CN111973666B

CN111973666B - Preparation method and application of total alkaloids of aerial part of radix Aconiti lateralis

Info

Publication number: CN111973666B
Application number: CN202010755742.0A
Authority: CN
Inventors: 张晓丹; 梁宗锁; 杨东风; 陈盛良; 张岩
Original assignee: Zhejiang University Of Science And Technology Shaoxing Biomedical Research Institute Co ltd
Current assignee: Zhejiang University Of Science And Technology Shaoxing Biomedical Research Institute Co ltd
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2023-02-17
Anticipated expiration: 2040-07-31
Also published as: CN111973666A

Abstract

The invention discloses a method for extracting total alkaloids from monkshood, which comprises the following steps: sun drying and pulverizing the stems and leaves of radix Aconiti lateralis, mixing the pulverized radix Aconiti lateralis aerial parts with an extracting agent, performing ultrasonic extraction, and taking the filtrate as an extracting solution; concentrating the extracting solution, and adding petroleum ether for extraction; respectively obtaining an organic layer positioned at the upper layer and a water layer positioned at the lower layer; the water layer contains total alkaloids. The invention also provides a method for separating the water layer by using the D101 macroporous resin. The total alkaloid has antioxidant and acetylcholinesterase inhibiting effects.

Description

Preparation method and application of total alkaloids of overground part of monkshood

Technical Field

The invention belongs to the field of alkaloid extraction, and relates to a preparation method and application of total alkaloid extracted and separated from the overground part of monkshood.

Background

Aconitum carmichaeli Debx is a well-known Chinese medicinal material in China from ancient times, and is a product of the root of Aconitum carmichaeli Debx of Ranunculaceae, which is also called radix Aconiti Kusnezoffii, radix Aconiti lateralis Preparata, and semen Toosendan. It is strongly hot in nature, sweet and pungent in flavor, and enters heart, kidney and spleen meridians. Monkshood generally grows in a warmer and humid environment facing the sun, is cold-resistant, mostly grows in hilly grass slopes or shrubs, and is mainly spread in Sichuan, shaanxi, hubei, hunan, yunnan and other places in China. The root of the plant is generally used for medicine, the best picking period is from late June to late August every year, but the root of the plant is not completely used, the mother root, the root hair and the like need to be removed, and only the seed root is remained. The medicinal materials are generally processed with Danba and salt, and the application method is different for different products, such as salt aconite, black aconite, rhizoma typhonii, etc. From ancient times to present, monkshood is used as a good medicine, has wide clinical application, has the function of treating severe internal cold syndrome, and can play a good role in warming up after taking a proper amount of monkshood. However, the traditional Chinese medicine is extremely toxic, and if the preparation process is improper or the dosage is not well controlled, poisoning can be easily caused by people, so that although the medicinal efficacy is good, few people are afraid of using the traditional Chinese medicine, the traditional Chinese medicine is evaluated as the medicine which is the most useful and the most difficult to use, and the toxicity is listed as the next-class medicine in Shen nong Ben Cao Jing. Nevertheless, fu Zi has been regarded as the first herb of rescuing from collapse by restoring yang, the good general of herbs and the four-dimensional of herbs for treating diseases and protecting life because of its remarkable efficacy, and it has been the hot research to minimize the toxicity of Fu Zi.

Aconite contains not only alkaloid but also many other compounds, such as some basic compounds, steroids, ceramides, and other components such as protein, enzyme, amino acid, organic base, and some trace elements, which have other effects. Therefore, many good efficacies of aconite are directly related to various substances contained in it.

The efficacy of aconite:

although Fu Zi is toxic, its special action can be exerted if the application method is controlled. The ingredients are analyzed for their medicinal properties, and the main content of aconite is alkaloid, so many medicinal effects are exerted in alkaloid. Benzylisoquinoline alkaloids have been found to play a useful role in the treatment of cardiovascular shock, heart failure and cardiac arrhythmias; the alkaloid can also protect ischemic myocardium, and the alkaloid can regulate the ischemic myocardium by improving energy metabolism and signal transduction defects; has good treatment effect on the symptoms of analgesia, rheumatoid arthritis, enteritis and the like. In addition, researches find that aconitine has quite good effects on resisting aging, resisting Alzheimer's disease and the like, and has good inhibition effect on acetylcholinesterase due to good oxidation resistance. Besides alkaloid, the aconite polysaccharide and the like also play medicinal roles, and the polysaccharide has the degradation effect on blood sugar and cholesterol, has the effects of inhibiting tumors and improving the immunity of organisms, and can relieve the pain of patients caused by toxic and side effects of chemotherapy. The effects of the total alkaloids of radix Aconiti lateralis have been researched and developed correspondingly, while the overground part has been proved to have alkaloid, but the biological activity of the total alkaloids of radix Aconiti lateralis has not been studied, so that the problem is to discuss the activity of the total alkaloids of the overground part of radix Aconiti lateralis, mainly study the antioxidant capacity and the acetylcholinesterase inhibition capacity of the extract of crude alkaloids of the overground part of radix Aconiti lateralis, and hopefully provide reference for further study on the medicinal value of the overground part.

