CN116474050A

CN116474050A - Compound Munich Cuminum cyminum prescription and application of extraction part thereof in preventing and treating dysmnesia

Info

Publication number: CN116474050A
Application number: CN202310498070.3A
Authority: CN
Inventors: 王长虹; 程雪梅; 赵祥; 李曼琳; 管辉达
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2023-05-04
Filing date: 2023-05-04
Publication date: 2023-07-25

Abstract

The invention discloses application of a compound wooden cumin prescription total extract and/or a compound wooden cumin prescription extraction part in preparation of a medicament for preventing and/or treating dysmnesia. According to the invention, a scopolamine (intraperitoneal injection: 1 mg/kg) induced C57BL/6J mouse memory injury model is adopted, and the influence of a compound Munich Cuminum formula (FFMN) macroporous resin enriched total Extract (EXT) and total Alkaloids (ALK), total Flavonoids (FLA) and total Saponins (SAP) on the memory injury model mouse behavior is researched through Morris Water Maze (MWM) experiments, so that from the curative effect, FFMN total saponins > FFMN total alkaloids > FFMN total extracts > FFMN total flavonoids.

Description

Compound Munich Cuminum cyminum prescription and application of extraction part thereof in preventing and treating dysmnesia

Technical Field

The invention relates to the technical field of medicines, in particular to a compound Munich cumin prescription and application of an extraction part thereof in preventing and treating dysmnesia.

Background

Memory impairment refers to a condition in which an individual is unable to remember or recall information or skills, and is manifested clinically as impaired memory, amnesia, misconfiguration, fiction, and latent memory. Clinically, the causes of the dysmnesia are complex, and the common diseases at present are mainly dementia (including senile dementia, vascular dementia or dementia caused by Lewy bodies), alzheimer disease, brain trauma, cerebral ischemia or dysmnesia caused by stress, and the dysmnesia has serious influence on the normal life of patients.

Dementia is a chronic progressive intellectual impairment syndrome, characterized clinically by slow onset of impaired intelligence, a core symptom of which is hypomnesis, and early onset of memory impairment. With the acceleration of aging of the population, the incidence of senile dementia increases, and the senile dementia has become a fourth cause of death of the elderly after tumor, heart disease and cerebrovascular disease.

Alzheimer's Disease (AD) occurs mainly in elderly people over 65 years old and is a chronic and progressive neurodegenerative disease. There is no clear conclusion on the current study of the pathogenesis of AD, and cholinergic system abnormalities, oxidative stress and neuroinflammation have been considered to be associated with AD.

Cholinergic system abnormalities are one of the mainstream hypotheses for AD. Cholinergic synapses have a high density in the central nervous system and learning and memory functions are not separated from cholinergic transmission. As an important neurotransmitter in the body, acetylcholine (ACh) is synthesized primarily by choline acetyltransferase (ChAT). Acetylcholinesterase (AChE) can degrade ACh, stop its transmission, and ensure transmission of nerve signals. However, abnormal increases in AChE activity in the brain of AD patients lead to abnormal decreases in ACh levels. Therefore, ACh hydrolysis by AChE is significantly reduced in the brain of AD patients, which may be one of the key factors in the production of AD. Studies have shown that sustained overactive neuroinflammation is intimately linked to AD. As a first line of defense against immune defenses in the brain, microglial cells have a main function of controlling various neuronal activities including neuronal apoptosis, formation of synapses, and the like. Excessive deposition of beta-amyloid (aβ) activates microglia, which in turn release neurotoxic components and inflammatory factors such as TNF- α, NO, etc. These components can further induce chronic inflammation and destroy the nervous system, while also promoting the accumulation of aβ, causing cognitive impairment. Oxidative stress is also a hypothesis for AD, and has a close relationship with other AD hypotheses. NADPH oxidase is activated by aβ, leading to an increase in ROS content, thereby causing oxidative stress. Peroxidation of lipids can lead to the generation of harmful substances such as MDA, which can damage cell membranes and cause damage to nerve cells. Studies have shown that the activity of SOD and GSH in the brain of AD patients is significantly lower than in healthy groups. Oxidative stress caused by ROS is also closely related to neuroinflammation, and activated microglia also release ROS in brain-sustained neuroinflammation, resulting in neurotoxicity.

Currently, the majority of drugs approved by the FDA for the treatment of memory disorders such as AD are cholinesterase inhibitors. These drugs are poorly effective, often with significant differences in patient response to certain specific drugs, and are accompanied by resistance and toxic side effects, which prevent their use. Thus, there is an urgent need to find new natural drug therapies to treat memory disorders such as AD patients.

In the prior art, no report is found on the application of the compound Munich Cuminum and its extraction part in preventing and treating dysmnesia.

Disclosure of Invention

Based on the above, the invention provides the application of the total extract of the compound Munich cumin and/or the extraction part of the compound Munich cumin in preparing medicines for preventing and/or treating dysmnesia.

According to another aspect of the invention, there is provided the use of a pharmaceutical composition comprising a total extract of compound manicure cumin and/or an extraction site of compound manicure cumin in the manufacture of a medicament for the prevention and/or treatment of memory disorders.

Further, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Further, the auxiliary material is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, slow release agents, retention aids, lubricants, dispersants, plasticizers, opacifiers and antioxidants.

Further, the pharmaceutical composition is in the form of tablet, dripping pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

Further, the pharmaceutical composition further comprises one or more drugs and/or extracts for preventing and/or treating memory disorders.

Further, the drug is a cholinesterase inhibitor, a glutamate receptor antagonist and/or mannite.

Further, the cholinesterase inhibitor is donepezil, rivastigmine and/or galantamine.

Further, the glutamate receptor antagonist is memantine.

Further, the memory disorder is a dementia, alzheimer's disease, brain trauma, cerebral ischemia and/or a memory disorder caused by stress.

Further, the dementia is senile dementia, vascular dementia or dementia with lewy bodies.

Further, the extraction site is a total alkaloid site and/or a total saponin site.

Further, the total extract of the compound manicure Cuminum cyminum and/or the extraction part of the compound manicure Cuminum cyminum are prepared by a method comprising the following steps:

(1) Pretreatment of macroporous resin: sequentially soaking macroporous resin in ethanol solution, washing and soaking in hydrochloric acid solution, washing and soaking in sodium hydroxide solution, washing with water until the pH is neutral, and obtaining pretreated macroporous resin;

(2) And (3) column loading: loading the pretreated macroporous resin into a column by a wet method to obtain a first macroporous resin adsorption column and/or a second macroporous resin adsorption column;

(3) Loading: precisely weighing a proper amount of medicinal materials, sieving, extracting, reflux-extracting, filtering, mixing filtrates, concentrating under reduced pressure until no alcohol smell exists, adding water to a certain crude drug concentration, centrifuging, and collecting supernatant to obtain macroporous resin sample liquid, allowing the sample liquid to pass through the first macroporous resin adsorption column at a certain flow rate to enable the sample liquid to be adsorbed on the first macroporous resin adsorption column, and optionally collecting the sample liquid which is not adsorbed to obtain leakage liquid; and

(4) Obtaining total alkaloid fraction and/or total saponin fraction: and (3) using a certain volume of 5% -15% ethanol to pass through the first macroporous resin adsorption column according to a certain flow rate for impurity removal, sequentially using 25% -35% ethanol, 35% -45% ethanol and 65% -75% ethanol to elute according to a certain flow rate, and collecting eluent to obtain the total alkaloid fraction and/or the total saponin fraction.

Further, in step (1), the macroporous resin is an H20 resin, an H60 resin, an HPD100 resin, an LSA-5B resin, a D101 resin, or an AB-8 resin.

Further, the macroporous resin is a D101 resin or an H20 resin.

Further, the concentration of the ethanol solution was about 95%, and the soaking time of the ethanol solution was about 24 hours.

Further, the volume of the hydrochloric acid solution was about 2BV, the concentration of the hydrochloric acid solution was about 5%, and the soaking time of the hydrochloric acid solution was about 2 hours.

Further, the volume of the sodium hydroxide solution was about 2BV, the concentration of the sodium hydroxide solution was about 2%, and the soaking time of the sodium hydroxide solution was about 2 hours.

Further, the water is ultrapure water.

Further, in the step (3), the medicinal material includes peganum harmala, black seed grass, pimpinella anisum, fennel root bark, chamomile, celery root, chicory seed, chicory root, citronella, moldavica dragonhead, licorice, basil seed and hollyhock seed.

Further, the screen is a No. 1 screen.

Further, the leaching is to add about 5 times of about 70% ethanol of the medicinal material quality to leach the medicinal material.

Further, the reflux extraction is to extract the medicinal material by reflux with about 70% ethanol added to about 8 times the mass of the medicinal material.

Further, the number of times of the reflux extraction was 2 times, each for about 1 hour.

Further, the crude drug concentration was about 0.3g/mL.

Further, the rotational speed of the centrifugation is 7000r/min to 9000r/min, and the time of the centrifugation is 5min to 15min.

Further, the rotational speed of the centrifugation is about 8000r/min, and the time of the centrifugation is about 10min.

Further, the pH of the sample loading solution is 3-7.

Further, the pH of the loading solution was about 5.

Further, the mass concentration of the sample loading liquid is 0.15 g/mL-0.5 g/mL.

Further, the mass concentration of the loading liquid is 0.3g/mL.

Further, the flow rate of the loading solution was about 3BV/h.

Further, the loading solution has a volume of 10BV to 15BV, for example, about 11BV or about 14BV.

Further, the step (4) is replaced with the following step (4'): and (3) using 5% -15% ethanol with a certain volume to remove impurities through the first macroporous resin adsorption column according to a certain flow rate, using 65% -75% ethanol to elute, and collecting eluent to obtain the total extract of the compound Munich cumin.

Further, in the step (4) or the step (4'), the flow rate was about 3BV/h.

Further, 3BV to 5BV, for example about 4BV, of water is passed through the first macroporous resin adsorption column or the second macroporous resin adsorption column at a flow rate of about 3BV/h before the impurity removal.

Further, the concentration of the ethanol in the removal of impurities is about 10%.

