A method for extracting and preparing total icariin and its application in drugs for treating Alzheimer's disease

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to a method for extracting and preparing total glycosides of icariin and its application in drugs for treating Alzheimer's disease. The total glycosides of icariin are prepared as follows: using icariin as raw material, using a 3%-5% polyamide-amine dendrimer aqueous solution as solvent, 70°C negative pressure wall breaking extraction, secondary filtration, collecting polyamide-amine dendrimer precipitates, 10% ethanol elution to remove impurities, and then 70% ethanol elution, concentration and drying, to obtain icariin with a purity of more than 95%, and it plays an important role in the treatment of Alzheimer's disease. The present invention provides a simple, effective, low-cost large-scale production process technology for total glycosides of icariin.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to a method for extracting and preparing total icariin and an application of the same in drugs for treating Alzheimer's disease. Background Art

[0002] Epimedium, also known as Xianlingpi and Gangqian, is pungent, sweet and warm in nature. It enters the liver and kidney meridians. It has the effects of tonifying kidney yang, strengthening tendons and bones, and removing rheumatism. It is used for impotence and spermatorrhea, tendon weakness, rheumatic pain, numbness and cramps caused by kidney yang deficiency. It is a commonly used Chinese medicine in clinical practice. There are 40 varieties of Epimedium in China, and there are 15 main types of medicinal products. Among them, Epimedium brevicornum Maxim., Epimedium pubescens Maxim., Epimedium sagittatum Maxim. and Epimedium koreanum Nakai are included as the main original species in the 2015 edition of the Pharmacopoeia of the People's Republic of China. Epimedium: The periclidinal walls of the upper and lower epidermal cells are deeply wavy, and there are heterocytic cells arranged longitudinally along the leaf veins, containing 1 to more calcium oxalate columnar crystals; the lower epidermal stomata are numerous, indefinite, and sometimes non-glandular hairs can be seen. Sagittaria: The cells of the upper and lower epidermis are relatively small; the stomata of the lower epidermis are denser, with many warty protrusions formed by the shedding of non-glandular hairs, and sometimes non-glandular hairs can be seen. Soft-haired Epimedium: The stomata of the lower epidermis are sparse, with many slender non-glandular hairs. Korean Epimedium: The stomata and non-glandular hairs of the lower epidermis are easy to see.

[0003] Epimedium extract is a product made from the dried stems and leaves of Epimedium brevicornum and other plants. There are many specifications of commercial extracts, and icariin or total flavonoids are usually used as standardized indicators.

[0004] At present, the commonly used extraction and preparation methods include decoction, water extraction and alcohol precipitation or alcohol extraction and water precipitation, enzyme-assisted extraction and supercritical extraction, etc. These methods are only suitable for laboratory-level extraction and preparation due to factors such as limited extraction rate or expensive equipment, and are difficult to use for large-scale industrial production. In addition, the efficacy of the traditional Chinese medicine epimedium is characterized by multiple targets and multiple pathways, which is based on the diversity of material components. For example, the synergistic effect of multiple icariin components has a significant synergistic enhancement effect than the efficacy of a single component icariin. Therefore, studying an extraction and preparation method of total icariin suitable for large-scale industrial production is a problem that needs to be solved at present. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the present invention provides a method for extracting and preparing total icariin and application of the total icariin in drugs for treating Alzheimer's disease.

[0006] The present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for extracting and preparing total icariin, comprising the following steps:

[0008] (1) Take the herbal medicine Epimedium, crush it, and prepare the herbal medicine Epimedium decoction slices;

[0009] (2) Add an aqueous solution of polyamide-amine dendrimers as a solvent, the amount of which is 16 times the weight of the boiled and powdered Epimedium slices, control the temperature at 70°C, and after soaking, use negative pressure to break the wall for extraction, that is, each time: the 70°C aqueous solution of the boiled and powdered Epimedium slices is first depressurized, and the pressure is rapidly reduced from normal pressure to -20 MPa---40 MPa within 1 minute, and the pressure is kept constant for 1 minute, and then the pressure is increased, and the pressure is rapidly increased from -20 MPa---40 MPa to normal pressure within 2 seconds, and stirred for 10 minutes. The liquid is filtered through a 75-150 μm ceramic membrane, and the residue is set aside; Studies have shown that under appropriate pressure, the selective adsorption of total glycosides of epimedium is enhanced, and there is no effect on polyamide-amine dendrimers.

