographic column packing material is cross-linked dextran with a particle size of 30~50μm, preferably 33~48μm, more preferably 35~45μm, and even more preferably 38~42μm; The eluent is a mixture of ethanol and water; The flow rate of the eluent is 1~3 mL / min, preferably 1.5~2.5 mL / min, and more preferably 2 mL / min.

[0036] In this invention, the gradient elution procedure in step 4) is as follows: Ethanol:water = 0:100, 10~15 min; Ethanol:water = 10:90, 5~10 min; Ethanol:water = 25:75, 5~10 min; Ethanol:water = 50:50, 8~12 min; Ethanol:water = 70:30, 5~10 min; Ethanol:water = 90:10, 3~8 min; Ethanol:water = 100:0 10~20 min; The ratio of ethanol to water mentioned above is a volume ratio.

[0037] In this invention, the mixed plant extract is the effluent from a gradient elution process where the ratio of ethanol to water is between 25:75 and 90:10.

[0038] In this invention, step 4) separation and purification also includes distilling the eluent under reduced pressure to remove ethanol from the system. The temperature is controlled at 75~80℃ during the reduced pressure distillation process, and a vacuum pump is connected.

[0039] Liquid chromatography (LC) utilizes the differences in adsorption and partition coefficients between the stationary phase (cross-linked dextran) and the mobile phase (ethanol-water) to achieve efficient separation of components in complex mixtures. By optimizing gradient elution programs, target components with cognitive impairment-improving activities can be precisely separated from other impurities, thereby obtaining high-purity active fractions. The purified extract removes ineffective components and impurities, not only improving the bioactivity per unit mass of the extract but also reducing potential side effects.

[0040] The cyclic freeze-thaw process utilizes the formation and melting of ice crystals to disrupt the cell wall structure of soybeans, creating conditions for subsequent microwave extraction. The first microwave extraction rapidly vaporizes the volatile active ingredients in soybeans, which are then collected via condensation. In the second microwave extraction, the microwave-treated soybeans are pulverized and subjected to a second water extraction. The thermal and non-thermal effects of microwaves further promote the release and dissolution of intracellular non-volatile active ingredients, thereby maximizing the extraction of effective components from soybeans. Mixing extracts A and B integrates multiple volatile and non-volatile active ingredients from soybeans, achieving full utilization of soybean resources. Its comprehensive effect is superior to products obtained by single extraction methods.

[0041] In this invention, the mass ratio of the mixed plant extract to the soybean extract is 1:1 to 3.

[0042] The present invention also provides a composition for improving cognitive impairment prepared by the above-described method. The technical solutions provided by the present invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1 Preparation of precursor powder: Weigh oats, sea buckthorn, okra, green tea and purple sweet potato in a mass ratio of 2:1:1:2:1, mix them and crush them to 40 mesh, then freeze dry to obtain precursor powder.

[0044] Plasma treatment: The precursor powder was mixed with a 0.2 mol / L sodium hydroxide solution to achieve a water content of 10%, and then treated using a dielectric barrier discharge plasma device. The device parameters were set as follows: discharge voltage 12 kV, discharge frequency 6 kHz, treatment time 0.5 min, discharge gap 1 mm, treatment temperature 5 °C, and the treatment atmosphere was ozone and inert nitrogen (volume ratio 1:8) at a gas flow rate of 3 L / min.

[0045] Extraction process: The processed precursor powder was mixed with water at a ratio of 1g:3mL, and the pH was adjusted to 6.5 with 0.5mol / L acetic acid solution. The mixture was then subjected to sonication (frequency 20kHz, time 10min, temperature 0℃), centrifugation, and filtration to obtain a mixed plant extract.

[0046] Separation and purification: The mixed plant extracts were eluted using gradient HPLC. Column parameters: length 50 cm, inner diameter 2.5 cm, packing material: cross-linked dextran, particle size 30 μm; eluent: a mixture of ethanol and water, flow rate 1 mL / min. Gradient elution program: ethanol:water = 0:100 (10 min) → 10:90 (5 min) → 25:75 (5 min) → 50:50 (8 min) → 70:30 (5 min) → 90:10 (3 min) → 100:0 (10 min). The eluent with an ethanol:water ratio between 25:75 and 90:10 was collected and subjected to vacuum distillation (75℃, 10 kPa) to obtain the mixed plant extracts.