Antioxidant-alkaloid relationship:

natural plants are considered as important sources of antioxidant substances, antioxidants achieve antioxidant effects by scavenging free radicals, atoms, molecules or ions are called free radicals if electrons of the atoms, the molecules or the ions are unpaired, a plurality of chemical reaction paths in a body and external adverse environments can enable the body to generate a plurality of free radicals, and the accumulation of the free radicals in the body is a source of aging and diseases of the body, such as chronic diseases of diabetes mellitus, hyperlipidemia, vascular diseases, tumors and the like. Many natural drug extracts such as alkaloid, polyphenol, vitamin, saponin, polysaccharide, polypeptide all have the function of scavenging free radicals. There are many methods for measuring the oxidation resistance of a substance, and the operation process is also simple and quick, and the common detection methods include an ABTS method and a DPPH method by measuring the radical clearance, and a FRAP method by measuring the reduction of ferric ions to ferrous ions.

ABTS solution and potassium persulfate solution react for 12-16 hours to form ABTS ⁺ Free radical, the solution turned green. When contacted with antioxidant, the formation of free radicals is inhibited, the color is lightened, and the absorbance value at 734nm is measured to calculate the clearance. The higher the clearance rate, the stronger the ability to inhibit the generation of free radicals, i.e., the stronger the antioxidant ability.

DPPH is a fatty radical, is not as active as other radicals, and shows a purple-black color when dissolved in ethanol, and can be measured at a wavelength of 517 nm. The free electron at the DPPH N position can capture an electron pair from the oxide solution, so the antioxidant capacity of the sample can be calculated according to the principle. The more the color of DPPH is faded away, the more electrons are captured by the free electrons on the N position, the stronger the antioxidant activity of the antioxidant is, and the clearance can be calculated according to the light absorption value, so that the antioxidant activity can be measured.

Total antioxidant capacity measurement (FRAP) is a measurement method of antioxidant capacity which is different from the principle of radical scavenging, which is a redox reaction and thus is a measurement of total antioxidant capacity. The principle is that ferric ion can be combined with TPTZ to form Fe ³⁺ TPTZ complex, and is readily reduced to Fe ²⁺ TPTZ, the conditions which promote this reaction being such that the oxide of the oligoacid is present. And Fe ²⁺ The highest absorption value at 593nm can be obtained according to the light absorption value, the expression value is the FRAP value, the total reduction capacity of the sample can be judged according to the FRAP value, and the reduction capacity corresponds to the oxidation resistance capacity.

The double-target effect of the acetylcholinesterase resistance and the antioxidant activity in the AD drug screening is as follows:

alzheimer's Disease (AD) is a degenerative disease of the nervous system, and patients often develop slowly and even insidiously. The degenerative diseases of the nervous central system, which are characterized by the occurrence of physical dysfunction, brain memory loss and impairment of cognitive functions of things, are important threats to the health of people at present, and are usually seen in the elderly. It is generally accepted that AD is a disease caused by multiple etiologies, of which the cholinergic and oxidative stress theories are widely accepted.

The theory of cholinergic hypothesis is that information transmission by the brain needs to be transmitted through a neurotransmitter called acetylcholine (Ach), i.e., to serve as a conscious actor to maintain brain consciousness. However, the brain contains an enzyme that hydrolyzes these neurotransmitters, acetylcholinesterase (AchE) located in the postsynaptic membrane of the neuromuscular tissue. Therefore, if AD patients can effectively inhibit AchE and prevent hydrolytic enzyme from hydrolyzing Ach, the AD patients can effectively recover the function of the nervous system. Physicians therefore generally treat AD patients with anti-AChE actives.