[0010] (3) In order to break the cell walls multiple times until all cells are broken and promote the dissolution of all effective ingredients, the drug residue filtered out in step (2) is further added with an aqueous solution of polyamide-amine dendrimers in an amount of 8-12 times the weight of the Epimedium decoction pieces as a solvent, and the extraction is repeated 3-5 times by the negative pressure cell wall breaking extraction method in step (2), stirring for 10 minutes after each extraction, and filtering the drug solution through a 75-150 μm ceramic membrane, and collecting the drug solution after the ceramic membrane filtration;

[0011] (4) The drug solution filtered through the ceramic membrane in step (3) is filtered through a 10-30 μm hollow fiber filter membrane, and the retained polyamide-amine dendrimer precipitate is washed with water for 3 times, each time with a water amount of 1-2 times the weight of the polyamide-amine dendrimer precipitate, and then eluted with 10% ethanol for 3 times, each time with a 10% ethanol amount of 3 times the weight of the polyamide-amine dendrimer precipitate, and finally eluted with 70% ethanol adjusted to pH 7.0-8.0 with ammonia water for 3-5 times, each time with a 70% ethanol amount of 7.0-8.0 being 3 times the weight of the polyamide-amine dendrimer precipitate, and the 70% ethanol eluate is collected, concentrated, and dried to obtain total icariin.

[0012] Furthermore, in step (2), the polyamidoamine dendrimer It is 28826g / mol, and the number of terminal amino groups is 128.

[0013] Furthermore, the particle size of the Epimedium decoction pieces in step (1) is 0.5-2.0 mm, and the aqueous solution of the polyamide-amine dendrimer in step (2) is 3%-5%.

[0014] Furthermore, the specific components of total icariin are: icariin, epimedin B, epimedin C, epimedin A, and baohuoside-I, among which the content of icariin is more than 95%.

[0015] In a second aspect, the present invention provides the use of total icariin prepared by the above method in the preparation of drugs for treating Alzheimer's disease.

[0016] Furthermore, the drug is made into tablets, granules or capsules for oral administration.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. The present invention provides a method for extracting and preparing total icariin from epimedium. The total icariin extracted by the method has a content of more than 95%, stable quality, and a reasonable method. It can realize large-scale, rapid, and efficient extraction of total icariin from epimedium. At the same time, the production cycle of the product is short, the energy consumption is low, the environmental pollution is small, and it is suitable for large-scale production;

[0019] 2. The present invention prepares total icariin at one time, wherein the icariin content can reach more than 95%, and the impurities are mainly epimedin B, epimedin C, epimedin A, and baohuoside-I, which is the first preparation in this field; in addition, studies have shown that the icariin total glycosides extract prepared in the present invention has better therapeutic effect on the treatment of Alzheimer's disease than single icariin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings.

[0021] Figure 1 This is a diagram of the tablet of the total glycosides extract of Epimedium of the present invention;

[0022] Figure 2 This is a diagram of the granules of the total glycosides extract of epimedium of the present invention;

[0023] Figure 3 This is a diagram of the icariin total glycosides extract capsule of the present invention;

[0024] Figure 4 The effect of the total icariin of the Chinese medicinal component of the present invention on the escape latency of AD rats;

[0025] Figure 5 The effect of the total glycosides of icariin, a Chinese medicinal ingredient of the present invention, on the number of AD rats crossing the platform;

[0026] Figure 6 The effect of the total glycosides of icariin, a Chinese medicinal ingredient of the present invention, on the quadrant distance of AD rats;

[0027] Figure 7 is the icariin standard curve of the present invention;

[0028] Figure 8 This is the liquid phase spectrum for determining the content of total icariin in the present invention. DETAILED DESCRIPTION

[0029] The preparation method of the icariin total glycosides extract is further described below through specific experimental examples and embodiments, but is not limited to the present invention.

