CN111366680B

CN111366680B - Substance content determination method and application thereof

Info

Publication number: CN111366680B
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 Pharmaceuticals Co ltd
Current assignee: Shaanxi Buchang Pharmaceuticals Co ltd
Priority date: 2020-02-29
Filing date: 2020-02-29
Publication date: 2022-09-23
Anticipated expiration: 2040-02-29
Also published as: CN111366680A

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 loranthus parasiticus 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 pole 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, Compound Pallas pit Viper venom Capsule antithrombin Activity quality Standard evaluation Studies, journal of practical medicine, 2019-01, reported in the literature, and the antithrombin Activity of Compound Pallas pit Viper venom Capsule was determined by the Thrombin titration method. The research result preliminarily draws up that the anticoagulant activity of the compound viper venom capsule is 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, and the measuring method of the ingredient mainly comprises a thin layer scanning method, an HPLC method and a near infrared spectrum method. For example, Jiafu Huai, Luwei et al, an improvement of a method for detecting the content of astragaloside in astragalus decoction pieces, Asia Pacific traditional medicine, 2019-10, the article compares several different pretreatment extraction methods, and 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 measure 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 Xuan; the content of astragaloside in urinary calculus removal particles is measured by a high performance liquid chromatography-evaporative light scattering detection method, the strait pharmaceutical is 2019-09, and the content of the astragaloside serving as an effective ingredient of astragalus in the urinary calculus removal 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. The near infrared spectroscopy method for rapidly detecting the astragaloside IV and the total solid content in the astragalus injection by Baixintao, Hobojun, Zhang et al, Chinese herbal medicines 2012-10, adopts a transmission spectrum sampling system to determine the near infrared transmission spectrum (NIRS) of a sample, and the established NIRS prediction model is feasible for rapidly determining the astragalus injection. However, the content detection method has the defects of complex 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, paresthesia (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 is about 1% in people over 60 years old, and the number of the Parkinson disease is increased year by year due to the current aging in China.

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 the apoplexy involving the channels and collaterals with symptoms of hemiplegia, hemianesthesia, facial distortion, language disorder and the like in the recovery period of the arteriosclerotic cerebral infarction based on the traditional Chinese medicine differentiation of qi deficiency and blood stasis. 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 this regard, the applicant has conducted a great deal of systematic studies on the product in terms of drug formulation, process improvement, pharmacodynamic substance composition, pharmacological and pharmacodynamic tests, etc., and has laid out a series of patent applications, wherein the application numbers are: 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 quality control index component of the existing leech capsule only takes astragaloside IV as the product, the effective substance component and the internal quality are difficult to effectively characterize. And the application publication 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 IV, 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 and the like in the Longsheng leech capsule. 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's 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 detection index content, 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 substance is surprisingly found to be capable of treating cardiovascular and cerebrovascular diseases of patients suffering from cardiovascular and cerebrovascular diseases and concurrently suffering from Parkinson's disease after taking the medicine, 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, 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 ^-1 Swirling 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 ～0.02g·mL ^-1 A sample of concentration gradient, a negative control solution of 10 concentration gradients;

(5) preparation of buffer solution

Taking 0.2 mol.L ^-1 Tris solution with 0.1 mol. L ^-1 Adding 40mL of hydrochloric acid solution into 100mL of water, 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 titration end point time 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: 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, complementing the weight loss with 2% potassium hydroxide methanol solution, shaking uniformly, filtering, accurately taking 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 an ammonia test solution for 2 times, 20ml each time, discarding the ammonia solution, evaporating the chloroform-n-butanol solution to dryness, dissolving the residue with methanol, transferring to a measuring flask, adding methanol to dilute to scale, shaking uniformly to serve as a test 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 crossed on the same silica gel G thin-layer plate which takes 0.3 percent sodium carboxymethyl cellulose as adhesive, 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 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, 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.

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, wherein the fillers include: 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 value statistical analysis of the leech capsule and the leech medicinal material is carried out when the thrombin concentration is 10 U.mL ^-1 The method has no obvious difference, which shows that the production conditions of the preparation have no obvious influence on the measured 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.

The hard capsule of the invention is applied to the clinical treatment of cardiovascular and cerebrovascular diseases and apoplexy diseases in a large amount, and a large amount of basic research on drug effect substances is carried out, so that the hard capsule is discovered accidentally to have a new application in treating Parkinson's disease.