The theory of oxidative stress hypothesis is also a widely recognized pathogenic theory, and is caused by the disruption of the equilibrium between free radical generation and the oxidative defense system in the body, resulting in the induction of molecular oxidation in cellular tissues, thereby causing tissue damage. The aerobic metabolism activity in the cell generates a free radical, namely Reactive Oxygen Species (ROS), which is a representative of active free radicals in the body and is a participant of many important reactions in the cell, such as lipid peroxidation in the cell, protein denaturation and modification, and also participates in important working processes of DNA, genes and the like. ROS in cells are also harmful to cells, and can gradually over-oxidize functional protein lipid on cell membranes to cause dysfunction, thereby causing apoptosis. The brain is an important organ of the human body, is an important oxidative metabolism site, and is also an important generation site of free radicals, and if a brain cell oxidative defense system is damaged, the free radicals are accumulated, so that the brain cell is aged and killed, and even chromosome mutation in the cell can cause AD.

In recent years, many researchers have found that alkaloids extracted from natural plants have inhibitory activity on acetylcholinesterase, and such alkaloids as steroids, triterpenes, huperzine, isoquinolines, indoles and the like all have strong inhibitory effect. The overground part of monkshood also contains similar alkaloids, so the potential medicinal effect of the overground part of monkshood in resisting senile dementia can be explored by measuring the acetylcholinesterase resistance and antioxidant activity of monkshood.

The activity of resisting acetylcholinesterase is measured by an Ellman method commonly, the reaction mechanism is that AchE can decompose iodothio-Acetylcholine (ATCI) to generate thiocholine and acetic acid, the thiocholine and a color developing agent DTNB generate a yellow substance, the light absorption value is measured at 412nm, the inhibition rate of the enzyme can be calculated according to the light absorption value, and the higher the inhibition rate is, the stronger the activity is. This is a commonly used method, and the experimental operation process is determined by experiments, but the principle is not changed.

Purpose, meaning and content of research

The monkshood has good pharmacodynamic function and large using amount, but always utilizes the radicles, has high toxicity, and the overground part cannot be reasonably utilized, so that huge waste is caused.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for extracting, separating and purifying total alkaloids from the overground part of monkshood.

In order to solve the technical problems, the invention provides a method for extracting total alkaloids from monkshood, which comprises the following steps:

1.1 Sun drying the stems and leaves of the monkshood until the water content is less than or equal to 15 percent (weight percent), and crushing to obtain crushed overground part of the monkshood;

1.2 600g of the ground aconite overground part crushed material and an extracting agent are mixed according to the material-liquid ratio of 1 g/8-12 ml, and then ultrasonic extraction is carried out at 50 ℃ and the ultrasonic power of 400-800W, wherein the extraction time is 2-4 hours; then filtering and separating to respectively obtain filtrate and filter cake;

the filter cake replaces the ground part crushed substance of the monkshood to repeat the ultrasonic extraction and the filtration for 1 to 3 times (namely, the ground part crushed substance of the monkshood is subjected to the ultrasonic extraction for 2 to 4 times), and all filtrate obtained by the filtration is combined to be used as an extracting solution;

the extractant is 85% (volume percent) ethanol solution;

1.3 Concentrating the extracting solution to (1 +/-0.1) L to obtain a concentrated solution; then adding petroleum ether for extraction; the volume usage ratio of the petroleum ether to the concentrated solution is 1-1.5: 1; respectively obtaining an organic layer positioned at the upper layer and a water layer positioned at the lower layer;

taking a water layer; the aqueous layer (about 850 mL) contained total alkaloids.

The improvement of the method for extracting the total alkaloid from the aconite further comprises the following steps of: the aqueous layer was separated using D101 macroporous resin.

As a further improvement of the method for extracting the total alkaloids from the aconite, the separation and purification of the total alkaloids are sequentially carried out by the following steps:

2.1 D101 macroporous resin pretreatment:

soaking in 95% ethanol for 24 hr, removing ethanol, and washing with distilled water (the distilled water is clear and has no ethanol taste);

then soaking the mixture for 3 hours by using a 5% hydrochloric acid solution, and washing the mixture by using distilled water (washing the mixture to be neutral by using the distilled water); soaking in 4% sodium hydroxide solution for 3 hr, and washing with distilled water (washing with distilled water to neutral);

2.2 Freeze-drying the water layer to obtain freeze-dried powder (generally, 25mL of water layer, 2.6-2.7 g of freeze-dried powder is obtained);

adding water into the lyophilized powder to make the concentration of the lyophilized powder in the obtained suspension be (0.2 + -0.02) g/mL, and adjusting pH to 3-4 with hydrochloric acid (36% hydrochloric acid solution) to obtain a sample solution;

description of the drawings: the purpose of adjusting the pH value of the hydrochloric acid solution is to acidify the alkaloid into salt;

