CN115837040B - Application of ligusticum chuanxiong hort extract in preparation of medicine for preventing and treating cognitive dysfunction - Google Patents

Abstract

The invention discloses an application of a ligusticum chuanxiong hort extract in preparing a medicine for preventing and treating cognitive dysfunction, and belongs to the field of medicines, wherein the ligusticum chuanxiong hort extract contains 10-20% of senkyunolide J, 10-20% of senkyunolide N and 40-60% of senkyunolide I by mass fraction; the ligusticum chuanxiong hort extract has a remarkable improvement effect on short-term memory disorder, long-term memory disorder, learning disorder and space memory disorder of model animals; in addition, the ligusticum wallichii extract disclosed by the invention has stability and safety obviously superior to those of ligusticum wallichii volatile oil; compared with pure senkyunolide I, the method has the advantages that the improvement effect on cognitive dysfunction is obviously improved, separation and purification of monomer compounds are not needed, and the method is easier to obtain and lower in cost.

Description

Application of ligusticum chuanxiong hort extract in preparation of medicine for preventing and treating cognitive dysfunction

Technical Field

The invention relates to an application of a ligusticum chuanxiong hort extract in preparing a medicine for preventing and treating cognitive dysfunction, and belongs to the field of medicines.

Background

Cognition is the process of receiving external information by human brain, converting the external information into intrinsic psychological activities through processing, thereby obtaining knowledge or applying knowledge, and comprises the aspects of memory, language, vision space, execution, calculation, understanding judgment and the like. Cognitive disorders refer to diseases in which one or more of the above cognitive functions are impaired and affect the daily or social abilities of an individual.

Vascular cognitive disorders are clinical strokes or subclinical vascular lesions caused by cerebrovascular lesions and their risk factors, involving at least one clinical syndrome of impaired cognitive domain. It encompasses all forms of cognitive disorders ranging from mild to severe vascular cognitive disorders, as well as mixed dementias that incorporate other pathologies such as Alzheimer's disease.

Severe vascular cognitive impairment, also known as vascular dementia, refers to hypoperfusion in the brain regions such as memory, cognition, behavior, etc., caused by ischemic stroke, hemorrhagic stroke, cerebrovascular disease, etc., characterized by memory loss, cognitive impairment, and cerebrovascular disease. The incidence rate of vascular dementia is only inferior to that of Alzheimer disease, the incidence rate of people in China is 1.1-3.0%, and the annual incidence rate is 5-9/1000 people.

Post-stroke cognitive impairment refers to a clinical syndrome characterized by cognitive impairment that occurs after a stroke event and persists for up to 6 months, including cognitive impairment caused by stroke events such as multiple infarction, critical-part infarction, subcortical ischemic infarction, and cerebral hemorrhage, as well as cognitive impairment caused by degenerative brain disorders such as Alzheimer's disease that progress within 6 months after stroke. Cognitive dysfunction occurs in 50% -70% of patients after stroke, and can be classified into non-dementia and post-stroke dementia according to the severity of cognitive impairment.

Brain trauma mainly refers to traumatic brain injury, including craniocerebral injury caused by traffic accidents, high falls, sports, war, etc. The mechanism of cognitive dysfunction caused by brain trauma is complex, and factors such as neurotransmitter release, free radical surge, inflammatory storm, calcium mediated injury, mitochondrial dysfunction and coagulation dysfunction can cause secondary injury of organisms, and bad results such as cognitive dysfunction, dyskinesia, sensory dysfunction, emotional instability and the like are often caused.

There is no recognized specific medicine for treating the cognitive disorder diseases at home and abroad. The current clinical application drugs mainly comprise cholinesterase inhibitors (donepezil, rivastigmine, galantamine and the like), NMDA receptor antagonists (memantine), nimodipine, butylphthalide, brain active agents, ginkgo biloba extract, calf blood deproteinized extract and the like. Butylphthalide (NBP) is a natural component extracted from celery seed, and can significantly improve microcirculation and blood flow in cerebral ischemia area. The synthesized butylphthalide racemate (trade name: endopril) is mainly used for treating the light and medium ischemic stroke. Clinical studies in recent years show that butylphthalide can delay the course of vascular dementia patients caused by cerebrovascular diseases and improve the overall cognitive function and life ability of the patients.