The invention has the beneficial effects that:

because the operation cost and the cost of the detection method in the prior art of the product are more steps and higher, 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 because the monarch drug of the product treats both symptoms and root causes by removing blood stasis with leeches, the product can take the effect of treating both symptoms and root causes, and the anticoagulant component is mainly hirudin and has the pharmacological effects of anticoagulation, antithrombotic and cardiovascular treatment. The hirudin content of the preparation is measured by adopting a thrombin titration method and is taken 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 effects on patients with Parkinson's disease besides the treatment of 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 the mice and obviously shorten the pole climbing time of the mice. The grip force experiment result shows that the drug can enhance the grip force of the mouse, obviously enhance the muscle force 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 process of inducing mouse PD by MPTP. 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 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 without limiting the invention to the right.

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

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 astragaloside content in the composition;

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 medicament of the invention

1. Instrument and reagent

Analytical balance (Switzerland Mettler), 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: 181005), leech (provided by stepsize, Inc.), physiological saline (Shijiazhuang Siyao, Inc.), fibrinogen (Lot: P06J10Y92294, Beijing Byeldi Biotech, Inc.), tris, hydrochloric acid, thrombin T8020 (Lot: 417G062, Beijing Sorbero Tech, 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 ^-1 And (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 (provided by Hairhiya Corp.) is 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 ^-1 10 concentration gradients of negative control solution.

2.1.3 preparation of buffer

Taking 0.2 mol.L ^-1 25mL of tris solution and 0.1 mol. L ^-1 Hydrochloric acid solution about 40mL, water to 100mL, adjusting pH to 7.4.

2.1.4 preparation of Thrombin titration solution

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

2.2 assay

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 the filament can be picked out from the test tube or the small ball is observed as the titration end point is known. 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 dropping is carried out for 1 time every 1min, wherein each time is 5 mu L; 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 ^-1 2. mu.L of the solution was added dropwise every 4 min. 2 determination of anticoagulant ActivityThe steps of (a) are substantially identical, but the difference in thrombin activity units per minute dropped by a factor of 40 on average in both methods. 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 measurement of anticoagulation Activity

Method I, Thrombin 40 U.mL ^-1 5 μ L each time, method II, thrombin 10 U.mL ^-1 2 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, in either method one or method two, the anticoagulant activity measured at 4min intervals is much less than the value measured at 1min intervals, indicating that the titration interval has a greater effect on the measurement of the anticoagulant activity value

2.4 Effect of Thrombin concentration on measurement of anticoagulation Activity

By adopting a thrombin titration method, respectively using 20, 10 U.mL ^-1 Dropping 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 steplength Co., Ltd.), 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 ^-1 When 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 sample concentration is 0.02-0.06 g/ml ^－1 In the linear range of (1), the thrombin concentrations are 20 U.mL respectively ^－1 And 10 U.ml-1, the titration interval time is 1min, and the measured anticoagulant activity is 11.6 +/-4.76 U.g ^-1 And 13.49. + -. 2.35 Ug ^-1 RSD were 2.44% and 5.74%, respectively. Wherein the thrombin concentration is 10 U.ml ^－1 The measured activity values are relatively close, and the accuracy is better; and the thrombin concentration is 20 U.mL ^-1 The measured values all have large errors (fig. 1).

TABLE 3 anticoagulant 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 ^－1 In the linear range of (1), the thrombin concentrations are 20 U.mL respectively ^-1 And 10 U.mL ^-1 The anticoagulant activity measured was 8.12. + -. 2.84 Ug ^-1 And 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 variance homogeneity test values (significance level 0.05) variance statistical test results 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 ^－1 And 10 U.ml ^－1 When 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 equal 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 5T-test results of medicinal materials of leech and capsule of leech

As is clear from Table 5, when the thrombin concentrations were 10 U.ml, respectively ^－1 When 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 content determination of the leech capsule by the existing method in pharmacopoeia is feasible, the method is simple and quick, the determination result is accurate and reliable, and the method can be used for quality control of the leech capsule.