2.3 Loading the pretreated D101 macroporous resin (500 mL) obtained in the step 2.1) into a column, adding a sample solution, standing the sample solution for 2h, discharging the sample solution, and then adding 2BV of distilled water to wash the sample solution according to the flow rate of 1.5mL/min so as to remove water-soluble macromolecular components (polysaccharide, protein and the like);

eluting with 10% ethanol at a flow rate of 4BV/h, and then eluting with 80% ethanol at a flow rate of 4BV/h, wherein the dosage of the 10% ethanol is 2-3 BV, and the dosage of the 80% ethanol is 3-4 BV;

concentrating the eluates corresponding to 10% and 80% ethanol, and vacuum freeze drying to obtain two crude alkaloids eluted with 10% and 80% ethanol.

The method comprises the steps of crushing overground parts (stems and leaves) of monkshood, extracting with an ethanol solution on an ultrasonic crusher, purifying total alkaloids (removing impurities such as lipid and protein) by D101 macroporous resin, concentrating, and freeze-drying to obtain a crude alkaloid sample. Then, the antioxidant capacity of the sample is comprehensively measured by adopting an ABTS method (or a DPPH method or an FRAP method), the AChE inhibiting activity of the sample is measured by adopting an improved Ellman method, and the active ingredients of the crude alkaloid sample are identified by UPLC-Q-TOF-MS/MS spectrum qualitative analysis, so that the total alkaloids of the overground part of the monkshood are integrally evaluated.

The stem and leaf of aconite root adopted by the invention are collected from Shaanxi Han and are identified as ranunculaceae and aconite plants.

In conclusion, the invention researches the extraction optimization and the determination of the biological activity of the total alkaloids of the overground part of the monkshood, namely, provides the application value of the total alkaloids; the invention comprises the steps of measuring the oxidation resistance and the acetylcholinesterase inhibition capacity, and qualitatively analyzing and identifying the active ingredients of a crude alkaloid sample through an UPLC-Q-TOF-MS/MS map, and the obtained result provides a basis for the medicine effect and functional research of the overground part of monkshood.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a graph of a 10%, 80% and VC fit to ABTS free radical clearance as determined by the ABTS method.

FIG. 2 is a bar graph of 10%, 80% versus ABTS free radical clearance as determined by the ABTS method.

Figure 3 is a 10%, 80% ethanol elution site and VC versus AchE clearance fit.

Figure 4 is a bar graph of the clearance of AchE by 10% and 80% ethanol elution sites.

Fig. 5 is a 10% total ion flow diagram.

Fig. 6 is a mass to charge ratio plot of the 10% main peak.

Fig. 7 is an 80% total ion flow diagram.

Fig. 8 and 9 are graphs of 80% main peak mass-to-charge ratios.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1, a method for obtaining total alkaloids from the aerial parts of aconite, comprising the following steps:

1. extraction:

1.1 Sun drying the stems and leaves of the overground part of the monkshood (the water content is less than or equal to 15 percent), cutting into small segments, mixing, and crushing by using an electric crusher (the powder can pass through a 50-mesh sieve) to obtain the crushed overground part of the monkshood.

The ground part of radix Aconiti lateralis can be collected in self-sealing bag, and sealed for storage.

1.2 600g of pulverized aerial parts of aconite (i.e., pulverized stem and leaf mixture) are weighed, 6 liters of 85% ethanol solution is added, the temperature of an ultrasonic cleaner is adjusted to 50 ℃, and then ultrasonic extraction is carried out for 3 hours under the ultrasonic power of 400-800W and the ultrasonic frequency of 40 kHz.

After the ultrasonic extraction is finished, separating the ultrasonic extraction product by using a suction filtration device to respectively obtain filtrate and filter cake; collecting filtrate;

repeating the ultrasonic extraction and separation for 2 times by replacing the ground aconite ground material with the filter cake, namely, continuously adding 6 liters of 85% ethanol solution into the filter cake, and carrying out the ultrasonic extraction and suction filtration separation for 2 times according to the technological parameters of the ultrasonic extraction;

therefore, the times of the total ultrasonic extraction are 3 times, and the filtrate obtained by the first extraction is found to be dark greenish black, the color of the filtrate obtained by the second extraction is lightened, and the filtrate obtained by the third extraction basically has no color, which indicates that the total volume of the extracting solution is about 18L.

