CN111366680A

CN111366680A - Substance content determination method and application thereof

Info

Publication number: CN111366680A
Application number: CN202010132575.4A
Authority: CN
Inventors: 王伟; 赵步长; 孙晓丽; 赵涛; 赵超; 王益民
Original assignee: Shaanxi Buchang Pharma Co ltd
Current assignee: Shaanxi Buchang Pharma Co ltd
Priority date: 2020-02-29
Filing date: 2020-02-29
Publication date: 2020-07-03
Anticipated expiration: 2040-02-29
Also published as: CN111366680B

Abstract

The invention relates to a method for measuring the content of a substance and application thereof, wherein the prescription of the substance comprises 225 parts of astragalus, 100 parts of leech, 90 parts of ligusticum wallichii, 90 parts of angelica, 90 parts of safflower, 113 parts of peach kernel, 90 parts of red paeony root, 90 parts of costustoot, 90 parts of rhizoma acori graminei, 60 parts of earthworm, 90 parts of parasitic loranthus and 35 parts of acanthopanax extract. The content detection method has the characteristics of simple and rapid operation, and good repeatability and accuracy. The pharmacological experiment of the substance shows that the compound can obviously improve the coordination ability of mouse behaviors, obviously shorten the climbing time of the mouse, enhance the holding power of the mouse and the swimming time of the mouse, and can obviously relieve MPTP-induced neuron damage. The content determination method of the substance can effectively control the internal quality of the medicine, and the substance has new therapeutic application, thereby expanding the clinical medication and disease treatment range of the medicine.

Description

Substance content determination method and application thereof

Technical Field

The invention relates to a substance content determination method and application thereof, belonging to the field of quality detection content methods.

Background

The leech contained in the ministerial drug of the invention contains various active ingredients, such as hirudin, thrombin and the like, and has the effects of anticoagulation, antithrombotic, fibrinolysis and the like. The research shows that the plasma antithrombin (AT-III) level is obviously reduced in the plasma of a cerebral infarction patient and is highly associated with the severity of cardiovascular and cerebrovascular diseases. Wuyang, Yan Ge et al, Fufang Pallas pit Viper venom capsule antithrombin Activity quality Standard evaluation research, journal of practical medicine, 2019-01, reported in literature, and used thrombin titration to determine the antithrombin activity of Fufang Pallas pit Viper venom capsule. The research results preliminarily draw up the anticoagulant activity of the compound viper venom capsule not less than 300.0U/g. The method is accurate, simple, convenient and rapid, and can realize quality control of the preparation. Wangwangjian 31054, study on heavy metal residue and anticoagulant activity of leech, a university of Beijing Chinese medicine, 2016-05, which adopts a determination method of antithrombin activity to evaluate antithrombin potency of leech.

The carding analysis is carried out aiming at the method for measuring the content of the active ingredient astragaloside in the monarch drug astragalus contained in the substance of the invention, and the measuring method of the ingredient mainly comprises a thin layer scanning method, an HPLC method and a near infrared spectroscopy method. For example, Jiafu Huai, Luwei et al, an improvement of the method for detecting the content of astragaloside in astragalus decoction pieces, a traditional Asia-Pacific medicine, 2019-10, and the article compares several different pretreatment extraction methods, finally determines a pretreatment method combining reflux extraction and ultrasonic extraction and extracting and purifying n-butanol and ammonia test solution, and combines an HPLC-ELSD method to determine the content of astragaloside. The improved method is accurate, efficient and good in reproducibility, and is suitable for detecting the content of astragaloside in the astragalus decoction pieces. Song Zeng dazzling; the content of astragaloside in urinary calculus removing particles is measured by a high performance liquid chromatography-evaporative light scattering detection method, the strait pharmaceutical is 2019-09, and the content of astragaloside serving as an effective ingredient of astragalus in the urinary calculus removing particles is measured by the high performance liquid chromatography-evaporative light scattering detection method, and the content limit is determined. The method can effectively control the quality of urinary calculus removing granule. According to the method, astragaloside and total solid content in the astragalus injection are rapidly detected by near infrared spectroscopy, 2012-10, Chinese herbal medicine, in Baixintao, Hoobao et al. However, the content detection method has the defects of complicated detection steps and high cost in the practical production process.

Parkinson's disease PD is a progressive degenerative disease of the nervous system with major clinical manifestations of motor and non-motor symptoms. Non-motor symptoms are manifested in a variety of forms, including affective disorders (e.g., apathy, depression, anxiety, and the like), cognitive impairment (e.g., dementia), autonomic dysfunction (e.g., bladder dysfunction), sleep disorders, sensory abnormalities (e.g., pain), language disorders, and feelings of fatigue. According to related reports, the number of Parkinson disease patients is increased by more than one time to more than 600 ten thousand in the whole world from 1990 to 2016. The incidence rate of the Parkinson disease in the population over 60 years is about 1 percent, and the number of the Parkinson disease in China tends to increase year by year due to the current aging aggravation.

The pathogenesis of the disease is very complex, and the clinical treatment is mainly based on drugs. The common monoamine oxidase inhibitors, anticholinergic drugs, levodopa and the like are clinically proven, and after a part of patients adopt independent drugs, certain symptoms can be improved, but the long-term curative effect is not ideal, but the long-term administration of the drugs can cause dyskinesia and mental symptoms, and the curative effect of the drugs can be obviously reduced.

The material is the leech capsule which is produced by Shanxi step size pharmaceutical company Limited and is mainly prepared from the components of astragalus, leech, ligusticum wallichii, angelica sinensis, safflower, peach kernel, red paeony root, costus root, grassleaf sweelflag rhizome, earthworm, Chinese taxillus twig, acanthopanax extract and the like. The medicine has the following effects: tonify qi, activate blood, remove blood stasis and dredge collaterals. Is used for treating hemiplegia, hemianesthesia, facial distortion, language disorder and the like in the stroke with qi deficiency and blood stasis type apoplexy in the recovery period of arteriosclerotic cerebral infarction. The national drug standard number executed nowadays is WS3-375 (Z-040) -2005(Z), and the drug is clinically used for treating cardiovascular and cerebrovascular diseases such as cerebral infarction, cerebral apoplexy and the like. Since the product comes into the market, the product has obvious and reliable clinical curative effect and is deeply favored by consumers. In contrast, the applicant has conducted a great deal of systematic research on the product in terms of drug composition, process improvement, pharmacodynamic substance components, pharmacological and pharmacodynamic tests, and has laid out a series of patent applications, wherein the application numbers are as follows: 02114551.2, 200510041937.4, 200510043066.X, 201010213671.8 and 201210228789.7 the subject matters of the patent protection contents mostly focus on the aspects of the formula proportion, the preparation method, the quality component detection method and the new clinical application of the medicine in diabetic diseases. Aiming at the problem that the effective substance components and the internal quality of the existing Longsheng leech capsule are difficult to effectively characterize by only taking astragaloside as the quality control index component of the product. And the application No. CN 109307721A of my company discloses that HPLC-QQQ/MS method is used for detecting active substance components of traditional Chinese medicines such as astragaloside A, hydroxysafflor yellow A, calycosin glucoside, calycosin, ferulic acid, syringin, n-butyl phthalide, ligustilide, senkyunolide A, senkyunolide I, senkyunolide H, ligustrazine, isofraxidin, dehydrocostuslactone, amygdalin, eleutheroside E, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, etc. in the capsule containing the Longsheng leech. However, the above detection method has a lot of operation cost steps, which is obviously far from sufficient for controlling the internal quality of the Chinese medicinal preparation. In a large number of clinical use processes, the product is unexpectedly found to have remarkable treatment effects on the Parkinson disease besides the treatment of the stroke disease.

