Application of berberis polystachya L. steroid saponin in preparation of medicine for treating vascular dementia

Abstract

The invention discloses application of steroid saponin of trillium tschonoskii maxim to preparation of a medicine for treating vascular dementia, and relates to the technical field of medicines. Animal experiments prove that the berberis apiacea steroid saponin can improve the spatial reference learning function of cerebral ischemia rats, improve the spatial reference memory function of the cerebral ischemia rats and enhance the long-term memory function of the cerebral ischemia rats. In addition, brain tissue damage and white matter structure damage of rats suffering from cerebral ischemia can be relieved, axonal myelin sheath microstructure reconstruction and white matter fiber reconstruction of the rats suffering from cerebral ischemia are promoted, and myelin sheath regeneration of rats suffering from vascular dementia is promoted. Network pharmacological analysis finds that the age grass total steroid saponin has a wide regulation effect on OPCs and OLs cell components and molecular functions, and further, OPCs differentiation myelin sheath inhibition related factors are regulated and controlled, and white matter myelin sheath regeneration is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and relates to a vascular dementia drug, in particular to application of a top one bead steroidal saponin in preparation of a vascular dementia drug. BACKGROUND

[0002] Vascular dementia (VaD) is the second most common subtype of dementia, and its incidence increases dramatically with age, accounting for about 30% of the elderly population in China and other Asian countries. Pathological damage such as ischemic stroke, hemorrhagic stroke and cerebral ischemia and hypoxia causes cognitive impairment after VaD. Previous studies on treatment targets for vascular dementia have mainly focused on apoptosis, inflammation and autophagy of neurons and endothelial cells. However, recent studies have shown that severe damage to white matter structure is an important reason for the high incidence of VaD, which is widespread among elderly patients worldwide. Causes of cerebral infarction, cerebral hemorrhage and cerebral hypoperfusion, including cardiac arrest and anesthesia accidents, induce stenosis and obstruction of deep white matter blood vessels, causing acute white matter damage, and infarction in key areas can directly cause VaD; risk factors for VaD such as hypertension, diabetes and hyperlipidemia cause cerebral vascular lesions, causing chronic white matter damage, and showing multi-cavity and extensive white matter damage. White matter damage is an important pathological feature after VaD, and diffuse lesions in white matter are caused by occlusion of blood vessels supplied by cerebral perforating arteries, followed by demyelination and axon dysfunction. Patients with VaD sequelae have gait abnormalities and limb disabilities such as hemiplegia, and the degree of recovery is significantly related to the integrity of the brain white matter; patients with cognitive decline show more severe white matter structural abnormalities.

[0003] White matter damage disrupts signal transmission between neurons, cortex and subcortical centers, leading to white matter damage, further damaging the gray matter and distant sites of white matter fiber projections, and clinically presenting as decreased sensory motor, visual spatial, psychomotor, and associative functions. White matter adjacent to the infarction site is a key biological treatment target for preventing VaD. The course of VaD is mostly slow, with a long treatment time window, but there is currently no ideal drug or therapy to effectively improve or even reverse the progression of VaD in the later stages. Therefore, there is an urgent need to develop effective treatment drugs to protect the integrity of brain white matter, promote repair after white matter damage, and delay the progression of the disease, which undoubtedly has great clinical significance for the neurological rehabilitation of VaD patients.

[0004] Traditional Chinese medicine prevents and treats VaD by promoting blood circulation for removing blood stasis and dredging meridians. Toudingyazhu is the rhizome of Trillium tschonoskii Maxim, also known as Tu'erqi, Foshouqi and Huanghua Sanqi. There are three species of Trillium in China, including T. tschonoskii, T. govanianum wall. ex Ruyle and T. kamtschicum Pall. ex puroch. Toudingyazhu belongs to the lily family and has a bitter taste, a slightly bitter taste and a warm nature. It is mildly toxic and belongs to the liver and kidney meridians. It has the effects of promoting blood circulation, relieving pain, calming the nerves and stabilizing the mind. It is commonly used to treat dizziness, headache, lower back and leg pain, irregular menstruation, metrorrhagia, external hemorrhage, traumatic injury and external use for treating boils. Modern pharmacological studies have shown that Toudingyazhu can protect gray matter neurons and treat migraine in the acute phase. Chemical composition analysis shows that the main active ingredient of Toudingyazhu is steroidal saponin.

[0005] The present application finds that steroidal saponin of Toudingyazhu can promote the proliferation and maturation of oligodendrocyte precursor cells (OPCs) in the peripheral tissue of the ischemic infarction of rats 30 days after the model of old infarction (MCAO) and promote myelin regeneration. Based on the above research results, the application of steroidal saponin of Toudingyazhu in the preparation of a drug for treating vascular dementia is proposed. SUMMARY

[0006] The present application provides the application of steroidal saponin of Toudingyazhu in the preparation of a drug for treating vascular dementia. Based on the key link of the inhibition signal Jagged / Notch / Hes of OPC differentiation and maturation, the molecular mechanism of white matter repair after vascular dementia is studied to provide sufficient theoretical basis for providing a new idea for targeted clinical treatment of vascular dementia. It is clear that steroidal saponin of Toudingyazhu can prevent and treat vascular dementia, reduce white matter ischemic injury and promote myelin regeneration, which provides an important theoretical basis for the clinical application of steroidal saponin of Toudingyazhu.

[0007] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0008] The present application provides the application of steroidal saponin of Toudingyazhu in the preparation of a drug for treating vascular dementia.

[0009] Preferably, the steroidal saponin of Toudingyazhu at least includes Chonglou Saponin VII, PRRG and Chonglou Saponin VI, and the content of the steroidal saponin of Toudingyazhu is not less than 40%.

[0010] Preferably, the content of Chonglou Saponin VII is not less than 9%, the content of PRRG is not less than 15%, and the content of Chonglou Saponin VI is not less than 16%.

[0011] Preferably, the medicinal material of the trillin is derived from the rhizome of Trillium tschonoskii Maxim, Trillium govanianum wall. ex Ruyle or Trillium kamtschicum Pall. ex puroch of the Liliaceae Trillium genus.

[0012] Preferably, the trillin is formulated into a preparation of a clinically used medicine as a sole active ingredient or in combination with other drugs.

[0013] Preferably, the preparation of the medicine is in the form of an oral liquid, a capsule, a tablet, a powder, a pill, a granule, an atomized agent, a microemulsion, a nano preparation, a film, a paste, a tincture or an injection.