1.3 Concentrating the extractive solution to about 1L with rotary evaporator (temperature is set to 45 deg.C) until no ethanol smell is obtained;

taking all the concentrated solutions, adding 1000ml of petroleum ether into the concentrated solutions for extraction, and then removing the petroleum ether on the upper layer, thereby primarily removing chlorophyll, lipid, steroids and the like; keeping the lower water layer (containing extract) in refrigerator for refrigeration; the volume of the water layer is about 850ml; the water layer contains total alkaloids.

Preparing the aqueous layer into lyophilized powder (by conventional operation of freeze dryer); for 25mL of the aqueous layer, the dry weight (weight of lyophilized powder) was 2.636g, a yield of 14.94%, which was calculated as: (850 ml/25ml 2.636g)/600g =14.94%.

2. Separation and purification of total alkaloid

2.1 D101 macroporous resin pretreatment:

the resin was soaked thoroughly with 95% (vol%) ethanol for 24h, the ethanol was aspirated, and the resin was washed with distilled water until the water stream was clear and free of the ethanol taste. Then soaking the mixture for 3 hours by using a 5 percent (weight percent) hydrochloric acid solution, and washing the mixture to be neutral by using distilled water; then soaking the mixture in 4 wt% sodium hydroxide solution for 3 hr, and washing the soaked mixture with distilled water to neutrality.

The using amounts of 95% ethanol, 5% hydrochloric acid solution and 4% sodium hydroxide solution are respectively 4 times of the volume of the D101 macroporous resin.

2.2 Adding distilled water into the freeze-dried powder obtained in the step 1) to ensure that the concentration of the freeze-dried powder in the obtained suspension is 0.2g/mL, and then adjusting the pH value to 3-4 by utilizing hydrochloric acid solution (hydrochloric acid solution with the mass concentration of 36%) to obtain sample solution;

description of the drawings: the purpose of the above-mentioned hydrochloric acid solution to adjust the pH is to acidify the alkaloid into a salt.

2.3 Taking 500mL of the pretreated D101 macroporous resin obtained in the step 2.1), loading the column, slowly adding the sample solution, standing for 2h, and discharging the solution; at the flow rate of 1.5mL/min, then adding 2BV of distilled water to wash and remove water-soluble macromolecular components such as polysaccharide, protein and the like;

eluting with 10% ethanol at flow rate of 4BV/h, wherein the amount of 10% ethanol is 3BV; collecting all eluent corresponding to the 10% ethanol as crude biological alkali liquor removed by the 10% ethanol;

the resin is found to be still colored, namely part of the substances are not eluted, and then 80% ethanol is used for eluting (the flow rate is 4 BV/h), the use amount of the 80% ethanol is 4BV, the color of the resin is changed to be colorless, and all eluent corresponding to the 80% ethanol is collected to be used as crude biological alkali liquor removed by the 80% ethanol.

Concentrating the crude biological alkali solution with 10% ethanol by rotary evaporator (at 45 deg.C) to obtain paste, and freeze-drying with vacuum freeze-drying apparatus to constant weight to obtain lyophilized powder I; the weight and yield of lyophilized powder I are shown in Table 1 below.

Concentrating the crude biological alkali liquor removed by 80% ethanol to paste by using a rotary evaporator (at the temperature of 45 ℃), and freeze-drying the paste to constant weight by using a vacuum freeze-drying instrument to obtain freeze-dried powder II. The weight and yield of the obtained lyophilized powder II are shown in the following table 1.

The calculation formula of the yield is as follows: the mass of the freeze-dried powder/the total weight of the medicinal materials is multiplied by 100 percent.

TABLE 1 macroporous resin purification results

Experiment one, the measurement of oxidation resistance,

ABTS method of determination

The assay was performed on a microplate reader using 96-well plates. Accurately weighing 20.3mg of ABTS reagent and 3.51mg of potassium persulfate, respectively adding 5mL of distilled water to dissolve, mixing, placing at room temperature, reacting in the dark for 12-16 hours, and diluting with absolute ethyl alcohol by 40 times for later use.

Weighing appropriate amount of two crude alkaloid lyophilized powders, respectively, and preparing into series of sample solutions with gradient concentration with anhydrous ethanol, namely 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2mg/mL. The experimental methods are shown in Table 2.

TABLE 2 ABTS operation

Finally, the radical clearance rate E is calculated by using the following formula 1 ₀ The calculation result is used for calculating IC ₅₀ Value, VC, served as a positive control.