Disclosure of Invention

The invention aims to provide a method for measuring the content of a substance and application thereof, wherein the substance takes hirudin as the content of a detection index, the content detection method has the characteristics of simple and quick operation and good repeatability and accuracy, and the method can effectively control the internal quality of the substance.

The invention aims to provide a new clinical application of a substance, and relates to an application of the substance in preparing a medicament for treating Parkinson's disease.

In a large amount of clinical medication processes, the surprising discovery shows that the substance can treat cardiovascular and cerebrovascular diseases of patients suffering from the cardiovascular and cerebrovascular diseases and also suffering from Parkinson's disease after the patients take the medicament, and the symptoms of the Parkinson's disease are obviously improved.

The technical scheme of the invention is as follows:

1. a method for detecting the content of a substance, comprising the steps of:

(1) the material comprises the following raw material medicines in percentage by weight: 225 parts of astragalus, 100 parts of leech, 90 parts of ligusticum wallichii, 90 parts of angelica sinensis, 90 parts of safflower, 113 parts of peach kernel, 90 parts of red paeony root, 90 parts of costustoot, 90 parts of rhizoma acori graminei, 60 parts of earthworm, 90 parts of parasitic loranthus and 35 parts of acanthopanax extract;

(2) the preparation method comprises the following steps: pulverizing Hirudo, Lumbricus, and radix Acanthopanacis Senticosi dry extract into fine powder; decocting radix astragali, rhizoma Ligustici Chuanxiong, radix Angelicae sinensis, Carthami flos, semen Persicae, radix Paeoniae Rubra, radix aucklandiae, rhizoma Acori Graminei and herba Taxilli with water twice (2 hr for the first time and 1.5 hr for the second time), filtering, mixing decoctions, concentrating the filtrate to relative density of 1.1-1.2 at 60 deg.C, spray drying into powder, adding Lumbricus, Hirudo and radix Acanthopanacis Senticosi powder, and mixing well to obtain the final product;

(3) preparing a test solution:

taking the mixed powder obtained in step (2) as a sample, and adding physiological saline respectively to obtain a solution with a concentration of 0.02 g/mL^-1～0.2g·mL^-1Samples of concentration gradient, i.e. samples, were vortexed for 30min, centrifuged for 10min, 5000rpm, taking supernatant, refrigerating for later use, and treating the leech medicinal material by the same method;

(4) negative control solution preparation

The leech-free sample is obtained according to the preparation process in the step (2), and is prepared according to the method in the step (3), namely 0.02 g/mL is obtained^-1～0.02g·mL^-1A sample of concentration gradient, a negative control solution of 10 concentration gradients;

(5) preparation of buffer solution

Taking 0.2 mol.L^-1Tris solution with 0.1 mol. L^-1Adding 40mL of hydrochloric acid solution, adding water to 100mL, and adjusting the pH value to 7.4;

(6) preparation of Thrombin titration solution

Adding 1000U thrombin into 5mL physiological saline, subpackaging according to 1mL, storing at-20 deg.C, collecting thrombin, and preparing into 10 U.mL-1, 20 U.mL-1 and 40 U.mL^-1；

(7) Determination of content

Precisely measuring a sample solution to be tested, placing the sample solution into an EP (EP) tube, adding 200 mu L of trihydroxymethyl aminomethane hydrochloric acid buffer solution containing 0.5% bovine fibrinogen, uniformly mixing, placing the sample solution into a water bath, soaking for 5min, starting to dropwise add thrombin titration solution, starting timing at the moment of dropwise addition, titrating until the sample solution to be tested is solidified or an end point state appears, recording the time of the end point of titration and the volume of consumed thrombin solution, and calculating the content of the thrombin solution.

The invention also provides a method for measuring the content of astragaloside, which comprises the following steps: the content detection method comprises the following steps:

taking 6g of the content of the product, accurately weighing, placing in a conical flask, accurately adding 50ml of 2% potassium hydroxide methanol solution, weighing, heating, refluxing and extracting for 1 hour, cooling, weighing, supplementing the weight loss with 2% potassium hydroxide methanol solution, shaking up, filtering, accurately weighing 30ml of subsequent filtrate, placing in an evaporator, adding 20ml of water into the residue after evaporation to dissolve, adding a chloroform-n-butanol mixed solution with a volume ratio of 2:1 to extract for 5 times, 10ml each time, combining the extract, washing with ammonia test solution for 2 times, 20ml each time, discarding ammonia solution, evaporating chloroform-n-butanol solution to dryness, dissolving the residue with methanol, transferring into a measuring flask, adding methanol to dilute to scale, shaking up to obtain a sample solution. Taking appropriate amount of astragaloside IV reference substance, precisely weighing, and adding methanol to obtain 1ml reference substance solution containing 0.5 mg. According to the experiment of appendix VIB of version 2000 of thin-layer chromatography, 4 mu L and 6 mu L of sample solution and 3 mu L and 5 mu L of reference solution are respectively absorbed on the same silica gel G thin-layer plate with 0.3 percent sodium carboxymethylcellulose as adhesive at cross points, and the layered chloroform-methanol-water lower layer solution is placed below 10 ℃ as developing solvent, wherein the volume ratio of chloroform-methanol-water is 65: 35: spreading, taking out, air drying, spraying 10% sulfuric acid ethanol solution, drying at 100 deg.C until the spots are clearly developed, taking out, covering a glass plate with the same size on a thin-layer plate, fixing the periphery with adhesive tape, scanning by thin-layer chromatography, measuring the absorbance integral value of the sample and the control sample at wavelength λ s of 530nm and λ R of 650nm, and calculating.

