HK1114350B - An extract for preventing or treating thrombotic diseases - Google Patents

Description

An extract for preventing or treating thrombotic diseases

Technical Field

The invention relates to an extract for preventing or treating thrombotic diseases, in particular to an extract with the molecular weight of leech and/or earthworm of which the molecular weight is less than 5800 daltons, a pharmaceutical composition thereof, a preparation method thereof and application thereof in preparing a medicament for preventing or treating thrombotic diseases.

Background

Leeches are taken as a classic blood breaking and stasis removing medicament and are collected by Chinese pharmacopoeia. The leech medicinal material variety collected in the Chinese pharmacopoeia is three kinds, namely leech Whitmania pigra Whitman, leech Hirudo mpponica Whitman or leech willow Whitmania ac-ranulata Whitman, which is the main medical variety of leech Whitman. The Hirudo mainly comprises protein, polypeptide, amino acids, nucleic acid, enzyme, saccharide and trace elements. Qualitative and quantitative researches on components such as protein, amino acid, saccharide and trace elements in leeches are reported, and the main methods are thin-layer chromatography, high-performance liquid chromatography, electrophoresis and the like. Wherein the small molecule components mainly comprise hypoxanthine, xanthine, uracil, uridine, etc., and pyrimidine and purine are unsaturated heterocyclic compounds containing N atoms.

Earthworm is taken as a traditional Chinese medicine, has the efficacies of clearing heat and arresting convulsion, dredging collaterals, relieving asthma and promoting urination, and is collected by Chinese pharmacopoeia. There are four kinds of earthworm medicinal material varieties in Chinese pharmacopoeia, namely Pheretima aspergillum (E.Perrier), Pheretima vulgaris Chen, Pheretima guilliermon guillelmi (Michaelsen), or Pheretima aspergillum Michaelsen, which are Megascolecidae, and the main medicinal variety is Pheretima aspergillum (E.Perrier), which is called Guangdong earthworm. The main components of Lumbricus include protein, amino acids, nucleic acid, enzyme, saccharide, etc. Qualitative and quantitative research on components such as protein, amino acid and the like in earthworm has been reported. Wherein the small molecule components mainly comprise hypoxanthine and nitrogen-containing compounds.

A clinically used medicine, Shuxuetong injection, is a traditional Chinese medicine injection taking water extracts of two medicinal materials of leech and earthworm as active pharmaceutical ingredients, and is a high-efficiency and quick-acting injection for treating cardiovascular and cerebrovascular diseases. Chinese patent applications 03148281.3, 200410101538.8 and 200510000266.7, which are incorporated herein by reference in their entirety, disclose respectively Shuxuetong injection and a process for preparing the same.

The substance having immunogenicity is generally a macromolecular substance, and the larger the relative molecular weight, the more immunogenic. Small molecule polypeptides are less or non-immunogenic. Possible reasons for antigens being generally macromolecular substances are: (1) the larger the relative molecular weight of the antigen is, the more surface special chemical genes (antigenic determinants) are, so that the more effective the stimulation of immune cells is, and the immune response is generated; (2) the macromolecular antigen substance has complex chemical composition, stable structure, difficult damage and removal and long retention time in vivo, thereby continuously stimulating the immune system to generate immune response. In medicine, human insulin (human insulin standard molecular weight of 5800 daltons) consisting of 51 amino acids is generally used as a partition, and substances having a molecular weight of more than 5800 daltons are collectively called high molecular weight substances and are strictly limited in injection.

Disclosure of Invention

The invention aims to provide an extract for preventing or treating thrombotic diseases, in particular to an extract of leeches and/or earthworms with the molecular weight of less than 5800 daltons, a preparation method thereof and application thereof in preparing medicaments. Compared with the prior art, the extract is safer and more reassuring in clinical application.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an extract for preventing or treating thrombotic diseases, which is prepared by extracting leech and/or earthworm as raw materials, and is characterized in that the molecular weight of the extract is less than 5800 daltons (dalton).

The extract can be an extract of leech, an extract of earthworm or an extract of leech and earthworm which are mixed together, wherein the extract of leech and earthworm which are mixed together can be an extract of leech and earthworm which are respectively extracted and mixed together, or an extract of leech and earthworm which are mixed together.

The leech may be Whitmana leech Whitman, Hirudo mpponica Whitman or Whitmana albuginea ac-ranulata Whitman belonging to the family Hirudinidae, and the earthworm may be Pheretima giganticum (E.Perrier), Pheretima vulgaris Mullgaris Chen, Pheretima wilfordii Gillenii Guillel (Michaelsen) or Pheretima pectinifera Michaelsen belonging to the family Pheretima. Among them, preferably, the leech is a dried or fresh leech (Whitmania pigra Whitman) belonging to the family Hirudinidae, and the lumbricus is a dried or fresh lumbricus (Pheretima aspergillum (E.Perrier)) belonging to the family Megasphaeaceae.

