WO2021168855A1 - Content determination method for material and application thereof - Google Patents

Abstract

A content determination method for a material and an application thereof. The material is prepared from 225 parts of Radix Astragali, 100 parts of Hirudo, 90 parts of Rhizoma Chuanxiong, 90 parts of Radix Angelicae Sinensis, 90 parts of Flos Carthami, 113 parts of Semen Persicae, 90 parts of Radix Paeoniae Rubra, 90 parts of Radix Aucklandiae, 90 parts of Rhizoma Acori Graminei, 60 parts of Pheretima, 90 parts of Herba Taxilli, and 35 parts of Acanthopanacis Senticosi extract. The content determination method has the characteristics of being simple in operation and quick, and having high repeatability and accuracy. Pharmacological experiments of the material show that the material can significantly improve the behavior coordination capability of mice, significantly shorten the pole climbing time of mice, increase the grip strength of mice and the swimming time of mice, and can significantly alleviate MPTP-induced neuronal damage. Moreover, the content determination method for a material can effectively control the inner quality of the drug, develop novel therapeutic use of the material, and expand the range of clinical use of the drug to treat diseases.

Description

A method for measuring substance content and its application Technical field

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

Background technique

The medicinal leech contained in the substance of the present invention contains multiple active ingredients, hirudin and thrombin, etc., which have the effects of anticoagulation, antithrombosis, fibrinolysis and the like. Xinyan Meng, Chengzhi Yang, Study on the correlation between plasma antithrombin levels and cerebral infarction, Journal of Beihua University, 2018-11, the study found that plasma antithrombin (AT-Ⅲ) levels were significantly reduced in the plasma of patients with cerebral infarction , And has a high degree of correlation with the severity of cardiovascular and cerebrovascular diseases. Wu Yang, Yan Ge, et al. Evaluation of quality standards for antithrombin activity of compound agkistrodon venom capsules, Journal of Practical Medicine, 2019-01, reported in the literature, using thrombin titration to determine the anticoagulant activity of compound agkistrodon venom capsules. The research results preliminarily proposed that the anticoagulant activity of compound Agkistrodon venom capsules is not less than 300.0U/g. The method is accurate, simple and rapid, and can realize the quality control of the preparation. Wang Wenyi, Study on the heavy metal residues and anticoagulant activity of leech medicinal materials, Journal of Beijing University of Traditional Chinese Medicine, 2016-05, this paper uses the method of measuring antithrombin activity to evaluate the antithrombin titer of leech medicinal materials.

The method for determining the content of the effective component astragaloside IV in the monarch drug Astragalus contained in the substance of the present invention is combed and analyzed. The determination methods of the component mainly include thin-layer scanning method, HPLC method and near-infrared spectroscopy. Such as Jia Fuhuai, Lu Wei, etc., Improvement of the detection method of astragaloside IV in astragalus decoction pieces, Asia Pacific Traditional Medicine, 2019-10, this article compares several different pre-treatment extraction methods, and finally determines the phase of reflux extraction and ultrasonic extraction. Combined with the pretreatment method of n-butanol and ammonia test solution extraction and purification, combined with the HPLC-ELSD method to determine the content of astragaloside IV. The improved method is accurate, efficient, and reproducible. It is suitable for the detection of astragaloside IV in astragalus decoction pieces. Song Zengxuan; Deng Yingming, Determination of Astragaloside IV in Urinary Paishi Granules by High Performance Liquid Chromatography-Evaporative Light Scattering Detection, Strait Pharmacy, 2019-09, This article uses high performance liquid chromatography-evaporative light scattering detection method for urinary discharge The content of Astragaloside IV, the effective component of Astragalus in the granules, was determined and the content limit was determined. This method can effectively control the drug quality of Urinary Paishi Granules. Bai Xintao, Huo Baojun, Zhang Bo et al. Rapid detection of astragaloside IV and total solids in astragalus injection by near infrared spectroscopy, Chinese herbal medicine, 2012-10, this article uses a transmission spectrum sampling system to determine the near infrared transmission spectra (NIRS) of samples ), the NIRS prediction model established by him is feasible for rapid determination of Huangqi injection. The analysis is fast, simple and accurate. However, the detection steps included in the above-mentioned content detection method are relatively complicated, and the application in the actual production process still has the disadvantage of higher cost.

Parkinson's disease PD is a progressive neurodegenerative disease, the main clinical manifestations are motor and non-motor symptoms. There are various manifestations of non-motor symptoms, including affective disorders (such as apathy, depression, and anxiety, etc.), cognitive impairment (such as dementia), autonomic dysfunction (such as bladder dysfunction), sleep disorders, and paresthesia (such as pain), Language barriers and fatigue. According to related reports, from 1990 to 2016, the number of patients with Parkinson's disease in the world more than doubled, reaching more than 6 million. The incidence rate in people over 60 years old is about 1%. Due to the current intensification of aging in our country, the number of Parkinson's disease is increasing year by year.

The pathogenesis of this disease is very complicated, and clinical treatment is mainly based on drugs. Commonly used are monoamine oxidase inhibitors, anticholinergic drugs, and levodopa. It has been clinically proven that some patients can improve certain symptoms after using the drug alone, but the long-term effect is not ideal, but long-term use can cause dysfunction Symptoms and psychiatric symptoms, the efficacy of the drug will also be significantly reduced.

The substance of the present invention is Longshengzhi Capsules, which are produced by Shaanxi Buchang Pharmaceutical Co., Ltd., which are mainly composed of astragalus, leeches, chuanxiong, angelica, safflower, peach kernel, red peony, woody, calamus, calamus It is prepared with ingredients such as dragon, mulberry, and Acanthopanax senticosus extract. The efficacy of this medicine is: invigorating qi and activating blood, dispelling blood stasis and dredging collaterals. It is used for patients with atherosclerotic cerebral infarction in the recovery period of the Chinese medicine dialectical syndrome of qi deficiency and blood stasis type stroke in the meridian, symptom of hemiplegia, partial numbness, skewed mouth, and poor language. The current national drug standard number is WS3-375(Z-040)-2005(Z), which is clinically used to treat cerebral infarction, stroke and other cardiovascular and cerebrovascular diseases. Since the product went on the market, it has significant and reliable clinical effects, and is well received by consumers. In this regard, the applicant has conducted a large number of systematic researches on the product in terms of drug formulation, process improvement, medicinal ingredients, pharmacological efficacy tests, etc., and laid out a series of patent applications. The application numbers are: 02114551.2 , 200510041937.4, 200510043066.X, 201010213671.8, 201210228789.7 The subject matter of the above-mentioned patent protection mostly focuses on the formulation of medicines, preparation methods, quality component detection methods, and new clinical uses in diabetic diseases. At present, only astragaloside IV in Longshengzhi capsules is used as the quality control index component of the product, and it is difficult to effectively characterize its active substance components and internal quality. And the publication number CN 109307721A applied by our company discloses the use of HPLC-QQQ/MS to detect astragaloside IV, hydroxysafflor yellow A, verbasil glucoside, verbasil, ferulic acid, and syringin in Longshengzhi capsules. , N-butylphthalide, ligustilide, ligustride A, ligustride lactone I, ligustride lactone H, ligustrazine, isopperidine, dehydrocostus lactone, amygdalin, acanthopanax Glycoside E, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin and other active ingredients of traditional Chinese medicine. However, the operation cost of the above detection method is many steps, which is obviously far from enough to control the internal quality of traditional Chinese medicine preparations. In the course of a large number of clinical use, it was unexpectedly discovered that its product has not only the treatment of stroke disease, but also the significant therapeutic effect on Parkinson's disease.