[0014] Preferably, the vascular dementia is any one of cerebral infarction, cerebral hemorrhage and cerebral tissue hypoperfusion, hypertension, diabetes and hyperlipidemia caused by abnormality of white matter structure in the brain.

[0015] Preferably, the preparation method of the trillin comprises the following steps:

[0016] Step 1: extraction of trillin

[0017] The medicinal material of trillin is taken, and the medicinal material is extracted by reflux with an ethanol solution with a volume fraction of 70% to 80%. The ethanol in the extract is recovered under reduced pressure to obtain a concentrated solution.

[0018] Step 2: extraction of trillin

[0019] The concentrated solution is extracted with petroleum ether for 3 to 5 times until the petroleum ether layer is nearly colorless. The aqueous layer solution is collected and extracted with water-saturated n-butanol for 2 to 3 times. The n-butanol reagent is recovered under reduced pressure to obtain an extracted extract.

[0020] Step 3: purification of trillin

[0021] Part of the extracted extract is taken and dissolved with water to obtain a sample solution. After enrichment through a macroporous resin column, the sample solution is eluted with 5BV of distilled water, 4 to 5BV of an ethanol solution with a volume fraction of 20% to 25%, 2 to 3BV of an ethanol solution with a volume fraction of 40% to 45% and 6 to 8BV of an ethanol solution with a volume fraction of 70% to 80%, respectively. The eluent of the part with a volume fraction of 70% to 80% ethanol is collected, and the ethanol is recovered under reduced pressure. After drying and crushing, a purified trillin powder is obtained.

[0022] Preferably, in step 1, the volume fraction of 70%~80% ethanol solution and the volume-mass ratio of the head of a pearl medicine material is 8~12 ml:1 g, the extraction times is 2~3 times, 1~1.5 h / time; the solid-liquid ratio in the concentrated solution is 2 g:2~3 ml.

[0023] Preferably, in step 2, the volume ratio of petroleum ether and concentrated solution is 2~3:2, and the volume ratio of water-saturated n-butanol and water layer solution is 2~3:2.

[0024] Preferably, in step 3, the mass of the extracted dry extract is 55~65 g, and 0.05~0.1 g of the medicine material is contained in 1 mL of the loading solution, and the flow rate of loading and elution is 0.5 mL / min.

[0025] Preferably, the macroporous resin is AB-8, D101 or HPD-100.

[0026] Preferably, the column volume of the macroporous resin is 2.5~3 L, and the diameter-height ratio is 1:5~6.

[0027] Compared with the prior art, the present scheme has the following beneficial effects:

[0028] The animal experiment proves that the steroidal saponin of the head of a pearl can improve the spatial reference learning function of cerebral ischemia rats, improve the spatial reference memory function of cerebral ischemia rats, and enhance the long-term memory function of ischemia rats. In addition, it can also reduce the brain tissue damage and white matter structure damage of cerebral ischemia rats, promote the reconstruction of axon myelin microstructure and white matter fiber of cerebral ischemia rats, and promote the myelin regeneration of vascular dementia rats. And through the cell experiment, it is verified that the steroidal saponin of the head of a pearl can up-regulate the expression levels of mature oligodendrocyte marker CNPase and myelin-related protein PLP, and also can down-regulate Notch1, Jagged1 and NICD protein expression, so as to clarify the mechanism of the steroidal saponin of the head of a pearl in treating vascular dementia, that is, the steroidal saponin of the head of a pearl inhibits the signal Jagged / Notch / Hes, so as to promote the differentiation and maturation of OPCs, and then promotes the white matter myelin regeneration. And the network pharmacology analysis also verifies this mechanism, that is, the total steroidal saponin of the head of a pearl has a wide regulation on OPCs, oligodendrocyte (OL) composition and molecular function, and further regulates the differentiation of OPCs, inhibits related factors, and promotes the white matter myelin regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1Statistical graphs of the path and related parameters of each group of rats in the water maze (A, path of the positioning navigation experiment; B, path of the quadrant exploration experiment; C, swimming distance before getting on the platform; D, swimming speed; E, swimming distance ratio and time ratio of rats in quadrant I before getting on the platform; F, platform switching experiment; G, swimming distance, distance ratio and time ratio of rats in quadrant II before getting on the platform; H, swimming distance, distance ratio and time ratio of rats in quadrant III before getting on the platform; I, swimming distance, distance ratio and time ratio of rats in quadrant IV before getting on the platform; TSTT represents a top one spirostanol saponin. Compared with the sham operation group, ## P<0.01; compared with the model group, ** P<0.01, * P<0.05);

[0030] Figure 2 Statistical graphs of T2 mapping and cerebral infarction volume of each group of rats (A, infarction area of each group of rats from the anterior fontanel to 5.0~0.6 mm; B, infarction volume statistics; C, ratio of cerebral parenchyma volume of the affected side to the healthy side; D, ratio of cerebral ventricle volume of the affected side to the healthy side; compared with the sham operation group, ## P<0.01; compared with the model group, ** P<0.01; the red dotted line represents the infarction volume);

[0031] Figure 3 Statistical graphs of the white matter brain area DTI and DTT of each group of rats (A, fractional anisotropy index (color FA); B, apparent diffusion coefficient (ADC); C, axial diffusion rate (AD); D, radial diffusion rate (RD); E, external capsule (EC) nerve fiber bundle tracing; F, internal capsule (IC) nerve fiber bundle tracing; G, H, I and J are the relative DTI numerical statistics of the external capsule, corpus callosum (CC), internal capsule and hippocampal umbrella (Fi), respectively. K and L are the relative nerve fiber length and density in the external capsule and internal capsule positions, respectively; compared with the sham operation group, ## P<0.01, # P<0.05; compared with the model group, ** P<0.01, * P<0.05; the white dotted line area represents the position of axonal myelin damage);

[0032] Figure 4 Statistical graphs of SMI32 / MBP immunofluorescence staining and integral optical density of the external capsule of each group of rats (A and B are DAPI + , SMI32 + , MBP + and SMI32 + / MBP +Immunofluorescence staining, DAPI: nuclear marker, SMI32: non-phosphorylated neurofilament heavy chain marker, MBP: myelin basic protein marker, 400x, bar=50μm; C, D and E are the relative integrated optical density statistics of SMI32, MBP and SMI32 / MBP in the affected side and the healthy side, respectively; compared with the sham operation group, ## P<0.01, # P<0.05; compared with the model group, ** P<0.01, * P<0.05;).