The application of a substance in preparing a medicament for treating Parkinson's disease comprises the following raw material medicaments in part by weight: 225 parts of astragalus membranaceus, 100 parts of leech, 90 parts of ligusticum wallichii, 90 parts of angelica sinensis, 90 parts of safflower, 113 parts of peach kernels, 90 parts of red paeony roots, 90 parts of elecampane, 90 parts of rhizoma acori graminei, 60 parts of earthworms, 90 parts of parasitic loranthus and 35 parts of acanthopanax extract.

The raw material medicaments of the substance comprise the following components in percentage by weight: pulverizing Hirudo, Lumbricus, and radix Acanthopanacis Senticosi dry extract into fine powder; decocting radix astragali, rhizoma Ligustici Chuanxiong, radix Angelicae sinensis, Carthami flos, semen Persicae, radix Paeoniae Rubra, radix aucklandiae, rhizoma Acori Graminei and herba Taxilli with water twice, 2 hr for the first time and 1.5 hr for the second time, filtering, mixing decoctions, concentrating the filtrate to relative density of 1.1-1.2 at 60 deg.C, spray drying to obtain powder, adding Lumbricus, Hirudo and radix Acanthopanacis Senticosi powder, mixing, adding medicinal adjuvants, and making into various pharmaceutically acceptable common dosage forms.

In the application of the medicine, the dosage forms of the medicine are capsules, tablets, pills, granules and the like. In order to make the above dosage forms possible, pharmaceutically acceptable excipients are added in the preparation of the above medicaments, such as: fillers, disintegrants, lubricants, binders, flavoring agents, and the like, including: starch, lactose, mannitol, chitin, microcrystalline cellulose and the like, and disintegrating agents include: starch, microcrystalline cellulose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose and the like, and the lubricating agent comprises: magnesium stearate, sodium lauryl sulfate, talc, and the like, binders including starch slurry, polyvinylpyrrolidone, hydroxypropylmethylcellulose, and the like, and sweeteners including: saccharin sodium, aspartame, sucrose, etc., and flavoring agents include: sweeteners, and the like.

The experimental mean statistical analysis of the leech capsule and the leech medicinal material is that the thrombin concentration is 10 U.mL^-1The time and the duration of the preparation have no obvious difference, which shows that the preparation production conditions have no obvious influence on the tested substances in the medicinal materials, and the method can be used for one of the quality control of the content determination of the Longsheng leech capsule.

We apply the hard capsules of the invention to clinical treatment of cardiovascular and cerebrovascular diseases and apoplexy in a large number, do a large number of drug effect substance basic researches, and find that the hard capsules also have new application in treating Parkinson's disease by accident.

The invention has the beneficial effects that:

because the detection method of the product in the prior art has more operation cost steps and higher cost, the company selects the hirudin active ingredient with obvious blood circulation promoting and blood stasis removing components as the quality control index of the product, and the monarch drug leech of the product treats both principal and secondary aspects of disease by removing blood stasis, the anticoagulant component is mainly hirudin, and the hirudin has the pharmacological effects of anticoagulation, antithrombotic and cardiovascular treatment. The thrombin titration method is adopted to measure the hirudin content in the preparation as one of the quality standards of the preparation. In a large number of clinical use processes, the product is unexpectedly found to have remarkable treatment effect on patients with Parkinson's disease besides treating stroke diseases. Therefore, corresponding pharmacological and pharmacodynamic tests are carried out to verify, so as to expand the range of diseases treated by clinical medication of the medicine and provide a new treatment idea and clinical medication selection for treating the diseases.

The invention also provides pharmacological tail suspension experiments and pole climbing experiments, and the medicament can obviously improve the behavior coordination ability of mice and obviously shorten the pole climbing time of the mice. The grip strength experiment result shows that the medicament can enhance the grip strength of the mouse, obviously enhance the muscle strength of four limbs of the mouse, prolong the swimming time of the mouse and obviously improve the behavior coordination capability. Serum biochemistry shows that the high-dose group of the medicine can obviously improve SOD activity, the high-dose and medium-dose groups can obviously reduce MDA content, and the high, medium and low doses of the medicine can obviously improve the activity of glutathione peroxidase (GSH-px). The drug can reduce oxidative stress injury in the MPTP induced mouse PD process. The immunohistochemical determination result shows that the medicine can obviously relieve MPTP-induced neuron damage.

From the results, the medicine provided by the invention develops a new treatment application, expands the range of diseases treated by clinical medication of the medicine, and provides a new medication choice for treating the Parkinson's disease. The medicine has the characteristics of safety and strong pharmacological activity, and has good market application prospect.

The present invention is further illustrated by the following exemplary embodiments in order that the practice of the invention may be more fully understood. The beneficial therapeutic effects of the drug are demonstrated below and by pharmacodynamics.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the comparison of anticoagulant activity of a hirudo product measured with thrombin at different concentrations (2. mu.L each time at 1min intervals);

FIG. 2-anticoagulant activity of hirudo drugs measured at different concentrations of thrombin (2. mu.L each time every 1 min);

FIG. 3 is a thin-layer chromatogram of the content of astragaloside in the substance;

FIG. 4-photograph of immunohistochemistry on paraffin sections of mouse brain tissue, which includes: a blank control group, a model control group, a medoba control group, a high-dose group of the medicament of the invention, a medium-dose group of the medicament of the invention, and a low-dose group of the medicament of the invention.

Detailed Description

In order to better understand the present invention, two experimental examples are listed below to illustrate its new use in the pharmaceutical field. The following experiments are intended to illustrate the invention and not to limit it.

EXAMPLE 1 method for detecting the content of hirudin in the drug of the invention

1. Instrument and reagent

Analytical balance (Switzerland Meiteler), HW.SY11-KP2 intelligent constant temperature water bath (Beijing Changfeng instruments and meters), 3-18KS centrifuge (German Sigma), KQ-500E ultrasonic cleaner (Kunshan ultrasonic instruments, Inc.), Longsheng leech capsule (Shaanxi stepsize, lot number: 181005), leech (provided by stepsize, Inc.), physiological saline (Shijiazhuang Siyao, Inc.), fibrinogen (lot number: P06J10Y92294, Beijing Byeldi Biotech, Inc.), trimethylol aminomethane, hydrochloric acid, thrombin T8020 (lot number: 417G062, Beijing Sorbebao, Inc)

2. Method and results

2.1 preparation of the solution

2.1.1 preparation of test solutions

0.4g, 0.3g, 0.2g, 0.1g were taken out, and 2mL, 4mL, 4.44mL, 5mL, 5.7mL, 6.67mL, 7.5mL, 6.67mL, 5mL of physiological saline were added to the sample to prepare a solution having a concentration of 0.2 g/mL^-1、0.1g·mL^-1、0.09g·mL^-1、0.08g·mL^-1、 0.07g·mL^-1、0.06g·mL^-1、0.05g·mL^-1、0.04g·mL^-1、0.03g·mL^-1、0.02g·mL^-1And (3) vortexing the samples with 10 concentration gradients, namely the samples from No. 1 to No. 10 for 30min, centrifuging the samples for 10min at 5000rpm, taking supernatant, and refrigerating the supernatant for later use. Hirudo materials (provided by Hairhiki Kaisha) are processed by the same method.