Further, the volume of the ethanol in the removal of impurities is about 5BV.

Further, eluting with about 30% ethanol, about 40% ethanol and about 70% ethanol at a certain flow rate, and collecting the eluate to obtain the total alkaloid fraction and/or the total saponin fraction.

Further, the 4 th BV to 10 th BV of the eluate of about 30% ethanol and the 1 st BV of the eluate of about 40% ethanol were pooled as the total alkaloid fraction.

Further, the 4 th BV to 9 th BV of the eluent of about 40% ethanol and the 1 st BV to 3 rd BV of the eluent of about 70% ethanol were combined as the total saponin fraction.

Further, the eluate was collected using about 5BV of about 70% ethanol to obtain a total extract of the compound genicumin.

The invention has the beneficial effects that:

according to the invention, a scopolamine (intraperitoneal injection: 1 mg/kg) induced C57BL/6J mouse memory injury model is adopted, and the influence of a compound Munich Cuminum formula (FFMN) macroporous resin enriched total Extract (EXT) and total Alkaloids (ALK), total Flavonoids (FLA) and total Saponins (SAP) on the memory injury model mouse behavior is researched through Morris Water Maze (MWM) experiments, so that from the curative effect, FFMN total saponins > FFMN total alkaloids > FFMN total extracts > FFMN total flavonoids.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it will be apparent that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings by those skilled in the art without departing from the scope of the claimed invention.

FIG. 1 is a schematic diagram of leakage curves for HAL and total flavonoids. Wherein A: HAL leakage rate; b: total Flavone (FLA) leakage rate.

FIG. 2 is a schematic diagram showing the removal of solids from ultrapure water elution of different column volumes.

FIG. 3 is a schematic diagram of gradient elution profile of different ethanol. Wherein A: total Alkaloid (ALK) elution amount; b: total Saponin (SAP) elution amount; c: total Flavone (FLA) elution amount.

FIG. 4 is a graph showing the elution profile of 70% ethanol. Wherein A: elution curves of the components; b: the elution rate was accumulated for each component.

FIG. 5 is a flow chart for separating the square (FFMN) ALK, FLA and SAP sites of a compound Munich.

Fig. 6 shows the intention of animal experiment time.

FIG. 7 is a graph showing the effect of FFMN and its different parts on SCO-induced dysmnesia mice. Wherein A is the track of the mouse in the space detection test of the MWM, B is the path length in the hidden platform test, C is the escape delay in the hidden platform test, and D is the frequency of the mouse passing through the platform position. In comparison with the control group, ^### P<0.001； ^* P<0.05， ^** P<0.01， ^*** P<0.001 sum ^**** P<0.0001。

FIG. 8 is a graph showing the effect of FFMN and its various parts on protein expression. Wherein A is the expression of ChAT and AChE proteins in the cortex and B is the expression of NF- κ B p65, p-NF- κ B p65 and p-IκBα proteins in the cortex. In comparison with the control group, ^## P<0.01 and ^### P<0.001； ^* P<0.05， ^** P<0.01 and ^*** P<0.001。

FIG. 9 is a graph showing the effect of FFMN and its various parts on biochemical markers. Wherein A-D is the activity level of ChAT and AChE in cortex and Hippocampus, E-F is the activity level of MPO in cortex and Hippocampus, G is the level of TNF- α in cortex, H is the level of NO in cortex, I is the level of MDA in cortex, J is the activity level of SOD in cortex, and G is the level of BDNF in cortex and Hippocampus. In comparison with the control group, ^# P<0.05 sum ^### P<0.001； ^* P<0.05， ^** P<0.01， ^*** P<0.001 sum ^**** P<0.0001。

FIG. 10 is a schematic diagram showing the result of Nile staining in animal experiments.

FIG. 11 is a schematic diagram of immunofluorescence results of animal experiments.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains or to which this term applies. Although any methods, conditions, materials, or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, the preferred methods, conditions, materials, or materials are described herein.

The invention is intended to cover all alternatives, modifications and equivalents, which may be included within the art of the invention as defined by the appended claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The invention is in no way limited to the description of methods and materials.

To the extent that the terms "includes," including, "and" has "or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term" comprising.

As described in the background section, prior art drugs for treating memory disorders such as AD have poor therapeutic effects, and patients often have significant differences in their responses to certain specific drugs, with concomitant problems of resistance and toxic side effects. In order to solve the problems, the invention provides an application of a compound wooden cumin prescription total extract and/or a compound wooden cumin prescription extraction part in preparing a medicament for preventing and/or treating dysmnesia.

In a preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

In a preferred embodiment, the pharmaceutical formulation of the invention contains 0.00001 to 50wt.%, or 0.0001 to 10wt.%, or 0.0001 to 5wt.%, or 0.005 to 1wt.%, or 0.1 to 20wt.%, or 0.5 to 15wt.%, or 1 to 5wt.% of at least one pharmaceutically acceptable auxiliary material, calculated to the weight of the pharmaceutical formulation.

In the present invention, the term "pharmaceutically acceptable" refers herein to a material, such as a carrier or diluent, that does not diminish the biological activity or properties of the compound and is relatively non-toxic, e.g., the administration of a material to an individual does not cause unwanted biological effects or interact in a deleterious manner with any of the components thereof in which it is contained.

In the present invention, the term "pharmaceutically acceptable excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without eliciting any undesired local or systemic effects), as is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995).

In a preferred embodiment, the adjuvant is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, slow release agents, retention aids, lubricants, dispersants, plasticizers, opacifiers and antioxidants.

The person skilled in the art will know how to select a specific chemical within the above-mentioned auxiliary material category. For example, the dispersant may be selected from one or more of the following: croscarmellose sodium, sodium starch glycolate and pregelatinized corn starch. The diluent may be selected from one or more of the following: sugar powder, starch, compressible starch, lactose, dextrin, mannitol, sorbitol, microcrystalline cellulose, calcium sulfate and calcium carbonate. The slow release agent can be selected from one or more of the following: sodium carboxymethyl cellulose, low substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, hydroxyethyl cellulose, acacia, gelatin, and shellac. The wetting agent may be selected from one or more of the following: polyoxymethylene stearate, poloxamer, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene alkyl ethers, polysorbates such as polysorbate 80, cetyl alcohol, glycerin fatty acid esters (such as triacetin, glycerin monostearate and the like), polyoxyethylene fatty acid esters, polyethylene glycol fatty acid esters, sodium lauryl sulfate, sorbitol fatty acid esters, sucrose fatty acid esters, polyoxyethylene ethers, benzalkonium chloride, polyoxyethylene castor oil, and sodium docusate. The flavouring agent may be selected from one or more of the following: sorbitol, glucose, mannose, sucrose and lactose. The lubricant may be selected from one or more of the following: calcium stearate, talc, magnesium stearate, stearic acid and colloidal silicon dioxide. The binder may be selected from one or more of the following: polyvinylpyrrolidone, hydroxypropyl cellulose, polyethylene glycol and methylcellulose. The disintegrant may be selected from one or more of the following: carboxymethyl cellulose, carboxymethyl cellulose calcium salt, and sodium carboxymethyl cellulose. The plasticizer may be dibutyl sebacate and/or various citrates. The antioxidant may be selected from one or more of the following: sodium bisulphite, sodium metabisulfite, sodium sulfite and sodium thiosulfate.

The auxiliary materials are preferably inert to drugs or can have synergistic or additive effects to enhance the therapeutic activity of the pharmaceutical composition, and the auxiliary materials are only listed, so that the auxiliary materials actually used in the invention are not limited to the auxiliary materials and can be adjusted according to the actual situation, and the effects of the invention can be achieved.

In a preferred embodiment, the pharmaceutical composition is in the form of a tablet, drop pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

In a preferred embodiment, the pharmaceutical composition further comprises one or more drugs and/or extracts for preventing and/or treating memory disorders.

In a preferred embodiment, the drug is a cholinesterase inhibitor, a glutamate receptor antagonist and/or mannite.

In a preferred embodiment, the cholinesterase inhibitor is donepezil, rivastigmine and/or galantamine.

In a preferred embodiment, the glutamate receptor antagonist is memantine.

In a preferred embodiment, the memory disorder is dementia, alzheimer's disease, brain trauma, cerebral ischemia and/or stress induced memory disorder.

In a preferred embodiment, the dementia is senile dementia, vascular dementia or dementia with lewy bodies.

In a preferred embodiment, the extraction site is a total alkaloid fraction and/or a total saponin fraction.

In a preferred embodiment, the total extract of the compound manicure and/or the extraction site of the compound manicure is prepared by a method comprising the steps of:

In the present invention, when a concentration, volume, time, rotational speed, pH, flow rate, pressure, ratio, equivalent weight, concentration, or other value or parameter is expressed as a range, preferred range, or a range bounded by a list of upper preferable values and lower preferable values, this should be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when ranges of "5 to 15" are disclosed, the ranges described should be interpreted to include ranges of "5 to 15", "5 to 14", "5 to 13", "5 to 12", "5 to 11", "5 to 10", "5 to 9", "5 to 8", "5 to 7", "5 to 6", "6 to 15", "6 to 14", "6 to 13", "6 to 12", "6 to 11", "6 to 10", "6 to 9", "6 to 8", "6 to 7", "7 to 15", and the like. When numerical ranges are described herein, unless otherwise stated, the ranges are intended to include the endpoints thereof and all integers and fractions within the range, and the technical effects of the invention are achieved within the ranges described above.

In a preferred embodiment, in step (1), the macroporous resin is an H20 resin, an H60 resin, an HPD100 resin, an LSA-5B resin, a D101 resin, or an AB-8 resin.

In a preferred embodiment, the macroporous resin is of the type D101 resin or H20 resin.

In a preferred embodiment, the concentration of the ethanol solution is about 95% and the soaking time of the ethanol solution is about 24 hours.

In a preferred embodiment, the volume of the hydrochloric acid solution is about 2BV, the concentration of the hydrochloric acid solution is about 5%, and the soaking time of the hydrochloric acid solution is about 2 hours.