The extract according to the invention can be prepared by a process comprising a filtration step with an ultrafiltration membrane or column having a molecular weight cut-off of less than or equal to 6000 daltons, preferably a molecular weight cut-off of 6000 daltons. For example, the starting material according to the invention is filtered through an extract (semi-finished product or intermediate) obtained by conventional methods of the prior art, using an ultrafiltration membrane or column with a molecular weight cut-off of less than or equal to 6000 daltons, preferably with a molecular weight cut-off of 6000 daltons, in order to obtain a leech and/or earthworm extract with a molecular weight of less than 5800 daltons.

Among them, preferably, the extract of the present invention is prepared by a method comprising the steps of:

(1) cleaning the raw materials with water for injection or normal saline, soaking at low temperature, and filtering to obtain filtrate and residue;

(2) crushing the dregs of a decoction, mixing the crushed dregs of a decoction with the filtrate obtained in the step (1) to form homogenate, repeatedly freezing and thawing the homogenate, and performing centrifugal separation to obtain supernatant;

(3) carrying out ultrafiltration on the supernatant obtained in the step (2) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of 10000-100000 daltons to obtain a permeate or a filtrate;

(4) and (4) filtering the permeate or filtrate obtained in the step (3) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 daltons, preferably with the molecular weight cut-off of 6000 daltons.

Wherein, if necessary, the filtration can be repeatedly carried out by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 Dalton, preferably with the molecular weight cut-off of 6000 Dalton. And, if necessary, hot pressing treatment can be carried out before filtration, for example, the filtrate is hot pressed for 10 to 45 minutes at a temperature of 105 to 136 ℃.

Among them, the extract of the present invention is preferably also prepared by a method comprising the steps of:

(1) cleaning raw materials with water for injection or normal saline, crushing to obtain homogenate, repeatedly freezing and thawing, centrifuging, and collecting supernatant;

(2) carrying out ultrafiltration on the supernatant obtained in the step (1) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of 10000-100000 daltons to obtain a permeate or a filtrate;

(3) treating the permeate or filtrate obtained in the step (2) by ion exchange column chromatography;

(4) desalting the eluent obtained in the step (3), and filtering by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 daltons, preferably the molecular weight cut-off of 6000 daltons; or filtering the eluent obtained in the step (3) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 daltons, preferably with the molecular weight cut-off of 6000 daltons, and desalting.

Wherein, if necessary, the filtration can be repeatedly carried out by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 Dalton, preferably with the molecular weight cut-off of 6000 Dalton.

The ultrafiltration membrane or ultrafiltration column can be selected from common ultrafiltration membranes or ultrafiltration columns in the technical field, such as hollow fiber ultrafiltration column made of polysulfone material, and has a cut molecular weight of less than or equal to 6000 daltons, preferably 6000 daltons; and during rough filtration, a hollow fiber ultrafiltration column with cut molecular weight of 10000-100000 daltons is used, and 10000 daltons or 50000 daltons is preferred.

The ion exchange column may be a cation exchange column or an anion exchange column. Wherein the cation exchange column is selected from CM-Sephadex, CM-Sepharose, CM-cellulose, SP-Sephadex, SP-Sepharose, SP-cellulose, etc.; as the anion exchange column, for example, DEAE-Sephadex, DEAE-Sepharose, DEAE-cellulose, Q-Sephadex, Q-Sepharose, Q-cellulose and the like can be used.

The extract of the invention contains polypeptide, amino acid and hypoxanthine.

The leech and/or earthworm extract can be dried or fresh leech and/or earthworm. The leech and the earthworm can be respectively extracted to obtain a leech extract and an earthworm extract, or the leech and the earthworm can be mixed together for extraction to obtain an extract of the leech and the earthworm mixed together.

The extract can be an extracting solution, and can also be in a solid form after dehydration and drying.

The invention also provides a preparation method of the extract, which comprises the step of filtering by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 daltons, and preferably with the molecular weight cut-off of 6000 daltons. For example, the leech and/or earthworm can be obtained by extracting and separating the raw leech and/or earthworm by a conventional method in the prior art, and finally filtering the obtained extract (also called semi-finished product or intermediate) by an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 daltons, preferably the molecular weight cut-off of 6000 daltons, so as to obtain the leech and/or earthworm extract with the molecular weight of less than 5800 daltons.

Preferably, the method for preparing the extract of the present invention comprises:

(1) cleaning the raw materials with water for injection or normal saline, soaking at low temperature, and filtering to obtain filtrate and residue;

(2) crushing the dregs of a decoction, mixing the crushed dregs of a decoction with the filtrate obtained in the step (1) to form homogenate, repeatedly freezing and thawing the homogenate, and performing centrifugal separation to obtain supernatant;

(3) carrying out ultrafiltration on the supernatant obtained in the step (2) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of 10000-100000 daltons to obtain a permeate or a filtrate;

(4) and (4) filtering the permeate or filtrate obtained in the step (3) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 daltons, preferably with the molecular weight cut-off of 6000 daltons.