[0030] Experimental Example 1 Preparation of total glycosides from Epimedium.

[0031] The present invention uses the stems and leaves of Epimedium and Epimedium sagittatum of the Berberaceae family;

[0032] (1) 15 kg of Epimedium medicinal materials were crushed and made into Epimedium decoction pieces with a particle size of 2.0 mm;

[0033] (2) Add an aqueous solution containing 5% polyamide-amine dendrimers as solvent, the amount of which is 16 times the weight of the Epimedium decoction pieces, control the temperature at 70°C, soak for 2 hours, and use negative pressure wall breaking extraction, that is, each time: the 70°C aqueous solution of the Epimedium decoction pieces is first depressurized, and the pressure is rapidly reduced from normal pressure to -20 MPa within 1 minute, and the pressure is kept constant for 1 minute, and then the pressure is increased, and the pressure is rapidly increased from -20 MPa to normal pressure within 2 seconds, and stirred for 10 minutes. The liquid medicine is filtered through a 75 μm ceramic membrane, and the residue is reserved for later use;

[0034] (3) The residue after filtration in step (2) is further added with an aqueous solution containing 3% polyamide-amine dendrimers in an amount of 8 times the weight of the Epimedium decoction pieces as a solvent, and the extraction is repeated 5 times using the negative pressure wall-breaking extraction method in step (2). After each extraction, the drug solution is stirred for 10 minutes, and the drug solution is filtered through a 75 μm ceramic membrane, and the drug solution after filtration by the ceramic membrane is collected;

[0035] (4) After the ceramic membrane filtration in step (3), the drug solution is filtered through a 10 μm hollow fiber filter membrane and retained. The retained polyamide-amine dendrimer precipitate is washed with water three times, each time with a water amount of 1 times the weight of the polyamide-amine dendrimer precipitate, then eluted with 10% ethanol three times, each time with a 10% ethanol amount of 3 times the weight of the polyamide-amine dendrimer precipitate, and finally eluted with 70% ethanol adjusted to pH 7.0 with ammonia water three times, each time with a 70% ethanol amount of 3 times the weight of the polyamide-amine dendrimer precipitate, and the 70% ethanol eluate is collected, concentrated, and dried to obtain 100 mg of total icariin with a purity of more than 95%.

[0036] Example 2

[0037] (1) 15 kg of Epimedium medicinal materials were crushed and made into Epimedium decoction pieces with a particle size of 1.0 mm;

[0038] (2) Add an aqueous solution containing 3% polyamide-amine dendrimers as solvent, the amount of which is 16 times the weight of the Epimedium decoction pieces, control the temperature at 70°C, soak for 2 hours, and use negative pressure wall breaking extraction, that is, each time: the 70°C aqueous solution of the Epimedium decoction pieces is first depressurized, and the pressure is rapidly reduced from normal pressure to -40 MPa within 1 minute, and the pressure is kept constant for 1 minute, and then the pressure is increased, and the pressure is rapidly increased from -40 MPa to normal pressure within 2 seconds, and stirred for 10 minutes. The drug solution is filtered through a 100 μm ceramic membrane, and the drug residue is reserved;

[0039] (3) The residue after filtration in step (2) is further added with an aqueous solution containing 5% polyamide-amine dendrimers in an amount of 8 times the weight of the Epimedium decoction pieces as a solvent, and the extraction is repeated 4 times using the negative pressure wall-breaking extraction method in step (2). After each extraction, the mixture is stirred for 10 minutes, and the mixture is filtered through a 100 μm ceramic membrane, and the mixture after filtration is collected;