Summary of the invention

The purpose of the present invention is a method for determining the content of a substance and its application. The substance uses hirudin as a detection index content. The content detection method has the characteristics of simple operation, rapidity, repeatability and accuracy, and the method It can effectively control the intrinsic quality of the substance.

The purpose of the present invention is to provide a new clinical application of a substance, which relates to the application of the substance in the preparation of drugs for treating Parkinson's disease.

After a large number of clinical medications, we accidentally discovered that the substance can not only treat the cardiovascular and cerebrovascular diseases of patients suffering from both cardiovascular and cerebrovascular diseases and Parkinson’s disease after taking the drug of the present invention, but also make The symptoms of Parkinson's patients have also improved significantly.

The technical scheme of the present invention is specifically as follows:

1. A method for detecting the content of a substance, which includes the following steps:

(1) The raw material composition and weight ratio of the substance are: 225 parts of astragalus, 100 parts of leech, 90 parts of Ligusticum chuanxiong, 90 parts of angelica, 90 parts of safflower, 113 parts of peach kernel, 90 parts of red peony, 90 parts of Muxiang, 90 parts of Shichangpu, 60 parts of Earthworm, 90 parts of Morus sylvestris, 35 parts of Acanthopanax senticosus extract;

(2) The preparation method is: take leech, earthworm, acanthopanax senticosus dry paste and pulverize into fine powder; astragalus, chuanxiong, angelica, safflower, peach kernel, red peony, woody, calamus, mulberry, decocted in water for two The first time is 2 hours, and the second time is 1.5 hours. Filter and combine the decoction. When the filtrate is concentrated to 60℃, the relative density is 1.1-1.2, spray-dried to powder, add earthworm, leech and Acanthopanax senticosus powder to mix Evenly, get it;

(3) Preparation of test solution:

Take the mixed powder prepared in (2) as the test product, add physiological saline separately to prepare ^{a sample with a concentration gradient of 0.02g·mL -1} to 0.2g·mL ^-1 , that is, the sample, vortex for 30 minutes, and centrifuge for 10 minutes , 5000rpm, take the supernatant, refrigerate for later use, treat leeches in the same way;

(4) Preparation of negative control solution

The sample without leeches is obtained according to the preparation process in (2), and prepared according to the method of step (3) above, and ^{a sample with a concentration gradient of 0.02g·mL -1} to 0.02g·mL ^-1 is obtained, and 10 concentration gradients are negative. Control solution

(5) Preparation of buffer

Take 0.2mol·L ^-1 Tris solution with a hydrochloric acid solution 0.1mol·L ^-1 40mL, add water to 100mL, adjust the pH to 7.4;

(6) Preparation of thrombin titrant

Add 1000 U of thrombin to 5 mL of normal saline, and store in 1 mL aliquots at -20°C. Take the thrombin and make 10U·mL-1, 20U·mL-1 and 40U·mL ^-1 ;

(7) Assay

Precisely measure the test solution, put it in an EP tube, add 200μL of 0.5% bovine fibrinogen-containing tris hydrochloric acid buffer, mix well, place in a water bath, warm soak for 5 minutes, and start dripping thrombin titration Start timing when the solution is dripped, and titrate until the sample solution to be tested has solidified or the end-point state appears. Record the end-point time of the titration and the volume of thrombin solution consumed, and calculate its content.

The present invention also provides a method for determining the content of astragaloside IV: the steps of the method for content detection are as follows:

Take 6g of the content of this product, accurately weigh it, place it in an Erlenmeyer flask, accurately add 50ml of 2% potassium hydroxide methanol solution, weigh it, heat and reflux for 1 hour, let it cool, weigh it, and use 2% hydroxide Potassium methanol solution to make up the lost weight, shake well, filter, accurately measure 30ml of the subsequent filtrate, place in the evaporation, evaporate the residue and add 20ml of water to dissolve, add a mixture of chloroform-n-butanol with a volume ratio of 2:1 Extract 5 times, 10ml each time, combine the extracts, wash 2 times with ammonia test solution, 20ml each time, discard the ammonia solution, evaporate the chloroform-n-butanol solution, and dissolve the residue with methanol, transfer to a measuring flask, add Dilute methanol to the mark, shake well, and use it as the test solution. Take an appropriate amount of astragaloside IV reference substance, accurately weigh it, and add methanol to make 1ml of reference substance solution containing 0.5 mm. According to an appendix VIB experiment of the 2000 edition of thin layer chromatography, draw 4μL and 6μL of the test solution, 3μL and 5μL of the reference solution, and respectively cross the same silica gel G with 0.3% methylcellulose sodium as the binder. On a thin layer plate, place a layered chloroform-methanol-water lower layer solution below 10°C as a developing agent, where the volume ratio of chloroform-methanol-water is 65:35:10, unfold, take out, dry, and spray 10% sulfuric acid ethanol solution, bake at 100℃ until the spots are clearly colored, take it out, cover the glass plate of the same size on the thin layer plate, fix the surrounding with tape, scan according to thin layer chromatography, wavelength λs=530nm, λR=650nm , Measure the integral value of the absorbance of the test substance and the integral value of the reference substance, calculate, and get.

The application of a substance in the preparation of a medicine for treating Parkinson's disease. The raw material composition and weight ratio of the substance are: 225 parts of astragalus, 100 parts of leech, 90 parts of Ligusticum chuanxiong, 90 parts of angelica, 90 parts of safflower, and peach kernel 113 parts, 90 parts of red peony root, 90 parts of Muxiang, 90 parts of Shichangpu, 60 parts of Earthworm, 90 parts of Morus sylvestris, 35 parts of Acanthopanax senticosus extract.