In a preferred embodiment, the volume of the sodium hydroxide solution is about 2BV, the concentration of the sodium hydroxide solution is about 2%, and the soaking time of the sodium hydroxide solution is about 2 hours.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 95" includes 95 ± 5%, or from 90.25 to 99.75; "about 24" includes + -5% of 24, or from 22.8 to 25.2; "about 2" includes + -5% of 2, or from 1.9 to 2.1; "about 5" includes + -5% of 5, or from 4.75 to 5.25.

In a preferred embodiment, the water is ultrapure water.

In a preferred embodiment, in step (3), the medicinal material comprises peganum harmala, nigella sativa, pimpinella anisum, fennel root bark, chamomile, celery root, chicory seed, chicory root, citronella, moldavica dragonhead, licorice, basil seed, and hollyhock seed.

In a preferred embodiment, the screen is a No. 1 screen.

In a preferred embodiment, the leaching is of the drug material with about 5 times the mass of about 70% ethanol.

In a preferred embodiment, the reflux extraction is reflux extracting the drug with about 70% ethanol added to about 8 times the mass of the drug.

In a preferred embodiment, the number of reflux extractions is 2, each of about 1 hour.

In a preferred embodiment, the crude drug concentration is about 0.3g/mL.

In a preferred embodiment, the rotational speed of the centrifugation is 7000r/min to 9000r/min and the time of the centrifugation is 5min to 15min.

In a preferred embodiment, the speed of centrifugation is about 8000r/min and the time of centrifugation is about 10min.

In a preferred embodiment, the pH of the loading solution is from 3 to 7.

In a preferred embodiment, the loading solution has a pH of about 5.

The nature of alkaloid, flavone and saponin components in FFMN has a certain difference, which is particularly obvious in the pH investigation process. Similar to the result of the acid extraction and alkali precipitation method, alkaloid components in FFMN are easy to form a free state under the weak alkaline condition and are more easily adsorbed by resin, flavone and saponin components are easy to hydrolyze under the acidic environment to form the free state and are easily adsorbed by resin, so that three components are required to be replaced to be enriched to the greatest extent, and finally, the pH=5 (namely, the pH of the stock solution) is selected through the investigation of the pH, so that the three components are enriched to the greatest extent at the same time, other substances are not introduced, interference is reduced, and convenience is provided for subsequent industrialized production.

In a preferred embodiment, the loading solution has a mass concentration of 0.15g/mL to 0.5g/mL.

In a preferred embodiment, the loading solution has a mass concentration of 0.3g/mL.

In a preferred embodiment, the flow rate of the loading solution is about 3BV/h.

In a preferred embodiment, the loading solution has a volume of 10BV to 15BV, such as about 11BV or about 14BV.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 5" includes ±5% of 5, or from 4.75 to 5.25; "about 70" includes + -5% of 70, or from 66.5 to 73.5; "about 8" includes + -5% of 8, or from 7.6 to 8.4; "about 1" includes + -5% of 1, or from 0.95 to 1.05; "about 0.3" includes + -5% of 0.3, or from 0.285 to 0.315; "about 8000" includes 8000 + -5%, or from 7600 to 8400; "about 10" includes + -5% of 10, or from 9.5 to 10.5; "about 3" includes + -5% of 3, or from 2.85 to 3.15; "about 11" includes + -5% of 11, or from 10.45 to 11.55; "about 14" includes + -5% of 14, or from 13.3 to 14.7.

In a preferred embodiment, step (4) is replaced by the following step (4'): and (3) using 5% -15% ethanol with a certain volume to remove impurities through the first macroporous resin adsorption column according to a certain flow rate, using 65% -75% ethanol to elute, and collecting eluent to obtain the total extract of the compound Munich cumin.

In a preferred embodiment, in step (4) or step (4'), the flow rate is about 3BV/h.

In a preferred embodiment, 3BV to 5BV, for example about 4BV, of water is passed through the first macroporous resin adsorption column or the second macroporous resin adsorption column at a flow rate of about 3BV/h prior to the removal of impurities.

In a preferred embodiment, the concentration of the ethanol in the removal of impurities is about 10%.

In a preferred embodiment, the volume of the ethanol in the removal of impurities is about 5BV.

In a preferred embodiment, the total alkaloid fraction and/or the total saponin fraction is obtained by eluting with about 30% ethanol, about 40% ethanol, and about 70% ethanol at a flow rate and collecting the eluate.

In a preferred embodiment, the total alkaloid fraction is the 4 th BV to 10 th BV of an eluate of about 30% ethanol and the 1 st BV of an eluate of about 40% ethanol.

In a preferred embodiment, the total saponin fraction is combined from about 40% ethanol eluent from 4 th BV to 9 th BV and from about 70% ethanol eluent from 1 st BV to 3 rd BV.

In a preferred embodiment, the elution is performed using about 5BV of about 70% ethanol and the eluate is collected to obtain a total extract of the reconstituted Munich cumin.

In the present invention, "about" means a value within + -5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 10" includes + -5% of 10, or from 9.5 to 10.5; "about 5" includes + -5% of 5, or from 4.75 to 5.25; "about 30" includes + -5% of 30, or from 28.5 to 31.5; "about 40" includes 40.+ -. 5%, or from 38 to 42; "about 70" includes + -5% of 70, or from 66.5 to 73.5; "about 8000" includes 8000 + -5%, or from 7600 to 8400.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not noted, are generally according to conventional conditions or conditions suggested by manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this patent specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

Examples

1. Experimental instrument

An Agilent 1260 high performance liquid chromatograph, agilent 1260 information evaporative light scattering detector (Agilent company, usa); milli-Q pure water apparatus (Millipore, mass., USA); sartorius BSA 124S-CW electronic analytical balance (beijing cerdolis instrument systems limited); TU-1901 double beam ultraviolet visible spectrophotometer (Beijing general analysis general Instrument Co., ltd.); DHG-9053A type electrothermal blowing dry box, HWS26 type electrothermal thermostatic water bath (Shanghai-constant scientific instrument limited); KQ-250DB model digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.); SHB-1000 circulating water type multipurpose vacuum pump (Shanghai Chengzhu instruments and equipment company); medical 4℃refrigerator, BC/BD-429H-20℃refrigerator (Qingdao sea Co., ltd.); an electric heating constant temperature oscillation water tank (Shanghai Jing laboratory equipment Co., ltd.), an N-1000 rotary evaporator, an OSB-2200 water bath kettle and a CCA-1112A type cooling water circulation device. Morris water maze (Shanghai number of migration information technologies Co., ltd.); high throughput tissue milling apparatus (Shanghai Mo Bai organism); transferring TS-8S to a decolorizing shaker; a Tanon gel image analysis system (Shanghai Technical Co., ltd.); bio-Rad Mini-PROTEAN Tetra Cell miniature electrophoresis system.

2. Experimental medicine, material and experimental animal

Control: harmine (HAL), harmine (HAR), rutin (RUT) and eleutheroside (Sie.A) are all from Shanghai traditional Chinese medicine standardization research center (purity is no less than 98%), and Glycyrrhizic Acid (GA) is purchased from Chengdu Desmost Biotechnology Co., ltd (batch number DST210620-060, purity is no less than 98%); sample: the peganum harmala (lot number Y2001032), fennel fruit (lot number Y1912001), fennel root bark (lot number Y2005056), black grass seed (lot number Y2009019), moldavica (lot number Y1911023), hollyhock seed (lot number Y2003001), chamomile (lot number Y2005054), chicory root (lot number Y2010002), licorice (lot number Y2005050), celery root (lot number Y2009014), chicory seed (lot number Y2001017), citronella (lot number Y2009033), and basil seed (lot number Y1909015) are all provided by Xinjiang Uygur pharmaceutical company, and are identified as genuine by the teaching of the university of Shanghai Chinese medicine Wang Changhong. Acetonitrile and glacial acetic acid are chromatographic purities; other reagents are all analytically pure; the water is ultrapure water. Macroporous resins H20, H60, HPD100, LSA-5B, D101, AB-8 were purchased from Tianjin Wako resin Co., ltd. Scopolamine hydrobromide was purchased from Chengdu Derst Biotechnology Co., ltd; sodium carboxymethylcellulose (CMC-Na), tween-20, PVDF membrane was purchased from national pharmaceutical group chemical agents limited; donepezil hydrochloride tablet (chinese sanitation and pharmaceutical company limited); 4% paraformaldehyde, toluidine blue, normal saline and PBS are purchased from Seville technologies and biology company; glycine, sodium lauryl sulfate, tris (hydroxymethyl) aminomethane, bovine Serum Albumin (BSA), separation gel (ph=8.8), concentrated gel (ph=6.8) were purchased from st-in-a-holy biosciences; hypersensitive ECL chemiluminescent kit, SDS-PAGE protein loading buffer (5X), APS subunit, protease phosphatase inhibitor cocktail (general purpose, 50X), 30% Acr-bis, TEMED were purchased from Shanghai Biyun Tian Biotechnology Co; GADPH, AChE, chAT, phospho-IκBα, NF- κ B p65, phospho-NF- κ B p65, available from Abcam Technology.

Experimental animals: 180C 57BL/6 mice weighing 20-25 g were supplied by Shanghai SLAC laboratory animal Co. The ethical number is PZSHUTCM220613006. All animals were served with enough food and water, were housed in a well lit conditioned room (25.+ -. 1 ℃) and maintained for 12 hours of light and dark cycles (lights on from 7:00 to 19:00).

3. Compound Munich Cuminum its prescription macroporous resin purification process experiment

3.1, experimental methods

3.1.1 preparation of macroporous resin sample solution

Weighing 13 medicinal materials according to a prescription proportion, wherein the peganum harmala, fennel root bark and fennel fruit are 120g, the other medicinal materials are 60g, sieving the mixture with a No. 1 sieve, adding 70% ethanol with the mass of 5 times of the medicinal materials for fully leaching the medicinal materials, then adding 70% ethanol with the mass of 8 times of the medicinal materials for reflux extraction for 1h, extracting for 2 times, filtering, combining the filtrates, concentrating under reduced pressure until no alcohol taste exists, and adding ultrapure water until the concentration of the crude drug is 0.3 g.mL ^-1 Concentrate at 8000 r.min ^-1 Centrifuging for 10min, and collecting supernatant to obtain macroporous resin sample solution. Storing at-20deg.C for use to prevent deterioration of medicinal liquid.