[0033] Figure 5 A is the myelin regeneration of cells in each group (A is PLP + Cell immunofluorescence staining; B is CNPase + Cell immunofluorescence staining; C is the number of PLP and CNPase positive cells).

[0034] Figure 6 A is the change of Notch1, Jagged1, NICD and F3 protein expression in each group of cells;

[0035] Figure 7 A is a Venn diagram (the green area is the treatment target of one bead steroidal saponin on the head; the pink area is the disease target; the gray intersection area is the target related to ischemic stroke disease in one bead steroidal saponin components);

[0036] Figure 8 A is the effective component of one bead on the head-Oligodendrocyte-potential target network (11 active saponins in one bead are from pennogenin and diosgenin, gray-green nodes represent active compounds, light pink nodes represent various cells, blue nodes represent oligodendrocyte precursor cells and oligodendrocytes, peach pink nodes represent targets);

[0037] Figure 9 A is the GO analysis of high expression target genes of OPCs;

[0038] Figure 10 A is the GO analysis of high expression target genes of OLs;

[0039] Figure 11 A is the GOBP enrichment analysis of OPCs (GOBP enrichment analysis of OPCs, showing the number of genes and P value related to each biological function item; x axis represents the number of genes enriched in each biological function, y axis represents different biological function categories);

[0040] Figure 12GOBP enrichment analysis of OLs (GOBP enrichment analysis of OLs, showing the number of genes associated with each biological function item and P value; x axis: the number of genes enriched in each biological function, y axis: different biological function classification). DETAILED DESCRIPTION

[0041] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0042] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0043] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0044] Example 1

[0045] A preparation method of a cerebrostid saponin, comprising the following steps:

[0046] Step 1: Extraction of cerebrostid saponin

[0047] Take 8 kg of cerebrostid medicinal materials (from Hubei Shennongjia, 18 g of cerebrostid medicinal materials containing 1 g of cerebrostid saponin) and use 80 L (10 times the amount) of 70% ethanol solution to reflux extract 3 times, 1.5 h / time, combine the extract, and recover ethanol under reduced pressure (65°C, 0.8 Mpa) to obtain a concentrated solution with a solid-liquid ratio of 1 g:1 ml.

[0048] Step 2: Extraction of cerebrostid saponin

[0049] Add an equal volume of petroleum ether to the concentrated solution and extract 3 times until the petroleum ether layer is nearly colorless, separate the layers, collect the aqueous layer solution, and continue to add an equal volume of water-saturated n-butanol to extract 3 times, separate the layers, collect the n-butanol extract layer, and recover the n-butanol under reduced pressure to obtain an extractive extract.

[0050] Step 3: Purification of cerebrostid saponin

[0051] Take the pretreated AB-8 resin and load it into a 5 L glass chromatography column (column volume 3.03 L, diameter-height ratio 1:6), wash with distilled water and equilibrate for standby use.

[0052] Take the extracted extract (the mass of dry matter is 60.00g), add water to dissolve, prepare into a solution containing 0.05g of medicinal materials per mL, pass through a macroporous resin column, and carry out dynamic adsorption and elution at a volume flow rate of 0.5mL / min, 5BV (5 column volumes) of distilled water are washed until colorless, the distilled water washing volume is discarded, and 5BV, 3BV, 7BV and 5BV of 20%, 40%, 70% and 95% ethanol solutions respectively are sequentially used for elution. The eluent of the part of 70% ethanol is collected, and this part is the purified Tetrastigma hemsleyanum steroidal saponin. The ethanol is recovered under reduced pressure until no alcohol smell (55℃, 0.9Mpa), and then continuously concentrated under reduced pressure (80℃, 1.0Mpa) until the extract state, and then placed in a 60℃ oven for drying, and then pulverized to obtain the purified Tetrastigma hemsleyanum steroidal saponin powder (named as TSTT) 32.40g.

[0053] The total steroidal saponin in TSTT is calculated based on the total content of Chonglou saponin VI, Pennogenin-3-O-α-L-rha-(1→4)-[O-α-L-rha-(1→2)]-O-β-D-glu (PRRG) and Chonglou saponin VII, and the HPLC detection shows that the total steroidal saponin content in TSTT is 43.88% (Chonglou saponin VI: 17.54%, PRRG: 16.29% and Chonglou saponin VII: 10.05%).

[0054] Example 2

[0055] The difference between this example and Example 1 is only that:

[0056] In Step 1, 8 times the amount of 80% ethanol solution is used for reflux extraction for 2 times, 1.5h / time; the solid-liquid ratio of the concentrated solution is 2g:3ml.

[0057] In Step 2, the volume ratio of petroleum ether to concentrated solution is 3:2, and petroleum ether is extracted for 5 times; the volume ratio of water-saturated n-butanol to water layer solution is 3:2; water-saturated n-butanol is extracted for 2 times.

[0058] In Step 3, the macroporous resin is D101, 0.1g of medicinal materials is contained in every 1ml of sample solution, 4BV, 2BV and 6BV of 25%, 45% and 80% ethanol solutions are sequentially used for elution, and the eluent of the part of 80% ethanol is collected; finally, 27.86g of TSTT is collected.

[0059] The HPLC detection shows that the total steroidal saponin content in TSTT is 46.25% (Chonglou saponin VI: 17.82%, PRRG: 16.66% and Chonglou saponin VII: 11.77%).

[0060] Example 3

[0061] The difference between this example and Example 1 is only that:

[0062] In Step 1, 2 times of 75% ethanol solution were used for reflux extraction, 1 h / time;

[0063] In Step 3, the macroporous resin was HPD-100, and 4BV of 20% ethanol solution, 2BV of 40% ethanol solution, and 8BV of 75% ethanol solution were used for elution in sequence, and the eluate of the 75% ethanol solution was collected; finally, 30.96g of TSTT was collected.

[0064] HPLC detection showed that the total content of steroidal saponins in TSTT was 44.13% (Chonglou Saponin VI: 17.61%, PRRG: 16.78%, and Chonglou Saponin VII: 9.64%).

[0065] The method for determining the content of the main components in the Top One Pearl in Examples 1-3 is as follows:

[0066] High performance liquid chromatography conditions: Agilent Poroshell C18 chromatographic column (100mm x 4.6mm, 2.7μm); mobile phase: acetonitrile (A pump) - water (B pump), elution gradient: 0-20min, 30%-50% (A is, 20-35min, 50%-100% (A is; flow rate: 0.8mL / min; injection volume: 10μL; detection wavelength: 203nm; column temperature: 30℃.