The traditional Chinese medicine also plays a unique role in preventing and treating cognitive dysfunction. Ligusticum wallichii is the dry rhizome of Ligusticum wallichii Ligusticum chuanxiong Hort of Umbelliferae, has the effects of promoting blood circulation, activating qi-flowing, dispelling wind and relieving pain, and has high use frequency in treating cognitive dysfunction and related diseases in traditional Chinese medicine. The research shows that the volatile oil is one of main pharmacodynamic substance groups showing the efficacy of the ligusticum wallichii, and has the functions of relieving inflammatory injury, anticoagulation, inhibiting platelet aggregation, dilating blood vessels and the like. The volatile oil of rhizoma Ligustici Chuanxiong contains ligustilide and senkyunolide A, and also contains small amount of butylphthalide, butenyl phthalide, neocnidilide, etc.

At present, more researches on the activity of the ligusticum chuanxiong volatile oil are reported, but the ligusticum chuanxiong volatile oil is difficult to prepare medicines due to the problems of unstable physicochemical properties, potential safety risks and the like.

In addition, phthalides are a class of compounds found in ligusticum wallichii that have been shown to be active in cardiovascular, blood and smooth muscle. Among them, butylphthalide, which has been studied more, has many activities such as anti-cerebral ischemia and protecting brain trauma tissue, and can be considered as one of the substance bases of the efficacy of Ligusticum chuanxiong. Therefore, more reports on the application of phthalide compounds in ligusticum wallichii in preparing medicines for preventing and treating cerebral apoplexy are available at present. Among them, the application of senkyunolide H (CN 102144999A), senkyunolide I (CN 102144998A) and senkyunolide J (CN 102144997A) in preventing and treating cerebral apoplexy is reported in the beginning of the research center of modern preparation technology of Zhangjiang traditional Chinese medicine in Shanghai.

However, these reports all require complicated separation and purification steps to prepare the related monomer compounds, and have complicated processes and high cost; moreover, it was confirmed that the effect of senkyunolide I was better than that of senkyunolide J under the same test conditions from the viewpoint of effect, but there may be room for further improvement in the effect.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and the ligusticum chuanxiong hort extract prepared by adopting a specific method, which is safe, effective and stable in property, is used for preparing the medicine for treating cognitive dysfunction.

The invention adopts the technical scheme that: the application of the ligusticum wallichii extract in preparing a medicament for preventing and treating cognitive dysfunction, wherein the sum of mass fractions of senkyunolide J, senkyunolide N and senkyunolide I in the ligusticum wallichii extract is more than or equal to 60 percent.

A large number of experiments show that the ligusticum chuanxiong extract prepared by adopting a specific preparation method and containing specific components and content has obvious improvement effects on short-term memory disorder, long-term memory disorder, learning disorder and space memory disorder of model animals, and is expected to be prepared into medicines for preventing and treating cognitive disorder.

As a preferable technical scheme: the mass fraction of senkyunolide J in the ligusticum wallichii extract is 10% -20%, the mass fraction of senkyunolide N is 10% -20%, and the mass fraction of senkyunolide I is 40% -60%.

Those skilled in the art will appreciate that: the ligusticum wallichii is taken as a raw material, and a specific extraction, transformation and purification process is adopted, so that the proportion of each main component in the obtained extract has a floating range, and 70-130% of the change is generally acceptable for Chinese medicine.

As a preferable technical scheme: the mass ratio of senkyunolide J to senkyunolide N in the ligusticum wallichii extract is 1:1.

as a preferred technical scheme, the preparation method of the ligusticum chuanxiong hort extract comprises the following steps of:

(1) Reflux extracting rhizoma Ligustici Chuanxiong crude drug or decoction pieces with 5-15 times of ethanol with volume concentration of 50% -75% for at least 1 time, each time for at least 0.5 hr, mixing extractive solutions, concentrating under reduced pressure, dispersing the obtained extract with water, extracting with ethyl acetate, mixing extractive solutions, concentrating under reduced pressure to obtain ethyl acetate extract;

(2) Adding the ethyl acetate extract obtained in the step (1) into ethanol, uniformly stirring, dissolving potassium hydrogen peroxymonosulfate composite salt, adjusting the pH value to be 5.5-6.5, adding the solution into the solution, and stirring at room temperature for at least 6 hours for reaction;

(3) Filtering the reaction liquid in the step (2), removing ethanol from the filtrate under reduced pressure, extracting the residual water liquid with ethyl acetate, merging ethyl acetate extracts, concentrating under reduced pressure, adding water into the extract, separating by column chromatography, eluting, and removing the solvent from the eluent under reduced pressure.