The composition of the raw materials of the substance and its weight ratio are: take leech, earthworm, acanthopanax senticosus dry ointment and pulverize into fine powder; Parasitic, add water to decoct twice, the first time is 2 hours, the second time is 1.5 hours, filter, combine the decoction, the relative density of the filtrate is 1.1-1.2 when concentrated to 60℃, spray dried into powder, add earthworm and leech , Acanthopanax senticosus powder is mixed evenly, and medicinal excipients are added to make various common pharmaceutical dosage forms permitted in pharmacy, and it is ready.

In the application of the medicine of the present invention, the dosage form of the medicine is capsules, tablets, pills, granules and the like. In order to realize the above dosage forms, pharmaceutically acceptable excipients, such as fillers, disintegrants, lubricants, adhesives, flavoring agents, etc., are added to the preparation of the above drugs. Fillers include starch, lactose, and mannitol. , Chitin, microcrystalline cellulose, etc., disintegrants include: starch, microcrystalline cellulose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, etc., lubricants include: magnesium stearate, lauryl sulfate Sodium, talcum powder, etc., binders include starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc., sweeteners include: saccharin sodium, aspartame, sucrose, etc., flavoring agents include: sweetness剂 etc.

The statistical analysis of the experimental mean values of Longshengzhi Capsules and Leech medicinal materials ^{showed no significant difference when the thrombin concentration was 10U·mL -1} , indicating that the preparation production conditions had no significant effect on the tested substances in the medicinal materials. The method can be used for the content of Longshengzhi Capsules One of the quality control measures.

We applied the hard capsule of the present invention to clinical treatment of cardiovascular and cerebrovascular diseases and apoplexy diseases, and did a lot of pharmacodynamic material basic research, and accidentally discovered that it also has a new application for treating Parkinson's disease.

The beneficial effects of the present invention:

Because the above-mentioned detection method of the product of the present invention has many operating cost steps and high cost, our company chooses the active ingredient of hirudin with obvious ingredients for promoting blood circulation and removing blood stasis as the quality control index of the product. Leech eliminates silt to treat symptoms, and can collect symptoms and root causes. The main anticoagulant component is hirudin, which has anticoagulant, antithrombotic and cardiovascular pharmacological effects. We use the thrombin titration method to determine the content of hirudin in the preparation as one of the quality standards of the preparation. In the course of a large number of clinical use, it was unexpectedly discovered that its products not only can treat stroke diseases, but also have significant therapeutic effects on Parkinson's disease patients. Therefore, we have carried out corresponding pharmacological and pharmacodynamic tests for verification, in order to expand the scope of clinical medication of the drug to treat diseases, and provide a new treatment idea and clinical medication choice for the treatment of the disease.

According to the pharmacological tail suspension experiment and pole climbing experiment, the present invention can significantly improve the behavior coordination ability of mice and shorten the climbing time of mice. The results of the grasping force experiment show that the drug can increase the grasping force of mice, significantly increase the muscle strength of the mice’s limbs, prolong the swimming time of the mice, and significantly improve the behavioral coordination ability. Serum biochemistry shows that the high-dose group of the present invention can significantly improve SOD activity, high-dose and medium-dose can significantly reduce MDA content, high, medium and low doses of the present invention can significantly increase glutathione peroxidase (GSH -px) Vitality. It shows that the drug can reduce the oxidative stress damage in the process of MPTP-induced PD in mice. The results of immunohistochemical determination show that the drug of the present invention can significantly alleviate the neuronal damage induced by MPTP.

From the above results, it can be seen that the drug of the present invention explores a new therapeutic use, expands the clinical use of the drug to treat diseases, and provides new drug options for the treatment of Parkinson's disease. The drug has the characteristics of safety, strong pharmacological activity, and good market application prospects.

In order to fully understand the implementation of the present invention, the following will further illustrate the present invention through typical embodiments. The following and through pharmacodynamics, to prove the beneficial therapeutic effect of the drug.

Description of the drawings

The drawings described here are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and the description thereof are used to explain the present invention, and do not constitute an improper limitation of the present invention.

Figure 1-Comparison of the anticoagulant activity of Longsheng Leek products measured by different concentrations of thrombin (1min interval, 2μL each time);

Figure 2-The anticoagulant activity of leech medicinal materials measured at different concentrations of thrombin (every 1 min, 2 μL each time);

Figure 3-Thin layer chromatogram of the content of astragaloside IV in the substance;

Figure 4-A picture of immunohistochemistry of paraffin sections of mouse brain tissue, including: blank control group, model control group, medobar group control group, high-dose drug group of the present invention, medium-dose drug group of the present invention, and the present invention Low-dose drug group.

Detailed ways

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 present invention but not to limit the present invention.

Example 1 Method for detecting the content of hirudin in the drug of the present invention

1. Instruments and reagents

Analytical balance (Switzerland METTLER Instrument Co., Ltd.), Longshengzhi Capsules (Shaanxi Buchang Co., Ltd., batch number: 181005), Leech (provided by Buchang Co., Ltd.), physiological saline (Shijiazhuang Siyao Co., Ltd.), fibrinogen (batch number: P06J10Y92294, Beijing Bai Erdi Biotechnology Co., Ltd.), tris, hydrochloric acid, thrombin T8020 (batch number: 417G062, Beijing Soleibao Technology Co., Ltd.)

2. Methods and results

2.1 Solution preparation

2.1.1 Preparation of test solution

Take 0.4g, 0.4g, 0.4g, 0.4g, 0.4g, 0.4g, 0.4g, 0.3g, 0.2g, 0.1g, respectively, add 2mL, 4mL, 4.44mL, 5mL, 5.7mL, 6.67 mL, 7.5mL, 6.67mL, 5mL of physiological saline, and the concentration is 0.2g·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 gradient samples, namely samples 1-10, vortex Spin for 30min, centrifuge for 10min, 5000rpm, take the supernatant, refrigerate for later use. Leech medicinal materials (provided by Buchang Company) are treated in the same way.

2.1.2 Preparation of negative control solution

The sample without leeches was obtained according to the preparation process of Longshengzhi capsules, and prepared according to the above method, namely 0.2g·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 gradient negative control solutions .

2.1.3 Preparation of buffer

Take 25 mL of 0.2 mol·L ^-1 tris solution and about 40 mL of 0.1 mol·L ^-1 hydrochloric acid solution, add water to 100 mL, and adjust the pH to 7.4.

2.1.4 Preparation of Thrombin Titration Solution

Add 1000 U of thrombin to 5 mL of normal saline, and store in 1 mL aliquots at -20°C. Take 1 mL of thrombin to prepare 10 U·mL ^-1 , 20 U·mL ^-1 and 40 U·mL ^-1 .