3.1.2 pretreatment with macroporous resin

After 6 kinds of resins including H20, H60, HPD100, LSA-5B, D and AB-8 were soaked in 95% ethanol for 24 hours, the resins were washed with 2BV HCl (5%) and NaOH (2%) in sequence and soaked for 2 hours respectively, then washed with ultrapure water until the pH was neutral, and the resins were stored in absolute ethanol for use. Before use, the ethanol in the resin is replaced by ultrapure water, and the resin can be loaded and adsorbed after the flushing liquid has no smell of ethanol.

3.1.3 static adsorption experiments

3.1.3.1 screening of macroporous resin type

In a 100mL Erlenmeyer flask, 1g of each of the 6 resins treated was added. 30mL of an aqueous sample solution (0.3 g crude drug mL) was added ^-1 ) Oscillating for 24 hours at 30 ℃, weighing before oscillation, and compensating for weight loss after oscillation. Measuring HAL, HAR, GA and Sie.A content in the liquid medicine after adsorption equilibrium by HPLC, and measuring total by ultraviolet spectrophotometryThe flavone content was calculated as adsorption rate and adsorption amount. Then 6 kinds of resin after adsorption are respectively added into a conical flask filled with 30mL of 90% ethanol, the mixture is oscillated for 24 hours at 30 ℃, the mixture is weighed before oscillation, the mixture is complemented after oscillation, the supernatant is taken, the content of each component is measured according to the method, and the desorption rate and the desorption amount are calculated.

Adsorption amount= (C ₀ -C ₁ ) V/m; adsorption rate= (C ₀ -C ₁ )/C ₀ ；

Desorption amount=c ₂ V/m; desorption rate=c ₂ /(C ₀ -C ₁ )。

Wherein C is ₀ : the concentration of each component in the sample loading liquid; c (C) ₁ : the concentration of each component in the solution after adsorption equilibrium; c (C) ₂ : the concentration of each component in the desorption liquid; v: the volume of the adsorption liquid and desorption liquid; m: the mass of the macroporous resin.

Wherein HPLC determination is based on the following references (Wei Yue, cheng Juanjuan, cheng Xuemei, et al. Uyghur drug compound Munich cumin granule quality control method study [ J ]]Journal of pharmaceutical analysis, 2017,37 (10): 1799-1809.) for improvement, specific methods: the chromatographic column is Diamond Plus C ₁₈ -a (250 x 4.6mm,5 μm); acetonitrile (A) -ammonium acetate buffer salt (B) is selected as a mobile phase, the column temperature is 30 ℃, and the gradient elution process is 0-10 min:19% of A, 10-20 min: 19-30% of A, 20-35 min:30% A; the flow rate is 1 mL/min ^-1 The ELSD evaporator tube and drift tube temperatures were 50 c, the gas flow rate was 1.6, and the gain value was set to 8.

The ultraviolet spectrophotometry measurement is based on the following references (Zhao Xiang, ring culvert, cheng Xuemei, etc.) the compound Munich cumin alcohol extraction process optimization [ J ]. Chinese patent, 2023,45 (03): 907-910).

3.1.3.2 influence of pH value of sample solution on adsorption Rate

1g of D101 resin was weighed out, and 30mL of an aqueous sample solution (0.3 g crude drug. ML) was added to a 100mL Erlenmeyer flask ^-1 ) The pH was adjusted to 1, 3, 5 (stock solution) and 9 with 0.1M hydrochloric acid and 0.1M sodium hydroxide, respectively, and the adsorption rate was calculated by the procedure described under "3.1.3.1".

3.1.3.3 influence of the mass concentration of the sample solution on the adsorption rate of the resin

Adding 5 parts of pretreated D101 resin 1g into 100mL conical flask, and adding crude drug with pH of 5 at concentrations of 0.067, 0.15, 0.3, 0.5, and 1.0 g.mL ^-1 After the sample solution 30mL of (C) was subjected to the step of shaking adsorption under item "3.1.3.1", the content of each component was measured and the static adsorption rate was calculated.

3.1.4 dynamic adsorption experiments

3.1.4.1 influence of the sample volume flow on the adsorption of the resin

About 25g (wet weight) of the pretreated D101 resin was taken and wet packed (column volume about 40 mL). Thus, 1BV is 40mL. Will be 0.3 g.mL ^-1 Sample solutions with pH of 5 were 1, 2, 3 BV.h, respectively ^-1 Is added to the resin column, and a sample volume and a leak ratio are respectively plotted on the horizontal and vertical axes for each 40mL (1 BV) of the resin column to determine a suitable sample flow rate and volume.

3.1.4.2 Water washing volume investigation

The purpose of the water washing is to remove macromolecular impurities such as proteins, sugars, etc., so that adsorption is carried out as described above according to the determined process parameters, followed by 3 BV.h with ultrapure water ^-1 Is eluted at a flow rate of 10BV. The effluent was collected 1 time every 2 BV. The mass of solids in the wash solution was measured to determine the wash volume.

3.1.4.3, impurity removal and ethanol elution volume fraction investigation

After loading according to the above determined process parameters, eluting with 4BV ultrapure water to remove impurities, and then eluting with 3 BV.h ^-1 For eluting flow rate, sequentially eluting with 10%, 30%, 50%, 70% and 95% ethanol of 3BV, mixing the 3BV eluents of each concentration, measuring the elution amounts of alkaloid, flavone and saponin components, calculating the elution amounts of three components in the ethanol eluents of different concentrations, taking the ethanol concentration as the abscissa, taking the elution amount as the ordinate, and drawing a gradient elution curve of the ethanol of different concentrations. To determine the elution volume fraction of the purified and eluted ethanol.

3.1.4.4 investigation of the amount of eluted ethanol

After loading according to the above-identified process parameters, 5BV of 10% ethanol was used to remove the impurities, and then 70% ethanol was used to elute 5BV, and 1 part was collected every 1BV. The content of alkaloid, flavonoid and saponin components is measured to determine the amount of ethanol eluted.

3.1.5 verification test

According to the above-identified procedure, 25g (wet weight) of the pretreated D101 resin was packed. Ph=5 of FFMN loading solution, mass concentration 0.3g·ml ^-1 The loading flow rate and the elution flow rate are both 3 BV.h ^-1 The loading volume was 11BV. After dynamic adsorption, macromolecular impurities are removed by using 5BV of 10% ethanol, and then 70% ethanol eluent is collected for 5BV, the content of alkaloid, flavone and saponin components in the eluent is measured, and the amount of ointment is measured, wherein the amount is 3 parts in parallel.

3.2 experimental results

3.2.1 static adsorption experiments

3.2.1.1 determination of type of macroporous resin

By combining static adsorption data of 6 resins, the D101 and H20 resins have better adsorption and desorption effects on alkaloids, flavones and saponins, the D101 resin has better adsorption and desorption effects on saponins than the H20 resin, and the adsorption effects on alkaloids and flavones are better than the H20 resin, but the desorption effect is slightly weaker than that of the H20 resin. In combination with the factory production cost, the D101 resin with lower price and wide application is selected for subsequent experiments. The static adsorption results are shown in tables 1 and 2.

TABLE 1 adsorption and desorption rates of different types of resins

/>

Wherein, alkaloids: the sum of the content of harmine and harmine; saponins: the sum of the content of glycyrrhizic acid and the content of the eleutheroside; flavonoids: total flavone content.

TABLE 2 static adsorption and desorption amounts of different types of resins

3.2.1.2 determination of the pH of the sample solution

When the pH value is lower, the D101 resin has better adsorption effect on flavone and saponin components, and the adsorption capacity of the resin on the flavone and saponin components is weakened along with the increase of the pH value, but the adsorption capacity of the resin on the alkaloid components is enhanced along with the increase of the pH value. When the pH=5 (stock solution) is combined, the D101 resin has good adsorption effect on the three components. Therefore, the pH was selected to be 5, i.e., the pH of the stock solution, as the pH of the loading solution. The results of the effect of pH on the adsorption rate are shown in Table 3.

TABLE 3 influence of pH on adsorption Rate

3.2.1.3 determination of the Mass concentration of the sample solution

When the concentration of the sample loading liquid is from 0.067g.mL ^-1 Raise to 1 g.mL ^-1 When the concentration of the sample solution is increased, the adsorption rates of the alkaloid and the saponin components are gradually reduced, and the adsorption rate of the flavonoid components is increased along with the increase of the concentration of the sample solution, when the mass concentration of the sample solution is more than 0.3 g.mL ^-1 After that, the adsorption rate starts to decrease instead. Therefore, according to the experimental result and the consideration of manpower and time cost in process production, the mass concentration of the sample loading liquid is finally selected to be 0.3 g.mL ^-1 . The effect of the loading solution mass concentration is shown in Table 4.

TABLE 4 influence of sample liquid mass concentration on adsorption Rate

Mass concentration/(g.mL) ^-1 )	Total alkaloid/(%)	Total flavone/(%)	Total saponins/(%)
				0.067	100	24.82	100
0.15	100	31.07	100
				0.3	67.56	57.29	100
0.5	52.57	15.38	60.43
				1	46.88	13.99	44.67

3.2.2 dynamic adsorption experiments

3.2.2.1 determination of the flow rate of the sample fluid

Through earlier investigation, it was determined that HAL is the first leaking component in HAL, HAR, sie.a and GA. Therefore, when the concentration of the HAL in the effluent liquid after being adsorbed by the macroporous resin reaches 10 percent of the concentration of the HAL in the sample liquid, the adsorption of the total alkaloids and the total saponins can be determined to be saturated; similarly, the loading amount of the total flavonoids in the adsorption saturation is determined according to the standard of the HAL adsorption saturation. The results show that the loading flow rate has less influence on alkaloid, flavone and saponin components in FFMN, so that the maximum loading flow rate, namely 3 BV.h, is selected for saving time cost ^-1 . When the flow rate is 3 BV.h ^-1 When HAL leaks at 21BV, flavonoid component starts to leak at 11BV, so that the optimal loading flow rate is 3 BV.h ^-1 The maximum loading was 11BV. The leakage curves for HAL and total flavonoids are shown in figure 1.