[0040] (4) After the ceramic membrane filtration in step (3), the drug solution is filtered through a 10 μm hollow fiber filter membrane and retained. The retained polyamide-amine dendrimer precipitate is washed with water three times, each time with a water amount of 1 times the weight of the polyamide-amine dendrimer precipitate, then eluted with 10% ethanol three times, each time with a 10% ethanol amount of 3 times the weight of the polyamide-amine dendrimer precipitate, and finally eluted with 70% ethanol adjusted to pH 8 with ammonia water three times, each time with a 70% ethanol amount of 3 times the weight of the polyamide-amine dendrimer precipitate, and the 70% ethanol eluate is collected, concentrated, and dried to obtain 105 mg of total icariin with a purity of more than 95%.

[0041] Example 3 Preparation of total glycosides from Epimedium.

[0042] (1) 15 kg of Epimedium medicinal materials were crushed and made into Epimedium decoction pieces with a particle size of 1.0 to 2.0 mm;

[0043] (2) Add an aqueous solution containing 4% polyamide-amine dendrimers as solvent, the amount of which is 16 times the weight of the Epimedium decoction pieces, control the temperature at 70°C, soak for 2 hours, and use negative pressure wall breaking extraction, that is, each time: the 70°C aqueous solution of the Epimedium decoction pieces is first depressurized, and the pressure is rapidly reduced from normal pressure to -35 MPa within 1 minute, and the pressure is kept constant for 1 minute, and then the pressure is increased, and the pressure is rapidly increased from -35 MPa to normal pressure within 2 seconds, and stirred for 10 minutes. The drug solution is filtered through a 150μm ceramic membrane, and the drug residue is reserved for later use;

[0044] (3) The residue after filtration in step (2) is further added with an aqueous solution containing 4% polyamide-amine dendrimers in an amount of 10 times the weight of the Epimedium decoction pieces as a solvent, and the extraction is repeated three times using the negative pressure wall-breaking extraction method in step (2). After each extraction, the mixture is stirred for 10 minutes, and the mixture is filtered through a 150 μm ceramic membrane, and the mixture after filtration is collected;

[0045] (4) After the ceramic membrane filtration in step (3), the drug solution is filtered through a 20 μm hollow fiber filter membrane and retained. The retained polyamide-amine dendrimer precipitate is washed with water three times, each time with a water amount of 1.5 times the weight of the polyamide-amine dendrimer precipitate, then eluted with 10% ethanol three times, each time with a 10% ethanol amount of 3 times the weight of the polyamide-amine dendrimer precipitate, and finally eluted with 70% ethanol adjusted to pH 7.5 with ammonia water for four times, each time with a 70% ethanol amount of 3 times the weight of the polyamide-amine dendrimer precipitate, and the 70% ethanol eluate is collected, concentrated, and dried to obtain 110 mg of total icariin with a purity of more than 95%.

[0046] Experimental Example 4 Determination of the content of total glycosides in icariin.

[0047] 1. Preparation of standard solution

[0048] Preparation of icariin standard solution: accurately weigh 10 mg of icariin, place it in a 100 ml volumetric flask, add methanol to dissolve, shake well, and make up to volume to obtain a icariin standard solution with a mass concentration of 0.1 mg / ml;

[0049] 2. Make a standard curve

[0050] Take 0.5ml, 2ml, 5ml, 10ml, 15ml and 30ml of icariin standard solution and place them in 50ml volumetric flasks respectively, add methanol to dissolve, shake well, make up to volume, and inject 10μl of each according to the content determination method. Take the mass concentration of icariin as the abscissa and the peak area as the ordinate for linear regression, and get the regression equation: y=25965.6531x+7598.2270, R 2 =0.9997. The experimental results show that icariin has a good linear relationship with the peak area in the mass concentration range of 1μg / ml~60μg / ml.

[0051] 3. Content Determination

[0052] Determined according to high performance liquid chromatography (General Rule 0512).