The extract prepared by the specific method is analyzed by the inventor, and contains senkyunolide J and senkyunolide N which are optical isomers, wherein the senkyunolide N has the following structure:

and the inventor analysis tests show that the content of the two components is about 1:1.

As a further preferred technical scheme: in the step (3), the column chromatography is a reversed phase C18 silica gel column, and is sequentially eluted with 10% ethanol and 50% ethanol by volume concentration, and the 50% ethanol eluted part is collected.

As a preferable technical scheme: the cognitive disorder comprises vascular cognitive disorder, cognitive disorder after stroke and cognitive disorder caused by brain trauma.

As a preferable technical scheme: the cognitive disorder includes vascular dementia.

As a preferable technical scheme: the cognitive disorder comprises post-stroke cognitive disorder non-dementia and post-stroke dementia.

Compared with the prior art, the invention has the advantages that: experimental results show that the ligusticum chuanxiong hort extract disclosed by the invention has remarkable improvement effects on short-term memory impairment, long-term memory impairment, learning impairment and space cognition impairment of cognition dysfunction animals; compared with the ligusticum chuanxiong hort volatile oil, the ligusticum chuanxiong hort extract disclosed by the invention has better stability and safety; compared with pure senkyunolide I, the method has the advantages that the improvement effect on cognitive dysfunction is obviously improved, separation and purification of monomer compounds are not needed, and the method is easier to obtain and lower in cost.

Drawings

FIG. 1 is an HPLC chart (C18 column separation) of the Ligusticum wallichii extract of example 1;

FIG. 2 shows senkyunolide J ¹ H NMR spectrum (solvent: deuterated DMSO);

FIG. 3 shows senkyunolide N ¹ H NMR spectrum (solvent: deuterated DMSO);

FIG. 4 is an HPLC chart (chiral column separation) of the Ligusticum wallichii extract of example 1;

FIG. 5 is a flow chart of a new object identification experiment of example 3;

FIG. 6 is a neurobehavioral assessment experimental timeline for example 3;

FIG. 7 shows the short term new object identification index (P <0.01 compared to Model group);

FIG. 8 shows long-term new object identification index (compared to Model group, # P <0.01; compared to QBT and NBP groups, # P <0.01; compared to QBT and SI groups, # P < 0.05);

fig. 9, 10, 11 and 12 show the time course of the water maze escape latency (P <0.01 compared to Model group);

FIGS. 13 and 14 show the number of water maze passes and the effective quadrant ratio (P <0.05 compared to Model group, < P <0.01; comparison between QBT and NBP groups, #P <0.05; comparison between QBT and SI groups, #P < 0.05);

FIG. 15 is a graph showing the protective effect of Model rat neurons (compared to Model group, # P <0.01 compared to between NBP and QBT groups);

FIG. 16 shows the stability of Ligusticum wallichii extract (senkyunolide I, J and N) compared to Ligusticum wallichii volatile oil (ligustilide, senkyunolide A).

Detailed Description

The invention will be further described with reference to the accompanying drawings.

Example 1 preparation and detection of Ligusticum chuanxiong Hort extract

(1) Reflux-extracting rhizoma Ligustici Chuanxiong decoction pieces with 10 times of 70% ethanol (v/v) for 2 times each for 1 hr. Mixing the extractive solutions, concentrating under reduced pressure, dispersing the obtained extract with water, extracting with ethyl acetate, mixing the extractive solutions, and concentrating under reduced pressure to obtain ethyl acetate extract.

(2) 20g of ethyl acetate extract and 200ml of 95% ethanol were added to a 1000ml flask and stirred well. 80g of potassium hydrogen peroxymonosulfate was dissolved in 300ml of water, and the pH was adjusted to 6 by adding sodium carbonate, then the mixture was added to the flask, and the mixture was stirred at room temperature for 16 hours.