3.2.2.2 determination of the Water washing volume

The water washing result showed that most of the macromolecular impurities were removed when the water washing volume was 4BV, and thus the water washing volume was determined to be 4BV. The results of solids removal for different water wash volumes are shown in figure 2.

3.2.2.3 determination of volume fraction of impurity-removed and eluted ethanol

From the results, it was found that 10% ethanol had little elution effect on the three components, and therefore 10% ethanol was selected instead of ultrapure water for impurity removal. In addition, 70% ethanol can elute almost most of the target components, and thus, the elution curves of the respective components are shown in fig. 3 using 70% ethanol as an eluting solvent.

3.2.2.4 determination of the amount of eluted ethanol

The elution test result shows that the elution amount of each component gradually increases with the increase of the amount of the eluting ethanol, and when the elution volume is 5BV, the cumulative elution rate of the three components is close to 100%, so that the optimal amount of the eluting ethanol is 5BV. The 70% ethanol elution profile is shown in figure 4.

3.2.2.5, optimum process

The optimal process is that the pH of the loading solution=5,the flow rate was 3 BV.h ^-1 The mass concentration is 0.3 g.mL ^-1 The loading volume of FFMN is 11BV, after dynamic adsorption, 5BV of 10% ethanol is used for removing impurities, and finally 5BV of 70% ethanol is used for preparing 3 BV.h ^-1 Eluting at the flow rate of the solution to obtain the final product.

3.2.2.6 verification test

The results of the verification test show that the total alkaloid desorption rate is 74.15%, the total saponin desorption rate is 83.28%, the total flavone desorption rate is 92.2%, the extract yield is reduced from 18.08g to 2.32g before purification, the purity of the total alkaloid is improved by 5.80 times, the purity of the total saponin is improved by 6.52 times, the purity of the total flavone is improved by 7.20 times (when the FFMN alcoholic extract is not enriched by the optimal process, the purities of the alkaloid, the saponin and the flavone in sequence are 2.59%,1.05% and 10.75%, and the purities of the alkaloid, the saponin and the flavone in sequence are 15.02%, 6.85% and 77.38% after the FFMN alcoholic extract is enriched by the optimal process), so that the purification process is stable, the repeatability is good, and the separation and purification effects on the total alkaloid, the total flavone and the total saponin components in the compound Munich cumin the compound woody cumin are better. The results of verifying the content of each component in the total part obtained by the above "3.2.2.5" best process after macroporous resin enrichment in the test are shown in table 5.

TABLE 5 content of various Components in the Total site after enrichment

4. Activity experiment of each part of compound Munich Cuminum cyminum

4.1, experimental methods

4.1.1 preparation of different parts of FFMN

As is known from "3.2.2.1", the optimum flow rate is 3 BV.h ^-1 The flavone leakage rates were 9% and 14% for 10BV and 12BV respectively, and 9.6% and 13.2% for 21BV and 22BV respectively. Thus the flavonoid component is in11BV leaks, while alkaloid components leak at 21 BV. The macroporous resin enrichment process determines the loading amount to be 11BV from the angle of reducing the leakage of flavonoid components. In the process of exploring and separating all parts, the polarities of the flavone and alkaloid components are near, the flavone and alkaloid components are doped with each other, the flavone and alkaloid components cannot be eluted by 10% ethanol, and the flavone and alkaloid components can be eluted by 30% ethanol simultaneously. When the flavone part is to be separated, the content of alkaloid and saponin components in the flavone part is reduced as much as possible, and the separation principle of other parts is the same. Therefore, the total flavone part can be prepared according to the leakage sequence of the flavone and alkaloid components, so that the flavone in the effluent liquid absorbed by the macroporous resin (for example, the flavone leaked from the 11BV sample when the FFMN alcohol extract is enriched, the effluent liquid absorbed by the macroporous resin is the flavone leaked from the allowable leakage range of the enrichment process, and therefore, the flavone in the total part after enrichment) is more. Most of the total saponins are eluted at 40% -70% ethanol concentration. Therefore, when alkaloid, flavone and saponin parts are separated, the enrichment process of the FFMN alcoholic extract is slightly improved, the loading amount is increased to 14BV, the rest loading conditions are unchanged, and effluent liquid (without alkaloid and saponin components) containing the flavone components after being adsorbed by macroporous resin is collected and used for enriching the flavone components. After adsorption of the sample liquid, eluting with 10BV 30% ethanol, 9BV 40% ethanol and 3BV 70% ethanol in sequence, collecting the eluent, and detecting the content of each component respectively. According to the result of '3.1.4.3', most of the flavonoid components can be eluted by eluting 3BV with 30% ethanol. Therefore, in order to reduce the flavonoid components of the non-flavonoid part as much as possible, the first 3BV of 30% ethanol is discarded, 30%4-10BV and 40%1BV are combined as alkaloid parts according to the content of each component in each BV, and 40%4BV-70%3BV are combined as saponin parts. The alkaloid and saponin parts are rotated until no alcohol smell exists, and then are frozen at-80 ℃, and freeze-dried powder is prepared by a freeze dryer, so that the total alkaloid and total saponin parts are obtained and are stored at-20 ℃. Centrifuging the flavone effluent, collecting the sample solution, loading for adsorption, removing impurities with 5BV 10% ethanol, eluting with 30% ethanol, collecting eluate, rotating until no ethanol smell, freezing at-80deg.C, and lyophilizing And (3) obtaining the total flavone part by freeze-drying, and storing at-20 ℃. The flow chart is shown in fig. 5.

4.1.2 preparation of solutions

(1) Configuration of 0.5% CMC-Na solution: weighing 5g of CMC-Na powder, dissolving in 1L of ultrapure water, and heating by ultrasonic until the powder is completely swelled to obtain the final product.

(2) Configuration of 5% BSA: about 2.0g of BSA powder was weighed, 40mL of PBST (1X) was added, and the mixture was sonicated to dissolve completely, thereby obtaining the BSA powder, which was stored at-20 ℃.

(3) Configuration of 5x electrophoretic fluid: weighing 30.2g of Tris, 10g of SDS, 188g of glycine in a 2L beaker, adding 2L of ultrapure water, and carrying out ultrasonic treatment until the solution is completely dissolved to obtain the compound.

(4) Preparation of 10x transfer solution: weighing 60.6g of Tris, 288g of glycine in a 2L beaker, adding 2L of ultrapure water, and performing ultrasonic treatment until the solution is completely dissolved to obtain the product.

(5) Configuration of 10% APS solution: about 100mg of APS powder was weighed out respectively, and 1mL of ultrapure water was added to 10 1.5mL centrifuge tubes, and the mixture was completely dissolved to obtain the APS powder, which was stored at-20 ℃.

4.1.3 configuration of antibodies

(1) GADPH (1:5000): 6 mu L of GADPH antibody stock solution is taken and placed in 29.995mL of PBST (1 x) solution containing 0.5% BSA, and the mixture is shaken until the mixture is clear, thus obtaining the GADPH antibody stock solution which is preserved at the temperature of minus 20 ℃.

(2) NF- κ B p65 (1:1000): taking 10 mu L of NF-kappa B p65 antibody stock solution in 9.99mL of PBST (1 x) solution containing 0.5% BSA, shaking uniformly until the solution is clear, and preserving at-20 ℃.

(3) p-NF- κ B p65 (1:1000): taking 10 mu L of p-NF-kappa B p antibody stock solution in 9.99mL of PBST (1 x) solution containing 0.5% BSA, shaking uniformly until the solution is clear, and preserving at-20 ℃.

(4) p-IκBα (1:1000): taking 10 mu L of p-IκBalpha antibody stock solution, shaking uniformly in 9.99mL of PBST (1 x) solution containing 0.5% BSA until the solution is clear, and preserving at-20 ℃.

(5) ChAT (1:1000): taking 10 mu L of ChAT antibody stock solution in 9.99mL of PBST (1 x) solution containing 0.5% BSA, shaking until the solution is clear, and preserving at-20 ℃.

(6) AChE (1:1000): taking AChE antibody stock solution 10 μl in 9.99mL of PBST (1 x) solution containing 0.5% BSA, shaking until clear, and preserving at-20deg.C.

4.1.4 preparation of the medicament

(1) FFMN different site solutions: and (3) taking a proper amount of lyophilized powder of a total part (EXT), total Alkaloids (ALK), total Flavonoids (FLA) and total Saponins (SAP) after enrichment of FFMN macroporous resin, and suspending with a proper amount of CMC-Na with the concentration of 0.5% to prepare an EXT group (low dose: L, medium dose: M and high dose: H), an ALK group (L, M and H dose), a FLA group (L, M and H dose) and an SAP group (L, M and H dose).

(2)0.5mg·mL ^-1 Donepezil Ji Rongye: taking 4 pieces of donepezil hydrochloride, grinding into fine powder by using a mortar, and completely dissolving by 40mL of 0.5% CMC-Na to obtain the finished product.

(3)0.1mg·mL ^-1 Scopolamine hydrobromide solution: accurately weighing scopolamine hydrobromide 50.0mg, adding 500mL physiological saline, and dissolving completely by ultrasound.

4.1.5 grouping and administration of animals

180 mice were randomly divided into 15 groups of 12, i.e. control group (CON, 0.5% CMC-Na), scopolamine group (SCO, 1 mg. Kg) ^-1 ) Donepezil group (DON, positive control, 5 mg.kg) ^-1 ) EXT-L, M and H dose groups, ALK-L, M and H dose groups, FLA-L, M and H dose groups and SAP-L, M and H dose groups. The total alkaloid content in ALK part, the total flavone content in FLA part and the total saponin content in SAP part are consistent with the corresponding component content in EXT. Each of EXT, ALK, FLA and SAP was administered with the corresponding concentration of drug 7 days prior to the water maze (MWM) test, except for the control and model groups which were intragastrically 0.5% CMC-Na. From day 8 to day 16, all groups were intraperitoneally injected with SCO 0.5h after dosing, except for the control group. Subsequently, behavioral tests were performed daily 0.5h after intraperitoneal injection of SCO or 0.9% NaCl. The experimental schedule is shown in fig. 6. The doses administered are shown in table 6.

