1. An in-vitro biological activity determination method for leech medicinal materials, decoction pieces and processed products is characterized by comprising the following specific determination steps:

(1) preparation of a test solution: taking leeches, crushing and screening the leeches through a third sieve, weighing 2.5g, precisely weighing, adding 20ml of water, weighing, heating at 85 ℃ for 15 minutes, cooling to 40 ℃, adjusting the pH to 2.0 by using dilute hydrochloric acid, adding pepsin with the ratio of 1% of enzyme base, carrying out enzymolysis at 40 ℃ for 1 hour, adjusting the pH to 8.0 by using 20% sodium hydroxide solution, adding trypsin with the ratio of 1% of enzyme base, carrying out enzymolysis at 40 ℃ for 3 hours, adjusting the pH to be nearly neutral by adding dilute hydrochloric acid, heating at 85 ℃ for 15 minutes, cooling to room temperature, carrying out high-speed centrifugation, placing supernate into a 25ml measuring flask, adding 5ml of water into precipitate, washing, carrying out high-speed centrifugation, adding the supernate into the measuring flask, adding water to scale, mixing uniformly, and filtering to obtain the leech extract;

(2) the determination method comprises the following steps: precisely measuring 100 mu l of a sample solution, placing the sample solution in a test tube, adding 200 mu l of trihydroxymethylaminomethane hydrochloric acid buffer solution containing 0.5 percent of fibrinogen (calculated by solid matters), shaking up, placing the sample solution in a water bath at 37 ℃ for 5 minutes, dropwise adding thrombin solution containing 10 units in each 1ml (dropwise adding 1 time every 4 minutes, 2 mu l each time, and gently shaking up while dropwise adding) until coagulation is realized, recording the volume of the consumed thrombin solution, and calculating the antithrombin activity according to the following formula:

U=C₁V₁/（C₂V₂）

in the formula: u-each 1g of leech contains antithrombin activity unit, U/g;

C₁concentration of thrombin solution, U/ml;

C₂-the concentration of the test solution, g/ml;

V₁-the volume of thrombin solution consumed, μ Ι;

V₁-the amount of sample solution added, μ l.

2. The method for measuring in vitro biological activity of leech medicinal materials, decoction pieces and processed products according to claim 1, wherein the pepsin activity is 1:15000 and the trypsin activity is 2500U/mg.

Background

The leech is an animal medicinal material, is salty, bitter, neutral in nature and enters liver channel, has the effects of breaking blood and removing stasis, is mainly clinically orally administered, is mainly used for treating cerebrovascular diseases, angina pectoris caused by coronary heart disease, hemiplegia caused by apoplexy and the like, is collected in various Chinese pharmacopoeias, and is dried by leech (Whitmania pigra Whitman) or leech (Japanese leech) Hirudado nipponica Whitman or Whitmania acranulata Acaranta Whitman of leech family, is captured in summer and autumn, killed by boiling water, dried in the sun or dried at low temperature; the decoction pieces are cleaned, cut into sections and dried; the processed product is scalded Hirudo, which is prepared by collecting purified Hirudo segments, and scalding with pulvis Talci according to the scalding method (Tonghe 0213 in Chinese pharmacopoeia) until it is slightly swollen.

The animal medicine has the characteristics of complex components, undefined drug effect substance basis, weak association between quality control indexes and drug effect and the like. At present, most of animal traditional Chinese medicines mainly depend on methods of character identification, microscopic identification and physicochemical identification, the quality control is carried out by directly measuring the protein content in most of content measurement items, and the quality control method lacks comprehensiveness, pertinence and specificity, which is one of the difficulties and the key points for constructing and perfecting an animal medicinal material quality evaluation system. The biological activity measuring method is developed mainly for medicinal materials with definite clinical or pharmacological medicinal effect, but more components and undefined medicinal material basis. Therefore, the biological activity determination method is very fit for the characteristics of animal traditional Chinese medicines and can be used as a consensus direction for quality evaluation and control development.