2.2 Assay

Precisely measure 100μL of the test solution, put it in a 2mL EP tube, add 200μL of Tris Hydrochloric Acid Buffer (pre-use preparation) containing 0.5% (bovine) fibrinogen (calculated as coagulum), and mix well Put it in a water bath (37±0.5)℃ for 5 minutes, start dripping thrombin, start timing when dripping, and titrate until the sample solution to be tested has solidified or the end-point state appears. Record the end-point time of the titration and the volume of thrombin solution consumed.

2.3 Factors affecting content determination

2.3.1 Investigation of extraction methods

The same batch of samples were extracted by ultrasonic for 15min, 30min, vortex for 30min, and 60min. The anticoagulant activity was determined by titration (10U·mL-1, 2μL per minute), and the results showed that the anticoagulant activity was consistent. In order to fully agitate and be consistent with the extraction time in the Pharmacopoeia, vortex for 30min was selected.

2.3.2 Titration temperature

Temperature has a great influence on the titration process. The same batch of samples were titrated at 25°C, 37°C, 50°C, and 70°C. The results showed that the activity of hirudin at 50°C and 70°C decreased significantly. The activity results at 25°C and 37°C are consistent. Therefore, the original standard titration temperature of 37°C can be retained.

2.3.3 The influence of the pH of the test solution on the content determination result

The pH value of the test solution is between 4.5 and 7.0, and its anticoagulation activity is basically stable. When the pH value of the test solution is lower than 4.0, the solution is not clear and the coagulation phenomenon cannot be observed. If the pH value is too high, the anticoagulation activity will be reduced. After the pH value of the test solution is between 4.5 and 7.0, the pH of the test solution is measured to be 5-6 (pH test paper)

2.3.4 Determination of titration end point

The same batch of samples were vortexed for 30 minutes and titrated at 37°C. The results are summarized according to the phenomenon. It can be seen that during the titration process, the end point of the titration is the observation that the silk or small ball can be picked out in the test tube. It is observed that the titration process takes too long, shaking too frequently (more than 15min), and it is not easy to observe silk or small balls. The repeatability of the titration method is better when it is completed within 10 minutes.

2.3.5 Titration concentration and interval time of thrombin

In the 2015 edition of the Chinese Pharmacopoeia, two methods for the determination of anticoagulant activity are proposed. One is blood-sucking leeches. The concentration of thrombin is 40U·mL-1, which is added once every 1min, 5μL each time; One is non-blood-sucking leeches (wide-body golden thread leech) and willow-leaf leeches, with a thrombin concentration of 10U·mL ^-1 instilled, once every 4min, 2μL each time. The steps of the two methods for determining anticoagulant activity are basically the same, but the average thrombin activity unit dropped by the two methods is 40 times different per minute. Therefore, it can be seen that the titration interval and the thrombin concentration are important factors for the determination of anticoagulant activity.

2.3.5.1 The effect of thrombin titration interval on the determination of anticoagulant activity

Method 1: Thrombin 40U·mL ^-1 , 5μL each time, Method 2: Thrombin 10U·mL ^-1 , 2μL each time, using two different intervals of 1min and 4min to determine different concentrations of the same test product The anticoagulant activity focuses on the influence of the titration interval on the measured value. The results are shown in Table 1.

Table 1 The effect of the titration interval of Longshengzhi Capsules on the anticoagulant activity (x±s, n=3)

It can be seen from Table 1 that whether it is Method 1 or Method 2, the anticoagulant activity measured at an interval of 4 minutes is much smaller than the value of 1 minute interval, indicating that the titration interval has a greater influence on the determination of the anticoagulant activity value.

2.4 The influence of thrombin concentration on the determination of anticoagulant activity

Using the thrombin titration method, use ^{the thrombin solution with two concentrations of 20 and 10 U·mL -1} respectively, drip once every 1 min, and add 2 μl each time to titrate the samples of Longshengzhi capsules and leech medicinal materials of different concentrations (step Provided by Chang Co., Ltd.), calculate its anticoagulant activity value (Table 2 and Table 3)

Table 2 Anticoagulant activity of Longsheng Lee products measured by titration of different concentrations of thrombin concentration

Note: "-" means no response within 30 minutes, "--" means empty

It can be seen from Table 2 that the negative control group does not interfere with the determination of anticoagulant activity. When the concentration is 0.2g·mL ^-1 , the thrombin titration process does not occur within 30 minutes, and the concentration is measured when the concentration is 0.07～0.1g·mL -1 The anticoagulant activity is unstable, and the measured value cannot be used to calculate the anticoagulant activity; when the sample concentration is ^{within the linear range of 0.02～0.06g·ml -1} , the thrombin concentration is 20U·mL ^-1 and 10U·ml － At 1 o'clock, the titration interval was 1 min, and the measured anticoagulant activities were 11.6±4.76 U·g ^-1 and 13.49±2.35 U·g ^-1 , and the RSD were 2.44% and 5.74%, respectively. Among them, the thrombin concentration is 10U·ml ^{-1 and} the measured activity value is relatively close, and the accuracy is better; while the thrombin concentration is 20U·mL ^{-1 and} the measured value has a large error (Figure 1).

Table 3 Anticoagulant activity of leech medicinal materials measured by the titration of different concentrations of thrombin

Note: "-" means no response within 30 minutes, "--" means empty

It can be seen from Table 3 that when the concentration of leech ^{is within the linear range of 0.03～0.08g·ml -1} and the thrombin concentration is 20U·mL ^-1 and 10U·mL ^-1 respectively, the measured anticoagulant activity is 8.12±2.84U·g ^-1 , RSD is 2.86%. The anticoagulant activity measured when the sample concentration is too high or too low is unstable.

2.5 Data processing

2.5.1 Variance homogeneity test value (significance level 0.05) Variance statistical test results are shown in Table 4

Table 4 Variance statistical test results of Longshengzhi Capsules and Leech medicinal materials

It can be seen from Table 4 that when the thrombin concentration is 20U·mL ^-1 and 10U·ml ^-1 , the F values are F=0.5595<3.45 and F=0.305<3.45, respectively. It is considered that the Longshengzhi Capsules and Leech medicinal materials during the experiment The variance of the experimental values is uniform, and the t-test between the means can be performed.

2.5.2 Test for the mean content of the preparations and medicinal materials for the t test, the results are shown in Table 5.

Table 5 T-test results of Longshengzhi Capsules and Leech medicinal materials

It can be seen from Table 5 that when the thrombin concentration is 10U·ml ^-1 , t=1.63<2.145, it can be considered that the experimental mean values of Longshengzhi Capsules and Leech medicinal materials have no significant difference within the 95% confidence interval. Therefore, it is considered feasible to determine the content of Longshengzhi capsules by the current method in the Pharmacopoeia. This method is simple, rapid, accurate and reliable, and can be used for the quality control of Longshengzhi capsules.