2.1.2 negative control solution preparation

The leech-free sample is prepared according to the preparation process of the capsule of the leech, and the preparation method is adopted to obtain 0.2 g.mL^-1、0.1g·mL^-1、0.09g·mL^-1、0.08g·mL^-1、0.07g·mL^-1、0.06g·mL^-1、0.05g ·mL^-1、0.04g·mL^-1、0.03g·mL^-1、0.02g·mL^-110 concentration gradients of negative control solution.

2.1.3 preparation of buffer

Taking 0.2 mol.L^-125mL of tris solution and 0.1 mol. L^-1About 40mL of hydrochloric acid solution, water was added to 100mL, and the pH was adjusted to 7.4.

2.1.4 preparation of Thrombin titration solution

Adding 1000U thrombin into 5mL physiological saline, subpackaging according to 1mL, storing at-20 deg.C, taking 1mL thrombin, and preparing into 10 U.mL thrombin^-1、20U·mL^-1And 40 U.mL^-1。

2.2 measurement of content

Precisely measuring 100 mu L of sample solution, placing the sample solution into a 2mL EP tube, adding 200 mu L of trihydroxymethyl aminomethane hydrochloric acid buffer solution (prepared for clinical use) containing 0.5 percent (bovine) fibrinogen (calculated by coagulum), uniformly mixing, placing the sample solution into a water bath (37 +/-0.5) DEG C for warm immersion for 5min, starting to dropwise add thrombin, starting timing at the moment of dropwise addition, titrating until the sample solution to be detected is coagulated or an end point state appears, and recording titration end point time and the volume of consumed thrombin solution.

2.3 factors influencing the determination of the content

2.3.1 examination of extraction methods

The same batch of samples are extracted by 4 extraction modes of ultrasonic treatment for 15min,30min, vortex treatment for 30min and 60 min. The anticoagulation activity is determined by titration (10U. mL-1, 2 μ L per minute), and the result shows consistent anticoagulation activity, and the anticoagulation activity is fully stirred and extracted for 30min by selecting vortex in accordance with the extraction time in pharmacopoeia.

2.3.2 titration temperature

The temperature has a great influence on the titration process, and the same batch of samples are titrated at 25 ℃, 37 ℃, 50 ℃ and 70 ℃. The results show that the hirudin activity is significantly reduced at a temperature of 50 ℃ and 70 ℃. The activity results were consistent at 25 ℃ and 37 ℃. Therefore, the original standard titration temperature can be kept at 37 ℃.

2.3.3 Effect of test solution pH on assay results

The pH value of the test solution is between 4.5 and 7.0, the anticoagulation activity of the test solution is basically stable, when the pH value of the test solution is lower than 4.0, the solution is not clear, the coagulation phenomenon cannot be observed, when the pH value is too high, the anticoagulation activity is reduced, the pH values of the test solution dissolved by normal saline are all between 4.5 and 7.0, and the pH value of the test solution is 5-6(pH test paper)

2.3.4 titration endpoint determination

The same batch of samples was vortexed for 30min, titrated at 37 ℃ and summarized according to the phenomenon. In the titration process, the fact that a filament or a small ball can be picked out from the test tube is observed as the titration endpoint. The titration process is observed to be too long, the shaking is too frequent (more than 15min), the silk or the small balls are not easy to be observed, and the repeatability of the titration method within 10min is better.

2.3.5 Thrombin titration concentration and Interval time

Two methods for measuring anticoagulant activity are provided in the 'Chinese pharmacopoeia' 2015 edition, one is blood-sucking leech, thrombin concentration is dropped into the leech at 40 U.mL < -1 >, and the thrombin is dropped for 1 time every 1min and 5 mu L of thrombin is dropped for each time; the other is non-hematophagous leech (Whitmania pigra Whitman) or Whitmania acranulata Whitman, the thrombin concentration is dropped into the mixture to be 10 U.mL^-1Dripping 1 time every 4min, 2 μ L each time. The 2 procedures for determining anticoagulant activity were essentially identical, but the two methods differed by an average of 40 fold per minute units of thrombin activity dropped. Therefore, it is known that the titration interval time and the thrombin concentration are important factors for measuring the anticoagulation activity.

2.3.5.1 Effect of Thrombin titration Interval time on anticoagulation Activity determination

Method I, Thrombin 40 U.mL^-15 μ L each time, method two, thrombin 10 U.mL^-12 mu L each time, respectively adopting two different intervals of 1min and 4min to determine the anticoagulant activity of the same test sample with different concentrations, and mainly investigating the influence of the titration interval on the measured value, wherein the results are shown in Table 1.

Table 1 effect of titration interval time of leech capsules on anticoagulant activity (x ± s, n ═ 3)

As can be seen from Table 1, whether the method I or the method II is adopted, the anticoagulant activity measured by the method with the interval time of 4min is far less than the value measured by the interval time of 1min, which indicates that the titration interval time has great influence on the measurement of the anticoagulant activity value

2.4 Effect of Thrombin concentration on anticoagulation Activity measurement

By thrombin titration, 20, 10 U.mL of the reagent was used^-1Dropping two concentrations of thrombin solution 1 times every 1min, each dropping 2 μ l, titrating different concentrations of Hirudo capsule sample and Hirudo medicinal material (provided by step-size Limited company), and calculating its anticoagulation activity value (tables 2 and 3)

TABLE 2 anticoagulation Activity of Syngnathus leeches, measured by titration of thrombin concentrations at various concentrations

Note: "-" indicates no reaction within 30min, and "-" indicates null

As can be seen from Table 2, the negative control group did not interfere with the measurement of anticoagulation activity, when the concentration was 0.2 g/mL^-1When the method is used, the thrombin titration process has no phenomenon in 30min, the measured anticoagulation activity is unstable when the concentration is 0.07-0.1 g.mL < -1 >, and the measured value cannot be used for calculating the anticoagulation activity; when the concentration of the sample is 0.02-0.06 g/ml^－1In the linear range of (1), the thrombin concentrations are 20 U.mL respectively^－1And 10 U.ml-1, the titration interval time is 1min, and the measured anticoagulant activity is 11.6 +/-4.76 U.g^-1And 13.49. + -. 2.35 Ug^-1RSD were 2.44% and 5.74%, respectively. Wherein the thrombin concentration is 10 U.ml^－1The measured activity values are relatively close, and the accuracy is better; and the thrombin concentration is 20 U.mL^-1The measured values were all in large error (fig. 1).