The practical operation is as follows:

adopting 96-well plate method, adding sample 20 μ L reaction system 200mL, calculating ABTS free radical scavenging rate according to absorbance, and calculating total alkaloid sample (hereinafter referred to as 10%, lyophilized powder I) eluted by 10% ethanol, total alkaloid sample (hereinafter referred to as 80%, lyophilized powder II) eluted by 80% ethanol and half scavenging concentration (IC) of VC on ABTS free radical by GraphPad Prism ₅₀ Values), a graph is fitted as in fig. 1, and a bar graph is made as in fig. 2. IC for calculating 10%, 80% and VC ₅₀ The values were about 17.99. Mu.g/mL, 20.21. Mu.g/mL, 2.265. Mu.g/mL. Because of the IC ₅₀ The smaller the value, the greater the antioxidant capacity. Therefore, the three components have the oxidation resistance of VC>10％>80 percent. As can be seen from FIG. 2, the clearance rate increases with the concentration of the sample, 0.1 mg. Multidot.mL ^-1 The removal rates of 10% and 80% are respectively 98.31% and 99.04%, and the two can be almost completely removed, and the removal capacities of the two are similar and are about 10 times smaller than that of VC.

Description of the drawings: when the DPPH method is adopted for detection, the obtained result is as follows: the clearance rate is higher along with the increase of the sample concentration, and the concentration is 0.5mg/mL ^-1 In this case, the clearance rates of 10% and 80% are 88.93% and 97.22%. Therefore, the difference between the 10% and 80% DPPH free radical scavenging capacity is small, and is about 10 times smaller than VC;

when the detection is carried out by using the FRAP method, the obtained result is as follows: the concentration of the clear sample increased and increased, 0.1 mg. Multidot.mL ^-1 The removal rates of 10% and 80% are respectively 98.31% and 99.04%, and the two can be almost completely removed, and the removal capacities of the two are similar and are about 10 times smaller than that of VC.

Experiment two, determination of anti-AChE activity:

improved Ellman method for measuring activity of anti-acetylcholinesterase

Respectively dissolving the freeze-dried powder I and the freeze-dried powder II into sample solutions for activity determination: concentration gradients of 0.1mg/mL, 0.5mg/mL, 0.75mg/mL, 1mg/mL, 2mg/mL, 2.5mg/mL, 5mg/mL and 10mg/mL were formulated with Buffer pH = 8.

Adding 140 μ L of acetylcholinesterase solution with Buffer pH =8 and 15 μ L of sample solution into a 96-well plate, mixing uniformly, placing in an ice box, incubating for 30 minutes in dark, adding 10 μ L of DTNB solution and 10 μ L of ATCI solution sequentially, mixing uniformly, placing in an incubator at 37 ℃, turning off a lamp, culturing for 20min, measuring absorbance at 412nm and 37 ℃ immediately by using a microplate reader, and marking as A _{Sample set} Repeat the test 3 times; sample control group substituted acetylcholine solution with 15 μ L Buffer pH =8 and absorbance was labeled as a _{Sample control group} (ii) a Blank set sample solution was replaced with 20 μ L Buffer pH =8 and absorbance was labeled as a _{Blank group} (ii) a Replacing the sample solution with 20 μ L huperzine A solution as complete inhibition group, and marking the light absorption value as A _{Complete inhibition group} . From the absorbance, the inhibition ratio was calculated using equation 2. Half Inhibitory Concentration (IC) of sample against acetylcholinesterase ₅₀ ) The assay was performed using GraphPad Prism 5 software, and the positive control was made using huperzine A instead of the sample. The concentrations of the relevant reagents and the formulation methods are shown in table 3:

TABLE 3 preparation of the desired solutions

The results obtained were:

using a 96-well plate method, 20. Mu.L of the sample was placed in 175. Mu.L of the reaction system, and the inhibition ratio of acetylcholinesterase (AchE) was calculated from the absorbance values, and 10% and 80% and the half inhibition concentration (I) of huperzine A (Huperzin-A) against AchE were determined using GraphPad PrismC ₅₀ Values), a curve is fitted as in fig. 3, and a bar graph is made as in fig. 4. The IC of 10%, 80% and huperzine A were calculated from GraphPad Prism ₅₀ Values of 93.86. Mu.g/mL, 89.3. Mu.g/mL, 0.574. Mu.g/mL, IC ₅₀ The smaller the value, the stronger the anti-AchE ability. Therefore, the three have the anti-AchE capacity of huperzine A>80％>10 percent. As can be seen from FIG. 4, the inhibition rate increases with the increase of the sample concentration, when the sample concentrations are all 0.5mg/mL, the 10% inhibition rate is 94.22%, and the 80% inhibition rate is already completely inhibited and reaches 100%, while the positive control group huperzine A has a much stronger inhibition rate than the sample, which is about 150 times stronger.