[0053] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler (column length was 250 mm, inner diameter was 4.6 mm, particle size was 5 μm); methanol was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the provisions in Table 1; the flow rate was 1 ml per minute; the column temperature was 30 °C; the detection wavelength was 273 nm. The theoretical plate number calculated based on the icariin peak should be no less than 5000.

[0054] .

[0055] Preparation of reference solution Take an appropriate amount of icariin reference substance, weigh it accurately, add methanol to make a solution containing 25μg per 1ml. Preparation of test solution Take an appropriate amount of this product, about 1g, add 50ml of 70% methanol, ultrasonicate for 45 minutes (power 250w, frequency 40hz), cool, shake well, filter, and take the filtrate. Determination method Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, record the chromatogram, and obtain (the content of total icariin is calculated as icariin), see Figure 7 , Figure 8 .

[0056] Example 5 Preparation of Epimedium Total Glycosides Extract Tablets.

[0057] Take 40g of the total glycosides of epimedium extract of Example 1, add 45g of starch, mix well, granulate, dry, sieve, add 5g of microcrystalline cellulose and 0.5g of magnesium stearate, mix well, and press into 1000 tablets to obtain total glycosides of epimedium extract tablets, see attached Figure 1 .

[0058] Example 6 Preparation of Epimedium Total Glycosides Extract Granules.

[0059] Take 40g of total glycosides of epimedium extract of Example 2, add 60g of dextrin, mix, granulate, dry, and granulate to obtain total glycosides of epimedium extract granules, see attached Figure 2 .

[0060] Example 7 Preparation of Epimedium Total Glycosides Extract Capsules.

[0061] Take 50g of the total glycosides of epimedium extract of Example 3, add 50g of starch, mix well, granulate, dry, granulate, and fill 100 capsules to obtain total glycosides of epimedium extract capsules, see attached Figure 3 .

[0062] Example 8 Application of total icariin in drugs for treating Alzheimer's disease.

[0063] 1 Experimental Materials

[0064] 1.1 Animal experiments

[0065] Wistar rats, male, 90, weighing 160±10g, were obtained from Huateng Biotechnology Laboratory Animal Technology Co., Ltd. The temperature of the animal room was 20±2℃, the relative humidity was 65%-75%, and the feed was standard ordinary feed. During the experiment, the animals were free to drink water and eat.

[0066] 1.2 Drugs and reagents

[0067] (1) The Chinese herbal medicine component icariin was prepared by our laboratory, batch number 221203, for future use;

[0068] (2) Galantamine, batch number JLTM230123, Sichuan Weikeqi Biotechnology Co., Ltd.; icariin, Sichuan Hengcheng Zhiyuan Biotechnology Co., Ltd., purity 95%.

[0069] 1.3 Main instruments

[0070] (1) Morris water maze operating system, model ZH0065, Huaibei Zhenghua Biological Instrument Equipment Co., Ltd.;

[0071] (2) Digital single-arm stereotaxic apparatus, Shenzhen Ruiwode Biological Instrument Equipment Co., Ltd.;

[0072] (3) 1 / 10,000 electronic balance, model BT125D, Sartorius;

[0073] (4) Dental drills, surgical instruments, etc.;

[0074] 2 Experimental methods

[0075] 2.1 Establishment of AD composite animal model

[0076] 2.1.1 Animal grouping

[0077] A total of 105 male Wistar rats were adaptively fed for one week and randomly divided into 7 groups according to the body weight method, with 15 rats in each group, namely the sham operation group, the AD model group, the high-dose icariin group (0.05 mg kg -1 ), medium dose group (0.04 mg kg -1 ), low-dose group (0.03 mg kg -1 ) and icariin group (icariin 0.05 mg kg -1 ) Positive control group (galantamine 0.03 mg kg -1 Each group of rats underwent Morris water maze behavioral training every day.