3 analysis and summary of the results

3.1 methodological validation

Because the Longsheng leech capsule consists 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 ^－1 And 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 leech in 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 sources of the raw materials need to be accurately identified, the concentrations of the titration solutions used for content determination are different when the varieties are different, and the large error is caused when the high-concentration titration solution is used for determining the varieties 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 ^-1 In between, the concentration of thrombin is 20 U.mL, respectively, in a substantially linear relationship with the anticoagulation activity (FIG. 1) ^-1 And r values of 0.9026 and 0.9738 for 10 U.mL-1, wherein the thrombin concentration is at 10 U.mL ^-1 The measured anticoagulant activity value of the sample is 13.49 +/-2.35 U.g ^-1 The 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 was found herein that the thrombin concentration and the titration interval time have a major influence on the thrombin titration time and the measured value of the anticoagulant activity of the same sample. Dripping thrombin 40 U.mL-1 in 1min once in 5 μ L each time in pharmacopoeia for Hirudo nipponica with high activity; for low-activity whitmania pigra, the concentration difference of the two is 40 times by adopting a titration method of 10 U.mL < -1 > once every 4min and 2 mu L every time. 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 fibrin stock solution, which is not favorable for coagulation: secondly, the method comprises the following steps: shaking each drop once again can destroy the coagulation formed by the initial connection of some fibrin, which is usually invisible to eyes, and the repeated destruction can dilute the fibrin, which can result in permanent titration and no coagulation. The measurable range is narrow (0.02-0.06 g.mL) ^-1 ) (ii) a The concentration of thrombin was 20 U.mL ^-1 And 10 U.mL ^-1 The 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 from 1min, hourThe 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 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; 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 usage and dosage are as follows: is administered orally. 3-5 granules at a time, 3 times a day.

EXAMPLE 3 the drug is prepared as a tablet

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 powder and 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 2 hours, the second 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 powder and mixing homogeneously, charging right amount of hydroxypropyl methyl cellulose, low substituted hydroxypropyl cellulose and 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, 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 protein) by leech capsules

1.1 Experimental materials

1.1.1 Experimental animals

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

1.1.2 Experimental drugs

The specification of the capsule is 0.4 g/grain, and the batch number is as follows: 20171250, Shanxi stepsize pharmaceuticals, Inc.; medoba, specification 0.25 g/tablet, lot number: SH3288, Shanghai Roche pharmaceutical Co., Ltd., lipid oxidation (MDA) detection kit, manufacturer: Nanjing, product number: lot 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 AbMole BioScience, paraformaldehyde, manufacturer: wuhan google biotechnology limited, lot No.: g1101; paraffin, xylene (national chemical group, chemical agents limited), PBS (G0002), EDTA (G1203).

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, refrigerated centrifuge, manufacturer: heal Force; a photographic microscope: nikon Eclipse Ti-SR.

1.2 Experimental methods

1.2.1 Molding, grouping and administration

The adoption of MPTP subacute molding method: a PD model was prepared by intraperitoneal (ip) injection of MPTP (30 mg/k) to experimental mice once a day for 5 consecutive days at a fixed time (9:00am) daily. 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, the model is formed according to the method (the blank group is administered with the normal saline) on the 8 th day of administration, the model is formed, the administration is continued for 7 days, and the behavioural experiments (climbing rod 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 rod with a length of 50cm and a diameter of 1cm was used as a climbing rod for the mouse experiment. Fix the top at the pole of climbing with a diameter 2 cm's foamed plastic bobble, 5 layers of medical adhesive tapes are twined on the pole of climbing in order to prevent that the mouse from crawling the in-process and skidding, carry out the training of climbing the pole after each group mouse adaptability is raised. Performing a pole climbing experiment 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 enable the mouse to freely climb down, the platform at the bottom of the double forelimb contact rod of the mouse is considered as climbing to complete the full length, and the time required by the full length climbing of the mouse is recorded: firstly, the time for the mouse to climb downwards; secondly, the mouse climbs over the upper half part of the pole; and thirdly, the time for the mouse to climb the lower half part of the rod.

Scoring by time: the scoring method comprises the following steps: scoring 3 points within 3s, scoring 2 points within 6s, scoring 1 point more than 6s, and taking three parts 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, 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 force is timely applied and pulled backwards when the animal holds the plate forcefully, so that the maximum grabbing force of the animal is measured. The change of the holding power of the mice after administration is inspected, and the influence of the Longsheng leech capsule on the action of the muscle force of the forelimb of the mice with the cerebral apoplexy 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 of the mouse from the beginning to the falling platform, if the mouse does not fall after exceeding 2min, terminating the test, and recording the rotation tolerance time of the mouse as 120 s. The influence of the leech dragon capsule on the balance ability of the stroke mouse is examined by observing the rotation tolerance time of the mouse after administration.