TABLE 6 dosing amounts

Part(s)	Low dose/mg.kg ^-1	Medium dose/mg.kg ^-1	High dose/mg.kg ^-1
				Total part	28	48	80
Total alkaloids	18	30	50
				Total saponins	10	16	27
Total flavone	100	175	292

4.1.6 Morris Water maze test

4.1.6.1, adaptive training

On day 8 after dosing, an adaptation training was performed, with no platform in the water maze. The training purpose is to familiarize mice with the environment of a water maze, after injecting SCO into the abdominal cavity of each group of mice for 30min, putting the mice facing the pool wall into water, keeping the operation of each mouse as consistent as possible, enabling the mice to swim freely for 1min, and then fishing out the mice and wiping the mice.

4.1.6.2 visual platform experiment

On day 9 after dosing, a visual platform experiment was performed with the platform placed in the middle of the northeast quadrant (NE) of the water maze 1cm above the water surface. The training purpose is to let mice familiar with the position of the platform, after SCO is injected into the abdominal cavity of each group of mice for 30min, water is respectively injected from SE, SW and NW directions, and each mouse is trained for 3 times, and each time is 1min. If the mouse does not find the platform within 1min, slowly guiding the mouse to swim to the platform, and staying on the platform for 15s; if the platform is successfully found within 1min, the platform is allowed to stay on for 15s. After swimming, the mice are required to be wiped dry in time to ensure good state.

4.1.6.3, positioning cruising experiment

On days 10-14 after dosing, a positioning cruise test was performed with the platform also placed in the middle of the NE quadrant, 1cm below the water surface. Also, after SCO was intraperitoneally injected for 30min, the mice were each trained 3 times for 1min from SE, SW, NW orientations, respectively. If the mouse does not find the platform within 1min, slowly guiding the mouse to swim to the platform, and staying on the platform for 15s; if the platform is successfully found within 1min, the platform is allowed to stay on for 15s. The positioning and cruising experiment needs to analyze blank and model group data in time so as to judge whether the modeling is successful or not.

4.1.6.4 space exploration experiment

On day 15 post-dosing, a space exploration experiment was performed, and the platform was removed. Mice were water-injected in the NE quadrant, and the behavior trace of the mice was observed for 1min, and the experiment was performed 1 time.

4.1.6.5 sample collection

On day 16 post-dose, anesthesia with isoflurane followed by CO ₂ The mice were choked and their brains were removed rapidly. 9 brain tissues were randomly selected for each group and divided into left and right cerebral cortex and left and right hippocampus. These tissues were snap frozen with liquid nitrogen and immediately stored at-80℃for ELISA and Western blot experiments. 3 mice were randomly selected from each group, and after taking the whole brain, the whole brain was washed with pre-chilled physiological saline, stored in 4% paraformaldehyde, and fixed for 24 hours for Nib staining and immunofluorescence experiments.

4.1.6.6 protein concentration determination

The principle of BCA (Bicinchoninic acid) method is biuret reaction, which is widely used for measuring protein concentration, and has the advantages of simplicity, rapidness and stability.

(1) Using BSA protein mother liquor (2 mg. ML) ^-1 ) Targets, 0,0.1,0.2,0.4,0.6,0.8,1,2 mg.mL, arranged in sequence in a series of concentration gradients ^-1 。

(2) And preparing proper amounts of A liquid and B liquid according to the ratio of 50:1 to obtain BCA working solution.

(3) 20. Mu.L of each concentration standard and a sample of the appropriate concentration (typically diluted 10 times) were added to a 96-well plate to rapidly accelerate 200. Mu.L of BCA working fluid, with a water bath at 37℃for 30min.

(4) After cooling to room temperature, absorbance was measured at 562 nm.

(5) Standard curves were drawn from different concentrations of standard. The protein concentration of the samples was calculated from the standard curve.

4.1.6.7 Western Blot experiments

(1) Preparation of a loading sample: the protein concentration of the sample to be measured was determined according to the BCA method, the loading was set at 20 μg, the volume required to prepare one sample was v=20 μg/sample protein concentration, 4 μl of 5x SDS-PAGE was added, the sample was made up to 20 μl with RIPA, the prepared sample was boiled at 95 ℃ for 15min, cooled and stored at-80 ℃ for measurement.

(2) Preparation of 8% separation gel: 4 gel blocks were prepared, 40mL total, 19mL of ultrapure water, 10.6mL of 30% acrylamide, 10mL of 4 XTris-SDS gel (pH=8.8), 0.4mL of 10% APS, 24. Mu. LTEMED were added, the first three were mixed uniformly, the last two were added to avoid air bubbles as much as possible, then the mixture was injected into a gel-making tank, the gel was sealed with absolute ethyl alcohol, and polymerization was carried out at room temperature for about 30 minutes.

(3) Preparation of concentrated glue: 4 gel pieces were prepared, a total of 12mL, 6.84mL of ultrapure water, 2.04mL of 30% acrylamide, 3mL of 4 XTris-SDS separating gel (pH=6.8), 0.12mL of 10% APS, 12. Mu. LTEMED were used, and after the same procedure as in (2), the absolute ethanol on the separating gel was poured out, the remaining absolute ethanol was sucked dry with filter paper, the concentrated gel was poured in, and a 15-hole comb was slowly inserted therein so as not to generate bubbles, and the polymerization time was about 30 minutes.

(4) Electrophoresis: transferring the gel block into a vertical electrophoresis tank, taking out the comb, sucking 20 μl of each sample into the hole with a pipetting gun, adding the electrophoresis tank into the electrophoresis apparatus, adding 1x electrophoresis solution, and covering the cover (red to red and black to black). Setting electrophoresis parameters, S1 (80V, 30 min), S2 (120V, 120 min), starting electrophoresis, stopping electrophoresis when the blue line reaches a proper position, and transferring to pre-cooled film transfer liquid for standby.

(5) Transferring: the PVDF membrane was cut to the appropriate dimensions (5X 8 cm), and immediately before use, activated with methanol for 3min for use. Making a horizontal electrophoresis sandwich, placing a black foam pad, thick filter paper, glue-PVDF film, thick filter paper and black foam pad in sequence under a negative basal plane, avoiding air bubbles as much as possible when making the sandwich, and finally clamping by using a white clamp. The "sandwiches" were placed in a transfer tank (red to red, black to black), 1x transfer solution was poured, and an ice bag was added to the electrophoresis apparatus to maintain low temperature, and the transfer apparatus was covered with ice. Parameters are set, S1 (100 v,120 min), the switch is pressed, and film transfer is started.

(6) Closing: PVDF membrane (after completion of transfer) was placed in a 5% BSA blocking solution and placed on a decolorizing shaker at about 10rpm and blocked at room temperature for about 1h.

(7) Incubating primary antibodies: taking out the closed PVDF membrane, cutting the membrane according to the molecular weight of the target protein, placing the membrane into a 15mL centrifuge tube, adding a proper amount of corresponding primary antibody diluent, and placing the membrane in a refrigerator at 4 ℃ for overnight incubation at a speed of about 40rpm.

(8) Incubating a secondary antibody: the strips were removed and rinsed with PBST (1X) for 10min, repeated 3 times at approximately 80rpm. The strips were then placed in secondary antibodies and incubated on a shaker for 1h at room temperature at approximately 40rpm. After incubation, the incubation was repeated 3 times, again with PBST (1X) for 10min at approximately 80rpm.

(9) Development and analysis: taking out the strip, putting the strip into a developing solution (A solution: B solution=1:1), fully soaking for 10s, taking out the strip, and taking a photo by using a Tanon chemiluminescent imager (pre-cooling to-20 ℃ before use). The photo is saved in 8Bits format while the photo is taken under white light. The pictures were processed with ImageJ software and gray values were calculated.

4.1.6.8 Biochemical index determination

(1) Preparation of homogenate: taking out the hippocampus and cortex on the same side from-80 ℃, precisely weighing, adding 9 times of PBS (1 x) according to mass, adding grinding steel balls, using a high-flux tissue grinder to grind 60Hz and lower Wen Yunjiang s, centrifuging for 10min at 15000xg and 4 ℃, and taking supernatant.

(2) Measurement of biochemical indexes: the content or activity of AChE, chAT, superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor alpha (TNF-alpha), nitric Oxide (NO), myeloperoxidase (MPO) and brain derived trophic factor (BDNF) is determined according to the instruction of the kit.

4.1.6.9 Nishi dyeing

(1) Washing, dewatering, transparentizing, wax dipping and embedding: the fixed brain tissue was carefully removed, washed with ultrapure water to remove the fixing solution and its crystals, and then dehydrated with absolute ethanol. The dehydrated brain tissue was immersed in xylene until it became transparent, and then the brain tissue was immersed in a mixture of paraffin and xylene at 1:1 for 2 hours. The brain tissue was immersed with paraffin liquid for 3h, this operation was performed 2 times, and this operation should be performed in an oven at 3 ℃ above the melting point of paraffin. After the wax liquid on the surface layer of the brain tissue is cooled, the brain tissue is quickly transferred into cold water to finish embedding.

(2) Slicing, sticking and baking: cutting the embedded wax block into regular quadrangular tables, adhering the regular quadrangular tables to a small wood table by using a small amount of hot wax liquid, clamping the regular quadrangular tables by using a wax block clamp, and slicing the regular quadrangular tables with a blade parallel to the section of the wax block; the sections were approximately 5 μm thick, transferred to paper with a brush pen, and fixed flat on a glass slide with proteoglycerol. After the filter paper absorbs moisture, the filter paper is placed in an oven for drying, and the temperature is 45 ℃.

(3) Dewaxing and hydration: the dried sections were dewaxed and hydrated in the following order, with xylene I and II each 15 min-absolute ethanol I and II each 5min-85% and 75% ethanol each 5 min-distilled water wash 5min.