(3) The reaction solution was filtered, ethanol was removed from the filtrate under reduced pressure, and the remaining aqueous solution was extracted with ethyl acetate (100 ml. Times.3). The ethyl acetate extracts were combined, concentrated under reduced pressure, and the extracts were separated by reverse phase silica gel chromatography (phi 40 mm. Times.300 mm) after water addition, eluting with 1500ml of 10% ethanol followed by 1500ml of 50% ethanol. Collecting 50% ethanol eluate, and removing solvent under reduced pressure to obtain rhizoma Ligustici Chuanxiong extract (QBT) 5.8g. HPLC detection results show that the mass fraction of senkyunolide J in the prepared Ligusticum wallichii extract is 13.2%, the mass fraction of senkyunolide N is 13.2%, and the mass fraction of senkyunolide I is 48.5%.

(4) Conventional HPLC detection conditions: the column was a Waters BEH C18 (50 mm×2.1mm i.d.,1.7 μm); the mobile phase is acetonitrile (A) -0.1% formic acid (B); gradient elution is carried out for 0 to 0.5min (20 percent A), 0.5 to 3.0min (20 percent A to 70 percent A), 3.0 to 3.5min (70 percent A to 100 percent A) and 3.5 to 4.0min (100 percent A); the flow rate is 0.4mL/min; column temperature is 35 ℃; the detection wavelength is 215nm (senkyunolide J, senkyunolide N), 280nm (senkyunolide I), and the results are shown in figure 1,

(5) Chiral HPLC detection is carried out on the senkyunolide J and senkyunolide N mixed components in the figure 1, and the detection conditions are as follows: the chromatographic column is Chiralpak AS-3column (100×4.6mm,3 μm); the mobile phase is isopropanol-n-hexane (20:80, v/v); the flow rate is 1.0mL/min; column temperature is 30 ℃; detecting wavelength 215nm (senkyunolide J, senkyunolide N), chiral separating to obtain nuclear magnetic resonance hydrogen spectra of senkyunolide J, senkyunolide N as shown in figure 2 and figure 3, and determining peak positions of senkyunolide J, senkyunolide N as shown in figure 4;

as can be seen from FIGS. 1 and 4, the mass fraction of senkyunolide J in the prepared Ligusticum wallichii extract is 13.2%, the mass fraction of senkyunolide N is 13.2%, and the mass fraction of senkyunolide I is 48.5%.

Example 2 rat model and dosing regimen

(1) Experimental animals: SPF-grade SD rats, male, weighing 260+ -20 g, from Fukang Biotech Co., ltd., license number: SCXK (jing) 2019-0008. The ambient temperature is maintained at 24+/-1 ℃, the relative humidity is 50% -70%, and the water and food are drunk and ingested freely in 12/12h day and night alternation.

(2) And (3) establishing a model: a rat 2VO model was prepared according to the method of document [ Acta Pharmacol Sin,2019,40 (4): 425-440 ]. SD rats were anesthetized by intraperitoneal injection of 300mg/kg of 10% chloral hydrate and sterilized for sterile surgery. The median carotid incision, the bilateral common carotid artery was isolated, the bilateral common carotid artery was ligated with 5-0 surgical knots, and the incision was sutured. The Sham group isolated only the common carotid artery but not ligature.

(3) Grouping animals: the water maze hiding platform experiment is carried out for 14 days after operation for 5 continuous days, the average time required by each rat to reach the hiding platform in the acquisition phase of 5 continuous days is calculated, the average time of each 2VO rat is set as V1, the average time of all rats in the Sham group is set as V2, and screening standards (screening criterion, SC) are selected as indexes for evaluating cognitive impairment of each ischemic rat: sc= (V1-V2)/V1. The models were randomly grouped after screening, and they were classified into Sham group (Sham operation), model group (Model), NBP group (butylphthalide, 54 mg/kg), QBT group (Ligusticum wallichii extract obtained in example 1, 54 mg/kg), SI group (senkyunolide I,54 mg/kg), and n=12 for each group.