Wherein, if necessary, the filtration can be repeatedly carried out by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cut-off less than or equal to 6000 Dalton, preferably with the molecular weight cut-off of 6000 Dalton. And, if necessary, hot pressing treatment can be carried out before filtration, for example, the filtrate is hot pressed for 10 to 45 minutes at a temperature of 105 to 136 ℃.

The molecular weight of the substance in the extract of the present invention was measured by using human insulin (molecular weight 5800) as a reference substance and measuring the content of the substance having a molecular weight higher than that of human insulin as the content of the high molecular weight substance.

For example, the invention adopts high performance liquid chromatography for measurement, and the specific method is as follows:

test material and instrument

1. Standard protein molecular weight material: human Insulin (Insulin), molecular weight 5800, specification 1 mg/count, purchased from China institute for pharmaceutical and biological products.

2. Instrument for measuring the position of a moving object

Visible ultraviolet spectrophotometer (Japanese Hitachi U-3210)

High performance liquid chromatograph Waters 600 system

High performance liquid chromatograph Agilent Hp1100

3. Reagent

Trifluoroacetic acid chemical purity (college of chemical and materials university of science and technology)

Acetonitrile chromatogram (Dima company)

Second, chromatographic conditions

A chromatographic column: GEL chromatography column (TSK GEL 2000SWxl 7.8 mm. times.300 mm);

mobile phase: trifluoroacetic acid-acetonitrile-water according to the volume ratio of 0.025: 30: 70;

an ultraviolet detector with the detection wavelength of 214 +/-1 nm;

the flow rate was 0.7 ml/min.

Third, measurement

Precisely measuring a proper amount of a test sample, and preparing a test sample solution by using a mobile phase; accurately weighing appropriate amount of human insulin, and making into reference solution with mobile phase; precisely absorbing the test solution and the reference solution, respectively, injecting into a high performance liquid chromatograph, measuring by high performance liquid chromatography, recording chromatogram, and measuring the content of substances with molecular weight higher than that of human insulin, namely calculating the content of substances corresponding to chromatographic peaks with retention time shorter than that of human insulin peaks, and taking the content as the content of substances with high molecular weight (molecular weight higher than 5800 daltons) in the test sample.

The molecular weight distribution of the substances in the extract of the present invention can also be determined by mass spectrometry. The molecular weight distribution of the components in the extract is determined, for example, by the following method.

A suitable amount of the extract of the present invention (or desalting treatment as necessary) was taken and dissolved in a 0.5% aqueous solution of TFA (trifluoroacetic acid). After mixing the sample and the matrix in a ratio of 1: 1, the target was spotted and dried at room temperature. The matrix is as follows: DHB (2, 5-dihydroxybenzoic acid), CCA (a-cyano-4-hydroxycinnamic acid).

Measured with an Autoflex (Bruker co.) instrument. Mass spectrum conditions: (MALDI-TOF-MS); an N2 laser source with the wavelength of 337 nm; linear scanning (flight tube length 1.6m, acceleration voltage 20 kv); ion type: positive ions (or negative ions).

The measurement result shows that the molecular weight of the components contained in the extract is less than 5800 daltons.

The extract of the invention has no substance with molecular weight more than 5800 daltons determined by the method, so the extract with the function of preventing or treating thrombotic diseases has molecular weight not more than 5800 daltons.

The extract of the invention contains polypeptide, amino acid and hypoxanthine. Wherein the polypeptide and amino acid can be analyzed by a suitable amino acid analyzer or a suitable system of high performance liquid chromatograph. For example, the amino acid determination can be performed according to the high performance liquid chromatography method in appendix VI D of the first edition of Chinese pharmacopoeia 2005:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; using A, B liquid as mobile phase, gradient elution, detecting wavelength 338nm and 262nm, column temperature 30-40 deg. C. Wherein:

solution A: adding a proper amount of sodium acetate into water for injection to dissolve, adding a proper amount of triethylamine, uniformly mixing, adjusting the pH value to be neutral, adding a proper amount of tetrahydrofuran, and uniformly mixing.

And B, liquid B: adding proper amount of sodium acetate into water for injection to dissolve, adding proper amount of acetic acid to adjust pH to neutral, adding proper amount of mixed solution of acetonitrile-methanol (1: 1), and mixing.

Adding a proper amount of boric acid buffer solution with the pH value of 10.4 into a proper amount of amino acid reference substance, uniformly mixing, adding o-phthalaldehyde and a 9-fluorenylmethyl chloroformate derivative reagent, and uniformly stirring to fully react to generate o-phthalic acid-9-fluorenylmethyl chloroformate amino acid serving as a reference substance solution. The extract of the invention is used as a test sample, and the amino acid is determined by adopting a high performance liquid chromatograph according to the method operation.

The hypoxanthine in the extract can be measured according to VID high performance liquid chromatography which is an appendix of the first edition of Chinese pharmacopoeia 2005, octadecylsilane chemically bonded silica is used as a stationary phase, 0.1% disodium hydrogen phosphate aqueous solution is used as a mobile phase, and the detection wavelength is 254 nm. The number of theoretical plates is not less than 3000 calculated according to xanthine.