Leeches are the only animal traditional Chinese medicinal materials adopting a biological activity determination method in Chinese pharmacopoeia, the antithrombin activity is determined by a thrombin titration method in the standard of medicinal materials (leech decoction pieces and processed products are not subjected to biological activity determination), and specifically, the antithrombin activity is calculated by taking 0.9 percent sodium chloride solution as an extraction solvent, titrating thrombin until coagulation is generated and the amount of consumed thrombin, and the titer limit of the medicinal materials is specified: the antithrombin activity of the whitmania pigra is not less than 4.0U/g, and the antithrombin activity of the hirudo nipponica is not less than 16.0U/g. This test method has certain limitations: firstly, the raw medicinal materials can only be controlled, the drink slices and the processed products can not be measured, the leech decoction pieces and the processed products need to be heated, the protein is denatured after treatment (the hirudin components are also one kind of protein), the physiological saline can not be dissolved and extracted, and the antithrombin activity can not be detected; secondly, the correlation between the anticoagulation activity represented by the biological activity determination method and the in-vivo pharmacodynamic action is not strong, the anticoagulation activity of leeches (Whitmania pigra Whitman) and leeches (Whitmania pigra Whitman) determined in vitro by a thrombin titration method under the item of leech medicinal materials in pharmacopeia is more than 4 times, and a large number of animal in-vivo pharmacodynamic experiment results show that the in-vivo pharmacodynamic activities of Whitmania pigra Whitman and Whitmania pigra Whitman are basically equivalent, the effect difference of the results is not four times that of the biological activity determination results of the medicinal materials, the curative effects of the two in clinical oral preparations are also basically equivalent, the analysis reason is that the antithrombin activity determination under the item of leech medicinal materials is the action of detecting macromolecular hirudin substances extracted by physiological saline, but hirudin mainly exists in salivary glands of blood sucking leeches such as Whitmania Whitman, and hirudin is not found in non-sucking leeches, so the in-vitro activity detection is larger, after oral administration, large molecular protein substances such as hirudin in hirudo nipponica are degraded into small molecular peptides in gastrointestinal tracts to absorb blood, the hirudin structure of the hirudo nipponica is damaged, the anticoagulation activity is weakened, whitmania pigra is slightly affected, the antithrombin activity of the hirudo nipponica and the antithrombin activity of the hirudo nipponica in vivo are basically equivalent, and the correlation between the in vitro biological activity determination method of the hirudo and the in vivo drug effect is not strong.

The macromolecular protein is degraded into micromolecular peptide substances in the gastrointestinal tract, and the micromolecular peptide substances can be absorbed into blood through intestinal tract transmembrane to play the drug effect, so that the quality of the leech medicinal material is defined by the antithrombin activity of the macromolecular protein and is not scientific enough, and the quality of the leech medicinal material can be really evaluated by taking the in-vivo metabolite, namely the micromolecular peptide, as a quality control index.

The invention discloses a method for measuring leech in-vitro biological activity, wherein a to-be-measured sample solution is obtained by carrying out enzymolysis on leech macromolecular protein into small molecular peptide components in vitro by using protease, and then measuring the in-vitro biological activity of the small molecular peptide solution, the correlation between the in-vitro biological activity and the in-vivo pharmacodynamic activity is strong, and the to-be-measured sample solution can be used as an index for evaluating leech medicinal materials, decoction pieces and processed products.

Disclosure of Invention

The invention aims to provide a more effective and clinically relevant method for measuring the in vitro biological activity of leech medicinal materials, decoction pieces and processed products.

The purpose of the invention is realized as follows:

the inventor provides an in vitro biological activity determination method for leech medicinal materials, decoction pieces and processed products, which comprises the following steps:

(1) preparation of a test solution: taking leeches, crushing and screening the leeches through a third sieve, weighing 2.5g, precisely weighing, adding 20ml of water, weighing, heating at 85 ℃ for 15 minutes, cooling to 40 ℃, adjusting the pH to 2.0 by using dilute hydrochloric acid, adding pepsin with the ratio of 1% of enzyme base, carrying out enzymolysis at 40 ℃ for 1 hour, adjusting the pH to 8.0 by using 20% sodium hydroxide solution, adding trypsin with the ratio of 1% of enzyme base, carrying out enzymolysis at 40 ℃ for 3 hours, adjusting the pH to be nearly neutral by adding dilute hydrochloric acid, heating at 85 ℃ for 15 minutes, cooling to room temperature, carrying out high-speed centrifugation, placing supernate into a 25ml measuring flask, adding 5ml of water into precipitate, washing, carrying out high-speed centrifugation, adding the supernate into the measuring flask, adding water to scale, mixing uniformly, and filtering to obtain the leech extract;