TABLE 3 anticoagulation Activity of hirudo drugs measured by titration of Thrombin concentrations at different concentrations

Note: "-" indicates no reaction within 30min, and "-" indicates null

As shown in Table 3, when the concentration of leech is 0.03-0.08 g/ml^－1In the linear range of (1), the thrombin concentrations are respectively 20 U.mL^-1And 10 U.mL^-1The anticoagulant activity is measured to be 8.12 +/-2.84 U.g^-1And RSD of 2.86%. Where the anticoagulation activity measured at too high or too low a sample concentration is unstable.

2.5 data processing

2.5.1 the values of the homogeneity test of variance (significance level 0.05) statistical test of variance the results of the variance are shown in Table 4

TABLE 4 statistical test results of variance of LONGSHENGBIAN Capsule and Hirudo

As is clear from Table 4, when the thrombin concentrations were 20U. mL, respectively^－1And 10 U.ml^－1When F is 0.5595<3.45 and F-0.3005<3.45, considering that the experimental numerical variances of the capsule and the medicinal material of the leech are the same in the experimental process, the t test between the mean values can be carried out.

2.5.2 content mean value test the content mean values of the preparation and the medicinal materials were subjected to t test, and the results are shown in table 5.

TABLE 5 t test results of LONGSHENGZHI Capsule and Hirudo

As is clear from Table 5, when the thrombin concentrations were 10 U.ml, respectively^－1When t is 1.63<2.145, the experimental mean values of the capsule and the medicinal material of the leech have no significant difference within 95% of the confidence interval in the experimental process. Therefore, the current method in pharmacopoeia is considered to be to add the capsule of the leechThe method is simple and quick, has accurate and reliable measurement result, and can be used for quality control of the Longsheng leech capsule.

3 analysis and summary of the results

3.1 methodological validation

Since the Longsheng leech capsule is composed of 12 traditional Chinese medicines and auxiliary materials are added during processing, the study on specificity and detection limit is required. The specificity can be seen from a negative control group, and other interference components have no influence on the experiment; the detection range is 0.02-0.06 g/ml^－1And the anticoagulation activity is stable. As can be seen from the tests of precision, linear relation, specificity, detection limit and stability, the method for determining the anticoagulation activity of the Longsheng leech capsule is stable and reliable.

3.2 method applicability

The leech medicinal materials have different content differences at home and abroad, and the hirudin content of leeches reported in literatures is far higher than that of leeches on the current market. The titration solution used for measuring the content of different types of medicinal materials is specified in the Chinese pharmacopoeia 2015 edition. Before the raw materials are inspected, the variety and the source of the raw materials must be accurately identified, the concentration of the titration solution used for content determination is different when the variety is different, and the large error is caused when the high-concentration titration solution is used for determining the variety with low hirudin content, so that the deviation of a determination numerical value system is large.

3.3 discussion of the concentration determination of Longsheng leech capsule

The investigation shows that when the concentration of the sample of the leech capsule is 0.02-0.06 g.mL^-1In between, the thrombin concentration is 20 U.mL, respectively, in a substantially linear relationship with the anticoagulation activity (FIG. 1)^-1And r values of 0.9026 and 0.9738 for 10 U.mL-1, wherein the thrombin concentration is at 10 U.mL^-1The measured anticoagulant activity value of the sample is 13.49 +/-2.35 U.g^-1The RSD was 5.74%, and the measured activity values were relatively stable (FIG. 1).

3.4 discussion of titration interval time and titration thrombin concentration in determining anticoagulation activity of Hirudo nipponica Whitman

It is found herein that thrombin concentration and titration interval time are measured on a drop of thrombin from the same sampleTiming and the measured anticoagulant activity value have a greater effect. Dripping thrombin 40 U.mL-1 in 1min once in 5 μ L each time for highly active hirudo nipponia in pharmacopoeia; for low-activity whitmania pigra, a titration method of 10 U.mL-1 is adopted, the concentration of the two is different by 40 times, and the titration method is carried out once every 4min and every 2 mu L. When the thrombin concentration and the titration interval time of the preparation and the medicinal materials are determined by a pharmacopoeia method, a sample with higher concentration cannot be measured, and the reasons are as follows: firstly, the method comprises the following steps: multiple titrations dilute the fibrinogen stock, which is not conducive to coagulation: secondly, the method comprises the following steps: shaking up once per drop to destroy the coagulation formed by the initial connection of some fibrin, which is often invisible to eyes, and the repeated destruction to thin fibrin may result in permanent titration and no coagulation. The measurable range is narrow (0.02-0.06 g.mL)^-1) (ii) a The thrombin concentration is 20 U.mL^-1And 10 U.mL^-1The thrombin concentration is too high to be measured meaninglessly, so that the method is not suitable for measuring the contents of preparations and medicinal materials; the interval time is selected to be 1min, the time is too long, and the titration endpoint phenomenon is not easy to observe.

Example 2 the drug is prepared as a capsule

Prescription: 225g of astragalus, 100g of leech, 90g of ligusticum wallichii, 90g of angelica, 90g of safflower, 113g of peach kernel, 90g of red peony root, 90g of costustoot, 90g of rhizoma acori graminei, 60g of earthworm, 90g of parasitic loranthus and 35g of acanthopanax extract;

the preparation method comprises the following steps: pulverizing Hirudo, Lumbricus, and radix Acanthopanacis Senticosi dry extract into fine powder; decocting radix astragali, rhizoma Ligustici Chuanxiong, radix Angelicae sinensis, Carthami flos, semen Persicae, radix Paeoniae Rubra, radix aucklandiae, rhizoma Acori Graminei and herba Taxilli with water twice (2 hr for the first time and 1.5 hr for the second time), filtering, mixing decoctions, concentrating the filtrate to relative density of 1.1-1.2 at 60 deg.C, spray drying to obtain powder, adding Lumbricus, Hirudo and radix Acanthopanacis Senticosi powder, mixing, adding medicinal starch, and making into 1000 granules.

The application and dosage are as follows: is administered orally. 3-5 granules at a time, 3 times a day.