[0047] Preparation of soybean extract: Soybeans were frozen at -40℃ for 1 hour, then thawed at 25℃ for 0.5 hours, and this cycle was repeated twice to complete the freeze-thaw treatment. The soybeans were then placed in a microwave reactor, and microwave extraction was performed at a frequency of 2 GHz for 30 seconds per extraction. After microwave extraction, the gas in the microwave reactor was condensed at -5℃ and -0.1 MPa to obtain soybean extract A. The microwave-extracted soybeans were pulverized to a particle size of 0.5 mm, and then mixed with deionized water at a mass ratio of 1:4. The mixture was then heated at 40℃ and 50℃. Soybean slurry was obtained by wetting the soybeans at 0 rpm for 10 minutes. The soybean slurry was then placed in a microwave reactor, and nitrogen gas was introduced into the reactor at a flow rate of 5 L / min. The microwave frequency was then controlled at 1 GHz and the magnetic field strength at 150 mT for a second microwave extraction of 30 s. The soybean slurry after the second microwave extraction was filtered through a 0.1 μm ceramic membrane, and the filtrate was freeze-dried at -20℃ and -0.1 MPa to remove water, yielding soybean extract B. Soybean extract A and soybean extract B were then mixed to obtain the soybean extract.

[0048] The prepared soybean extract was mixed with mixed plant extracts at a mass ratio of 1:1 to obtain a composition for improving cognitive impairment.

[0049] Example 2 Preparation of precursor powder: Weigh oats, sea buckthorn, okra, green tea and purple sweet potato in a mass ratio of 4:2:2:5:3, mix them, crush them to 80 mesh, and freeze-dry them to obtain precursor powder.

[0050] Plasma treatment: The precursor powder was mixed with a 1 mol / L sodium hydroxide solution to achieve a water content of 20%, and then treated using a dielectric barrier discharge plasma device. Device parameters: discharge voltage 22 kV, discharge frequency 9 kHz, treatment time 5 min, discharge gap 3 mm, treatment temperature 15 °C, treatment atmosphere was ozone and inert nitrogen (volume ratio 2:10), gas flow rate 8 L / min.

[0051] Extraction process: The processed precursor powder was mixed with water at a ratio of 1g:5mL, and the pH was adjusted to 7 using 0.5mol / L acetic acid solution. The mixture was then subjected to sonication (frequency 50kHz, time 20min, temperature 10℃), centrifugation, and filtration to obtain a mixed plant extract.

[0052] Separation and purification: Liquid chromatography parameters: column length 100cm, inner diameter 5cm, packing material cross-linked dextran, particle size 50μm; eluent flow rate 3mL / min. Gradient elution program: ethanol:water = 0:100 (15min) → 10:90 (10min) → 25:75 (10min) → 50:50 (12min) → 70:30 (10min) → 90:10 (8min) → 100:0 (20min). Collect the eluent with an ethanol:water ratio between 25:75 and 90:10, and subject the collected eluent to vacuum distillation (temperature 75℃, pressure 10Kpa) to obtain a mixed plant extract.

[0053] Preparation of soybean extract: Soybeans were frozen at -40℃ for 1 hour, then thawed at 25℃ for 0.5 hours, and this cycle was repeated twice to complete the freeze-thaw treatment. The soybeans were then placed in a microwave reactor, and microwave extraction was performed at a frequency of 2 GHz for 30 seconds per extraction. After microwave extraction, the gas in the microwave reactor was condensed at -5℃ and -0.1 MPa to obtain soybean extract A. The microwave-extracted soybeans were pulverized to a particle size of 0.5 mm, and then mixed with deionized water at a mass ratio of 1:4. The mixture was then heated at 40℃ and 50℃. Soybean slurry was obtained by wetting the soybeans at 0 rpm for 10 minutes. The soybean slurry was then placed in a microwave reactor, and nitrogen gas was introduced into the reactor at a flow rate of 5 L / min. The microwave frequency was then controlled at 1 GHz and the magnetic field strength at 150 mT for a second microwave extraction of 30 s. The soybean slurry after the second microwave extraction was filtered through a 0.1 μm ceramic membrane, and the filtrate was freeze-dried at -20℃ and -0.1 MPa to remove water, yielding soybean extract B. Soybean extract A and soybean extract B were then mixed to obtain the soybean extract.