Experiment III, identification of active ingredients

UPLC-Q-TOF-MS/MS spectrogram analysis

The sample solution was made up to 10. Mu.g/mL with 80% methanol (chromatographic grade). The above sample solution was filtered through a 0.22 μm ultrafiltration membrane.

Chromatographic conditions are as follows: an Acqity HSSTS (100 mm. Times.2.1 mm, ID1.8 μm) column was used, mobile phase A was 0.1% formic acid-water solution, mobile phase B was 0.1% formic acid-acetonitrile solution, and gradient elution was as shown in Table 4.

TABLE 4 gradient elution

Mass spectrum conditions: and detecting in a positive ion V mode by using an electrospray ionization ion source ESI. The conditions are shown in Table 5.

TABLE 5 Mass Spectrometry conditions

UPLC-Q-TOF-MS/MS spectrogram

10% of total ion flux (FIG. 5) and mass to charge (FIG. 6), and 80% of total ion flux (FIG. 7) and mass to charge ratio (FIGS. 8 and 9) were determined by LC/MS.

From the mass-to-charge ratio results, the following table 6 was obtained by referring to a known analytical method (Zhoujis, etc.).

TABLE 6 qualitative analysis of alkaloid in aerial parts of monkshood

Thus, 10% of the alkaloids can be judged to be benzoylaconine and benzoylhypaconine, while 80% of the alkaloids are aconine, hypaconine, benzoylmesaconine, 10-OH-mesaconine, and Niaoling. The contents of the two components are different, and 80% contains more components than 10%. And the components of the two crude alkaloids are not mixed with each other, which indicates that the separation effect of the macroporous resin is good.

The summary conclusion of the invention is as follows:

1. the content of alkaloid in aerial part of radix Aconiti lateralis

After the extract of the overground part of the monkshood is separated and purified by D101 macroporous resin, the yields of crude alkaloid obtained by 10 percent ethanol elution and 80 percent ethanol elution are respectively 0.27 percent and 1.41 percent, and the crude alkaloid can be calculated to be 1.68 percent when the extract of the overground part of the monkshood is combined. The alkaloid content of the overground part of the monkshood is not small, and the overground part of the monkshood has development value because the stems and leaves of the overground part of the monkshood are abundant.

2. Oxidation resistance

The oxidation resistance of the crude alkaloid extract is comprehensively analyzed by an ABTS method, a DPPH method and an FRAP method, and the total oxidation resistance of crude alkaloid eluted by 10 percent ethanol is similar to that of crude alkaloid eluted by 80 percent ethanol. Besides the oxidation resistance of alkaloid, the product also has other substances with oxidation resistance. The total alkaloid of the overground part of the monkshood has good oxidation resistance, so the method has development value.

3. Anti-acetylcholinesterase Activity

The anti-acetylcholinesterase activity of the total alkaloids in the aerial parts of monkshood is measured by an improved Ellman method, so as to judge the medicinal value of the total alkaloids in the aspect of treating AD. The results show that 10% and 80% of the total alkaloids have almost the same inhibitory effect, and thus the total alkaloids from the overground part of monkshood have almost the same anti-acetylcholinesterase activity, and the activity is more remarkable after further purification.

4. UPLC-Q-TOF-MS/MS spectrogram analysis

Because of more impurities, the spectrum is not ideal. However, the comparison literature still can judge that the 80% ethanol elution of alkaloid species than 10% ethanol elution, and also show the difference of the two yields (80% yield is greater than 10%). And the components of the two crude alkaloids are not mixed with each other, which indicates that the separation effect of the macroporous resin is good. This can be demonstrated initially that the aerial part of aconite has various alkaloids.

In conclusion, according to the extraction yield of crude alkaloids in the overground part of monkshood, the oxidation resistance and the acetylcholinesterase resistance of crude alkaloid samples, the overground part of monkshood has the activities of oxidation resistance and acetylcholinesterase resistance, the pathogenesis of Alzheimer disease is closely related to cholinesterase and oxidative stress, anti-senile dementia drugs and active parts are usually screened by using the dual-target activities of oxidation resistance and acetylcholinesterase resistance, and the overground part of monkshood is used as a waste resource, so that the anti-senile dementia drugs and health care products have development values, and basis and reference are provided for further researching the chemical components and the activity mechanism of the overground part of monkshood. The product of the invention is used as a crude extract of a natural product, the antioxidant and anti-AChE activities of the product show good activities, and the overground part of monkshood can be used as a raw material resource of a medicine for preventing and treating senile dementia. Analyzing by UPLC-Q-TOF-MS/MS spectrogram to obtain aconitine, hypaconitine, benzoylmesaconine, benzoylaconine, benzoylhypaconine, 10-OH-mesaconine, and Niolan.