3 Analysis and summary of measurement results

3.1 Methodological verification

Since Longshengzhi Capsules are composed of 12 traditional Chinese medicines, supplementary materials are added during processing, so studies on specificity and detection limits are required. The specificity can be seen from the negative control group, other interference components have no effect on the experiment; the detection range is between 0.02～0.06g·ml ^-1 , and the anticoagulant activity is relatively stable. Through the precision, linear relationship, specificity, detection limit and stability test, it can be seen that the anticoagulant activity of Longshengzhi Capsules is stable and reliable by this method.

3.2 Applicability of the method

The content of leech medicinal materials varies greatly at home and abroad. According to the literature, the content of hirudin in leech is much higher than that of leech in the current market. The 2015 edition of the "Chinese Pharmacopoeia" stipulates the titrant used for the determination of the content of different kinds of medicinal materials. The origin of the raw materials must be accurately identified before the raw materials are tested. Different varieties will have different concentrations of the titrant used for content determination. Using high-concentration titrants to determine varieties with lower hirudin content will cause greater errors, then the determination The numerical system has a large deviation.

3.3 Discussion on the determination of the concentration of Longshengzhi Capsules

This study found that when the sample concentration of Longshengzhi capsule is between 0.02～0.06g·mL ^-1 , it is basically linearly correlated with the anticoagulant activity (Figure 1), and the thrombin concentration is 20U·mL ^-1 and 10U· The r value of mL-1 is 0.9026 and 0.9738. The anticoagulant activity value of the sample measured at a ^{thrombin concentration of 10 U·mL -1 is 13.49±2.35 U·g -1} , and the RSD is 5.74%. The measured activity value is relatively Stable (Figure 1).

3.4 Discussion on the titration interval and thrombin concentration in the determination of the anticoagulant activity of Longshengzhi Capsule Leech

This article found that the thrombin concentration and titration interval have a greater impact on the thrombin titration time of the same sample and the measured anticoagulant activity value. In the Pharmacopoeia, 40 U·mL-1 of thrombin is used for high-activity Japanese medicinal leech, and a titration method of 5μL each time is dripped once every 1min; for low-activity wide-body golden thread leech, 10U·mL-1 is used every 4min One time, each 2μL titration method, the difference between the two concentrations is 40 times. When preparations and medicinal materials use the thrombin concentration and titration interval of the pharmacopoeia method, the higher concentration samples cannot be measured. Reasons: first: multiple titrations, the fibrinogen solution becomes thinner, which is not conducive to coagulation: second: once per drop Shake well once, so that the coagulation formed by the initial connection of some fibrin is destroyed, and these coagulation is often invisible to the eyes. Due to repeated destruction, the fibrin becomes thinner, which may lead to permanent titration and no coagulation. . The measurable range is relatively narrow (0.02～0.06g·mL ^-1 ); the thrombin concentration is 20U·mL ^-1 and 10U·mL ^-1 , the thrombin concentration is too high to measure meaningless, and it is not suitable for the content of preparations and medicinal materials Determination; the interval time is 1min, if the time is too long, it is not easy to observe the end of the titration.

Example 2 Preparation of medicine as capsule

Prescription: Astragalus 225g, Leech 100g, Ligusticum chuanxiong 90g, Angelica 90g, Safflower 90g, Peach Kernel 113g, Red Peony 90g, Muxiang 90g, Shichangpu 90g, Earthworm 60g, Mulberry 90g, Acanthopanax Senticosus 35g;

Preparation method: take leech, earthworm, acanthopanax senticosus dry paste and crush into fine powder; astragalus, chuanxiong, angelica, safflower, peach kernel, red peony, woody, calamus, mulberry, decocted twice, first The second time is 2 hours, and the second time is 1.5 hours. Filter and combine the decoction. When the filtrate is concentrated to 60℃, the relative density is 1.1-1.2, spray-dried to powder, add earthworm, leech and Acanthopanax senticosus powder and mix well, add medicine Use starch to make 1000 grains and get it.

Usage and dosage: Oral. Take 3 to 5 capsules at a time, 3 times a day.

Example 3 Preparation of medicine into tablets

Prescription: Astragalus 225g, Leech 100g, Ligusticum chuanxiong 90g, Angelica 90g, Safflower 90g, Peach Kernel 113g, Red Peony 90g, Muxiang 90g, Shichangpu 90g, Earthworm 60g, Mulberry 90g, Acanthopanax Senticosus 35g;

Preparation method: take leech, earthworm, acanthopanax senticosus dry paste and crush into fine powder; astragalus, chuanxiong, angelica, safflower, peach kernel, red peony, woody, calamus, mulberry, decocted twice, first The second time is 2 hours, and the second time is 1.5 hours. Filter and combine the decoction. When the filtrate is concentrated to 60℃, the relative density is 1.1-1.2, spray-dried to powder, add earthworm, leech, and Acanthopanax senticosus powder and mix evenly, add appropriate amount The starch slurry and magnesium stearate are mixed, granulated and compressed into tablets.

Example 4 Preparation of medicine as a pill

Prescription: Astragalus 225g, Leech 100g, Ligusticum chuanxiong 90g, Angelica 90g, Safflower 90g, Peach kernel 113g, Red peony 90g, Muxiang 90g, Shichangpu 90g, Earthworm 60g, Mulberry 90g, Acanthopanax senticosus 35g;

Preparation method: take leech, earthworm, acanthopanax senticosus dry paste and crush into fine powder; astragalus, chuanxiong, angelica, safflower, peach kernel, red peony, woody, calamus, mulberry, decocted twice, first The second time is 2 hours, and the second time is 1.5 hours. Filter and combine the decoction. When the filtrate is concentrated to 60℃, the relative density is 1.1-1.2, spray-dried to powder, add earthworm, leech, and Acanthopanax senticosus powder and mix evenly, add appropriate amount The hydroxypropyl methylcellulose and low-substituted hydroxypropyl cellulose are mixed, granulated, and compressed into tablets.

Example 5 Preparation of medicine into granules

Prescription: Astragalus 225g, Leech 100g, Ligusticum chuanxiong 90g, Angelica 90g, Safflower 90g, Peach Kernel 113g, Red Peony 90g, Muxiang 90g, Shichangpu 90g, Earthworm 60g, Mulberry 90g, Acanthopanax Senticosus 35g;

Preparation method: take leech, earthworm, acanthopanax senticosus dry paste and crush into fine powder; astragalus, chuanxiong, angelica, safflower, peach kernel, red peony, woody, calamus, mulberry, decocted twice, first The second time is 2 hours, and the second time is 1.5 hours. Filter and combine the decoction. When the filtrate is concentrated to 60℃, the relative density is 1.1-1.2, spray-dried to powder, add earthworm, leech, and Acanthopanax senticosus powder and mix evenly, add appropriate amount The dextrin and lactose are mixed, granulated and dried to make granules.