[0054] The prepared soybean extract was mixed with mixed plant extracts at a mass ratio of 1:1 to obtain a composition for improving cognitive impairment.

[0055] Example 3 Preparation of precursor powder: Weigh oats, sea buckthorn, okra, green tea and purple sweet potato in a mass ratio of 3:1.5:1.5:3:2, mix them, crush them to 60 mesh, and freeze-dry them to obtain precursor powder.

[0056] Plasma treatment: The precursor powder was mixed with a 0.5 mol / L sodium hydroxide solution to achieve a water content of 15%, and then treated using a dielectric barrier discharge plasma device. Device parameters: discharge voltage 17 kV, discharge frequency 7.5 kHz, treatment time 3 min, discharge gap 2 mm, treatment temperature 10 °C, treatment atmosphere of ozone and inert nitrogen (volume ratio 1.5:9), gas flow rate 5 L / min.

[0057] Extraction process: The processed precursor powder was mixed with water at a ratio of 1g:4mL, and the pH was adjusted to 6.8 with 0.5mol / L acetic acid solution. The mixture was then subjected to sonication (frequency 34kHz, time 15min, temperature 4℃), centrifugation, and filtration to obtain a mixed plant extract.

[0058] Separation and purification: Liquid chromatography parameters: column length 70cm, inner diameter 3.8cm, packing material cross-linked dextran, particle size 40μm; eluent flow rate 2mL / min. Gradient elution program: ethanol:water = 0:100 (12min) → 10:90 (8min) → 25:75 (8min) → 50:50 (10min) → 70:30 (8min) → 90:10 (5min) → 100:0 (15min). Collect the eluent with an ethanol:water ratio between 25:75 and 90:10, and subject the collected eluent to vacuum distillation (temperature 75℃, pressure 10Kpa) to obtain a mixed plant extract.

[0059] Preparation of soybean extract: Soybeans were frozen at -40℃ for 1 hour, then thawed at 25℃ for 0.5 hours, and this cycle was repeated twice to complete the freeze-thaw treatment. The soybeans were then placed in a microwave reactor, and microwave extraction was performed at a frequency of 2 GHz for 30 seconds per extraction. After microwave extraction, the gas in the microwave reactor was condensed at -5℃ and -0.1 MPa to obtain soybean extract A. The microwave-extracted soybeans were pulverized to a particle size of 0.5 mm, and then mixed with deionized water at a mass ratio of 1:4. The mixture was then heated at 40℃ and 50℃. Soybean slurry was obtained by wetting the soybeans at 0 rpm for 10 minutes. The soybean slurry was then placed in a microwave reactor, and nitrogen gas was introduced into the reactor at a flow rate of 5 L / min. The microwave frequency was then controlled at 1 GHz and the magnetic field strength at 150 mT for a second microwave extraction of 30 s. The soybean slurry after the second microwave extraction was filtered through a 0.1 μm ceramic membrane, and the filtrate was freeze-dried at -20℃ and -0.1 MPa to remove water, yielding soybean extract B. Soybean extract A and soybean extract B were then mixed to obtain the soybean extract.

[0060] The prepared soybean extract was mixed with mixed plant extracts at a mass ratio of 1:1 to obtain a composition for improving cognitive impairment.

[0061] Example 4 Preparation of precursor powder: Weigh oats, sea buckthorn, okra, green tea and purple sweet potato in a mass ratio of 2.5:1.2:1.2:2.5:1.5, mix them and crush them to 50 mesh, then freeze dry to obtain precursor powder.

[0062] Plasma treatment: The precursor powder was mixed with a 0.3 mol / L sodium hydroxide solution to achieve a water content of 12%, and then treated using a dielectric barrier discharge plasma device. Device parameters: discharge voltage 14 kV, discharge frequency 6.5 kHz, treatment time 1 min, discharge gap 1.2 mm, treatment temperature 7 °C, treatment atmosphere of ozone and inert nitrogen (volume ratio 1.2:8.3), gas flow rate 4 L / min.