(2) the determination method comprises the following steps: precisely measuring 100 mu l of a sample solution, placing the sample solution in a test tube, adding 200 mu l of trihydroxymethylaminomethane hydrochloric acid buffer solution containing 0.5 percent of fibrinogen (calculated by solid matters), shaking up, placing the sample solution in a water bath at 37 ℃ for 5 minutes, dropwise adding thrombin solution containing 10 units in each 1ml (dropwise adding 1 time every 4 minutes, 2 mu l each time, and gently shaking up while dropwise adding) until coagulation is realized, recording the volume of the consumed thrombin solution, and calculating the antithrombin activity according to the following formula:

U＝C₁V₁/(C₂V₂)

in the formula: u-each 1g of leech contains antithrombin activity unit, U/g;

C₁concentration of thrombin solution, U/ml;

C₂-the concentration of the test solution, g/ml;

V₁-the volume of thrombin solution consumed, μ Ι;

V₁-the amount of sample solution added, μ l.

The enzyme activity of the pepsin used in the process is 1:15000, and the enzyme activity of the trypsin is 2500U/mg.

The invention is obtained by a large number of tests, and overcomes the defect that only the biological activity and the correlation between the biological activity and the body of the medicinal material can be measured under the item of Chinese pharmacopoeia.

Advantageous effects

In order to further verify the correlation between the leech bioactivity determination method and the in-vivo efficacy, the inventor carries out animal pharmacodynamic experiments:

modern researches show that the blood rheology, the blood circulation and the blood physicochemical change into the main biochemical basis of the occurrence of the blood stasis syndrome, and the laboratory diagnosis and evaluation indexes of the blood stasis syndrome have seven aspects of the blood rheology, the blood coagulation, the hemodynamics, the platelet aggregation and the like, wherein the blood coagulation index and the blood rheology become the main indexes of the research of the blood stasis syndrome diagnosis and the blood circulation promoting and blood stasis removing traditional Chinese medicines. The acute blood stasis model has the characteristics of strong clinical relevance, high success rate and wide application. Therefore, the in vivo pharmacodynamic research of the invention adopts an acute blood stasis rat model to research the influence of the whitmania pigra and the hirudo nipponia on a blood coagulation system, and truly reflects the in vivo blood circulation promoting and blood stasis removing effects of the whitmania pigra and the hirudo nipponia by measuring relevant indexes of blood coagulation, thereby laying a foundation for the establishment of the in vitro biological activity measuring method of the leech.

1. Laboratory apparatus

Xi1000 model full-automatic coagulation tester (Beijing Zhongshuwei science and technology development Co., Ltd.); model N6C full-automatic hemorheometer (manufactured by beijing prism instruments ltd); model AL-204 ten thousandth electronic balance (Mettler Toledo, Switzerland); a Centrifuge 5810R type high speed refrigerated Centrifuge (eppendorf gmbh, germany); FD-1A-50 type freeze drier (Beijing Bo Yi kang laboratory instruments Co., Ltd.).

2. Experimental Material

The adrenaline hydrochloride (batch number: Y03J7C15662, purity: not less than 99 percent) and the sodium citrate (batch number: R23M10P89097, purity: not less than 99.5 percent) are provided by Shanghai-sourced leaf Biotech Co., Ltd; chloral hydrate (Tianjin, Daozuo chemical reagent factory, lot number: 20190301, purity ≥ 99.5%); the Prothrombin Time (PT) assay kit (batch number: 2019-; aspirin (enteric coated tablet, lot number: 190411202, specification: 25 mg/tablet) and physiological saline (lot number: 1910242706) are all provided by Chengxin pharmaceutical industry, Inc.; the other reagents are analytically pure; the experimental water was deionized water.

3. Laboratory animal

SPF grade SD rats 54, female, body mass (200 ± 10) g, provided by experimental animal breeding limited, shandong ju nanpo, animal production license number: SCXK (lu) 20190003.

4. Experimental methods

4.1 preparation of leech samples

Leech (leech of Japanese medicine): taking fresh hirudo nipponia, homogenizing, freeze-drying, pulverizing, and sieving with No. 5 sieve.