The extract for preventing or treating thrombotic diseases is prepared into liquid medicine containing 0.5g/ml of crude drugs through a local irritation test of rabbits, 1ml of the extract is injected into the left leg, the right leg, the thigh and the four head of the rabbits respectively, the animals are killed 48 hours after the injection, the quadriceps femoris on two sides are dissected and taken out, the longitudinal incision is carried out, the injection local irritation reaction is observed, the fixation is carried out by putting 10 percent formaldehyde solution, and the pathological histology examination is carried out according to the routine. As a result: the naked eye examination shows that the quadriceps femoris of the rabbit has no hyperemia and necrosis, the score is calculated as O according to the score of the response grade in the stimulation reaction, namely, no obvious change exists, and the pathological examination proves that no obvious inflammatory reaction exists. The result is that the extract of the invention has no significant change in local irritant response.

The extract for preventing or treating thrombotic diseases is prepared into liquid medicine containing 0.5g/ml of crude drugs through a rabbit vascular irritation test, and is injected once per ear vein once a day and three times continuously according to 1g/kg dosage (equivalent to 25 times of clinical dosage). Fixing the injection part with ear, performing conventional histopathological examination, and observing whether the local injection vein has inflammatory reaction. As a result: the pathological examination shows that the ear vein of the domestic rabbit has no stimulation reaction of tissue degeneration or necrosis, which indicates that the extract of the invention has no obvious inflammatory reaction on local blood vessels after multiple intravenous injections.

The extract for preventing or treating thrombotic diseases is prepared into liquid medicine containing 0.5g/ml crude drugs by guinea pig anaphylaxis test, 6 healthy guinea pigs are taken and divided into the I group and the 2 group, and each group comprises 3 crude drugs. The guinea pigs in group 1 and 2 of this experiment were injected intraperitoneally with 0.5ml each of the extract injection of the present invention on day l, day 3 and day 5 of the experiment. Group 1 Each guinea pig was injected intravenously with the extract injection lml of the present invention on day 14 after the 1 st injection. Group 2 Each guinea pig was injected intravenously with the extract injection lml of the present invention on day 21 after the 1 st injection. Within 15 minutes after intravenous injection, each guinea pig was immediately observed for signs of hair erection, dyspnea, sneezing, retching or cough, or gong, convulsion, collapse or death. As a result: no piloerection, dyspnea, sneezing, coughing, convulsion, death, etc. occurred in the guinea pigs of group I14 and group 2 within 15 minutes after intravenous injection of the extract injection of the present invention on day 2. The experiment shows that no obvious allergic reaction phenomenon occurs in the guinea pigs on the 2 nd day of the last administration, which indicates that the extract of the invention has negative results of the allergic test of the guinea pigs.

In addition, the extract for preventing or treating thrombotic diseases passes the hemolytic test of rabbits. The test results show that no hemolysis and no erythrocyte agglutination are found. Indicating that the hemolysis test of the extract of the present invention is a negative result.

Therefore, the extract for preventing or treating thrombotic diseases according to the present invention can be used as a raw material for preparing various preparations. The present invention also relates to a pharmaceutical composition comprising the above-described extract as an active ingredient. The pharmaceutical composition can be tablets, capsules, granules, films, patches, suppositories, pills, powders, ointments, mixtures (such as oral liquid), syrups, tinctures, ophthalmic preparations, nasal preparations, injections, sterile powders for injections, concentrated solutions for injections or sustained-release and controlled-release preparations of the above. The preparation is a conventional pharmaceutical preparation, and thus, the preparation can be prepared by a conventional pharmaceutical technology in the field.

The extract or the pharmaceutical composition has the effects of preventing and treating thrombotic diseases, and can be used for preparing medicines for preventing and treating thrombotic diseases. For example, it can be used for preventing and treating diseases such as hyperlipidemia, atherosclerosis, myocardial infarction, angina pectoris, arteriosclerotic cerebral infarction, cerebral embolism, venous thrombosis, pulmonary embolism, pulmonary infarction, thromboangiitis obliterans, arteriosclerotic occlusion, Disseminated Intravascular Coagulation (DIC), thrombosis and thromboembolism complicated with surgical operation, hepatic infarction, renal infarction, gallbladder infarction, mesenteric infarction, gangrene of limbs, diabetic peripheral neuropathy, retinal vessel occlusion, or sudden deafness.

The extract for preventing or treating thrombotic diseases has the following outstanding advantages: the components have molecular weight distribution below 5800 daltons, and relatively small molecular weight (molecular weight), so that the immunogenicity is weak or non-immunogenic. Moreover, in terms of the number and size of insoluble particles, the number and size of insoluble particles in the leech and/or earthworm extract of the present invention are much smaller than those of the existing leech and/or earthworm extract, and thus the safety is significantly improved. Compared with the existing leech and/or earthworm extract, the safety of the medicament is obviously improved under the condition that the medicinal activity or the treatment effect is not reduced at all or is improved, and the injection prepared by the extract can be used without skin test, thereby saving the time for rescuing patients, relieving the pain of the patients and the like.