[0063] Extraction process: The processed precursor powder was mixed with water at a ratio of 1g:3.3mL, and the pH was adjusted to 6.6 with 0.5mol / L acetic acid solution. The mixture was then subjected to sonication (frequency 25kHz, time 12min, temperature 0℃), centrifugation, and filtration to obtain a mixed plant extract.

[0064] Separation and purification: Liquid chromatography parameters: column length 55cm, inner diameter 3cm, packing material cross-linked dextran, particle size 33μm; eluent flow rate 1.5mL / min. Gradient elution program was the same as in Example 1. The corresponding eluent was collected, and the collected eluent was subjected to vacuum distillation (temperature 75℃, pressure 10Kpa) to obtain mixed plant extracts.

[0065] Preparation of soybean extract: Soybeans were frozen at -40℃ for 1 hour, then thawed at 25℃ for 0.5 hours, and this cycle was repeated twice to complete the freeze-thaw treatment. The soybeans were then placed in a microwave reactor, and microwave extraction was performed at a frequency of 2 GHz for 30 seconds per extraction. After microwave extraction, the gas in the microwave reactor was condensed at -5℃ and -0.1 MPa to obtain soybean extract A. The microwave-extracted soybeans were pulverized to a particle size of 0.5 mm, and then mixed with deionized water at a mass ratio of 1:4. The mixture was then heated at 40℃ and 50℃. Soybean slurry was obtained by wetting the soybeans at 0 rpm for 10 minutes. The soybean slurry was then placed in a microwave reactor, and nitrogen gas was introduced into the reactor at a flow rate of 5 L / min. The microwave frequency was then controlled at 1 GHz and the magnetic field strength at 150 mT for a second microwave extraction of 30 s. The soybean slurry after the second microwave extraction was filtered through a 0.1 μm ceramic membrane, and the filtrate was freeze-dried at -20℃ and -0.1 MPa to remove water, yielding soybean extract B. Soybean extract A and soybean extract B were then mixed to obtain the soybean extract.

[0066] The prepared soybean extract was mixed with mixed plant extracts at a mass ratio of 1:1 to obtain a composition for improving cognitive impairment.

[0067] Example 5 Preparation of precursor powder: oats, sea buckthorn, okra, green tea and purple sweet potato were weighed in a mass ratio of 3.5:1.8:1.8:3.5:2.5, mixed and crushed to 70 mesh, and freeze-dried to obtain precursor powder.

[0068] Plasma treatment: The precursor powder was mixed with a 0.8 mol / L sodium hydroxide solution to achieve a water content of 18%, and then treated using a dielectric barrier discharge plasma device. Device parameters: discharge voltage 20 kV, discharge frequency 8.5 kHz, treatment time 4.5 min, discharge gap 2.8 mm, treatment temperature 13 °C, treatment atmosphere was ozone and inert nitrogen (volume ratio 1.8:9.8), gas flow rate 7 L / min.

[0069] Extraction process: The processed precursor powder was mixed with water at a ratio of 1g:4.7mL, and the pH was adjusted to 6.9 with 0.5mol / L acetic acid solution. The mixture was then subjected to sonication (frequency 45kHz, time 18min, temperature 8℃), centrifugation, and filtration to obtain a mixed plant extract.

[0070] Separation and purification: Liquid chromatography parameters: column length 90cm, inner diameter 4.5cm, packing material cross-linked dextran, particle size 48μm; eluent flow rate 2.5mL / min. Gradient elution program was the same as in Example 2. The corresponding eluent was collected, and the collected eluent was subjected to vacuum distillation (temperature 75℃, pressure 10Kpa) to obtain mixed plant extracts.