EXAMPLE 3 preparation of the drug into tablets

the preparation method comprises the following steps: pulverizing Hirudo, Lumbricus, and radix Acanthopanacis Senticosi dry extract into fine powder; astragalus root, Ligusticum wallichii, Chinese angelica root, safflower, peach kernels, radix paeoniae rubrathe, banksia rose, grassleaved sweetflag rhizome, Loranthus mulberry mistletoe, watering and boiling twice, the first time 2 hours, the second time 1.5 hours, filtering, merging the boiling liquid, concentrating the filtrate to relative density 1.1-1.2 at 60 deg.C, spraying and drying to powder, charging earthworm, leech, acanthopanax powder, mixing homogeneously, charging right amount of starch slurry and magnesium stearate, mixing homogeneously, granulating, pressing into tablets.

Example 4 the drug is prepared as a pill

the preparation method comprises the following steps: pulverizing Hirudo, Lumbricus, and radix Acanthopanacis Senticosi dry extract into fine powder; astragalus root, Ligusticum wallichii, Chinese angelica root, safflower, peach kernels, radix paeoniae rubrathe, banksia rose, grassleaved sweetflag rhizome, Loranthus mulberry mistletoe, watering and boiling twice, the first time 2 hours, the second time 1.5 hours, filtering, merging the grilling liquid, concentrating the filtrate to the relative density of 1.1-1.2 at 60 deg.C, spraying and drying to powder, charging earthworm, leech, acanthopanax root powder and mixing homogeneously, charging right amount of hydroxypropyl methyl cellulose and low substituted hydroxypropyl cellulose, mixing homogeneously, granulating, pressing into tablets.

Example 5 preparation of the drug into granules

Prescription: 225g of astragalus, 100g of leech, 90g of ligusticum wallichii, 90g of angelica sinensis, 90g of safflower, 113g of peach kernel, 90g of red paeony root, 90g of costustoot, 90g of rhizoma acori graminei, 60g of earthworm, 90g of parasitic loranthus and 35g of acanthopanax extract;

the preparation method comprises the following steps: pulverizing Hirudo, Lumbricus, and radix Acanthopanacis Senticosi dry extract into fine powder; astragalus root, Ligusticum wallichii, Chinese angelica root, safflower, peach kernels, radix paeoniae rubrathe, banksia rose, grassleaved sweetflag rhizome, Loranthus mulberry mistletoe, watering and boiling twice, the first time 2 hours, the second time 1.5 hours, filtering, merging the boiling liquid, concentrating the filtrate to relative density 1.1-1.2 at 60 deg.C, spraying and drying to powder, charging earthworm, leech, acanthopanax powder and mixing homogeneously, charging right amount of dextrin and lactose, mixing homogeneously, granulating, drying and making into granules.

Example 6 method for determining content of astragaloside in drug of the invention

EXAMPLE 7 pharmacological Activity test of the drug of the present invention

1. Mouse Parkinson model induced by MPTP (Multi-protein transfer point) by using leech capsules

1.1 Experimental materials

1.1.1 Experimental animals

C57BL/6J, SPF male mice, 72, about 23-25g, Beijing Wittiulihua laboratory animal technology, Inc.; license number: SCXK (Kyoto) -2016-.

1.1.2 Experimental drugs

The specification of the capsule is 0.4 g/granule, and the batch number is as follows: 20171250, Shanxi stepsize pharmaceuticals, Inc.; meiduoba, specification 0.25 g/tablet, batch number: SH3288, Shanghai Roche pharmaceutical Co., Ltd., lipid oxidation (MDA) detection kit, manufactured by Nanjing, with product number of batch No. 20180412; superoxide dismutase (SOD), manufacturer: building Nanjing; product number 20180418; glutathione peroxide (GSH-PX) determination kit, manufacturer, Nanjing Kangkui, product batch number 20180908.

1.1.3 Experimental reagent

MPTP (1-methyl-4-phenyl-1, 2,3, 6-tetrahydropyridine), batch No.: m9049-02, supplied by AbMoleBioScience, paraformaldehyde, manufacturer: wuhan google biotechnology limited, lot number: g1101; paraffin, xylene (chemical reagents of national drug group limited), PBS (G0002, google bio-technologies of wuhan), EDTA (G1203, google bio-technologies of wuhan).

1.1.4 Experimental instruments

SA417 rat grip strength tester, manufacturer: YLS-13A, a science and technology development Co., Ltd, Yiyan Ji. Experimental swimming box, manufacturer: self-made, freezing centrifuge, manufacturer: heal Force; a photographic microscope: nikon Nikoneclipse Ti-SR.

1.2 Experimental methods

1.2.1 Molding, grouping and administration

The adoption of MPTP subacute molding method: the experimental mice were injected intraperitoneally (ip) with MPTP at 30mg/k once daily for 5 consecutive days for a fixed period of time (9:00am) daily to prepare PD models. 72 mice were randomly divided into 6 groups, blank group, model group, positive drug medobba group (50mg/kg), Longsheng leech capsule high (1.6g/kg), medium (0.8g/kg), low (0.4g/kg) dose group. Except for the blank group and the model group, the administration group is administered with the corresponding drug for 1 time per day by intragastric administration, the model is formed according to the method (the blank group is administered with the physiological saline) on the 8 th day of administration, the model is formed, the administration is continued for 7 days, and the behavioural experiments (climbing pole experiment, tail suspension experiment (holding power) and swimming experiment) of each group of mice are detected.

1.2.2 behavioral testing

1.2.2.1 Pole climbing experiment

An iron pole 50cm in length and 1cm in diameter is used as a climbing pole for a mouse experiment, a foamed plastic ball 2cm in diameter is fixed at the top end of the climbing pole, 5 layers of medical adhesive tapes are wound on the climbing pole to prevent the mouse from skidding in the climbing process, climbing pole training is carried out after adaptive feeding of all groups of mice is finished, pole climbing experiments are carried out 1 day after the last administration for evaluating the movement coordination capacity of each group of mice, during detection, each mouse is placed on the top of the ball to freely climb down, the full length of the mouse is considered to be completely climbed by the fact that the two forelimbs of the mouse contact a pole bottom platform, and the time required by the mouse to completely climb is recorded, namely ① time for the mouse to start to climb down, ② time for the mouse to climb over the upper half part of the pole and ③ time for the mouse to climb over the lower half part of the pole.

Scoring by time: the scoring method comprises the following steps: score 3 was completed within 3s, score 2 was completed within 6s, score 1 was completed more than 6s, and three parts were taken from each mouse to complete the total score. Training was performed 3 times a day for 2 days prior to the experiment. In the official test, each mouse is measured 3 times at intervals of 1min, and the values are added and averaged.