(4) Dosing regimen: butylphthalide (NBP) was purchased from the stone pharmaceutical group Enpride pharmaceutical Co., ltd. Senkyunolide I (SI) was purchased from Chengdu Poisson Biotechnology. After grouping, the administration was performed by gavage at a volume of 1mL/100g, and an equal volume of physiological saline was administered once daily to the Sham group and the Model group for 28 consecutive days.

Example 3 neurobehavioral evaluation-New object identification experiment

(1) In the new object recognition experiment, a normal rat can take more time to search for a new object, and if the cognitive function of the rat is damaged, the time for searching for the new object and the old object is not different. The new object recognition experiment is divided into four stages, namely a adaptation stage, a training stage, a test I stage and a test II stage [ Antioxid Redox Signal,2014,21 (4): 533-550 ]. The specific operation is as follows:

the adaptation period is as follows: the rats were placed in a new object recognition box 24h in advance for free movement for 10min.

Training period: 2 identical objects are placed in parallel at the bottom of the box body, and rats are explored in the box body for 5min.

Test phase i: after 3 hours from the end of the training period (one object in the box is replaced by an object with different color and shape but equivalent size), the rats were explored in the box for 5 minutes.

Test phase II: the end of test I period was performed for 24 hours (one object in the box was replaced with a different color shape but a comparable size) and rats were explored in the box for 5 minutes.

Data recording and analysis: recording and analyzing the time (T1) for exploring new objects and the time (T2) for exploring old objects of each rat, and substituting the time into a cognitive index calculation formula: noi=t1/(t1+t2) ×100% to obtain the cognitive indices NOI (3 h) and NOI (24 h) for test phase i and test phase II, respectively.

The new object identification experimental flow is shown in fig. 5, and the neuro-behavioral evaluation experimental time line is shown in fig. 6.

(2) The cognition index of short-term test (3 h) is shown in FIG. 7, and the new object recognition index of the Sham group rats is 61.62 +/-7.98%, and the new object recognition index of the Model group rats is reduced to 39.47+/-5.23% (P < 0.01), which indicates that the short-term memory of the Model group rats is obviously reduced. Compared with the Model group, the new object recognition index of the NBP group rat is 51.44 +/-6.60% (P < 0.01), the QBT group is 55.25+/-3.11% (P < 0.01), and the SI group is 53.89+/-6.81% (P < 0.01). The ligusticum chuanxiong hort extract disclosed by the invention can obviously improve the short-term memory of a model rat.

(3) The cognitive index of the long-term test (24 h) is shown in FIG. 8, and the new object recognition index of the Sham group rats is 52.69+/-8.38%, and the new object recognition index of the Model group rats is reduced to 34.06+/-8.16% (P < 0.01), which indicates that the long-term memory of the Model group rats is obviously reduced. Compared with the Model group, the new object recognition index of the NBP group rat is 45.47+/-8.05% (P < 0.01), the QBT group is 56.49 +/-8.01% (P < 0.01), and the SI group is 48.27 +/-7.52% (P < 0.01). The results show that the ligusticum chuanxiong hort extract disclosed by the invention can obviously improve long-term memory of model rats and improve cognitive dysfunction, and the QBT group effect is superior to that of NBP group (P < 0.01) and SI group (P < 0.05).

Example 4 neurobehavioral evaluation-Water maze experiment

(1) Morris water maze (Morris water maze, MWM) was used to evaluate the learning and memory capacity of rats. In the experiment, the diameter of the MWM womb is 160cm, the height of the MWM womb is 60cm, the diameter of the safety platform is 12cm, the water level is 1.5cm higher than that of the safety platform, and the water temperature is 23+/-1 ℃. The uterus of the MWM is divided into 4 quadrants, namely a first quadrant, a second quadrant, a third quadrant and a fourth quadrant, the third quadrant is used as a target quadrant, a safety platform is placed in the central position of the quadrants, and the position of the platform is kept unchanged in the whole experimental process. MWM experiments were performed in two stages, the first stage being performed 14 days after 2VO surgery, to assess the extent of cognitive impairment in each ischemic rat. In the first stage, a water maze hiding platform experiment is carried out for 5 continuous days, the average time required by each rat to reach the hiding platform in the acquisition stage of 5 continuous days is calculated, the average time of each rat with 2VO is set as V1, the average time of all rats in a Sham group is set as V2, and a screening Standard (SC) is selected as an index for evaluating cognitive impairment of each ischemic rat: sc= (V1-V2)/V1. If SC > 20%, cognitive impairment is considered. The ratio of 20% < SC less than or equal to 30% is mild dementia, 30% < SC less than or equal to 40% is moderate dementia, and SC > 40% is severe dementia [ university of Chinese medical science, university, 2002,31 (3): 166-167 ]. The second phase was performed on days 41-46 after 2VO surgery and the experiment was divided into two parts: latent platform experiments and space exploration experiments.