[0071] Preparation of soybean extract: Soybeans were frozen at -40℃ for 1 hour, then thawed at 25℃ for 0.5 hours, and this cycle was repeated twice to complete the freeze-thaw treatment. The soybeans were then placed in a microwave reactor, and microwave extraction was performed at a frequency of 2 GHz for 30 seconds per extraction. After microwave extraction, the gas in the microwave reactor was condensed at -5℃ and -0.1 MPa to obtain soybean extract A. The microwave-extracted soybeans were pulverized to a particle size of 0.5 mm, and then mixed with deionized water at a mass ratio of 1:4. The mixture was then heated at 40℃ and 50℃. Soybean slurry was obtained by wetting the soybeans at 0 rpm for 10 minutes. The soybean slurry was then placed in a microwave reactor, and nitrogen gas was introduced into the reactor at a flow rate of 5 L / min. The microwave frequency was then controlled at 1 GHz and the magnetic field strength at 150 mT for a second microwave extraction of 30 s. The soybean slurry after the second microwave extraction was filtered through a 0.1 μm ceramic membrane, and the filtrate was freeze-dried at -20℃ and -0.1 MPa to remove water, yielding soybean extract B. Soybean extract A and soybean extract B were then mixed to obtain the soybean extract.

[0072] The prepared soybean extract was mixed with mixed plant extracts at a mass ratio of 1:1 to obtain a composition for improving cognitive impairment.

[0073] Comparative Example 1 This comparative example is the soybean extract in Example 1 that was not mixed with the mixed plant extracts.

[0074] Comparative Example 2 Preparation of precursor powder: Weigh oats, sea buckthorn, okra, green tea and purple sweet potato in a mass ratio of 2:1:1:2:1, mix them and crush them to 40 mesh, then freeze dry to obtain precursor powder.

[0075] Extraction process: The precursor powder was directly mixed with water at a ratio of 1g:3mL, and the pH was adjusted to 6.5 with 0.5mol / L acetic acid solution. The mixture was then subjected to sonication (frequency 20kHz, time 10min, temperature 0℃), centrifugation, and filtration to obtain a mixed plant extract.

[0076] Separation and purification: The mixed plant extracts were eluted using gradient HPLC. Column parameters: length 50 cm, inner diameter 2.5 cm, packing material: cross-linked dextran, particle size 30 μm; eluent: a mixture of ethanol and water, flow rate 1 mL / min. Gradient elution program: ethanol:water = 0:100 (10 min) → 10:90 (5 min) → 25:75 (5 min) → 50:50 (8 min) → 70:30 (5 min) → 90:10 (3 min) → 100:0 (10 min). The eluent with an ethanol:water ratio between 25:75 and 90:10 was collected and subjected to vacuum distillation (75℃, 10 kPa) to obtain the mixed plant extracts.

[0077] Preparation of soybean extract: Soybeans were frozen at -40℃ for 1 hour, then thawed at 25℃ for 0.5 hours, and this cycle was repeated twice to complete the freeze-thaw treatment. The soybeans were then placed in a microwave reactor, and microwave extraction was performed at a frequency of 2 GHz for 30 seconds per extraction. After microwave extraction, the gas in the microwave reactor was condensed at -5℃ and -0.1 MPa to obtain soybean extract A. The microwave-extracted soybeans were pulverized to a particle size of 0.5 mm, and then mixed with deionized water at a mass ratio of 1:4. The mixture was then heated at 40℃ and 50℃. Soybean slurry was obtained by wetting the soybeans at 0 rpm for 10 minutes. The soybean slurry was then placed in a microwave reactor, and nitrogen gas was introduced into the reactor at a flow rate of 5 L / min. The microwave frequency was then controlled at 1 GHz and the magnetic field strength at 150 mT for a second microwave extraction of 30 s. The soybean slurry after the second microwave extraction was filtered through a 0.1 μm ceramic membrane, and the filtrate was freeze-dried at -20℃ and -0.1 MPa to remove water, yielding soybean extract B. Soybean extract A and soybean extract B were then mixed to obtain the soybean extract.

[0078] The prepared soybean extract was mixed with mixed plant extracts at a mass ratio of 1:1 to obtain a composition for improving cognitive impairment.

[0079] This comparative example uses traditional extraction methods to prepare mixed plant extracts.