[0067] Prepare the reference solution: accurately weigh 0.00115g of Chonglou Saponin VII, 0.00104g of PRRG, and 0.00103g of Chonglou Saponin VI reference substances, respectively, dissolve in 70% ethanol, and prepare a reference solution with a concentration of 10mg / ml. Filter through a 0.45μm microporous filter, and take the filtrate. Dilute by a factor of 1.25, 1, 0.625, 0.5, 0.3125, 0.25, and 0.15625mg / ml, respectively, to prepare a reference solution with a concentration of 1.25, 1, 0.625, 0.5, 0.3125, 0.25, and 0.15625mg / ml, respectively, and store at 4℃ in a sealed refrigerator.

[0068] Prepare the test solution: accurately weigh 0.00101g of Top One Pearl steroidal saponin (powder) in triplicate, and dissolve in 0.2ml of 70% ethanol. Filter through a 0.45μm microporous filter, and take the filtrate. Inject 10μL of each of the reference and test solutions, measure the retention time and peak area of Chonglou Saponin VII, PRRG, and Chonglou Saponin VI, and calculate the content according to the standard curve method.

[0069] Application Example 1 Whole animal experiment

[0070] I. Experimental method

[0071] 1.1 Experimental animals and model preparation

[0072] MCAO cerebral ischemia animal model (model group): SPF male SD rats weighing 300-320 g were anesthetized with isoflurane inhalation (5% for induction of anesthesia, 2% for maintenance of anesthesia) and fixed supine. The rectal temperature was monitored by a temperature monitor to maintain the body temperature at (36.5±0.5) °C. The carotid artery was exposed by incision along the median line of the neck, and the bilateral carotid artery and vagus nerve were carefully separated and permanently ligated with 4-0 silk thread.

[0073] Sham operation group: the incision, approach and vessel exposure of the rats were the same as the model group, but the bilateral carotid artery was not ligated, and the same operation time was directly sutured after the same operation time, and penicillin sodium was used to prevent infection.

[0074] 1.2 Animal grouping and experimental design

[0075] After successful replication of the bilateral carotid artery ligation model, random grouping was performed according to the factorial design scheme (random number list: http: / / www.99cankao.com / numbers / random-number-generator.php.), and the grouping was as follows: sham operation group, model group and drug administration group, wherein the drug administration group included one bead of top steryl saponin group and Jin Nado group (60 mg / kg), and the one bead of top steryl saponin group included high, medium and low dose (120 mg / kg, 60 mg / kg and 30 mg / kg) groups, which were recorded as TSTT 120 mg / kg, TSTT 60 mg / kg and TSTT 30 mg / kg, respectively. Treatment was started at 24 hours after operation, and the sham operation group and the model group were given the same volume of normal saline (10 ml / kg / D) by gavage, once a day, and the body weight change and neurological score of the rats were recorded, and the indicators were detected for 30 days of continuous administration. According to the following calculation of clinical dose:d 大鼠 =d 人 x 0.71 / 0.11 (d 人 = 0.17 g / kg, d 大鼠 = 1.10 g / kg). The medium dose of TSTT prepared in Examples 1-3 was selected as 60 mg / kg / d. Therefore, the large, medium and small doses of TSTT were selected as 120 mg / kg, 60 mg / kg and 30 mg / kg, respectively, and the TSTT prepared in Example 1 was used in this application example.

[0076] 1.3 Main detection indicators and methods

[0077] 1.3.1 Neurobehavioral detection

[0078] Morris water maze test: The rats in each group were detected on the 28th-32nd day after modeling. The experiment process was divided into positioning navigation experiment and spatial exploration experiment, respectively, to evaluate the effect of a top bead steroidal saponin on the spatial learning and memory retention ability of vascular dementia rats.

[0079] 1.3.2 Magnetic resonance imaging detection

[0080] After the neurobehavioral test, magnetic resonance scanning was performed. T2 mapping detected the degree of white matter tissue structure damage: multi-slice multi-echo sequence was used, with the following parameters: 16 TE, TR=2500 ms, Flip angle=180°, FOV=3.3×3.3 cm 2 , Matrix=256×256, number of layers=5, layer thickness=1 mm. After scanning, the T2 relaxation time of the corpus callosum, external capsule and internal capsule was obtained using Paravision version 5.1 software, and the abnormal signal area of the image was analyzed using ImageJ software.

[0081] DTI detected the remodeling of brain white matter structure: axial single-shot spinecho-planar imaging sequence (EPI) was used, with the following parameters: TR=6300 ms, TE=25 ms, 30 diffusion directions, b value of 0 and 1000 s / mm 2 , Flip angle=90°, FOV=3.5×3.5 cm 2 , Number of excitation (NEX)=1, Matrix=128×128, number of layers=23, layer thickness=1 mm. Paravision version 5.1 software was used to reconstruct the data and obtain the anisotropy fraction, apparent diffusion coefficient, radial diffusion and axial diffusion values of the corpus callosum, external capsule and internal capsule. DiffusionToolkit and TrackVis software were used for fiber tract tracing (DTT).

[0082] 1.3.3 Neurological examination

[0083] Hematoxylin-eosin staining was used to observe the pathological morphological changes of the corpus callosum, external capsule and internal capsule under a light microscope; fast blue staining was used to observe the demyelination changes of the corpus callosum, external capsule and internal capsule under a light microscope, and ImageJ 8.0 was used to evaluate the demyelination area; transmission electron microscopy was used to observe the ultrastructure changes of the myelin sheath, and ImageJ 8.0 was used to analyze the G ratio=axial diameter / myelin sheath diameter and evaluate the degree of demyelination.