Drawings

FIG. 1: high performance liquid chromatography assay for human insulin (molecular weight 5800 daltons).

FIG. 2: the high performance liquid chromatography detection chart of the extract (leech earthworm extract) is provided.

FIG. 3: mass spectrometric detection of the extract (leech extract) of the invention.

Wherein: the matrix was CCA and detected as positive ions.

FIG. 4: mass spectrometry of the extract of the invention (earthworm extract).

Wherein: the substrate is DHB, and the detection is negative ions.

Detailed Description

Example 1

Soaking Hirudo (dried), and Lumbricus (dried) in normal saline or water for injection, spreading the surface sufficiently, washing with normal saline or water for injection, soaking in 2-4 times of normal saline or water for injection at low temperature of 0-4 deg.C for 24 hr, filtering, and storing the filtrate and residue respectively.

Mashing the residue with tissue triturator, homogenizing with colloid mill, grinding into homogenate with diameter less than 0.5 μm, freezing the homogenate at-15 deg.C below zero for 15-30 hr, thawing at low temperature of 0-4 deg.C, and repeatedly freezing and thawing for at least 2 times.

Centrifuging the frozen and melted solution in a centrifuge to obtain supernatant, coarsely filtering with ultrafiltration column with molecular weight of 5 ten thousand daltons, and filtering and purifying the filtrate with ultrafiltration column with molecular weight of 1 ten thousand daltons

Taking the filtrate which passes through an ultrafiltration column with the molecular weight of 1 ten thousand daltons, further purifying the filtrate by an ultrafiltration column with the molecular weight of 6000 daltons, carrying out hot pressing treatment on the filtrate at the temperature of 105 ℃ and 136 ℃ for 10-45 minutes, and filtering the filtrate by an ultrafiltration column with the molecular weight of 6000 to obtain the extracting solution.

If necessary, the extract obtained above may be dehydrated and dried to a solid.

The extractive solution contains polypeptide, amino acids and hypoxanthine. The extract has no substance with molecular weight greater than 5800 daltons, and the molecular weight distribution of the obtained extract is below 5800 daltons. The results of the chromatographic measurements are shown in FIGS. 1 and 2.

The data for each peak in FIGS. 1 and 2 are shown in tables 1 and 2, respectively.

Table 1 figure 1 human insulin test results

Table 2 figure 2 extract test results of the present invention

Example 2

Soaking Hirudo (dried or fresh) and Lumbricus (dried or fresh) in normal saline or water for injection respectively, spreading the surface sufficiently, washing with normal saline or water for injection repeatedly, soaking in 2-4 times of normal saline or water for injection at low temperature of 0-4 deg.C for 24 hr, filtering, and storing the filtrate and residue respectively.

Mashing the residue with tissue triturator, homogenizing with colloid mill, grinding into homogenate with diameter less than 0.5 μm, freezing the homogenate at-15 deg.C below zero for 15-30 hr, thawing at low temperature of 0-4 deg.C, and repeatedly freezing and thawing for at least 2 times.

Centrifuging the frozen and melted solution in a centrifuge to obtain supernatant, coarsely filtering with an ultrafiltration column with molecular weight of 50000 daltons, and filtering and purifying the filtrate with an ultrafiltration column with molecular weight of 10000 daltons to obtain filtrate.

Adjusting pH of the filtrate to 6.0-7.0 with phosphate buffer solution, passing through CM-sephadex cation exchange column, eluting with 5 times column volume of buffer solution, collecting eluate, adjusting pH to 6.0-7.0 with buffer solution, passing through DEAE-sephadex anion exchange column, washing with buffer solution, discarding eluate, eluting with buffer solution of pH6.0-7.0 and 0.2-0.5mol/L sodium chloride, collecting eluate, filtering with ultrafiltration column with molecular weight of 6000 daltons, and desalting to obtain extractive solution. Preparing leech extract and earthworm extract respectively, and mixing the two prepared extracts uniformly to obtain the leech and earthworm extract.

If necessary, the extract obtained above may be dehydrated and dried to a solid.

The above extractive solution contains polypeptide, amino acids and hypoxanthine. Through the detection of the high performance liquid chromatography, substances with molecular weight more than 5800 daltons are not found in the extract, so that the molecular weight distribution of the obtained leech earthworm extract is less than 5800 daltons.

Example 3

Soaking Hirudo (dried or fresh) and Lumbricus (dried or fresh) in normal saline or water for injection respectively, spreading the surface sufficiently, washing with normal saline or water for injection repeatedly, soaking in 2-4 times of normal saline or water for injection at low temperature of 0-4 deg.C for 24 hr, filtering, and storing the filtrate and residue respectively.