1.2.2.2, grip test

Mice were tested for grip strength 24h, 1 week, 2 weeks, and 4 weeks after surgery, respectively. When the mouse is held, the mouse should be lightly held to avoid the influence of the stimulation on the operation, the mouse is held by the right hand and then placed on the grabbing plate, the grabbing plate is pushed forwards by the left hand, then the right hand slides backwards to the tail of the mouse, the grabbing plate is slightly loosened by the left hand, the grabbing plate slides forwards along with the force of pulling the tail of the mouse by the right hand, and the animal should be timely stressed and pulled backwards when the animal grabs the plate with strength to measure the maximum grabbing force of the animal. The change of the gripping force of the mice after administration is examined, and the influence of the Longsheng leech capsule on the action of the muscle force of the forelimb of the stroke mice is determined.

1.2.2.3 spin tolerance

And (3) placing the mouse on a rotating platform of a balance rotator, setting the rotating speed to be 60r/min, recording the time length from the beginning of the rotation of the mouse to the falling of the platform, if the time length exceeds 2min and the mouse does not fall, terminating the test, and recording the rotation tolerance time of the mouse to be 120 s. By observing the rotation tolerance time of the mice after administration, the influence of the Longsheng leech capsule on the balance ability of the cerebral apoplexy mice is examined.

1.2.2.4 Tail suspension experiment

After the adaptive feeding of each group of mice, the two forepaws of the tested mice were suspended on a horizontal metal wire (diameter 1mm, 30cm from the ground) and stayed for 10s each day. Testing was performed until day 15 after dosing was complete. The scoring criteria were as follows: the test time is 10s, the mouse grabs the metal wire with two hind paws within the test time for 3 minutes, the mouse grabs the metal wire with one hind paw for 2 minutes, the mouse can not grab the metal wire with two hind paws for 1 minute, the mouse falls and marks 0 minute, and finally the score condition is calculated and is subjected to statistical analysis.

1.2.2.5 swimming test

A water tank with the specification of 20cm × 30, 30cm × 20cm is used as an experimental swimming tank, drinking water with the water depth of 10cm and the water temperature of (22-26) DEG C is filled in the swimming tank before the experiment, a mouse is placed in the water tank when the experiment starts, the scoring standard is that the test time is 10min, the mouse continuously swims for 30 minutes within the test time, the mouse swims for more than half the test time for 25 minutes, the mouse floats for more than half the test time for 20 minutes, the mouse occasionally swims for 15 minutes, the mouse floats on one side and occasionally swims for 10 minutes by using hind limbs, the detection and recording are carried out after the administration is finished on day 15, and finally, the scoring condition is calculated and is statistically analyzed.

1.2.3 serum Biochemical assays

After the blood was left to stand for 0.5h, it was centrifuged at 5000rpm at 4 ℃ using a refrigerated centrifuge, and the supernatant was aspirated and stored at-80 ℃. Detection of activity of MDA, SOD and GSH-Px

1.2.4 immunohistochemistry of brain tissue

After the animal tissue sample collection ethological detection is finished, anaesthetizing, cutting the chest cavity, exposing the heart, cutting the right atrium by an ophthalmic scissors, injecting 0.9% normal saline into the needle from the left ventricle, replacing blood through systemic circulation until the outflow liquid is no longer red, peeling the animal brain shell, carefully separating bilateral cortex until striatum is exposed, then taking out fresh striatum tissues at two sides by the ophthalmic forceps, separating bilateral hippocampus, taking out mesoencephalic substantia nigra, operating on ice in the whole process, and storing the taken out fresh tissues at-80 ℃; the whole brains of the other half of the experimental animals were stripped and stored in 4% paraformaldehyde for later use. And carrying out immunohistochemical detection on related indexes.

1.2.5 tissue Paraffin embedding section experiment procedure

(1) Material taking: fresh tissue was fixed in 4% paraformaldehyde for over 24 h. Taking out the tissue from the fixing solution, flattening the tissue of the target part in a fume hood by using a scalpel, and placing the trimmed tissue and the corresponding label in a dehydration box.

(2) And (3) dehydrating: and (5) putting the dehydration box into a hanging basket, and dehydrating by sequentially gradient alcohol in a dehydrating machine. 75% alcohol 4 h-85% alcohol 2 h-90% alcohol 2 h-95% alcohol 1 h-absolute ethanol I30 min-absolute ethanol II 30 min-alcohol benzene 5-10 min-xylene I5-10 min-xylene II 5-10 min-wax I1 h-wax II 1 h-wax III 1 h.

(3) Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, the melted wax is put into an embedding frame, the tissue is taken out from the dehydration box and put into the embedding frame according to the requirements of an embedding surface before the wax is solidified, and a corresponding label is pasted on the tissue. Cooling at-20 deg.C, solidifying wax, taking out the wax block from the embedding frame, and trimming the wax block.

(4) Slicing: the trimmed wax block was sliced on a paraffin slicer to a thickness of 4 μm. The slices are floated on warm water at 40 ℃ of a spreading machine to spread the tissues, the tissues are taken out by a glass slide and put into a 60 ℃ oven to be baked. Taking out after water baking and wax baking and roasting for standby at normal temperature.

1.2.6 immunohistochemical Experimental procedure on Paraffin section

(1) Paraffin section dewaxing to water: placing the slices in xylene I15 min-xylene II 15 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-85% alcohol 5 min-75% alcohol 5 min-distilled water washing.

(2) Antigen retrieval: the tissue slices are placed in a repairing box filled with EDTA antigen repairing buffer solution (pH9.0) to perform antigen repairing in a microwave oven, and excessive evaporation of the buffer solution is prevented in the process, so that dry slices are not cut. After natural cooling, the slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker.

(3) Blocking endogenous peroxidase: the sections were placed in 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH7.4) with shaking on a destaining shaker.

(4) Blocking with BSA or serum, i.e., spinning the section slightly, circling the tissue with a organizing pen (to prevent antibody from flowing away), dripping 3% BSA in the circle to uniformly cover the tissue, and blocking at room temperature for 30 min.

(5) Adding a primary antibody: gently removing the confining liquid, dripping PBS (phosphate buffer solution) on the slices to prepare primary antibodies according to a certain proportion, and flatly placing the slices in a wet box for incubation at 4 ℃ overnight. (Small amounts of water added in wet boxes to prevent evaporation of antibody).

(6) Adding a secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, the tissue is covered with a secondary antibody (HRP mark) of a primary antibody corresponding species in a group reagent box which is dripped into a ring, and the tissue is incubated for 50min at room temperature.

(7) DAB color development: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, a DAB developing solution which is prepared freshly is dripped into the ring, the developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop developing.

(8) Counterstaining cell nuclei: harris hematoxylin is counterstained for about 3min, washed with tap water, 1% hydrochloric acid alcohol is differentiated for several seconds, washed with tap water, returned to blue by ammonia water and washed with running water.