Leech (whitmania pigra): hanging the product, removing impurities from the Whitmania pigra Whitman, cutting into segments, pulverizing, and sieving with No. 5 sieve.

Scalding leech (scalding whitmania pigra whitman): the product is prepared by placing 50g of talcum powder in a pot, heating with strong fire to a flexible state, adding 100g of whitmania pigra section, continuously stirring, scalding to slightly bulge, taking out, sieving to remove talcum powder, cooling, pulverizing, and sieving with No. 5 sieve.

Leech production (production of whitmania pigra): in the item of leech processing product from Shandong province Chinese medicinal material processing standard, 100g of wide-body leech powder is taken, and is uniformly mixed with 10g of yellow wine and moistened until the medicinal materials are thoroughly soaked. Heating the pot with middle fire, uniformly spreading 10g of wheat bran into the pot, adding wine-soaked Hirudo segments when smoking, parching until the surface is yellow and special odor is released, quickly taking out, sieving out the parched wheat bran, cooling, pulverizing, and sieving with No. 5 sieve.

4.2 grouping, Molding and administration

After one week of adaptive feeding, all rats were randomly divided into 15 groups, namely a blank group, a model group, an aspirin group (positive control), a low, medium and high dose group of hirudo nipponensis, a low, medium and high dose group of hirudo broadloom, and a low, medium and high dose group of hirudo broadloom prepared, and 6 mice in each group. Except for the blank group of rats injected with the same volume of physiological saline subcutaneously, the other groups of rats were injected with adrenaline hydrochloride 0.9mg/kg (solvent is physiological saline) subcutaneously 2 times a day at 4h intervals, and after the 1 st subcutaneous injection for 2h, the rats were placed in an ice-water bath for 4min, and the above operation was repeated for 15d to replicate the acute blood stasis model. From the 8 th day of model building, the rats of each dose group are respectively irrigated with corresponding leeches according to 0.35, 1.4 and 3.5g/kg (based on crude drug, the solvent is physiological saline) after the 2 nd injection of adrenaline hydrochloride for 1h every day, the aspirin is irrigated with aspirin according to 0.2g/kg (the solvent is physiological saline) in the aspirin group in the same period of time, and the physiological saline with the same volume is irrigated in the blank group and the model group in the same period of time for 1 time every day for 8 consecutive days.

4.3 blood sample Collection

The next day after the last administration, all rats were fasted for 12h without water deprivation, anesthetized with 10% chloral hydrate, and blood was taken from the abdominal aorta, wherein part of the whole blood was packed in heparin anticoagulation tubes, and the rest of the whole blood was added to anticoagulation tubes containing 3.2% sodium citrate (blood to anticoagulant volume ratio 9: 1).

4.4 detection of coagulation System-related indices

Whole blood anticoagulated by 3.2% sodium citrate is taken and centrifuged at 3500r/min for 15min to prepare PPP. The prothrombin time (i.e., PT), activated partial prothrombin time (i.e., APTT), and thrombin time (i.e., TT) were determined using a fully automatic coagulometer, and the procedures were performed according to the instructions of the instrument and the kit.

4.5 statistical analysis

Statistical analysis was performed on the data using SPSS 20.0 statistical software, expressed as Mean + -SD, with single factor analysis of variance of the differences between groups of samples, and two-by-two comparison using Dunnett-t. P <0.05 indicates that the difference is statistically significant.

5. Results of the experiment

APTT, PT, TT reflect respectively the extrinsic, intrinsic and common pathways of blood coagulation. The results of the effect of whitmania pigra and hirudo nipponia on clotting time are shown in the following table.

Table blood coagulation index measurement results of rats in each group (x ± s, n ═ 6, s)

Note: in comparison with the blank set, the results,^*P＜0.05，^**p is less than 0.01; in comparison with the set of models,^#P＜0.05，^##P＜0.01

in the experiment, the body temperature of a rat is raised by injecting adrenaline hydrochloride, the heart rate is accelerated to simulate the change of the body during rage, and the body is simulated by combining ice water soaking to simulate cold evil, so that the body is jointly acted by the two, and the model is made by the method for causing blood stasis. In addition, the experiment utilizes the characteristics that female rats are irritable and the body change is greatly influenced by temperature, and selects the female rats for molding. Aspirin, which is a widely used clinical drug for treating cardiovascular diseases, is administered orally, and is more comparable to leeches in the same way, so that aspirin is regarded as a positive drug.