Mashing the residue with tissue triturator, homogenizing with colloid mill, grinding into homogenate with diameter less than 0.5 μm, freezing the homogenate at-15 deg.C below zero for 15-30 hr, thawing at low temperature of 0-4 deg.C, and repeatedly freezing and thawing for at least 2 times.

Centrifuging the frozen and melted solution in a centrifuge to obtain supernatant, coarsely filtering with an ultrafiltration column with molecular weight of 50000 daltons, and filtering and purifying the filtrate with an ultrafiltration column with molecular weight of 10000 daltons.

Taking the filtrate passing through an ultrafiltration column with the molecular weight of 10000 Dalton, further purifying by an ultrafiltration column with the molecular weight of 6000 Dalton, carrying out hot pressing treatment on the filtrate at the temperature of 105 ℃ and 136 ℃ for 10-45 minutes, and filtering by an ultrafiltration column with the molecular weight of 6000 Dalton to obtain the extracting solution. Preparing leech extract and earthworm extract respectively.

The mass spectrometry method is adopted for detection, and the molecular weight distribution of the components in the leech extract and the earthworm extract is less than 5800 daltons. The results are shown in FIGS. 3 and 4.

Mixing the two extractive solutions uniformly to obtain Hirudo Lumbricus extract. Adding 4% pharmaceutical adjuvants mannitol and 0.5-1% active carbon for removing pyrogen, filtering with microporous membrane, packaging into penicillin bottles, freeze drying, capping, detecting, and making into lyophilized powder for injection.

Example 4

Soaking Hirudo raw materials (dried or fresh) in normal saline or water for injection to fully develop its surface, repeatedly washing with normal saline or water for injection, soaking in 2-4 times of normal saline or water for injection at low temperature of 0-4 deg.C for 24 hr, filtering, and storing filtrate and residue respectively.

Mashing the residue with tissue triturator, homogenizing with colloid mill, grinding into homogenate with diameter less than 0.5 μm, freezing the homogenate at-15 deg.C below zero for 15-30 hr, thawing at low temperature of 0-4 deg.C, and repeatedly freezing and thawing for at least 2 times.

Centrifuging the frozen and melted solution in a centrifuge to obtain supernatant, coarsely filtering with an ultrafiltration column with molecular weight of 50000 daltons, and filtering and purifying the filtrate with an ultrafiltration column with molecular weight of 10000 daltons.

Taking the filtrate passing through an ultrafiltration column with the molecular weight of 10000 Dalton, carrying out hot pressing at the temperature of 105 ℃ and 136 ℃ for 10-45 minutes, and repeatedly filtering for 2-3 times through the ultrafiltration column with the molecular weight of 6000 Dalton to obtain an extracting solution.

Adding 5.4% of beta-cyclodextrin into the extract, stirring uniformly, soaking for 30 minutes at 45 ℃ to fully include, and spray drying to obtain dry powder;

adding 1.5% of silicon dioxide, 0.3% of PVP and 1% of carboxymethyl starch, mixing, granulating, and making into hard capsule containing 1g of crude drug.

Example 5

An extract was obtained by the same procedure as in example 1.

Adding 6.4% starch into the extractive solution, mixing, drying, and sieving to obtain medicinal powder.

Adding 0.1% of silica gel micropowder and 0.1% of magnesium stearate into the dried powder, mixing, and encapsulating.

Example 6: effect on clotting time in mice

Male Kunming mice of 20 + -2 g were divided into 3 groups at random according to body weight, 16 mice were each group, 10ml/kg of physiological saline was injected into the tail vein of each group, 2.5g/kg of ZL03148281.3 extract, 2.5g/kg of the extract of the present invention (example 1) was injected into the tail vein of each group, blood was collected from the orbit after 15 minutes, and the blood coagulation time was measured by the capillary method, and the results are shown in Table 3.

TABLE 3 Effect on clotting time in mice (X. + -. s)

Note:^*p is less than 0.01 compared with the normal saline group; comparison of # with ZL03148281.3 extract P > 0.05.

The results are shown in Table 3: the ZL03148281.3 extract can obviously prolong the blood coagulation time of mice (compared with a normal saline group, the Q value is 5.9637, and P is less than 0.01), and the extract can also obviously prolong the blood coagulation time of mice (compared with a normal saline group, the Q value is 6.4456, and P is less than 0.01), which indicates that the blood coagulation time can be obviously prolonged by both the extracts. The difference between the two extracts was not significant (Q value 0.4819, P > 0.05).

Example 7: effect on rat platelets and platelet adhesion Rate

1. Effects on platelets

30 male rats 220-270g were randomly divided into 3 groups, and injected with physiological saline 5.0ml/kg into the tail vein, 1.0g/kg of the extract of the present invention and 1.0g/kg of the ZL03148281.3 extract, and blood was collected from the orbit after 15 minutes and platelets were measured on eight blood cell counter instruments, and the results are shown in Table 4.