(9) Dewatering and sealing: placing the slices in 75% alcohol for 6 min-85% alcohol for 6 min-anhydrous alcohol I for 6 min-anhydrous alcohol II for 6 min-xylene I for 5min, dehydrating, taking out the slices from xylene, air drying, and sealing with neutral gum.

(10) Microscopic examination, image acquisition and analysis, and specific immune section images are shown in attached figures 1-4 of the specification.

1.3 Experimental results:

1.3.1 behaviours

1.3.1.1 mouse tail suspension experiment

TABLE 6 mouse Tail suspension test results

From the above results, the suspension test score of the model mice was significantly reduced compared to the blank control group (P < 0.01). Compared with the model group, the positive drug medoba and the Longsheng leech capsule in the low-dose and medium-dose and high-dose group mice have obviously increased mark (P <0.05) and obviously improved behavior coordination ability.

1.3.1.2 Pole climbing experiment

TABLE 7 mouse Pole climbing experiment score

From the above results, the pole-climbing time of the model group was significantly prolonged (P <0.01) compared to the blank control group. Compared with the model control group, the rod climbing time of the positive drug meduoba and the Longsheng leech capsule in the low, medium and high dose groups is obviously shortened (P is less than 0.05).

1.3.1.3 mouse grip test

TABLE 8 mouse grip test results

From the above results, the model group mice had significantly weaker holding power than the blank group (P < 0.01). Compared with the model group mice, the positive drug Meiduoba and the Longsheng leech capsule have obviously enhanced holding power (P is less than 0.01, and P is less than 0.05) in the high and low dose group mice, which indicates that the muscle strength of four limbs of the mice is obviously enhanced.

1.3.1.4 mouse spin tolerance test

TABLE 9 results of mouse spin experiments

From the above results, the model group mice swimming test score was significantly less than the blank group (P <0.05). Compared with the mice in the model group, the swimming time scores of the mice in the high, medium and low dose groups of the positive drug Meiduoba and the Longsheng leech capsule are obviously improved (P is less than 0.01, P is less than 0.05), and the behavior coordination ability is obviously improved.

1.3.1.5 mouse swimming test

TABLE 10 mouse swimming test Scoring results

3.2 mouse serum biochemistry

TABLE 11 Biochemical indices of mouse serum

From the above results, compared with the blank group, the activities of SOD and GSH-px in the model group are obviously reduced, and the MDA content is obviously increased (P <0.05), compared with the model group, the high-dose group of the Longsheng leech capsule can obviously improve the SOD activity (P <0.05), the high-dose and the medium-dose can obviously reduce the MDA content (P <0.05), and the high, the medium and the low doses of the Longsheng leech can obviously improve the GSH-px activity. The leech capsule can reduce oxidative stress injury in the MPTP-induced mouse PD process.

3.3 organ coefficients

TABLE 12 measurement results of organ changes in mice in the administration group

3.4 immunohistochemical determination of TH

TABLE-13 mouse brain tissue immunohistochemical determination of positive cell experimental results

From the above results, the number of TH positive cells in the substantia nigra region of the brain of the model group mice is obviously reduced (P <0.01) compared with that of the blank control group, and the number of TH positive cells in the substantia nigra region of the mice in the high-dose and medium-dose groups is obviously increased (P <0.05, P <0.01) after the stomach-lavage administration treatment of the Longsheng leech capsule. The result shows that the leech capsule can obviously relieve MPTP-induced neuron damage.

Claims

1. A method for detecting the content of a substance, characterized by comprising the steps of:

(3) preparing a test solution:

taking the mixed powder prepared in the step (2) as a test sample, and respectively adding physiological saline to prepare the mixed powder with the concentration of 0.02 g/mL^-1～0.2g·mL^-1Swirling the sample with concentration gradient for 30min, centrifuging for 10min, and 5000rpm, collecting supernatant, refrigerating for use, and processing Hirudo with the same method;

(4) negative control solution preparation

The leech-free sample is obtained according to the preparation process in the step (2), and is prepared according to the method in the step (3) to obtain 0.02 g.mL^-1～0.2g·mL^-1Sample of concentration gradient, negative control of 10 concentration gradientsA solution;

(5) preparation of buffer solution

(6) preparation of Thrombin titration solution

Adding 1000U thrombin into 5mL physiological saline, subpackaging according to 1mL, storing at-20 deg.C, collecting thrombin, and making into 10U. mL^-1、20U·mL^-1And 40 U.mL^-1；

(7) Determination of content

Precisely measuring a sample solution to be tested, placing the sample solution into an EP (EP) tube, adding 200 mu L of trihydroxymethyl aminomethane hydrochloric acid buffer solution containing 0.5% bovine fibrinogen, uniformly mixing, placing the sample solution into a water bath, soaking for 5min, starting to dropwise add thrombin titration solution, starting timing at the moment of dropwise addition, titrating until the sample solution to be tested is solidified or an end point state appears, recording the titration end point time and the volume of consumed thrombin solution, and calculating the content of the thrombin solution.

2. The application of a substance in preparing a medicine for treating Parkinson's disease is characterized in that the substance comprises the following raw material medicines in parts by weight: 225 parts of astragalus, 100 parts of leech, 90 parts of ligusticum wallichii, 90 parts of angelica sinensis, 90 parts of safflower, 113 parts of peach kernel, 90 parts of red paeony root, 90 parts of costustoot, 90 parts of rhizoma acori graminei, 60 parts of earthworm, 90 parts of parasitic loranthus and 35 parts of acanthopanax extract.

3. Use of a substance according to claim 2, wherein the substance is prepared by a method comprising: pulverizing Hirudo, Lumbricus, and radix Acanthopanacis Senticosi dry extract into fine powder; decocting radix astragali, rhizoma Ligustici Chuanxiong, radix Angelicae sinensis, Carthami flos, semen Persicae, radix Paeoniae Rubra, radix aucklandiae, rhizoma Acori Graminei and herba Taxilli with water twice (2 hr for the first time and 1.5 hr for the second time), filtering, mixing decoctions, concentrating the filtrate to relative density of 1.1-1.2 at 60 deg.C, spray drying to obtain powder, adding Lumbricus, Hirudo and radix Acanthopanacis Senticosi powder, mixing, adding medicinal adjuvants, and making into various pharmaceutically acceptable common dosage forms.

4. Use of a substance as defined in claim 2 or 3, wherein the substance is in the form of an oral preparation such as a capsule, tablet, pill, granule, etc.

5. The use of a substance as claimed in claim 4, wherein the substance is in the form of a hard gelatin capsule.

6. Use of a substance according to any one of claims 2 to 5 in the manufacture of a medicament for the treatment of parkinson's disease.