(4) Dyeing and running water immersion cleaning: after taking out the water on the slice, circling the tissue edge by using a grouping pen, dripping toluidine blue on the slice, and soaking the slice by using running water after placing the wet box for 15 seconds.

(5) Dewatering and sealing: the sections were immersed in 70% ethanol for about 3s, followed by a dehydration operation, immersing in 70%, 80% and 90% ethanol for 3s, respectively, treating with absolute ethanol I and II for 1min, and xylene I and II for 2min, respectively. The dehydrated slice is dripped on the tissue by using center gum, and a cover glass is taken and gradually covered at one end, thus obtaining the tissue.

(6) Photographing: the treated sections were photographed under a microscope, and brain tissue pictures were taken with a 100x and 400x microscope in sequence.

4.1.6.10 immunofluorescent staining

(1) Washing, dewatering, transparentizing, wax dipping, embedding, slicing, pasting, baking, dewaxing and hydrating operations are the same as "4.1.6.6".

(2) Antigen retrieval: the sections were placed in citric acid (ph=6.0) and repaired in a microwave oven. Sequentially stopping the middle fire for 8min and stopping the middle fire for 7min. After cooling, the mixture was placed in PBS and washed with shaking on a decolorizing shaker for 5min, and repeated 3 times.

(3) Fluorescence quenching: circling the tissue edge with a organizing pen, then adding an autofluorescence quenching agent, quenching for 5min, and washing for 10min.

(4) Closing: blocking with BSA was performed for 30min.

(5) Incubating primary antibodies: after removal of the blocking solution, an appropriate amount of AChE antibody diluent (1:1000) was added, placed in a wet box and incubated overnight at 4 ℃.

(6) Incubating a secondary antibody: after the sections were washed 3 times with PBS on a decolorizing shaker, secondary antibodies were added and incubated for 1h in the dark.

(7) DAPI counterstaining nuclei: after the sections were washed 3 times with PBS on a destaining shaker, DAPI dye was added and incubated at room temperature in the dark for 10min.

(8) Sealing piece: washing the slices with PBS for 3 times on a decolorizing shaking table, and sealing the slices with anti-fluorescence quenching sealing tablets.

(9) Photographing: sections were observed with a fluorescence microscope and photographed. DAPI counterstained nuclei were blue under uv laser and expressed positively as red.

4.2 experimental results

4.2.1 Morris water maze behavioural experiment results of each part of FFMN

The results of the effects of EXT, ALK, FLA and SAP sites on SCO-induced memory acquired impairment model mouse behaviours are shown in table 7, table 8, table 9 and fig. 7. In the course of the C57BL/6 mouse water maze positioning navigation experiment, the average swimming distance and the upper incubation period of each dose group of mice show a gradually shortened trend along with the increase of training days. On days 4, 5 of the pilot cruising experiment, the swimming distance and platform finding time (i.e. the platform latency) of the SCO mice were significantly increased compared to the control group, with significant statistical differences (P < 0.001). Among the three groups given EXT-L, M, H by intragastric administration, significant advantages were demonstrated on day 4 of the pilot cruising experiment, with statistical differences in the swimming distance and the upper latency of the mice compared to the model group (P <0.05, P <0.01 or P < 0.001). Mice in the M and H dose groups of ALK, FLA and SAP showed significant differences (P <0.05, P <0.01, or P < 0.001) on day 4 or 5, respectively, and the L dose group showed a trend of shortening of swimming distance and ascending latency with increasing drug treatment and training days. However, they were not statistically different from the model group (P > 0.05). Furthermore, there was no significant difference in swim speed between the experimental groups, indicating that swim speed did not interfere with the assessment of learning and memory.

In the last day of space exploration experiments, EXT, ALK, FLA and SAP sites had a significant increasing effect on the number of board passes of SCO-induced memory impaired mice. The number of times that the SCO group mice passed through the original platform region was significantly reduced compared to the control group, with statistical differences (P < 0.001). There was a significant increase in the number of passings (P <0.05, P <0.01 or P < 0.001) for both EXT and FLA in the L, M and H dose groups compared to the SCO group, and a dose dependency was shown. In addition, ALK and SAP groups showed a significant increase in number of passings (P <0.05 or P < 0.01) for both M and H dose groups, except for the L dose group, compared to the SCO group, and exhibited a certain dose dependence. In combination with the experimental results, EXT, ALK, FLA and SAP parts have obvious improving effect on learning and memory of mice with SCO-induced dysmnesia models.

At approximate dose, for swimming course index, SAThe P part is obviously superior to other groups, namely, the total saponins are added in high dosage (27 mg.kg) ^-1 ) The average swimming distance (5.59 m) in the fifth day is the same as that in EXT part, and the dosage is 50 mg.kg ^-1 Left and right; ALK site is required to achieve a comparable effect, and the dose is 40 mg.kg ^-1 Left and right; the FLA part is expected to achieve equivalent effect, and the administration dosage is 280 mg.kg ^-1 Left and right.

TABLE 7 influence of FFMN and parts on the swimming distance of C57BL/6 mice (mean.+ -. SD)

Wherein, compared with the control group, ^## P<0.01 and ^### P<0.001； ^* p compared with the model group<0.05； ^** P compared with the model group<0.01； ^*** P compared with the model group<0.001。

Under the approximate dosage, aiming at the index of the upper stage latency, the SAP part is obviously superior to other groups, namely, the SAP part is used for treating the diabetes mellitus with high dosage of total saponins (27 mg.kg) ^-1 ) The average stage latency (19.24 s) on the fifth day is the same as that on the EXT site, and the dose is 50 mg.kg ^-1 Left and right; ALK site is required to achieve a comparable effect, and the dose is 40 mg.kg ^-1 Left and right; the FLA portion is required to achieve a comparable effect, and the dosage is 280 mg.kg ^-1 Left and right.

TABLE 8 influence of FFMN and parts on the stage latency of C57BL/6 mice (mean.+ -. SD)

/>

Wherein, compared with the control group, ^## P<0.01 and ^### P<0.001； ^* p compared with the model group<0.05； ^** P compared with the model group<0.01； ^*** P compared with the model group<0.001)。

Under the approximate dosage, aiming at the index of the number of times of crossing the platform, the SAP part is obviously superior to other groups, namely, the total saponins are used in high dosage (27 mg.kg) ^-1 ) The EXT part is required to achieve a corresponding effect based on the number of crossing platforms (3.20 times), and the administration dosage is 40 mg.kg ^-1 Left and right; ALK site is required to achieve a comparable effect, and the dose is 30 mg.kg ^-1 Left and right; the FLA portion is required to achieve a comparable effect, and the dosage is 280 mg.kg ^-1 Left and right.

TABLE 9 influence of FFMN and parts on spatial exploration of C57BL/6 mice (mean.+ -. SD)

Group of	Dosage/mg.kg ^-1	Number of passes through the platform (times)
			Blank group	/	3.50±0.97
Model group	/	1.20±1.14 ^###
			Positive medicine group	5	3.50±0.71 ^***
High total part	80	3.80±1.03 ^****
			In the total part	48	3.10±1.10 ^**
Low total part	28	2.80±140 ^*
			High total alkaloid content	50	3.30±0.95 ^***
Among total alkaloids	30	3.10±1.20 ^**
			Low total alkaloids	18	2.40±0.84
High total flavone content	292	3.20±1.32 ^**
			Among the total flavonoids	175	3.10±1.29 ^**
Low total flavone content	100	2.60±1.17
			High total saponins	27	3.20±1.14 ^**
Among the total saponins	16	2.80±1.23 ^*
			Low total saponins	10	2.30±1.06

Wherein, compared with the control group, ^### P<0.001； ^* p compared with the model group<0.05； ^** P compared with the model group<0.01； ^*** P compared with the model group<0.001。

4.2.2 effects of FFMN and different sites on protein expression in the cortex

Protein expression of AChE, chAT, NF-kappa B p65, p-NF-kappa B p65 and p-Ikappa B alpha in mouse cortex is detected by using a Western blot technique. The results of AChE and ChAT protein expression show that compared with the control group, the SCO injection has obviously increased AChE expression in the cortex of SCO group mice, and obviously reduced ChAT protein expression (P < 0.0001). Indicating that the injection of SCO into the abdominal cavity causes cholinergic abnormality of the mice; whereas after EXT (L, M and H doses), ALK (M and H doses) and SAP (H doses) were given, AChE expression was significantly reduced (P <0.05, P <0.01, or P < 0.001) and dose-dependent, while the protein expression of AChE in the FLA panel cortex and SCO panel were not significantly different (P > 0.05). Both the EXT, ALK, FLA and SAP H dose groups enhanced the expression of the ChAT protein in the cortex (P <0.05, P <0.01, or P < 0.001). Protein expression results of NF- κ B P65, P-NF- κ B P65 and P-IκBα showed no significant difference (P > 0.05) between NF- κ B P65 protein expression and blank in each cortex after intraperitoneal injection of SCO. The protein expression of the SCO group p-NF-kappa Bp65 and p-I kappa Balpha is obviously increased, and after the SCO is injected into the abdominal cavity, I kappa Balpha phosphorylation in the brain of a mouse is caused, NF-kappa B p65 is released, nuclear translocation is caused, and NF-kappa B signal channels are activated to cause neuroinflammation. Protein expression of P-NF- κ B P65 and P-IκBα was reduced (P <0.05, P <0.01, or P < 0.001) following treatment with EXT, FLA and SAP groups. However, there was no significant difference (P > 0.05) in protein expression of P-NF- κ B P65 and P-IκBα after treatment in ALK group compared to SCO group. The results of protein expression are shown in FIG. 8.

4.2.3 effects of FFMN and different parts thereof on biochemical indicators in cortex or Hippocampus

Using ELISA kits we tested the activity or content of EXT, ALK, FLA and SAP in FFMN on cholinergic system-related enzymes (AChE, chAT), inflammatory factors (MPO, TNF- α and NO), oxidative stress factors (SOD, MDA) in the cortex or hippocampus of SCO-induced memory impairment model mice.