[0084] 1.3.4 Immunofluorescence detection of endogenous OPC proliferation and differentiation

[0085] Paraffin sections were placed in a 60°C oven for 60 minutes; deparaffinized and hydrated, washed with 0.01M PBS for 3 times, 3 minutes each time; citrate buffer (pH = 6.0-6.1) heating repair for 20 minutes, after cooling to room temperature, washed with 0.01M PBS for 2 times, 3 minutes each time; 10% goat serum blocking, 37°C incubator for 60 minutes; incubate the first antibody: rabbit anti-MBP (1:200) / mouse anti-SMI32 (1:200) 4°C for 40h; 37°C incubator for 60 minutes, 0.01M PBS for 10 minutes, a total of 4 times; incubate the second antibody: goat anti-mouse IgG / FITC (1:300) and goat anti-rabbit IgG / TRITC (1:400) 37°C incubate in the dark for 2h, 0.01M PBS for 10 minutes, a total of 6 times, distilled water for 5 minutes, a total of 3 times; DAPI mounting medium mounting, observation. Under the Nikon optical microscope: excitation / emission wavelength is 540 / 580nm, SMI32 (labeling axons of myelin fiber loss) single-labeled positive cells present red fluorescence; excitation / emission wavelength is 470 / 490nm, NG2 (labeling oligodendrocyte precursor cells), CNPase (labeling mature oligodendrocytes), MBP (labeling axonal myelin) single-labeled positive cells present green fluorescence; using NIS-Elements Basic Research image acquisition and analysis system, 40x objective lens to obtain single-labeled images of positive cells in 3 non-overlapping areas of the rat infarction lesion surrounding cortex and striatum of each section, after the system "merge" option, red and green fluorescence images are merged respectively, double-labeled positive cells show yellow fluorescence. Image analysis and statistics of SMI32 and MBP single-labeled positive cells integral optical density in both brain regions, the data is expressed as the calculated (ipsilateral / contralateral) SMI32 single-labeled positive cell integral optical density ratio, MBP single-labeled positive cell integral optical density ratio, SMI32 / MBP cell integral optical density ratio.

[0086] 1.3.5 Statistical analysis

[0087] SPSS 28.0 software (IBM Corporation, USA) was used for data processing, and the data was expressed as mean ± standard deviation (x ± SD). Repeated measures two-way ANOVA was used for neurobehavioral data, one-way ANOVA was used for other indicators, and Dunn-Bonferroni test was used for comparison between two groups; if not normal, rank sum test was used, P<0.05 was statistically significant. Mathematical modeling principal component analysis was used for comprehensive analysis of the data to find the correlation between the drug and each indicator.

[0088] II. Experimental results

[0089] 2.1 The effect of TSTT on learning and memory in cerebral ischemia rats

[0090] 2.1.1 The effect of TSTT on spatial reference learning and memory in cerebral ischemia rats

[0091] The effect of TSTT on spatial reference learning and memory in cerebral ischemia rats was investigated by water maze test. In the place navigation test, compared with the sham operation group, the distance of the model group before entering the platform increased significantly 21-24 days after ischemia (P<0.01). Compared with the model group, TSTT 60mg / kg significantly shortened the distance of the rats before entering the platform 21-24 days after ischemia (P<0.01 or P<0.05), and TSTT 120mg / kg and TSTT 30mg / kg significantly reduced the distance of the rats before entering the platform 22-24 days after ischemia (P<0.01 or P<0.05). Compared with the model group, the Jin Nai Duo group reduced the distance of the rats before entering the platform on the 22nd and 23rd days (P<0.01 or P<0.05). There was no statistical difference in swimming speed among the groups. It is suggested that TSTT improves the spatial reference learning function of cerebral ischemia rats.

[0092] In the spatial exploration test, compared with the sham operation group, the distance and time percentage of the model group in the target quadrant I were significantly reduced (P<0.01). Compared with the model group, TSTT 120mg / kg and TSTT 60mg / kg significantly increased the distance percentage of the ischemic rats in the target quadrant (P<0.01 or P<0.05), and TSTT increased the time percentage of the ischemic rats in the target quadrant (P<0.01 or P<0.05). Compared with the model group, the Jin Nai Duo group increased the distance and time percentage of the ischemic rats in the target quadrant (P<0.05). It is suggested that TSTT improves the spatial reference memory function of cerebral ischemia rats (see Figure 1 ).

[0093] 2.1.2 The effect of TSTT on working memory in cerebral ischemia rats

[0094] In the probe platform test (see Figure 1Compared with the sham-operated group, the model group rats showed a significant increase in the pre-platform distance for finding the target quadrants II, III, and IV platforms (P<0.01), and a significant decrease in the percentage of distance traveled and the percentage of time spent in target quadrants II and III (P<0.01). Compared with the model group, after drug treatment, TSTT 60 mg / kg significantly shortened the pre-platform distance for ischemic rats in target quadrants II, III, and IV (P<0.01), and increased the percentage of distance traveled and the percentage of time spent in target quadrants II, III, and IV (P<0.01 or P<0.05); TSTT 120 mg / kg and TSTT 30 mg / kg significantly shortened the pre-platform distance for ischemic rats in target quadrants II and III (P<0.01); TSTT 30 mg / kg significantly increased the percentage of distance traveled and the percentage of time spent in target quadrant III (P<0.01), and the percentage of time spent in quadrant IV (P<0.01). Compared with the model group, the Ginnado group shortened the distance to the target quadrant II before the platform for ischemic rats (P<0.01) and increased the percentage of distance and time in the target quadrants II and III for ischemic rats (P<0.01 or P<0.05).

[0095] In summary, the steroidal saponin in the head can enhance the long-term memory function of ischemic rats.

[0096] 2.2 Steroidal saponins from the top of the head reduce white matter structural damage in rats with cerebral ischemia.

[0097] T2 mapping coronal images showed that the brain tissue structure of the sham-operated group rats was intact (see...). Figure 2 Following middle cerebral artery embolization, abnormally bright signals appeared in the cortex and other brain regions supplied by the middle cerebral artery in the model group rats, indicating tissue ischemia and infarction. After treatment, the high signal intensity in the affected brain tissue region of the scalp-top steroidal saponin group decreased, and the infarction was alleviated. Statistical results showed that compared with the model group, TSTT 60mg / kg significantly reduced the total infarct volume in MCAO rats (P<0.01). Compared with the model group, Ginkgo biloba significantly reduced the total infarct volume in cerebral ischemia rats (P<0.01), suggesting that scalp-top steroidal saponin alleviates brain tissue damage in cerebral ischemia rats. After middle cerebral artery embolization, compared with the model group, TSTT 60mg / kg significantly reduced the ratio of ventricular volume and brain parenchyma volume on the affected and unaffected sides in cerebral ischemia rats (P<0.01). Compared with the model group, the Ginkgo biloba group significantly reduced the ratio of brain parenchyma volume on the affected and unaffected sides in cerebral ischemia rats (P<0.01). These results suggest that scalp-top steroidal saponin alleviates white matter structural damage in cerebral ischemia rats.