[0078] 2.1.2 Morris water maze training experiment

[0079] 2.1 Morris water maze behavioral experiment

[0080] Experimental principle: Rats will instinctively look for a place to rest in the water. This behavior can reflect the complexity of the memory process, collect information related to spatial positioning, and process this information. After sorting-memorizing-reinforcement-retrieval, they can finally successfully find the platform hidden in the water and escape from the water.

[0081] The water maze device is mainly composed of a constant temperature circular pool, a circular platform with adjustable height and position, a tracking camera, and a computer connected to the camera. The diameter of the circular pool is 90 cm, the height is 50 cm, the water depth is 40 cm, and the diameter of the circular platform is 10 cm. The water temperature is controlled at (25±2)℃. The platform is 3 cm below the liquid surface. The camera is located 2 m above the circular pool and can capture the animal's position in the pool, the swimming distance within a certain period of time, and the path it has passed. The Morris water maze experiment includes two tests: directional navigation and spatial exploration. The following behavioral training is first performed before the following experimental animals are tested.

[0082] The experimental procedure is as follows:

[0083] (1) Three marking points △, □, and ○ are set at equal distances on the upper edge of the pool as the entry points for the animals. The circular pool is divided into four quadrants. The platform can be placed in any quadrant. In this experiment, the platform was placed in the third quadrant of the pool as a target for the rats to search after entering the water.

[0084] (2) During training, the animals were allowed to enter the water from three different marked points. After the animals entered the water, the camera was clicked to start recording. The time required for the rat to find the platform within the specified time (here set to 150 seconds) from entering the water was recorded as the latency (i.e., escape latency). If the platform was not found within 150 seconds after entering the water, the animal was guided to the platform and stayed there for 10 seconds. The total distance the rat traveled to find the platform and the distance it traveled through the third quadrant were recorded, and its related swimming trajectory was recorded.

[0085] (3) Remove the animals and dry them. If necessary, dry the rats under a hair dryer and put them back in the cage. Each animal in each group was trained twice a day, once at 9 am and once at 3 pm. The training lasted for 5 consecutive days.

[0086] Starting from the fifth working day, the platform was removed and a space exploration experiment was conducted to record the animal's running trajectory and the number of times the animal crossed the platform position within 150 seconds (number of platform crossings).

[0087] 2.1.3 Modeling

[0088] The D-gal model was established by intraperitoneal injection: D-gal was prepared into 1.25% injection solution with normal saline and kept aside. Ten Wistar rats were selected and fed for one week. The injection was then adjusted to 4 mL kg according to the rats' body weight. -1 Intraperitoneal injections were performed once in the morning and afternoon every day. Modeling was completed after 4 consecutive weeks of injections.

[0089] 2.1.4 AD model detection

[0090] The rats were subjected to water maze behavioral tests according to the modeling sequence. First, the directional navigation test was performed, followed by the spatial exploration test. The escape latency in the directional navigation test and the number of times the rats crossed the platform in the spatial exploration were recorded. The rats that did not meet the requirements were eliminated, and the rats that met the requirements were selected in each group for the following tests.

[0091] 2.2 Administration

[0092] Each group of rats was given the drug by gavage, and the dosage was based on the rat body weight (10 mL·kg -1 ) and the high-dose group was calculated as 0.05 mg·mL -1 , medium dose group: 0.04 mg·mL -1 , low-dose group 0.03 mg·mL -1 Concentration administration: icariin and galantamine groups were given 0.05 mg mL -1 (equivalent to 6 times the daily clinical dosage for adults), the AD model group and the sham operation group were gavaged with the same dose of normal saline solution, each group was gavaged once a day for 30 consecutive days.