[0080] The composition prepared according to the embodiments and comparative examples of the present invention was tested for its efficacy and safety in improving cognitive function in elderly patients with mild cognitive impairment (MCI). The specific test methods and data are as follows: Experimental subjects: Inclusion criteria included participants aged 60-80 years; MMSE scores of 20-26, meeting the Petersen MCI diagnostic criteria; and stable cognitive function over the past 3 months (excluding those diagnosed with organic dementia such as Alzheimer's disease, those with severe liver or kidney dysfunction, those taking antidementia medications within the past 6 months, and those allergic to any component of the test substance). Participants were randomly assigned to four groups of 30 each, for a total of 120 participants.

[0081] The experimental groups are shown in Table 1.

[0082] Table 1 Experimental Groups

[0083] The study lasted for 24 weeks, including a 2-week screening period, a 20-week dosing period, and a 2-week follow-up period. Indicators were measured at baseline (week 0), week 12, and week 24.

[0084] The evaluation methods and standards are as follows: Overall cognitive function: The Mini-Mental State Examination (MMSE) was used, covering 7 dimensions including orientation and memory, with a total score of 30 points.

[0085] Memory function: The Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-Cog) focuses on testing word memory, instruction comprehension, etc. The lower the score, the better the cognitive function.

[0086] Episodic Memory: Auditory Vocabulary Learning Test (AVLT) assesses immediate and delayed recall abilities.

[0087] Table 2. Initial test results of various indicators for each experimental group (baseline values)

[0088] Table 3. Results of various indicators in each experimental group after 12 weeks of medication.

[0089] Table 4. Results of various indicators in each experimental group after 24 weeks of medication.

[0090] All the above data are averages.

[0091] All experimental groups (Examples 1-4) and comparative groups (1-2) in the placebo group and low / medium / high dose group showed highly similar baseline values ​​for MMSE, ADAS-Cog (word memory, instruction comprehension), and AVLT at the beginning of the experiment (week 0), with minimal fluctuations (e.g., MMSE scores were all between 23.0 and 23.2). There were no statistically significant differences between groups (P>0.05), which meets the balance requirements of randomization in clinical trials and ensures the validity and comparability of subsequent experimental results.

[0092] The following conclusions can be drawn from the data in Tables 2-4: Regarding overall cognitive function (MMSE), after 24 weeks of administration, the high-dose group showed significant improvement in all examples, with Example 3 showing the best results. The medium-dose group improved by 3.2 points from baseline, and the high-dose group improved by 4.1 points, which were significantly higher than the placebo group's 1.1 points. The low-dose group also showed stable improvement (Example 3: low-dose group improved by 2.2 points). All examples showed a clear dose-response relationship: high-dose > medium-dose > low-dose > placebo.

[0093] Regarding memory function (ADAS-Cog), a decrease in score indicates functional improvement. At 24 weeks, in Example 3, the high-dose group showed a 5.3-point decrease in word memory and a 2.7-point decrease in instruction comprehension compared to baseline, while the medium-dose group showed decreases of 3.8 and 2.0 points, respectively, which were significantly better than the 1.2 and 0.3 points in the placebo group. The improvement trend in each dose group was consistent with that of MMSE, with higher doses showing more significant improvements in memory function.

[0094] Regarding episodic memory (AVLT), in Example 3, the high-dose group showed an improvement of 4.2 words in delayed recall scores after 24 weeks compared to baseline, the medium-dose group showed an improvement of 3.6 words, the low-dose group showed an improvement of 2.2 words, and the placebo group showed an improvement of only 0.9 words. Moreover, the improvement effect of the high-dose group was significantly higher than that of the low-dose group, which fully verified the effect of the composition on episodic memory.