To elucidate the effect of FFMN and its different sites on cholinergic function of mouse brain tissue, the activity of AChE and ChAT in mouse brain cortex and hippocampus was assessed. After SCO (1 mg/kg) injection, the AChE activity level of cortex and Hippocampus was significantly improved and ChAT activity was significantly decreased (P < 0.0001) compared with the control group. Treatment with EXT (M and H doses), ALK (L, M and H doses), FLA (H doses) and SAP (M and H doses) significantly attenuated increases in AChE levels in cortex and Hippocampus (P <0.05, P <0.01, or P < 0.001). The EXT ALK, FLA and SAP-H dose groups significantly increased the ChAT activity of the cortex and hippocampus (P <0.01, P <0.001, or P < 0.0001). At the same time, ALK-M dose groups also increased the ChAT activity in the hippocampus (P < 0.05). The DON group also significantly reduced the levels of AChE in the cortex and hippocampus and significantly increased the activity of ChAT (P <0.05, P <0.01, respectively).

BDNF plays a bridging role in neurogenesis and neuroinflammation as an important role in promoting synaptic growth to prevent neurodegeneration. To further verify the mechanism by which FFMN modulates neuroinflammation through NF- κ B p65 signaling pathway, quantitative analysis of mouse cortex or hippocampal pro-inflammatory cytokines and inflammatory mediators (TNF- α, NO and MPO) and BDNF was performed. Compared with the control mice, the pro-inflammatory cytokines and inflammatory mediators of the cortex or hippocampus of the SCO mice are significantly increased, and BDNF is significantly decreased (P <0.05, P <0.01 or P < 0.001). After treatment with FFMN at different concentrations, levels of TNF- α, NO and MPO were significantly reduced in brain tissue and BDNF levels were significantly increased (P <0.05, P <0.01, P <0.001, or P < 0.0001) compared to SCO groups. Consistent with the Western blot results, ALK improves inflammatory factors downstream of NF- κB signaling pathway with less capacity than FLA and SAP.

To elucidate the effect of FFMN and its different sites on oxidative stress in mouse brain tissue, the effect of FFMN on MDA and SOD levels and activity in the cerebral cortex was assessed. SOD activity was significantly reduced in the cortex of SCO mice compared to control, while MDA levels were significantly increased. After DON and FFMN treatment, SOD activity is obviously increased, MDA level is obviously reduced (P is less than 0.05, P is less than 0.01, P is less than 0.001, or P is less than 0.0001). The results of the effect of FFMN and its various sites on biochemical indicators in the cortex or hippocampus of mice are shown in figure 9.

4.2.4 Nile stain results

The effect of SCO-induced degeneration of hippocampal neurons was studied using nisetum staining. Pathological features such as neuronal fixation, increased cell gap and neuronal cell damage were observed in the hippocampal CA3 region of the SCO group. After FFMN treatment, SCO-induced lesions are reduced and pathological lesions are ameliorated. In addition, the normal neuronal cell numbers in the CA3 region of the hippocampus of the SCO group were significantly reduced compared to the control group (P < 0.001). However, the number of normal neuronal cells increased following treatment of the FFMN components compared to the SCO group, indicating that it can alleviate pathological changes and protect neuronal cells. The results of the Nile staining are shown in FIG. 10.

4.2.5 AChE immunofluorescence results

The expression of AChE in the mouse cortex was studied by immunofluorescence experiments, and compared with the blank group, AChE protein expression in the SCO group mouse cortex was significantly increased (P < 0.0001). Treatment with EXT (M and H doses), ALK (M and H doses), FLA (H doses) and SAP (M and H doses) significantly attenuated AChE protein expression in the cortex (P < 0.05, P < 0.01, or P < 0.001). The DON group also significantly reduced AChE protein expression in the cortex (P < 0.001). The results of immunofluorescence are shown in FIG. 11.

The foregoing has outlined rather broadly the more detailed description of embodiments of the invention in order that the detailed description of the principles and embodiments of the invention may be implemented in conjunction with the detailed description of embodiments of the invention that follows. Meanwhile, based on the idea of the present invention, those skilled in the art can make changes or modifications on the specific embodiments and application scope of the present invention, which belong to the protection scope of the present invention. In view of the foregoing, this description should not be construed as limiting the invention.

Claims

1. The application of the total extract of the compound woody cumin and/or the extraction part of the compound woody cumin in preparing the medicine for preventing and/or treating the dysmnesia.

2. Use of a pharmaceutical composition comprising a total extract of compound manicure cumin and/or an extraction site of compound manicure cumin in the manufacture of a medicament for the prevention and/or treatment of a memory disorder.

3. The use according to claim 2, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients;

preferably, the auxiliary material is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, slow release agents, retention aids, lubricants, dispersants, plasticizers, opacifiers and antioxidants;

More preferably, the dosage form of the pharmaceutical composition is a tablet, a drop pill, a capsule, a powder, an injection, a film, a lozenge, a granule or an oral liquid.

4. The use according to claim 2, characterized in that the pharmaceutical composition further comprises one or more drugs and/or extracts for preventing and/or treating memory disorders;

preferably, the drug is a cholinesterase inhibitor, a glutamate receptor antagonist and/or mannite;

more preferably, the cholinesterase inhibitor is donepezil, rivastigmine and/or galantamine;

still preferably, the glutamate receptor antagonist is memantine.

5. The use according to any one of claims 1 to 4, wherein the memory disorder is dementia, alzheimer's disease, brain trauma, cerebral ischemia and/or stress-induced memory disorders;

preferably, the dementia is senile dementia, vascular dementia or dementia with lewy bodies;

more preferably, the extraction site is a total alkaloid site and/or a total saponin site.

6. The use according to claim 5, wherein the total extract of the compound manicure and/or the extraction site of the compound manicure is prepared by a process comprising the steps of:

(3) Loading: precisely weighing a proper amount of medicinal materials, sieving, extracting, reflux-extracting, filtering, combining filtrate, concentrating under reduced pressure until no alcohol smell exists, adding water to a certain crude drug concentration, centrifuging, and collecting supernatant to obtain macroporous resin sample liquid, allowing the sample liquid to pass through the first macroporous resin adsorption column according to a certain flow rate so that the sample liquid is adsorbed on the first macroporous resin adsorption column, and optionally collecting the sample liquid which is not adsorbed to obtain leakage liquid; and

7. The method of claim 6, wherein in step (1), the macroporous resin is an H20 resin, an H60 resin, an HPD100 resin, an LSA-5B resin, a D101 resin, or an AB-8 resin;

preferably, the macroporous resin is D101 resin or H20 resin;

more preferably, the concentration of the ethanol solution is about 95%, and the soaking time of the ethanol solution is about 24 hours;

still preferably, the volume of the hydrochloric acid solution is about 2BV, the concentration of the hydrochloric acid solution is about 5%, and the soaking time of the hydrochloric acid solution is about 2 hours;

particularly preferably, the volume of the sodium hydroxide solution is about 2BV, the concentration of the sodium hydroxide solution is about 2%, and the soaking time of the sodium hydroxide solution is about 2 hours;

particularly preferably, the water is ultrapure water.

8. The method of claim 6, wherein in step (3), the medicinal materials include peganum harmala, nigella sativa, pimpinella ania, fennel root bark, chamomile, celery root, chicory seed, chicory root, citronella, moldavica dragonhead, licorice, basil seed, and hollyhock seed;

preferably, the screen is a No. 1 screen;

more preferably, the leaching is leaching the drug material with about 70% ethanol added in an amount of about 5 times the mass of the drug material;

Still preferably, the reflux extraction is reflux extracting the medicinal material with about 70% ethanol added in an amount of about 8 times the mass of the medicinal material;

still preferably, the number of reflux extractions is 2, each for about 1 hour;

particularly preferably, the crude drug concentration is about 0.3g/mL;

particularly preferably, the rotational speed of the centrifugation is 7000 r/min-9000 r/min, and the time of the centrifugation is 5 min-15 min;

particularly preferably, the rotational speed of the centrifugation is about 8000r/min and the time of the centrifugation is about 10min;

particularly preferably, the pH of the loading solution is 3 to 7;

particularly preferably, the pH of the loading solution is about 5;

particularly preferably, the mass concentration of the loading liquid is 0.15 g/mL-0.5 g/mL;

particularly preferably, the loading solution has a mass concentration of 0.3g/mL;

very particularly preferably, the flow rate of the loading solution is about 3BV/h;

particularly preferably, the loading solution has a volume of 10BV to 15BV, for example about 11BV or about 14BV.

9. The method of claim 6, wherein step (4) is replaced with the following step (4'): and (3) using 5% -15% ethanol with a certain volume to pass through the first macroporous resin adsorption column according to a certain flow rate to remove impurities, using 65% -75% ethanol to elute, and collecting eluent to obtain the total extract of the compound Munich cumin.

10. The method according to claim 6 or 9, wherein in step (4) or step (4'), the flow rate is about 3BV/h;

preferably, 3-5 BV, e.g., about 4BV, of water is used to pass through the first macroporous resin adsorption column or the second macroporous resin adsorption column at a flow rate of about 3BV/h prior to the removing of the impurities;

preferably, the concentration of the ethanol in the removal of impurities is about 10%;

more preferably, the volume of the ethanol in the removal of impurities is about 5BV;

still preferably, eluting with about 30% ethanol, about 40% ethanol, and about 70% ethanol at a flow rate, and collecting the eluate to obtain the total alkaloids fraction and/or the total saponins fraction;

particularly preferably, the 4 th to 10 th BV of the eluate of about 30% ethanol and the 1 st BV of the eluate of about 40% ethanol are combined as the total alkaloid fraction;

particularly preferably, the total saponin fraction is combined with the 4 th to 9 th BV of the eluate of about 40% ethanol and the 1 st to 3 rd BV of the eluate of about 70% ethanol;

particularly preferably, about 5BV of about 70% ethanol is used for elution, and the eluent is collected to obtain a compound total extract of the Munich;

Particularly preferably, the rotation speed of the centrifugation is 7000 r/min-9000 r/min, and the time of the centrifugation is 5 min-15 min;

particularly preferably, the rotational speed of the centrifugation is about 8000r/min and the time of the centrifugation is about 10min.