2. Effect on platelet adhesion Rate

30 male rats 220-270g were randomly divided into 3 groups, and injected with physiological saline 5.0ml/kg in the tail vein, 1.0g/kg of the extract of the present invention and 1.0g/kg of the ZL03148281.3 extract, and blood was collected from the orbit after 15 minutes, and platelets were measured on an eight-item hemocytometer as the number of platelets before adhesion. Taking 1.5ml of blood, putting the blood into a test tube filled with 0.3ml of 3.8% sodium citrate solution, uniformly mixing, taking 1ml of blood from the test tube, putting the blood into a silicon tube ring, putting the blood into an in-vitro thrombosis-platelet adhesion instrument, rotating the blood at the speed of 17 rpm for 5min at 37 ℃, taking blood-measuring platelets as the number of the platelets after adhesion, and calculating the adhesion rate of the platelets, wherein the results are shown in a table 4. The calculation method of platelets is as follows:

the adherence ratio (%) - (number of platelets before adherence-number of platelets after adherence/number of platelets in blood before adherence. times.100%

TABLE 4 Effect on the number of platelets and the rate of platelet adhesion in rats (X. + -.s)

Note:^*compared with the normal saline group, P is less than 0.05,^**p is less than 0.01; comparison of # with ZL03148281.3 extract P > 0.05.

The results are shown in Table 4: the ZL03148281.3 extract can obviously reduce the number of the blood platelets of normal rats (compared with a normal saline group, the Q value is 2.9260, and P is less than 0.05), and the extract can also obviously reduce the number of the blood platelets of the normal rats (compared with the normal saline group, the Q value is 5.1399, and P is less than 0.01), which indicates that the two extracts can reduce the number of the blood platelets of the normal rats. However, in comparison of the two extracts, there was no significant difference in platelet count and platelet adhesion rate (Q value 2.2139, P > 0.05; t-1.6066, P-0.1186, P > 0.05). The extracts of the invention and ZL03148281.3 extract have the functions of reducing the number of platelets and the adhesion of the platelets after being administrated, and are beneficial to reducing the formation of thrombus.

Example 8: visible foreign matter and insoluble particle detection

And preparing leech earthworm extract (hereinafter, ZL03148281.3 extract) according to the method disclosed in the document ZL03148281.3 by taking equal amount of leech (dried product) and earthworm (dried product).

1. Detection of visible foreign matter: the extracts of the invention (example 1) and ZL03148281.3 were tested for visible impurities as specified in the general injection protocol of chinese pharmacopoeia (2005 edition) and the results are shown in table 5.

Table 5 results of foreign matter detection

Item inspection product	Qualification rate of lamp inspection	Short and small fiber
			Extract of the invention	92.7％	3.9％
ZL03148281.3 extract	63.7％	28.5％

The test results show that the extract of the invention has a significant difference with the ZL03148281.3 extract in the aspect of visible foreign matters. The light inspection qualification rate and short fibers of the extract are obviously better than those of ZL03148281.3 extract, so the extract is safer to apply.

2. Detection of insoluble particles: the extracts of the invention (example 1) and ZL03148281.3 extract were tested for insoluble particles as defined in the general injection protocol of the Chinese pharmacopoeia (2005 edition) and are shown in Table 6.

TABLE 6 insoluble particle detection results

Item inspection product	Particles of more than 10um (mean/count)	Particles of more than 25um (mean/count)
			Extract of the invention	196 pieces/branch	65/piece
ZL03148281.3 extract	250/piece	110/piece

The test results show that the extract of the invention also has a significant difference with respect to the ZL03148281.3 extract in terms of insoluble particles. The extract of the present invention has significantly less insoluble particles than ZL03148281.3 extract, and therefore the extract of the present invention is safer to use.

The above test results show that the extract of the invention and the ZL03148281.3 extract have no obvious difference in drug effect compared with ZL03148281.3 extract, and even the extract of the invention shows better results than ZL03148281.3 extract in some indexes. That is, the extract of the present invention remarkably improves the safety of the drug without decreasing the pharmaceutical activity, compared to the existing leech earthworm extract.

The invention has been described with reference to specific embodiments. It should be understood that the foregoing description and examples are only illustrative of the present invention. Numerous alternatives and modifications can be devised by those skilled in the art without departing from the spirit and scope of the present invention, which should be construed as within the scope of the invention.

Claims (23)

1. An extract for preventing or treating thrombotic diseases, which is prepared by extracting leech and earthworm as raw materials, and is characterized in that the molecular weight of the extract is less than 5800 daltons, and the extract is prepared by a method comprising the following steps:

(1) cleaning the raw materials with water for injection or normal saline, soaking at low temperature, and filtering to obtain filtrate and residue;

(2) crushing the dregs of a decoction, mixing the crushed dregs of a decoction with the filtrate obtained in the step (1) to form homogenate, repeatedly freezing and thawing the homogenate, and performing centrifugal separation to obtain supernatant;

(3) carrying out ultrafiltration on the supernatant obtained in the step (2) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of 10000-100000 daltons to obtain a permeate or a filtrate;

(4) and (4) filtering the permeate or the filtrate obtained in the step (3) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of less than or equal to 6000 daltons.