[0095] The composition of this invention can significantly improve overall cognition, memory, and episodic memory in elderly patients with mild cognitive impairment. The effect increases with increasing dosage and duration of administration, exhibiting a clear dose-dependent and time-dependent effect, with optimal improvement observed after 24 weeks of medium-to-high dose administration. The combination of oat and other mixed plant extracts with soybean extract shows a significant synergistic effect, greatly enhancing the cognitive improvement effect of soybean extract alone and solving the technical problem of limited improvement effect of pure soybean extract. The dielectric barrier discharge plasma cell disruption process is key to improving the composition's efficacy; compared to traditional extraction processes, it effectively improves the extraction efficiency of plant active ingredients, significantly enhancing the cognitive improvement effect of the composition. The composition of this invention is a natural plant extract preparation; no adverse reactions were observed in experiments, indicating high safety. It can serve as an effective natural preparation for the prevention and treatment of age-related cognitive impairment, providing experimental evidence for clinical dosage and treatment course settings.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a composition for improving cognitive impairment, characterized in that, Includes the following steps: 1) Mix oats, sea buckthorn, okra, green tea and purple sweet potato, crush and freeze dry to obtain precursor powder; 2) The precursor powder was mixed with sodium hydroxide solution and then processed using a dielectric barrier discharge plasma device; 3) Mix the processed precursor powder from step 2) with water and adjust the pH to 6.5-7. Then, perform ultrasonication, centrifugation, and filtration to obtain a mixed plant extract. 4) The mixed plant extract described in step 3) was subjected to gradient elution by liquid chromatography to separate and purify the mixed plant extract; 5) Soybeans are subjected to cyclic freeze-thaw treatment and microwave extraction to obtain soybean extract A, a gaseous condensate, and microwave-treated soybeans; the microwave-treated soybeans are crushed and mixed with water for microwave extraction again; after microwave extraction, the mixture is filtered and the filtrate is freeze-dried to obtain soybean extract B; soybean extract A and soybean extract B are mixed to obtain soybean extract. 6) Mix the mixed plant extracts obtained in step 4) with the soybean extract obtained in step 5) to obtain a composition for improving cognitive impairment.

2. The method for preparing a composition for improving cognitive impairment according to claim 1, characterized in that, The mass ratio of oats, sea buckthorn, okra, green tea, and purple sweet potato mentioned in step 1) is 2~4:1~2:1~2:2~5:1~3; The precursor powder has a particle size of 40-80 mesh.

3. A method for preparing a composition for improving cognitive impairment according to claim 2, characterized in that, The concentration of the sodium hydroxide solution mentioned in step 2) is 0.2~1 mol / L; The water content of the system after mixing the precursor powder with the sodium hydroxide solution is 10-20%.

4. A method for preparing a composition for improving cognitive impairment according to claim 3, characterized in that, The parameter settings for the dielectric barrier discharge plasma device described in step 2) are as follows: The discharge voltage is 12~22kV; The discharge frequency is 6~9kHz; The processing time is 0.5~5 minutes; The discharge gap is 1~3mm; The processing temperature is 5~15℃; The treatment atmosphere is a mixture of ozone and inert gas, wherein the volume ratio of ozone to inert gas is 1~2:8~10, and the gas flow rate is 3~8L / min.

5. A method for preparing a composition for improving cognitive impairment according to any one of claims 2 to 4, characterized in that, The mixing ratio of the processed precursor powder to water in step 3) is 1g: 3~5mL; The ultrasound frequency is 20~50kHz, the duration is 10~20min, and the temperature is 0~10℃.

6. A method for preparing a composition for improving cognitive impairment according to claim 5, characterized in that, The parameters for liquid chromatography described in step 4) are set as follows: The length of the chromatographic column is 50~100cm, and the inner diameter is 2.5~5cm; The chromatographic column packing material is cross-linked dextran with a particle size of 30~50μm; The eluent is a mixture of ethanol and water; The flow rate of the eluent is 1-3 mL / min.

7. A method for preparing a composition for improving cognitive impairment according to claim 6, characterized in that, The gradient elution procedure described in step 4) is as follows: Ethanol:water = 0:100, 10~15 min; Ethanol:water = 10:90, 5~10 min; Ethanol:water = 25:75, 5~10 min; Ethanol:water = 50:50, 8~12 min; Ethanol:water = 70:30, 5~10 min; Ethanol:water = 90:10, 3~8 min; Ethanol:water = 100:0 10~20min.

8. A method for preparing a composition for improving cognitive impairment according to claim 7, characterized in that, The mixed plant extract is the effluent from a gradient elution process where the ratio of ethanol to water is between 25:75 and 90:

10.

9. A method for preparing a composition for improving cognitive impairment according to claim 8, characterized in that, The mass ratio of the mixed plant extract to the soybean extract is 1:1 to 3.

10. The composition for improving cognitive impairment prepared by the method of any one of claims 1 to 9.

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