Comparative example 1, the "pretreated D101 macroporous resin" in example 1 was changed to "pretreated AB-8 weakly polar resin", and the rest was identical to example 1. The total yield of 2 kinds of freeze-dried powder is about 1.1%.

Comparative example 2-1, the extractant of step 1 of example 1 was changed from "85% ethanol" to "95% ethanol"; the rest is the same as example 1. The total yield of 2 kinds of freeze-dried powder is about 1.5%.

Comparative example 2-2, the extractant of step 1 of example 1 was changed from "85% ethanol" to "75% ethanol"; the rest is the same as example 1. The total yield of 2 lyophilized powders was about 1.4%.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. The method for extracting total alkaloids from monkshood is characterized by comprising the following steps:

1.1 Sun drying the stems and leaves of the monkshood until the water content is less than or equal to 15 percent, and crushing to obtain crushed overground part of the monkshood;

1.2 600g of ground aconite and an extracting agent are mixed according to a feed-liquid ratio of 1g/8 to 12ml, and ultrasonic extraction is carried out at 50 ℃ and under the ultrasonic power of 400 to 800W for 2 to 4 hours; then filtering and separating to respectively obtain filtrate and filter cake;

repeating the ultrasonic extraction and the filtration by using the filter cake instead of the ground aconite, wherein the repetition times are 1 to 3 times, and combining all filtrates obtained by filtration to obtain an extracting solution;

the extractant is 85% ethanol solution;

1.3 Concentrating the extracting solution to 1 +/-0.1L to obtain concentrated solution; then adding petroleum ether for extraction; the volume usage ratio of the petroleum ether to the concentrated solution is 1 to 1.5:1; respectively obtaining an organic layer positioned at the upper layer and a water layer positioned at the lower layer;

taking a water layer; the water layer contains total alkaloids;

2) And (3) separating and purifying the total alkaloids: separating the water layer by using D101 macroporous resin, and sequentially carrying out the following steps:

2.1 D101 macroporous resin pretreatment:

soaking in 95% ethanol for 24 hr, removing ethanol, and washing with distilled water;

then soaking the mixture for 3 hours by using a 5% hydrochloric acid solution, and washing the mixture by using distilled water; soaking in 4% sodium hydroxide solution for 3 hr, and washing with distilled water;

2.2 Lyophilizing the water layer to obtain lyophilized powder;

adding water into the lyophilized powder to make the concentration of the lyophilized powder in the obtained suspension be 0.2 + -0.02 g/mL, and adjusting pH to 3-4 with hydrochloric acid to obtain a sample solution;

2.3 Loading the pretreated D101 macroporous resin obtained in the step 2.1) into a column, adding a sample solution, standing the sample solution for 2 hours, discharging the sample solution, and washing the sample solution with 2BV of distilled water at a flow rate of 1.5mL/min to remove water-soluble macromolecular components;

eluting with 10% ethanol at a flow rate of 4BV/h, and eluting with 80% ethanol at a flow rate of 4BV/h, wherein the use amount of the 10% ethanol is 2 to 3BV, and the use amount of the 80% ethanol is 3 to 4BV;

concentrating the eluates corresponding to 10% and 80% ethanol, and vacuum freeze drying to obtain crude alkaloids 10% and crude alkaloids 80% ethanol eluates;

the crude alkaloid eluted by 10% ethanol contains: benzohypacotinine, benzophenononitine;

the crude alkaloid eluted by 80% ethanol contains: neoline, dehydrated benzoylmesaconine, hypacoconine, mesacconine, 10-OH-Mesacconine.

2. The application of the total alkaloids extracted by the method as claimed in claim 1 in preparing anti-oxidation drugs and acetylcholinesterase inhibiting drugs, wherein the total alkaloids extracted by the method as claimed in claim 1 are as follows: the crude alkaloid eluted by 10% ethanol and the crude alkaloid eluted by 80% ethanol respectively have the oxidation resistance and the acetylcholinesterase inhibition.