2. The extract of claim 1, wherein in step (4), the filtration is performed with an ultrafiltration membrane or column having a molecular weight cut-off of 6000 daltons.

3. The extract according to claim 1, wherein in the step (4), the filtration is repeatedly performed by the ultrafiltration membrane or the ultrafiltration column.

4. The extract according to claim 1, wherein in the step (4), the autoclaving treatment is performed before the filtration.

5. The extract according to claim 4, wherein the hot pressing treatment is a treatment of hot pressing the filtrate at a temperature of 105 to 136 ℃ for 10 to 45 minutes.

6. An extract for preventing or treating thrombotic diseases, which is prepared by extracting leech and earthworm as raw materials, and is characterized in that the molecular weight of the extract is less than 5800 daltons, and the extract is prepared by a method comprising the following steps:

(1) cleaning raw materials with water for injection or normal saline, crushing to obtain homogenate, repeatedly freezing and thawing, centrifuging, and collecting supernatant;

(2) carrying out ultrafiltration on the supernatant obtained in the step (1) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of 10000-100000 daltons to obtain a permeate or a filtrate;

(3) treating the permeate or filtrate obtained in the step (2) by ion exchange column chromatography;

(4) desalting the eluent obtained in the step (3), and filtering by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of less than or equal to 6000 daltons; or filtering the eluent obtained in the step (3) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of less than or equal to 6000 daltons, and then desalting.

7. The extract of claim 6, wherein in step (4), the filtration is performed with an ultrafiltration membrane or column having a molecular weight cut-off of 6000 daltons.

8. The extract as claimed in claim 6, wherein in the step (4), the filtration is repeatedly performed by the ultrafiltration membrane or the ultrafiltration column.

9. The extract according to any one of claims 1 to 8, which contains polypeptide, amino acid and hypoxanthine components.

10. The extract according to any one of claims 1 to 8, wherein the raw materials are dried or fresh products of leeches and earthworms.

11. The extract according to any one of claims 1 to 8, wherein the leech and the earthworm are extracted separately or mixed together.

12. The extract according to any one of claims 1 to 8, wherein the extract is an extract or a dehydrated and dried solid.

13. A process for preparing the extract of claim 1, comprising:

(1) cleaning the raw materials with water for injection or normal saline, soaking at low temperature, and filtering to obtain filtrate and residue;

(2) crushing the dregs of a decoction, mixing the crushed dregs of a decoction with the filtrate obtained in the step (1) to form homogenate, repeatedly freezing and thawing the homogenate, and performing centrifugal separation to obtain supernatant;

(3) carrying out ultrafiltration on the supernatant obtained in the step (2) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of 10000-100000 daltons to obtain a permeate or a filtrate;

(4) and (4) filtering the permeate or the filtrate obtained in the step (3) by using an ultrafiltration membrane or an ultrafiltration column with the molecular weight cutoff of less than or equal to 6000 daltons.

14. The method of claim 13, wherein in step (4), the filtration is performed with an ultrafiltration membrane or column having a molecular weight cut-off of 6000 daltons.

15. The method according to claim 13 or 14, wherein in the step (4), the filtration is repeatedly performed with the ultrafiltration membrane or the ultrafiltration column.

16. The method according to claim 13 or 14, wherein in step (4), the autoclaving is carried out before the filtration.

17. The method according to claim 16, wherein the hot pressing treatment is a treatment of hot pressing the filtrate at a temperature of 105-136 ℃ for 10-45 minutes.

18. A pharmaceutical composition comprising the extract of any one of claims 1 to 12 as an active ingredient.

19. The pharmaceutical composition according to claim 18, which is a tablet, a capsule, a granule, a film, a patch, a suppository, a pill, a powder, an ointment, a mixture, a syrup, a tincture, an ophthalmic preparation, a nasal preparation, an injection, a sterile powder for injection or a concentrated solution for injection.

20. The pharmaceutical composition of claim 18, which is an oral liquid.

21. The pharmaceutical composition of claim 18, which is a sustained release formulation or a controlled release formulation.

22. Use of the extract of any one of claims 1-12 or the pharmaceutical composition of any one of claims 18-21 for the manufacture of a medicament for the prevention or treatment of a thrombotic disorder.

23. The use according to claim 22, wherein the thrombotic disorder is selected from the group consisting of hyperlipidemia caused by or resulting in thrombosis, atherosclerosis, myocardial infarction, angina pectoris, arteriosclerotic cerebral infarction, cerebral embolism, venous thrombosis, pulmonary embolism, pulmonary infarction, thromboangiitis obliterans, arteriosclerotic occlusion, disseminated intravascular coagulation, surgical complications of thrombosis and thromboembolism, hepatic infarction, renal infarction, gall bladder infarction, mesenteric infarction, gangrene of the limbs, diabetic peripheral neuropathy, retinal vessel occlusion, sudden deafness.