[0098] 2.3 Steroidal saponins from the top of the head can promote the repair of white matter structure in rats with cerebral ischemia.

[0099] The effect of a single bead on white matter repair in rats with cerebral ischemia was observed using DTI combined with DTT (see...). Figure 3). The rats in sham operation group had clear gray-white matter structure without abnormal signals. After MCAO, the ischemic side of the model group rats showed low signal in FA images and bright signal in ADC, AD and RD images. Compared with the sham operation group, the rFA of the white matter regions of the model group rats, including the internal capsule, external capsule, hippocampal fimbria, and the rADC, rAD and rRD were significantly increased 30 days after MCAO (P<0.05 or P<0.01), and the rFA of the corpus callosum was significantly decreased, and the rADC and rRD were significantly increased (P<0.05 or P<0.01), indicating that the microstructure of the white matter regions of the rats after MCAO was abnormal, such as cell edema, axon disintegration, and myelin sheath rupture. Compared with the model group, the rFA of the external capsule of the TSTT 60mg / kg group was increased, the rAD and rRD were decreased, the rFA of the internal capsule was increased, the rADC, rAD and rRD were decreased, the rRD of the corpus callosum was decreased, and the rADC and rAD of the hippocampal fimbria were decreased (P<0.05 or P<0.01). Compared with the model group, the rFA of the internal capsule of the Jinnaruo group was increased, the rADC, rAD and rRD were decreased, and the rAD of the hippocampal fimbria was decreased (P<0.05 or P<0.01). It is worth noting that compared with the model group, TSTT 60mg / kg and Jinnaruo groups had significant effects on the four DTI parameters in the internal capsule. It is suggested that the top one echinacoside promotes the reconstruction of axonal myelin microstructure in cerebral ischemic rats.

[0100] The DTT results showed that the gray matter nerve fiber structure of the rats in the sham operation group was complete and closely arranged, and the relative fiber density and length of each gray-white matter region were about 1. Compared with the sham operation group, the model group rats had a large number of nerve fiber loss and disordered arrangement, and the quantitative results showed that the relative fiber density and length of the external capsule and internal capsule of the model group rats were significantly decreased (P<0.05 or P<0.01). Compared with the model group, TSTT 60mg / kg increased the nerve fiber length and density of the external capsule and internal capsule of the cerebral ischemic rats (P<0.05 or P<0.01). Compared with the model group, the Jinnaruo group increased the nerve fiber length of the external capsule and the nerve fiber density of the internal capsule (P<0.01). It is suggested that the top one echinacoside promotes the reconstruction of white matter fiber in cerebral ischemic rats.

[0101] 2.4 The top one echinacoside can promote the regeneration of myelin sheath in cerebral ischemic rats

[0102] The effect of the top one echinacoside on the regeneration of myelin sheath in vascular dementia rats was observed by SMI32 / MBP immunofluorescence double labeling. Figure 4 The results showed that compared with the sham operation group, the SMI32 + cell ratio increased, and the MBP +The cell ratio decreased, and the SMI32 / MBP ratio increased (P<0.01). Compared with the model group, the SMI32 ratio of the outer capsule in the TSTT 60mg / kg and Ginkgo biloba groups was significantly lower. + The cell ratio was significantly reduced, and MBP + The cell ratio was significantly increased, and the SMI32 / MBP ratio was significantly decreased (P<0.01). This suggests that scalp steroidal saponins promote myelin regeneration in rats with vascular dementia.

[0103] Application Example 2: In Vitro Cell Experiments

[0104] I. Experimental Methods

[0105] 1. Primary cell culture and identification of OPCs

[0106] Brain tissue was obtained from 24-hour newborn SD rats, and OPCs were isolated and identified. The cerebral cortex was gently dissected, washed three times with PBS, digested, and transferred to centrifuge tubes to form a single-cell suspension. The suspension was filtered through a 70 μm filter membrane, yielding approximately 1 × 10⁻⁶ cells. 7 Cells were seeded at high density in T75 poly-D-lysine (PDL, Sigma-Aldric H) coated flasks and grown for 7-10 days. 10% FBS and 1% penicillin-streptomycin antibiotics were added to DMEM. When mixed glia culture was observed, the flasks were first shaken at 180 rpm at 37°C for 45-60 minutes, then the microglia were removed, the culture medium was changed, and the cells were shaken for 16-20 hours to collect OPCs. The obtained OPCs were further purified in a 30-minute culture dish, and then seeded onto PDL-coated plates with the following proliferation medium: Neurobasal-A (Gibco, CA) with 2% B27 (Gibco), 10 ng / mL platelet-derived growth factor AA (PDGF-AA, Gibco), 10 ng / mL basic fibroblast growth factor (bFGF, Peprotech, NJ), and 2 mM glutamine (Gibco).

[0107] 2. Preparation of Oxygen-Glucose Deprivation OPCs Model

[0108] Oxygen-glucose deprivation (OGD) was used to establish a simulated ischemia-hypoxia injury model in OPCs. Third-generation OPCs were digested and prepared into a single-cell suspension, with the cell density adjusted to 2.0 × 10⁶ cells / cells. 5 100 μL of fibronectin-coated medium was seeded into 96-well plates and cultured in endothelial basal cell culture medium. After the cells adhered, the original culture medium was discarded, the cells were washed with PBS, and then replaced with sugar-free endothelial basal cell culture medium. The cells were then placed in a 37°C incubator with a mixed gas of 5% CO2 and 95% N2 for 6 h.