Example 6 Method for determining the content of astragaloside IV in the medicine of the present invention

Take 6g of the content of this product, accurately weigh it, place it in an Erlenmeyer flask, accurately add 50ml of 2% potassium hydroxide methanol solution, weigh it, heat and reflux for 1 hour, let it cool, weigh it, and use 2% hydroxide Potassium methanol solution to make up the lost weight, shake well, filter, accurately measure 30ml of the subsequent filtrate, place in the evaporation, evaporate the residue and add 20ml of water to dissolve, add a mixture of chloroform-n-butanol with a volume ratio of 2:1 Extract 5 times, 10ml each time, combine the extracts, wash 2 times with ammonia test solution, 20ml each time, discard the ammonia solution, evaporate the chloroform-n-butanol solution, and dissolve the residue with methanol, transfer to a measuring flask, add Dilute methanol to the mark, shake well, and use it as the test solution. Take an appropriate amount of astragaloside IV reference substance, accurately weigh it, and add methanol to make 1ml of reference substance solution containing 0.5 mm. According to an appendix VIB experiment of the 2000 edition of thin layer chromatography, draw 4μL and 6μL of the test solution, 3μL and 5μL of the reference solution, and respectively cross the same silica gel G with 0.3% methylcellulose sodium as the binder. On a thin layer plate, place a layered chloroform-methanol-water lower layer solution below 10°C as a developing agent, where the volume ratio of chloroform-methanol-water is 65:35:10, unfold, take out, dry, and spray 10% sulfuric acid ethanol solution, bake at 100℃ until the spots are clearly colored, take it out, cover the glass plate of the same size on the thin layer plate, fix the surrounding with tape, scan according to thin layer chromatography, wavelength λs=530nm, λR=650nm , Measure the integral value of the absorbance of the test substance and the integral value of the reference substance, calculate, and get.

Example 7 Pharmacological activity test of the drug of the present invention

1. Longshengzhi Capsule's effect on MPTP-induced mouse Parkinson's model

1.1. Experimental materials

1.1.1, experimental animals

C57BL/6J, SPF male mice, 72 mice, about 23-25g, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.; License number: SCXK (京)-2016-0006.

1.1.2, experimental drugs

Longshengzhi Capsules, specification 0.4g/capsule, batch number: 20171250, Shaanxi Buchang Pharmaceutical Co., Ltd.; Medoba, specification 0.25g/tablet, batch number: SH3288, Shanghai Roche Pharmaceutical Co., Ltd., lipid oxidation (MDA) detection reagent Box, manufacturer: Nanjing Jianshe, product number: batch number 20180412; Superoxide Dismutase (SOD), manufacturer: Nanjing Jianshe; product number: 20180418; Glutathione Peroxide (GSH-PX) determination kit, production Manufacturer: Nanjing built, product batch number 20180908.

1.1.3, experimental reagents

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), batch number: M9049-02, is provided by AbMole BioScience, paraformaldehyde, manufacturer: Wuhan Google Biotechnology Co., Ltd. Company, batch number: G1101; paraffin, xylene (China National Pharmaceutical Group Chemical Reagent Co., Ltd.), PBS (Wuhan Google Biotechnology Co., Ltd., G0002), EDTA (Wuhan Google Biotechnology Co., Ltd., G1203).

1.1.4, experimental equipment

SA417 rat and mouse grasping force tester, manufacturer: Jinan Yiyan Technology Development Co., Ltd., YLS-13A. Experimental swimming box, manufacturer: self-made, refrigerated centrifuge, manufacturer: Heal Force; photographic microscope: Nikon Eclipse Ti-SR.

1.2 Experimental method

1.2.1. Model building, grouping and drug delivery

The MPTP subacute modeling method was adopted: the experimental mice were intraperitoneally injected (ip) MPTP at a fixed time (9:00am) daily, 30mg/k, once a day for 5 consecutive days to prepare a PD model. 72 mice were randomly divided into 6 groups, blank group, model group, positive drug Medopa group (50mg/kg), Longshengzhi capsule high (1.6g/kg), medium (0.8g/kg), low (0.4g/kg) dose group. Except for the blank group and the model group by intragastric administration of normal saline every day, the administration group was given the corresponding drugs once a day. On the 8th day of administration, the model was created according to the above method (the blank group was given physiological saline). The drug was administered for 7 days, and the behavioral experiments (climbing rod experiment, tail suspension experiment (holding power) and swimming experiment) of each group of mice were tested.

1.2.2, behavioral testing

1.2.2.1、Climbing pole experiment

An iron rod with a length of 50 cm and a diameter of 1 cm was used as a climbing rod for the mouse experiment. A foam plastic ball with a diameter of 2 cm was fixed on the top of the climbing pole, and 5 layers of medical tape were wrapped around the climbing pole to prevent the mice from slipping during the crawling process. After the adaptive breeding of each group of mice, the climbing pole training was carried out. One day after the last administration, a pole climbing experiment was carried out to evaluate the motor coordination ability of each group of mice; during the test, each mouse was placed on the top of the ball and allowed to climb down freely, and the two forelimbs of the mouse were in contact with the sole of the pole. The platform is deemed to be fully crawling, and the time required for the mouse to crawl fully is recorded: ①the time when the mouse starts to crawl down; ②the time when the mouse crawls through the upper half of the pole; ③the time when the mouse crawls through the lower half of the pole.

Scoring according to time: Scoring method: 3 points for completion within 3s, 2 points for completion within 6s, 1 point for more than 6s, and each mouse takes the sum of the three parts to complete the score. Train for 2 days before the experiment, 3 times a day. In the formal test, each mouse was tested 3 times with an interval of 1 min. The values were added and the average value was taken.

1.2.2.2, grip test

After 24 hours, 1 week, 2 weeks, and 4 weeks after the operation, the mice were subjected to a grip test experiment. When measuring, it should be noted that the mouse should be handled gently when presiding, to avoid irritating it and affecting the operation. Grasp the mouse with the right hand and place it on the grip plate, push the grip plate forward with the left hand, and then slide the right hand back to At the tail of the mouse, open the grip plate with your left hand. The grip plate slides forward with the force of the right hand holding the tail. When the animal grasps the plate hard, it should be pulled back in time to make it measure the animal's maximum grip. Investigate the changes in the mice's grasping power after administration, and determine the effect of Longshengzhi Capsule on the muscle strength of the forelimbs of the stroke mice.

1.2.2.3, rotation tolerance

Place the mouse on the rotating platform of the balance rotator, set the rotation speed to 60r/min, record the time from the beginning of the rotation of the mouse to fall off the platform, if the mouse does not fall for more than 2 minutes, the test is terminated, the mouse’s rotation The endurance time was recorded as 120s. By observing the rotation tolerance time of mice after administration, the effect of Longshengzhi Capsule on the balance ability of stroke mice was investigated.