[0093] 2.3 Behavioral test of drugs on AD model rats

[0094] 2.3.1 Morris water maze test

[0095] After 29 days of drug administration, the above-mentioned "Morris water maze training experiment" was first performed. The test was divided into two parts: positioning navigation experiment and spatial exploration experiment. The time it took for the rats to find the platform from different water entry points within 120 seconds was recorded as the escape latency. After the positioning navigation experiment, the platform was removed and the entry point farther from the platform was selected as the water entry point. The number of times the rats crossed the corresponding position of the platform during this time was recorded, that is, the number of crossings, the total distance the animals traveled to find the platform, and the distance they traveled through the third quadrant. At the same time, the swimming trajectory of the animals after entering the water was recorded.

[0096] On the second day after the Morris water maze test, the rats in each group were placed in a dark-protected chamber to adapt to the environment.

[0097] 2.4 Statistical analysis

[0098] GraphPad Prism 5 software was used for data processing. One-way analysis and Newman-Keuls test were used for comparison among the groups. Data are expressed as ± s, and P < 0.05 was considered statistically significant.

[0099] 3 Experimental results

[0100] 3.1 General behavioral observations on rats

[0101] Before modeling, the rats in each group had shiny hair, good mental state, agile movements, and normal drinking and eating. After modeling, the rats in each group had dry and dull hair, reduced activity, slow movements, slow reactions, obvious drowsiness, curled up, reduced diet, reduced weight, severe pigmentation spots on the tail, and some rats died after modeling. After the administration of the component Chinese medicine, the above conditions of the rats in each group were alleviated to varying degrees, the hair was smoother, the activity increased, the mental state was improved, and the death of rats was reduced compared with that after modeling.

[0102] 3.2 Morris water maze test

[0103] The experimental results showed that in the positioning navigation experiment, compared with the sham operation group, the escape latency of each model rat was significantly increased, and the difference was extremely significant (P<0.01); compared with the model group, the escape latency of the rats in the drug-treated group was reduced, among which the positive drug and high-dose groups had more obvious effects, and the difference was significant to a certain extent (P<0.05). In the space exploration experiment, compared with the sham operation group, the model group had a significantly reduced number of platform crossings (P<0.05), and the percentage of distance crossing the third quadrant also decreased significantly (P<0.01); compared with the model group, the number of platform crossings in each drug-treated group increased, and the percentage of distance crossing the third quadrant also increased, with the positive drug and high- and medium-dose groups having more obvious effects (P<0.05). The results are shown in Table 2. Figure 4 , Table 3, Figure 5-Figure 6 .

[0104] Table 2 Effects on escape latency of AD rats (±s, n=10)

[0105]

[0106] Note: Compared with the sham operation group, △△ P<0.01, △ P<0.05; compared with the model group, *P<0.05

[0107] Table 3 Effects on the number of crossings and quadrant distance of AD rats (±s, n=10)

[0108]

[0109] Note: Compared with the sham operation group, △△ P<0.01, △ P<0.05; compared with the model group, *P<0.05

[0110] The Morris water maze test is an experimental method that forces experimental animals, such as rats and mice, to swim to find a platform hidden in the water. It was initially used to explore the effects of brain structure on learning and memory, and has now become one of the more commonly used methods for evaluating animal learning and memory. This experimental method has now been widely used in research fields in many disciplines, including learning and memory ability, hippocampus / extrahippocampus research, new drug development, screening and evaluation, and has been widely accepted in the world, becoming a classic choice for behavioral research, especially for experiments related to learning and memory.

[0111] The experimental method of learning and memory is based on conditioned reflexes. Avoidance conditioned reflexes are also very convincing in the application of animal models of memory disorders, because animals can learn to avoid electric shocks after training, consolidate the memory, and maintain it for a period of time. The dark avoidance experiment is one of the more commonly used methods, which is used to detect the passive escape response of mice and rats for the study of learning and memory functions. This method is simple to operate, can observe the effects of drugs on the learning and memory process of trained animals, has high sensitivity, and is especially suitable for initial drug screening. It is one of the necessary methods for the development and screening of various drugs for brain health, memory enhancement, anti-aging and health care products.