1.2.2.4 Tail suspension experiment

After the adaptive breeding of each group of mice is finished, two front paws of the tested mice are hung on a horizontal metal wire (the diameter is 1mm, and the distance from the ground is 30cm) every day and stay for 10 s. Detection was performed until the end of the 15 th day dosing. The scoring criteria were as follows: the test time is 10s, the mouse grabs the metal wire with two hind paws 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 for 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 a specification of 20cm × 30cm × 20cm is used as an experimental swimming tank. Before the experiment, drinking water with the water depth of 10cm and the water temperature of (22-26) DEG C is poured into the swimming tank, and when the experiment is started, the mouse is placed into the water tank. The scoring criteria were as follows: the test time is 10min, and the swimmers of the mice continuously count for 30 minutes in the test time; recording 25 minutes when the swimming time of the mice accounts for more than half of the tested time; recording 20 minutes when the floating time of the mice accounts for more than half of the tested time; mice occasionally swimmers scored 15 points; mice floated on one side and were scored 10 points occasionally with hind limb swimmers. And (5) detecting and recording after the administration on the 15 th day, and finally calculating the score condition and performing statistical analysis.

1.2.3 serum Biochemical assays

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

1.2.4 immunohistochemistry of brain tissue

After the animal tissue sample collection ethological detection is finished, anaesthetizing, cutting off the chest, exposing the heart, cutting off the right atrium by an ophthalmologic scissors, injecting 0.9% normal saline into a needle from the left ventricle, replacing blood through systemic circulation until the effluent liquid is no longer red, peeling off the animal brain shell, carefully separating bilateral cortex until striatum is exposed, then taking out fresh striatum tissues at two sides by an ophthalmologic forceps, separating bilateral hippocampus, taking out mesencephalon substantia nigra, operating on ice in the whole process, and storing the taken out fresh tissue at-80 ℃; the whole brains of the other half of the experimental animals were stripped and stored in 4% paraformaldehyde for further use. And carrying out immunohistochemical detection on related indexes. 1.2.5 tissue Paraffin embedding section Experimental 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 (4) putting the dehydration box into a hanging basket, and dehydrating by sequentially gradient alcohol in the dehydration machine. 75% alcohol 4 h-85% alcohol 2 h-90% alcohol 2 h-95% alcohol 1 h-anhydrous ethanol I30 min-anhydrous 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, molten 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 float on 40 ℃ warm water of a slice spreading machine to flatten the tissues, the tissues are taken out by a glass slide, and the slices are placed into a 60 ℃ oven to be baked. Taking out after water baking, wax baking and melting, and storing at normal temperature for later use.

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-anhydrous ethanol I5 min-anhydrous ethanol II 5 min-85% ethanol 5 min-75% ethanol 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) and subjected to 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 slide was 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 slices slightly, circling the tissue with a organizing pen (to prevent the 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, dropwise adding PBS (phosphate buffer solution) on the slices according to a certain proportion to prepare primary antibodies, 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: the 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 the ring, and the tissue is incubated for 50min at room temperature.

(7) DAB coloration: the 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 color developing solution which is prepared freshly is dripped into the ring, the color developing time is controlled under a microscope, the positive color is brownish yellow, and the section is washed by tap water to stop color developing.

(8) Counterstaining cell nuclei: re-staining with Harris hematoxylin for about 3min, washing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, washing with tap water, turning blue with ammonia water, and washing with running water.

(9) Dewatering and sealing: placing the slices in 75% alcohol for 6 min-85% alcohol for 6 min-anhydrous ethanol I6 min-anhydrous ethanol II 6 min-xylene I5 min, dehydrating, 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 mouse suspension test score of the model group was significantly decreased 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 medoba 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 grip of the model group mice was significantly weaker than that of the blank group (P < 0.01). Compared with the model group mice, the positive drug Meiduoba and the Longsheng leech capsule high and low dose group mice have obviously enhanced holding power (P is less than 0.01, and P is less than 0.05), which indicates that the muscle strength of the 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 score of the model group mouse swimming test was significantly less than that of the blank group (P <0.05). Compared with the model group mice, the swimming time scores of the positive drug meduoba and the mice with the high, medium and low doses of the Longsheng leech capsule are obviously improved (P is less than 0.01, and 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 experiment Scoring results

3.2 mouse serum biochemistry

TABLE 11 measurement results of various indicators of serum biochemistry of mice

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 process of inducing mouse PD by MPTP.

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 test results

From the above results, the number of TH positive cells in the substantia nigra region of the brain in the mice of the model group 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 of the high-dose and medium-dose groups is obviously increased (P <0.05, P <0.01) after the administration of the Longsheng leech capsule through gastric lavage. The leech and leech containing longsheng capsules can obviously relieve MPTP-induced neuron damage.

Claims

1. 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.

2. The use of a substance as claimed in claim 1, wherein the substance is in the form of an oral preparation such as capsules, tablets, pills, granules, etc.

3. Use of a substance as claimed in claim 2, wherein the substance is in the form of a hard gelatin capsule.

4. Use of a substance according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of parkinson's disease.