1.2.2.4, tail suspension experiment

After the adaptive breeding of the mice in each group, the two front paws of the test mice were hung on a horizontal metal wire (diameter 1mm, 30cm above the ground) for 10s every day. The test will be performed after the end of the administration on the 15th day. The scoring standards are as follows: the test time is 10s. Within the test time, the mouse grasps the metal wire with two hind paws to score 3 points, the mouse uses one hind paw to grasp the metal wire to score 2 points, and the mouse grasps both hind paws. 1 point is scored if the metal wire is not held, 0 points are scored for mice falling, and finally the scores are calculated and statistically analyzed.

1.2.2.5, swimming experiment

The 20cm×30cm×20cm water tank is used as the experimental swimming tank. Before the experiment, the swimming box was filled with drinking water with a depth of 10cm and a water temperature of (22～26)℃. At the beginning of the experiment, the mice were put into the water tank. The scoring standards are as follows: the test time is 10 minutes, and the mice that swim continuously during the test time are scored 30 points; the mice swimming for more than half of the test time are scored 25 points; the floating time of the mice accounts for half of the test time 20 points for those over time; 15 points for mice occasionally swimming; 10 points for mice floating on the side and occasionally swimming with hindlimbs. On the 15th day after the end of the administration, the test was recorded, and finally the score was calculated and statistically analyzed.

1.2.3, serum biochemical test

After the taken blood was placed for 0.5 h, it was centrifuged at 4°C in a refrigerated centrifuge at 5000 rpm, and the supernatant was aspirated and stored at -80°C. Determination of MDA, SOD, GSH-Px activity detection

1.2.4, brain tissue immunohistochemistry

After the animal tissue samples are collected and the behavioral test is completed, anesthetize the chest cavity and expose the heart. Use ophthalmic scissors to cut the right atrium. Insert a needle from the left ventricle to infuse 0.9% saline, and replace the blood through the systemic circulation until the fluid flows out. When there is no more redness, peel off the animal skull, carefully separate the bilateral cortex until the striatum is exposed, then use ophthalmic forceps to remove the fresh striatum tissue on both sides, separate the bilateral hippocampus, remove the substantia nigra of the midbrain, and put it on ice during the whole process In operation, the fresh tissue taken out was stored at -80°C; the whole brain of the other half of the experimental animals was peeled off and stored in 4% paraformaldehyde for later use. Perform immunohistochemical detection of related indicators. 1.2.5 Experimental procedures for tissue paraffin-embedded sections

(1) Material: Fresh tissue is fixed in 4% paraformaldehyde for more than 24 hours. Take the tissue out of the fixative solution and place it in a fume hood. Use a scalpel to trim the tissue at the target site. Put the trimmed tissue and the corresponding label in the dehydration box.

(2) Dehydration: Put the dehydration box into the hanging basket and dehydrate with gradient alcohol in the dehydrator. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h-anhydrous ethanol I 30min-anhydrous ethanol II 30min-alcohol benzene 5-10min-xylene I 5-10min-xylene II 5- 10min-wax I 1h-wax II 1h-wax III 1h.

(3) Embedding: Embed the wax-soaked tissue in an embedding machine. First put the melted wax into the embedding frame, and before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20°freeze table. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block.

(4) Sectioning: Put the trimmed wax block on a paraffin microtome for sectioning, with a thickness of 4μm. Float the slices on the spreader's 40℃ warm water to flatten the tissue, lift the tissue with a glass slide, and put it into a 60℃ oven to bake the slices. After the water is baked, the wax is baked, and then it is taken out and stored at room temperature for later use.

1.2.6, paraffin section immunohistochemistry experiment procedure

(1) Dewax the paraffin sections to water: sequentially put the sections in xylene Ⅰ 15min- xylene II 15min- anhydrous ethanol I 5min- anhydrous ethanol II 5min-85% alcohol 5min-75% alcohol 5min-distilled water wash.

(2) Antigen retrieval: The tissue section is placed in a retrieval box filled with EDTA antigen retrieval buffer (PH9.0) for antigen retrieval in a microwave oven. During this process, the buffer should be prevented from over-evaporating, and the slice should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed 3 times with shaking on a decolorizing shaker, each time for 5 minutes.

(3) Block endogenous peroxidase: put the slice into 3% hydrogen peroxide solution, incubate at room temperature for 25 minutes in the dark, put the slide in PBS (PH7.4) and wash 3 times with shaking on a decolorizing shaker. 5min each time.

(4) Blocking with BSA or serum: After the slices are dried a little, draw a circle around the tissue with a histochemical pen (to prevent the antibody from flowing away), drop 3% BSA into the circle to evenly cover the tissue, and seal at room temperature for 30 minutes.

(5) Add primary antibody: Gently shake off the blocking solution, add PBS to the slices with a certain proportion of the primary antibody, and place the slices flat in a humid box and incubate overnight at 4°C. (Add a small amount of water to the wet box to prevent the antibody from evaporating).

(6) Add secondary antibody: the slides were placed in PBS (PH7.4) and washed 3 times with shaking on a decolorizing shaker, each time for 5 minutes. After the sections were dried slightly, the tissue was covered with the secondary antibody (HRP labeled) corresponding to the primary antibody in the histochemical kit and incubated for 50 min at room temperature.

(7) DAB color development: the slides were placed in PBS (PH7.4) and washed 3 times with shaking on a decolorizing shaker, each time for 5 minutes. After the sections were dried slightly, freshly prepared DAB color developing solution was added dropwise to the circle, and the color development time was controlled under the microscope. The positive color was brownish yellow. Rinse the sections with tap water to stop the color development.

(8) Counter-stained cell nucleus: Harris hematoxylin counter-stained for about 3 minutes, washed with tap water, 1% hydrochloric acid and alcohol for a few seconds, rinsed with tap water, returned to blue with ammonia, rinsed with running water.

(9) Dehydration and mounting: Put the slices in 75% alcohol for 6min-85% alcohol for 6min-anhydrous ethanol I 6min-anhydrous ethanol Ⅱ 6min-xylene I for 5 minutes to dehydrate and transparent, take the slices out of the xylene and air dry , Neutral gum mounting film.

(10) Microscopic examination, image acquisition and analysis, the specific immunoslices are shown in Figures 1-6 in the manual.

1.3 Experimental results:

1.3.1 Behavior

1.3.1.1 Mouse tail suspension experiment

Table 6-Mouse tail suspension test results

It can be seen from the above results that the suspension test score of the model group was significantly lower than that of the blank control group (P<0.01). Compared with the model group, the tail suspension test scores of mice in the positive drug Medoba and Longshengzhi Capsule low, middle and high dose groups were significantly increased (P<0.05), and the behavioral coordination ability was significantly improved.