[0109] 3 Experimental grouping and processing method

[0110] 3.1 Effect of one-bead-on-head steroidal saponins on OPCs cell differentiation

[0111] Sterilized 24-well plates were added with 0.1 μg / ml of polylysine 500 μL, 37°C, 5% CO2 for 30 minutes. 0.01M PBS was washed twice, and dried under sterile conditions for standby. The logarithmic growth period of BV2 cells was taken, diluted to a cell density of 1.5 x 10 5 6 / ml, 500 μL / well of cell suspension was inoculated in 24-well plates, 37°C, 5% CO2 for 24 hours. The BV2 cells had adherent growth, the culture medium supernatant was discarded, 500 μL / well of drug pre-incubation was added for 6 hours, and 37°C, 5% CO2 was continued for 24 hours. 50 μL of LPS was added in the corresponding experimental wells to make the final concentration 1 μM, 37°C, 5% CO2 for 24 hours. Discard the culture medium, 0.01M PBS pre-cooled at 4°C was washed 3 times for 5 minutes each time. 4% paraformaldehyde was fixed at room temperature for 20 minutes, PBS was washed 3 times for 5 minutes each time. 0.5% triton-100 was permeated for 2 minutes, PBS was washed 3 times for 5 minutes each time. 5% BSA was blocked for 30 minutes. Incubate the first antibody 200 μL / well (PDGFR-α + / Olig2 + : 1 / 200 / 1:200, MBP + / Olig2 + : 1 / 200 / 1:200). 200 μL of 0.01M PBS was used to fill the experimental wells and the edge wells, and the plastic self-sealing bag was sealed and placed in the 4°C refrigerator overnight. The next day, the 24-well plate was taken out, PBS was washed 3 times for 5 minutes each time. Incubate the second antibody: goat anti-mouse IgG (H+L) / FITC (1:400), goat anti-rabbit IgG / TRITC (1:400). Room temperature, 2 hours of light protection, 0.01M PBS was washed 3 times for 5 minutes each time, 10 μL of DAPI was added for 5 minutes of light protection, and 0.01M PBS was washed 3 times for 5 minutes each time. Add 200 μL of 0.01M PBS per well and observe under an inverted fluorescence microscope.

[0112] The experimental groups were as follows: control + blank serum group, oxygen-glucose deprivation group, oxygen-glucose deprivation + 5% TSTT drug-containing serum group, oxygen-glucose deprivation + 10% TSTT drug-containing serum group, oxygen-glucose deprivation + 20% TSTT drug-containing serum group. Under a Nikon inverted microscope, the excitation / emission wavelength was 470 / 490 nm, PLP + and CNPase + (labeling mature oligodendrocytes) single positive cells were green fluorescent, DAPI +Nuclei were labeled with DAPI. Single-labeled positive cells were blue. Four experimental wells were selected from each group, and 100 cells were counted in three non-overlapping fields under 200x to calculate PLP + CNPase + Cell number.

[0113] 3.2 The regulation of Jagged / Notch / Hes signaling by a top-head-bead steroidal saponin

[0114] OPCs cells were washed twice with 1 ml / well pre-cooled PBS for 5 min each time. 2x10 6 Cell density was added to the prepared lysis solution (RIPA:PMSF=100:1), 60 μL / well, and shaken on a horizontal shaker for 15 min. The first time, the cells were scraped to the same position with a pre-cooled -20°C spatula. After shaking on the horizontal shaker for another 15 min, the cells were collected with a pipette into a 1.5 ml centrifuge tube. The tube was placed in a 4°C centrifuge, and centrifuged at 10000 r for 20 min. The supernatant was collected and transferred to a new 0.5 ml centrifuge tube, which was stored at -80°C for later use. The protein concentration of each sample was quantified by the BCA method (A:B=50:1). The BSA standard was diluted according to the table (0.4 mg / mL solution, BSA gradient solution was prepared. 200 μL of BCA solution was added to each well of a 96-well plate. 6 μL of sample supernatant was diluted with 54 μL of normal saline. 25 μL of sample and standard was added to each well, and mixed gently with BCA solution. The mixture was incubated at 37°C for 30 min. The OD value was measured at 560 nm on a microplate reader, and the protein concentration of each sample was calculated according to the standard curve. The final protein concentration of each sample was determined to be 3 μg / μL, and the mixed system consisted of protein supernatant, RIPA and loading buffer, which accounted for 20% of the total system. The mixed solution containing sample protein was placed in a 100°C metal bath for denaturation for 5 min.

[0115] Gel preparation (10% separation gel, 5% concentration gel), 7 μL per well, after constant voltage electrophoresis by SDS-PAGE, constant current electric transfer sample protein to PVDF membrane. The results of ponceau staining were cut into bands, and 5% BSA or skim milk powder was blocked at room temperature for 60 minutes. Incubate primary antibody Notch1 (1:2000), Jagged1 (1:2000), NICD (1:2000), F3 (1:2000), GAPDH (1:40000) 4°C overnight. The next day, the band was warmed at room temperature for 60 minutes, and washed 5 times with 1xTBST buffer for 5 minutes each time. Enter secondary antibody goat anti-rabbit peroxidase-conjugated IgG (1:20000), incubate at room temperature for 2 hours. Wash the membrane with 1xTBST buffer for 6 times, 5 minutes each time. Add 200 μL of hypersensitive chemiluminescence solution uniformly, and expose the gel chemiluminescence imaging analysis system. Image J 6.0 software was used to measure the gray value of each band, and the data was expressed as the ratio of the gray value of the target protein to the internal reference GAPDH, and the result represented the relative content of the corresponding target protein.

[0116] II. Experimental results.

[0117] Figure 5 The immunofluorescence results showed that compared with the control + blank serum group, the expression levels of mature oligodendrocyte markers CNPase and myelin-related protein PLP in the oxygen-glucose deprivation + blank serum group were significantly reduced (P<0.01 or P<0.001). After TSTT serum pretreatment, compared with the oxygen-glucose deprivation + blank serum group, the expression of CNPase and PLP in the 10% TSTT drug-containing serum group was significantly up-regulated (P<0.05), and the expression of CNPase in the 20% TSTT drug-containing serum group was also significantly increased (P<0.05).

[0118] Figure 6 The Western Blot detection results showed that compared with the control + blank serum group, the protein expression levels of Notch1, Jagged1 and NICD in the oxygen-glucose deprivation + blank serum group were significantly increased (P<0.01). After TSTT serum pretreatment, compared with the oxygen-glucose deprivation + blank serum group, the protein expression of Notch1, Jagged1 and NICD in the 10% TSTT drug-containing serum group was significantly reduced (P<0.05 or P<0.01), and the protein expression of Notch1 and NICD in the 5% TSTT drug-containing serum group was significantly reduced (P<0.05 or P<0.01). Compared with the control + blank serum group, the protein expression of F3 in the oxygen-glucose deprivation + blank serum group was significantly reduced (P<0.01), and compared with the oxygen-glucose deprivation + blank serum group, the expression of F3 in the oxygen-glucose deprivation + 5%, 10%, 20% TSTT drug-containing serum group was significantly increased (P<0.05 or P<0.01).