In vivo pharmacodynamic results show that the Whitmania pigra Whitman and the Whitmania pigra Whitman can both prolong TT obviously, and the dosage has a certain dose-effect relationship (trend) with TT, the Whitmania pigra Whitman has a certain prolonging effect on APTT, but is not obvious, the Whitmania pigra Whitman and the positive medicine aspirin have a slightly obvious prolonging effect on APTT, and each medicine has no good prolonging effect on PT, which shows that the Whitmania pigra Whitman is most sensitive to TT prolonging indexes, but has relatively low sensitivity on prolonging of APTT and PT.

In addition, the difference between the in-vivo medicinal effects of the whitmania pigra and the hirudo nipponica is small, which is consistent with the in-vivo medicinal effect research results of Guanshi swordsman, Wangzhou and the like. Under the item of leech medicinal materials in 'Chinese pharmacopoeia' 2020 edition, the anticoagulant activities of whitmania pigra and hirudo nipponica are detected in vitro by adopting a thrombin titration method, and the difference between the anticoagulant activities is more than 4 times, which shows that the correlation between the anticoagulant activity detection method of the leech in the current pharmacopoeia and the in-vivo drug effect is not strong.

Detailed Description

The invention is further illustrated by the following examples, which are intended to be illustrative only and not limiting:

example 1

Determining the antithrombin activity of different batches of whitmania pigra decoction pieces: 15 batches of whitmania pigra medicinal materials mainly produced by 3 whitmania pigra in Jiangsu Rugao City, Shandong Jinning city and Zhejiang Pinhu city are collected,

preparation of a test solution: taking leeches, crushing and screening the leeches through a third sieve, weighing 2.5g, precisely weighing, adding 20ml of water, weighing, heating at 85 ℃ for 15 minutes, cooling to 40 ℃, adjusting the pH to 2.0 by using dilute hydrochloric acid, adding pepsin (the enzyme activity is 1:15000) with the enzyme bottom ratio of 1%, carrying out enzymolysis at 40 ℃ for 1 hour, adjusting the pH to 8.0 by using a 20% sodium hydroxide solution, adding trypsin (the enzyme activity is 2500U/mg) with the enzyme bottom ratio of 1%, carrying out enzymolysis at 40 ℃ for 3 hours, adjusting the pH to near neutrality by using dilute hydrochloric acid, heating at 85 ℃ for 15 minutes, cooling to room temperature, carrying out high-speed centrifugation, placing the supernatant in a 25ml measuring flask, adding 5ml of water for washing after precipitation, carrying out high-speed centrifugation, merging the supernatant into the measuring flask, adding water to the scale, mixing uniformly, and filtering to obtain the leeches;

the determination method comprises the following steps: precisely measuring 100 mu l of a sample solution, placing the sample solution in a test tube, adding 200 mu l of trihydroxymethylaminomethane hydrochloric acid buffer solution containing 0.5 percent of fibrinogen (calculated by solid matters), shaking up, placing the sample solution in a water bath at 37 ℃ for 5 minutes, dropwise adding thrombin solution containing 10 units in each 1ml (dropwise adding 1 time every 4 minutes, 2 mu l each time, and gently shaking up while dropwise adding) until coagulation is realized, recording the volume of the consumed thrombin solution, and calculating the antithrombin activity according to the following formula:

U＝C₁V₁/(C₂V₂)

in the formula: u-each 1g of leech contains antithrombin activity unit, U/g;

C₁concentration of thrombin solution, U/ml;

C₂-the concentration of the test solution, g/ml;

V₁-the volume of thrombin solution consumed, μ Ι;

V₁-the amount of sample solution added, μ l.

The results of the measurements are shown in the following table.

Table leech in vitro antithrombin activity determination result table

As can be seen from the results in the table above, the determination range of the in vitro antithrombin activity of the whitmania pigra is 10U-14U, and the average value is 11.73U; the antithrombin activity of the hirudo nipponica is 16U, which is slightly higher than that of the hirudo formosana, and is basically equivalent to the conclusion of the in vivo drug effect.