[0112] The results of Morris water maze test showed that compared with the sham operation group, the latency of the AD model rats was significantly increased, the number of platform crossings and the percentage of crossing the third quadrant were also significantly reduced, indicating that intraperitoneal injection of D-gal modeling can cause learning and memory impairment in rats. After administration, the high-dose drug group (110.2 mg kg -1 ) can significantly shorten the escape latency of experimental animals, increase the number of rats crossing the platform, and increase the percentage of rats crossing the third quadrant. The results of the dark avoidance experiment showed that the escape latency of AD model rats was lower than that of the sham operation group, and the number of memory errors increased. After drug administration, the escape latency increased and the number of memory errors decreased. The high and medium dose groups (110.2 and 70.6 mg·kg -1 ) had a more obvious effect, indicating that the drug had a significant improvement on the spatial memory and learning ability of the model rats.

[0113] The results showed that the effects of total icariin on the escape latency, the number of platform crossings and the quadrant distance of AD rats were significantly better than those of icariin, and the statistical results were significantly different.

Claims

1. A method for extracting and preparing total icariin, characterized in that: The following steps are involved: (1) Taking the herbal medicine of Epimedium, crushing it, and preparing the herbal medicine of Epimedium decoction slices; (2) Adding an aqueous solution of polyamide-amine dendrimers as a solvent, the amount used is 16 times the weight of the epimedium decoction pieces, controlling the temperature to 70°C, and after soaking, using negative pressure wall breaking extraction, that is, each time: the 70°C aqueous solution of the epimedium decoction pieces is first depressurized, and the pressure is rapidly reduced from normal pressure to negative 20 MPa-negative 40 MPa within 1 minute, and the pressure is kept constant for 1 minute, and then the pressure is increased, and the pressure is rapidly increased from negative 20 MPa-negative 40 MPa to normal pressure within 2 seconds, and stirred for 10 minutes, and the drug solution is filtered through a 75-150 μm ceramic membrane, and the drug residue is reserved; the aqueous solution of polyamide-amine dendrimers is 3%-5%; wherein, the polyamide-amine dendrimers is 28826 g / mol, and the number of terminal amino groups is 128; (3) adding an aqueous solution of polyamide-amine dendrimers 8-12 times the weight of the Epimedium decoction pieces as a solvent to the filtered residues in step (2), and repeatedly extracting 3-5 times by the negative pressure wall-breaking extraction method in step (2), stirring for 10 minutes after each extraction, filtering the medicinal solution through a 75-150 μm ceramic membrane, and collecting the medicinal solution after the ceramic membrane filtration; (4) filtering the drug solution filtered by the ceramic membrane in step (3) through a 10-30 μm hollow fiber filter membrane, and intercepting the intercepted polyamide-amine dendrimer precipitate; washing the intercepted polyamide-amine dendrimer precipitate with water for 3 times, each time using water in an amount of 1-2 times the weight of the polyamide-amine dendrimer precipitate; eluting it with 10% ethanol for 3 times, each time using 10% ethanol in an amount of 3 times the weight of the polyamide-amine dendrimer precipitate; and finally eluting it with 70% ethanol adjusted to pH 7.0-8.0 with ammonia water for 3-5 times, each time using 70% ethanol adjusted to pH 7.0-8.0 in an amount of 3 times the weight of the polyamide-amine dendrimer precipitate; collecting the 70% ethanol eluate, concentrating it, and drying it to obtain total icariin.

2. The extraction and preparation method according to claim 1, characterized in that: The particle size of the Epimedium brevicornum powder slices in step (1) is 0.5-2.0 mm.

3. The extraction and preparation method according to claim 1, characterized in that: The specific components of the total icariin glycosides are: icariin, epimedin B, epimedin C, epimedin A, and baohuoside-I, among which the content of icariin is more than 95%.

4. Use of total icariin prepared by the extraction and preparation method according to claim 1 in preparing drugs for treating Alzheimer's disease.

5. The use according to claim 4, characterized in that: The medicine is made into tablets, granules or capsules for oral administration.

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