(2) Latent platform experiment: each rat was trained in two fixed quadrants at the same time each day, each morning and afternoon, for 5 consecutive days. The time 120s is measured, and the rat stays on the safe platform for more than 10s, if the rat is considered to find the platform, and if the rat does not find the safe platform within 120s, the rat is guided to stay on the platform for 10s. The escape latency is the time it takes the rat to find the platform, and if not, it is noted as 120s.

As shown in fig. 9, 10, 11 and 12, the latency time for four groups of rats to find the platform was reduced with the increase of the training time of rats, and the latency time was the shortest at the 5 th day of training, indicating that the four groups of rats obtained a certain learning and memory ability during training. Model rats found a hidden platform for 5 days in training period all had significantly increased latency time (P < 0.01) compared to Sham group; the latency time for NBP, QBT and SI rats to find the hidden platform 4 days after the training period was significantly reduced (P < 0.01) compared to Model group.

(3) Space exploration experiment: the platform was removed from the pool on day 6, rats were watered from the opposite quadrant of the quadrant where the safety platform was located, the time was measured for 120s, and the number of rat passes (the number of passes over the area where the platform was located) and the effective quadrant rate (the proportion of total time 120s for the time of the quadrant where the platform was located) were recorded.

The results are shown in FIG. 13 and FIG. 14, wherein the number of times of crossing of the Sham group rats is 6.8+ -1.7 times, and the effective quadrant rate is 47.3+ -7.8%; compared with the Sham group, the number of times of crossing of the Model group rats is obviously reduced to 3.3+/-1.7 times (P < 0.01), the effective quadrant rate is also reduced to 34.3+/-9.2% (P < 0.01), and the memory of the Model group rats on the space position of the safety platform is obviously reduced. The number of passes and effective quadrant rates were 5.2.+ -. 1.8 and 42.1.+ -. 10.6, respectively, for the NBP group, which were significantly different (P < 0.05) compared to the Model group. The number of passes and effective quadrant rates of QBT groups were 7.3+ -2.8 and 51.6+ -10.4, respectively, with significant differences (P < 0.01) compared to Model groups. The number of crossings and effective quadrant rates of rats in the SI group were 6.1+ -2.0 and 47.8+ -11.1%, respectively, which were significantly different from those in the Model group (P < 0.01). The experimental result shows that the model rat lavage QBT can obviously improve the cognitive dysfunction and the spatial learning and memory capacity of the model rat, and the effect is better than NBP and SI.

EXAMPLE 5 evaluation of nerve tissue pathology

(1) Brain tissue was taken after the rat was sacrificed, immunofluorescent stained, observed with a fluorescent microscope and pictures were taken for analysis. The results are shown in FIG. 15, where the number of neurons in the CPu region was significantly less in the Model group rats than in the Sham group (P < 0.01) after 2VO surgery, indicating that 2VO caused severe neuronal damage. The number of neuronal cells in the brain tissue CPu region of rats in the NBP group, QBT group and SI group was significantly greater than that in the Model group (P < 0.01), indicating that administration of NBP, QBT and SI by gavage had a protective effect on neuronal damage in the Model rat CPu region. Furthermore, the CPu zone neuronal cell numbers were significantly increased (P < 0.01) in the QBT group compared to the NBP group, indicating that the neuronal protection effect of QBT was superior to NBP.

Example 6 data analysis

Experimental data analysis used SPSS 26.0. Experimental data are expressed in mean±sd. When grouped into two groups, independent sample t-test was employed. When not less than three groups are grouped, single-factor analysis of variance is adopted, LSD test is adopted when the variances are uniform, and Tamhane's T2 test is adopted when the variances are not uniform. Statistical differences were expressed as P < 0.05.