[0119] Application Example 3 Network pharmacology analysis

[0120] In this application example, network pharmacology analysis was used to study the target points of the action of the cerebstatin in promoting white matter remyelination after cerebral ischemia.

[0121] First, 11 active compounds in the cerebstatin were screened out by searching the TCMSP database combined with literature retrieval, and a total of 448 predicted target points were obtained. A total of 1739 disease target points related to ischemic stroke were obtained from four disease databases (TTD, OMIM, GeneCards and DrugBank). The 448 predicted target points of the 11 active compounds in the cerebstatin were mapped into the 1739 disease target points related to ischemic stroke (see Figure 7 ), and a total of 91 potential target points were identified as the target points of the cerebstatin saponins in the treatment of ischemic stroke. The treatment target points of the cerebstatin saponins for stroke were obtained by AlzData database. The result analysis showed that 72 (79.1%) target genes were significantly expressed in OLs, and 59 (64.8%) target genes were significantly expressed in OPCs (see Figure 8 ).

[0122] The highly expressed target genes in each cell were subjected to biological process (GOBP), cell composition (GOCC) and molecular function (GOMF) enrichment using Metascape, and the top 10 enrichment information ranked by database comprehensive analysis was selected. The results showed that the cerebstatin saponins regulated various cell structures including receptor complex, vesicle membrane, cell substrate junction, etc. in OPCs, and regulated protein structure domain specific binding, protein kinase activity and transcription factor binding activity, etc. (see Figure 9 ); and regulated cell structures mainly including caveola, secretory granule membrane, and intracellular vesicle membrane in OLs, and regulated protein kinase binding, RNA polymerase II special DNA binding transcription factor activity (see Figure 10 ). Further, the highly expressed target genes in each cell were subjected to biological process enrichment using Metascape to find biological processes related to ischemic stroke. The results showed that the cerebstatin saponins mainly affected oligodendrocytes, oligodendrocyte precursor cell Notch signaling pathway regulation, tissue repair, glial cell generation, nuclear material transport and cognitive biological processes.

[0123] The top 20 biological processes in the GOBP category were selected to make a bubble chart. The results showed that the effect of one bead on the head of the cerebrosides on ischemic stroke was mainly related to hypoxic cell response, Notch signal regulation, learning and memory, behavior, etc. The research results show that one bead on the head of the cerebrosides has a wide range of regulatory effects on the composition and molecular function of OPCs and OLs cells, and further regulates the key link of inhibiting related factors of OPCs differentiation myelin, studies the molecular mechanism of one bead on the head of the cerebrosides to promote white matter remyelination, and has a reliable theoretical basis.

[0124] One bead on the head of the cerebrosides has multiple target points on OPCs and OLs after cerebral ischemia.

[0125] The above specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. Use of a top one bead of triterpene saponins in the preparation of a drug for treating vascular dementia.

2. Use according to claim 1, characterized in that, The top one bead of triterpene saponins at least includes rhizostavloside VII, PRRG and rhizostavloside VI, and the content of the top one bead of triterpene saponins is not less than 40%.

3. Use according to claim 2, characterized in that, The content of the rhizostavloside VII is not less than 9%, the content of the PRRG is not less than 15%, and the content of the rhizostavloside VI is not less than 16%.

4. Use according to claim 2, characterized in that, The rhizostavloside VII, the PRRG and the rhizostavloside VI are extracted from the rhizome of Trillium tschonoskii Maxim, Trillium govanianum wall. ex Ruyle or Trillium kamtschicum Pall. ex puroch.

5. Use according to claim 2, characterized in that, The top one bead of triterpene saponins is used as the only active component or is mixed with other drugs to prepare a preparation of a drug for clinical use.

6. Use according to claim 2, characterized in that, The preparation of the drug is in the form of oral liquid, capsule, tablet, powder, pill, granule, atomization, microemulsion, nano preparation, film, paste, tincture or injection.

7. The use according to claim 1, characterized in that, The vascular dementia is any one of vascular dementia caused by abnormality of white matter structure, cerebral infarction, cerebral hemorrhage, low perfusion of brain tissue, hypertension, diabetes and hyperlipidemia.

8. The use according to claim 1, characterized in that, The preparation method of the top one bead of triterpene saponins comprises the following steps: Step 1: extraction of the top one bead of triterpene saponins The top one bead of triterpene saponins is extracted from the rhizome of Trillium tschonoskii Maxim, Trillium govanianum wall. ex Ruyle or Trillium kamtschicum Pall. ex puroch. Step 2: extraction of the top one bead of triterpene saponins The concentrated solution is extracted with petroleum ether for 3-5 times until the petroleum ether layer is nearly colorless, and the water layer solution is collected and extracted with water-saturated n-butanol for 2-3 times, and the n-butanol is recovered under reduced pressure to obtain an extraction extract. Step 3: purification of the top one bead of triterpene saponins Part of the extraction extract is dissolved in water to obtain a sample solution, and the sample solution is enriched through a macroporous resin column, then washed with distilled water until colorless, and the volume of the distilled water is discarded, and then the sample solution is eluted with 4-5 BV of 20%-25% ethanol solution, 2-3 BV of 40%-45% ethanol solution and 6-8 BV of 70%-80% ethanol solution in sequence; the eluent of the 70%-80% ethanol solution is collected, the ethanol is recovered under reduced pressure, dried, and crushed to obtain a purified powder of the top one bead of triterpene saponins.

9. Use according to claim 8, characterized in that, In step 1, the volume-to-mass ratio of the 70%-80% ethanol solution to the top one bead of triterpene saponins is 8-12 ml:1 g, and the extraction is performed for 2-3 times, 1-1.5 h / time; the solid-to-liquid ratio in the concentrated solution is 2 g:2-3 ml.

10. Use according to claim 8, characterized in that, In step 2, the volume ratio of the petroleum ether to the concentrated solution is 2-3:2, and the volume ratio of the water-saturated n-butanol to the water layer solution is 2-3:

2.

11. Use according to claim 8, characterized in that, In step 3, the mass of the dry substance of the extraction extract is 55-65 g, 0.05-0.1 g of the medicinal material is contained in each 1 mL of the sample solution, and the flow rate of the sample solution and the eluent is 0.5 mL / min.

12. The use according to claim 8, characterized in that, The macroporous resin is AB-8, D101 or HPD-100.

13. The use according to claim 11, characterized in that, The column volume of the macroporous resin is 2.5-3 L, and the ratio of diameter to height is 1:5-6.

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