1.3.1.2 Climbing pole experiment

Table 7-Scores of mouse climbing pole experiment

From the above results, it can be seen that compared with the blank control group, the climbing time of the model group was significantly prolonged (P<0.01). Compared with the model control group, the pole-climbing time of the positive drug Medoba and Longshengzhi Capsules in the low, medium and high dose groups was significantly shortened (P<0.05).

1.3.1.3 Mouse grip experiment

Table 8-Mouse grip test results

It can be seen from the above results that the gripping power of the model group is significantly weaker than that of the blank group (P<0.01). Compared with the model group of mice, the grasping power of mice in the positive drug Metopa and Longshengzhi Capsule high, medium and low dose groups was significantly enhanced (P<0.01, P<0.05), indicating that the muscle strength of the mice's limbs was significantly enhanced.

1.3.1.4 Rotation tolerance experiment in mice

Table 9-Results of mouse rotation experiment

From the above results, it can be seen that the swimming test scores of mice in the model group were significantly lower than those in the blank group (P<0.05). Compared with mice in the model group, the swimming time scores of mice in the positive drug Medoba and Longshengzhi Capsule high, medium and low dose groups were significantly increased (P<0.01, P<0.05), and the behavioral coordination ability was significantly improved.

1.3.1.5 Mouse swimming experiment

Table 10-Scoring results of mouse swimming experiment

3.2 Mouse serum biochemistry

Table 11-Measurement results of various biochemical indicators of mouse serum

It can be seen from the above results that compared with the blank group, the SOD and GSH-px activities of the model group were significantly reduced, and the MDA content was significantly increased (P<0.05). Compared with the model group, the Longshengzhi Capsule high-dose group could significantly increase the SOD activity ( P<0.05), high and medium doses can significantly reduce the MDA content (P<0.05), high, medium and low doses of Longshengzhi can significantly increase the vitality of GSH-px. It shows that Longshengzhi Capsule can reduce the oxidative stress damage in the process of MPTP-induced PD in mice.

3.3 Organ coefficient

Table 12-Measurement results of organ changes in mice in the administration group

3.4 Immunohistochemical determination of TH

Table-13 Experimental results of immunohistochemical determination of positive cells in mouse brain tissue

It can be seen from the above results that compared with the blank control group, the number of TH-positive cells in the substantia nigra of the mouse model group was significantly reduced (P<0.01). The number of TH-positive cells in the substantia nigra of mice increased significantly (P<0.05, P<0.01). It shows that Longshengzhi Capsule can significantly alleviate the neuronal damage induced by MPTP.

Claims (6)

A method for detecting the content of a substance, characterized in that the method for detecting the content includes the following steps: (1) The raw material composition of the substance and its weight ratio are: 225 parts of astragalus, 100 parts of leech, 90 parts of Ligusticum chuanxiong, 90 parts of angelica, 90 parts of safflower, 113 parts of peach kernel, 90 parts of red peony, 90 parts of woody 90 parts of Acorus calamus, 60 parts of Earthworm, 90 parts of Morus sinensis, and 35 parts of Acanthopanax senticosus extract; (2) The preparation method is: take leech, earthworm, acanthopanax senticosus dry paste and pulverize into fine powder; astragalus, chuanxiong, angelica, safflower, peach kernel, red peony, woody, calamus, mulberry, decocted in water for two The first time is 2 hours, and the second time is 1.5 hours. Filter and combine the decoction. When the filtrate is concentrated to 60℃, the relative density is 1.1-1.2, spray-dried to powder, add earthworm, leech and Acanthopanax senticosus powder to mix Evenly, get it; (3) Preparation of test solution: Take the mixed powder prepared in step (2) as the test product, and add physiological saline to prepare ^{a sample with a concentration gradient of 0.02g·mL -1} to 0.2g·mL ^-1 , that is, the sample, vortex for 30 minutes, and centrifuge 10min, 5000rpm, take the supernatant, refrigerate for later use, treat leeches in the same way; (4) Preparation of negative control solution The sample without leeches is obtained according to the preparation process in (2), and is prepared according to the above step (3) to obtain a sample with a concentration gradient of ^{0.02g·mL -1} to 0.2g·mL ^{-1, and 10 negative concentration gradients} Control solution (5) Preparation of buffer Take 0.2mol·L ^-1 Tris solution with a hydrochloric acid solution 0.1mol·L ^-1 40mL, add water to 100mL, adjust the pH to 7.4; (6) Preparation of thrombin titrant Add 1000 U of thrombin to 5 mL of normal saline, and store in 1 mL aliquots at -20°C. Take the thrombin to prepare 10 U·mL ^-1 , 20 U·mL ^-1 and 40 U·mL ^-1 ; (7) Assay Precisely measure the test solution, put it in an EP tube, add 200μL of 0.5% bovine fibrinogen-containing tris hydrochloric acid buffer, mix well, place in a water bath, warm soak for 5 minutes, and start dripping thrombin titration Start timing when the solution is dripped, and titrate until the sample solution to be tested has solidified or the end-point state appears. Record the end-point time of the titration and the volume of thrombin solution consumed, and calculate its content. The application of a substance in the preparation of a medicine for the treatment of Parkinson's disease is characterized in that the raw material composition and weight ratio of the substance are: 225 parts of astragalus, 100 parts of leech, 90 parts of Ligusticum chuanxiong, 90 parts of Angelica sinensis, and safflower 90 parts, 113 parts of peach kernel, 90 parts of red peony root, 90 parts of Muxiang, 90 parts of Shichangpu, 60 parts of Earthworm, 90 parts of Morus sylvestris, and 35 parts of Acanthopanax senticosus extract. The application of the substance according to claim 2, wherein the preparation method of the substance is: taking leeches, earthworms, and acanthopanax senticosus dry paste and pulverizing into fine powder; astragalus, chuanxiong, angelica, safflower, peach kernel, red Paeonia, woody, calamus, mulberry, decocted twice with water, the first time is 2 hours, the second time is 1.5 hours, filtered and combined the decoction, the relative density of the filtrate is 1.1-1.2 when the filtrate is concentrated to 60℃, spray Dried into powder, mixed with earthworm, leeches, and Acanthopanax senticosus powder evenly, and added medicinal excipients to prepare various commonly used pharmaceutical dosage forms permitted in pharmaceutics, ready to be obtained. The use of the substance according to claim 2 or 3, characterized in that the dosage form of the substance is an oral preparation such as a capsule, a tablet, a pill, or a granule. The use of the substance according to claim 4, wherein the dosage form of the substance is a hard capsule. The use of the substance according to any one of claims 2 to 5 in the preparation of a medicine for treating Parkinson's disease.

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