Example 7 acute toxicity test

SPF-grade ICR mice, available from Chengdu laboratory animal Co., ltd, were each half and half, and were used as laboratory animal production license number SCXK 2020-030. The volatile oil of rhizoma Ligustici Chuanxiong is self-made and contains ligustilide 52.4% (w/w) and senkyunolide A22.1% (w/w). The test drug is injected through the tail vein, and the toxic reaction condition of the mice is observed and recorded.

The results show that the mice begin to die after single injection of NBP 80mg/kg, and the mice survive after the dose is reduced to 70 mg/kg; the death rate of mice after 100mg/kg injection of the ligusticum chuanxiong volatile oil is about 40%; mice did not die after 300mg/kg QBT injection, and continuous observation for 14 days had no significant effect on mice weight gain compared to the blank group, and no significant difference in weight change (P > 0.05) compared to the blank group. Thus, the maximum tolerance of QBT is >300mg/kg. The above results show that QBT has significantly better safety than Ligusticum chuanxiong volatile oil and NBP.

Example 8 stability test

Respectively taking the volatile oil of Ligusticum chuanxiong and QBT in proper amounts, placing into a 25ml transparent volumetric flask, dissolving with diluted ethanol, and diluting to scale to obtain 2mg/ml solution. The volumetric flask containing the solution was placed under room temperature natural light and sampled and tested on days 0, 5, 10, 20, 30, respectively. As shown in FIG. 16, the changes of senkyunolide J, senkyunolide N and senkyunolide I in QBT solution are less than 5%, while the changes of ligustilide and senkyunolide A in volatile oil solution of rhizoma Ligustici Chuanxiong are significantly degraded, and the peak area is reduced by more than 10%. The results show that the stability of QBT is obviously better than that of Ligusticum chuanxiong volatile oil.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (5)

1. An application of a ligusticum chuanxiong hort extract in preparing a medicament for preventing and treating cognitive dysfunction is characterized in that: the sum of the mass fractions of the senkyunolide J, the senkyunolide N and the senkyunolide I in the ligusticum wallichii extract is more than or equal to 60 percent, wherein the mass fraction of the senkyunolide J in the ligusticum wallichii extract is 10 to 20 percent, the mass fraction of the senkyunolide N is 10 to 20 percent, the mass fraction of the senkyunolide I is 40 to 60 percent,

the preparation method of the ligusticum chuanxiong hort extract comprises the following steps:

(1) Reflux extracting rhizoma Ligustici Chuanxiong crude drug or decoction pieces with 5-15 times of 70% ethanol for at least 1 time and 0.5 hr each time, mixing extractive solutions, concentrating under reduced pressure, dispersing the obtained extract with water, extracting with ethyl acetate, mixing extractive solutions, concentrating under reduced pressure to obtain ethyl acetate extract;

(2) Adding the ethyl acetate extract obtained in the step (1) into ethanol, uniformly stirring to obtain an ethanol solution, dissolving potassium hydrogen peroxymonosulfate composite salt in water, adjusting the pH value to be 5.5-6.5, adding the solution into the ethanol solution, and stirring at room temperature for at least 6 hours for reaction;

(3) Filtering the reaction liquid in the step (2), removing ethanol from the filtrate under reduced pressure, extracting the residual water liquid with ethyl acetate, combining ethyl acetate extracts, concentrating under reduced pressure, dispersing the extract with water, and separating by column chromatography, wherein the column chromatography is a reversed phase silica gel chromatographic column, eluting with 10% ethanol and 50% ethanol by volume in sequence, collecting the 50% ethanol elution part, and removing the solvent under reduced pressure.

2. Use according to claim 1, characterized in that: the mass ratio of senkyunolide J to senkyunolide N in the ligusticum wallichii extract is 1:1.

3. use according to claim 1, characterized in that: the cognitive disorder comprises vascular cognitive disorder, cognitive disorder after stroke and cognitive disorder caused by brain trauma.

4. Use according to claim 1, characterized in that: the cognitive disorder includes vascular dementia.

5. Use according to claim 1, characterized in that: the cognitive disorder comprises post-stroke cognitive disorder non-dementia and post-stroke dementia.