A leech-characteristic pteridine compound and its preparation method and application

Abstract

The present invention discloses a pteridine compound or a salt thereof, wherein the pteridine compound is a compound of formula I. The present invention enriches and prepares a reference substance for a characteristic pteridine compound from leeches by column chromatography, performs purity testing by HPLC, and identifies the structure using mass spectrometry and nuclear magnetic resonance data, resulting in SZ-1. This compound is a characteristic chemical component of leeches and a hallmark component for distinguishing leeches from leeches and their closely related counterfeits. Platelet aggregation assay results show that SZ-1 promotes platelet aggregation, providing a candidate standard substance for identifying different leech origin varieties and authenticity. Currently, there are no reports on the isolation and identification of SZ-1 from leeches as described in the present invention. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a leech-characteristic pteridine compound and a preparation method and application thereof. Background Art

[0002] Leeches, a traditional Chinese medicine, are derived from the dried body of Hirudinidae, a species of animal called the Hirudinidae. They are known for their ability to dissipate blood circulation, promote menstruation, and dissipate blood stasis. Modern pharmacological research has shown that leeches possess a variety of effects, including anticoagulant, antithrombotic, antiatherosclerotic, antiplatelet aggregation, antitumor, anti-inflammatory, improved hemorheology, and protective effects against cerebral ischemia-reperfusion injury.

[0003] Currently, most commercially available leeches in traditional Chinese medicine are leeches, which are relatively rare, and willow leeches are almost unseen. While all three are legally recognized leech origins, leeches have an anticoagulant activity 100 times higher than leeches. Therefore, effectively distinguishing leeches from leeches is crucial for ensuring medication safety. Currently, there is limited research on the differential components between different leech origin varieties and their adulterated counterparts, and there is no report on the characteristic components of leeches.

[0004] The leech-characteristic pteridine compound of the present invention, its preparation method and application have not been reported in the prior art. Summary of the Invention

[0005] Based on this, the present invention provides a pteridine compound or a salt thereof, wherein the pteridine compound is a compound of the following formula I:

[0006]

[0007] Furthermore, the pteridine compound is derived from the dried body of leech.

[0008] Furthermore, the leech is Hirudo nipponica Whitman of the Hirudinidae family.

[0009] Furthermore, the detection conditions of the pteridine compounds are as follows: using an LC-20A high performance liquid chromatograph, using a Kromasil (250×4.5 mm, 5 μm) chromatographic column; 10% acetonitrile isocratic elution for 40 minutes; a flow rate of 1 mL / min; and wavelengths of 244 nm and 254 nm.

[0010] According to another aspect of the present invention, a pharmaceutical composition comprising the above-mentioned pteridine compound or a salt thereof is provided.

[0011] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0012] Furthermore, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention aid, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0013] Furthermore, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

[0014] According to another aspect of the present invention, a method for preparing the above-mentioned pteridine compound is provided, which comprises: subjecting a leech extract to MCI column chromatography, gel column chromatography, silica gel column chromatography, and high performance liquid chromatography in sequence to obtain the pteridine compound.

[0015] Furthermore, the preparation method of the leech extract comprises: weighing an appropriate amount of leech powder, adding a first solvent for ultrasonic extraction, filtering, centrifuging the filtrate, taking the supernatant, spin-drying and concentrating to obtain a leech extract, and adding a second solvent to dissolve the leech extract to obtain the leech extract.

[0016] Furthermore, the first solvent is alcohol, such as ethanol.

[0017] Furthermore, the volume percentage concentration of the ethanol is 90% to 100%, for example, about 95%.

[0018] Furthermore, the mass / volume (g / mL) ratio of the leech powder to the first solvent is 0.01-1, such as 0.06-0.2, such as about 0.1.

[0019] Furthermore, the power of the ultrasonic extraction is 200-300W, for example, about 250W.

[0020] Furthermore, the frequency of the ultrasonic extraction is 30 to 60 kHz, such as 30 to 50 kHz, such as about 40 kHz.

[0021] Furthermore, the ultrasonic extraction time is 15 to 45 minutes, such as 20 to 40 minutes, such as about 30 minutes.

[0022] Furthermore, the ultrasonic extraction is performed 1 to 5 times, for example, 3 times.

[0023] Furthermore, the centrifugal rotation speed is 10000-15000 r / min, for example, about 12000 r / min.

[0024] Furthermore, the centrifugation time is 5 to 20 minutes, such as 5 to 15 minutes, such as about 10 minutes.

[0025] Furthermore, the end point of the spin-drying concentration is concentration to dryness.

[0026] Furthermore, the second solvent is an alcohol, such as ethanol.

[0027] Furthermore, the volume percentage concentration of the ethanol is 60% to 80%, for example, about 70%.

[0028] Furthermore, the volume of the second solvent is 5-20 mL, for example, about 10 mL.

[0029] Furthermore, the mass / volume (g / mL) ratio of the leech extract to the second solvent is 0.1-1, such as about 0.5.

[0030] Furthermore, the MCI column chromatography method comprises: adding the leech extract to a pretreated MCI chromatography column, eluting with a third solvent, collecting the eluate, concentrating, and drying to obtain a first dried product.

[0031] Furthermore, the MCI chromatography column is an MCI CHP20 chromatography column.

[0032] Furthermore, the ratio of the height (H) to the diameter (d) of the MCI CHP20 chromatography column is 10:1.

[0033] Furthermore, the volume ratio of the leech extract to the column filler is about 1:about 15.

[0034] Furthermore, the elution flow rate is 1 to 5 mL / min, for example, about 3 mL / min.

[0035] Furthermore, the third solvent is an alcohol, such as methanol.

[0036] Furthermore, the volume percentage concentration of the methanol is 10% to 30%, for example, about 20%.

[0037] Furthermore, the ratio of the volume of the third solvent to the column volume of the MCI chromatography column is 2-4, for example, about 3.

[0038] Furthermore, the gel column chromatography method includes: adding a fourth solvent to the first dried product to dissolve it to obtain a first sample solution, adding the first sample solution to the gel column, eluting it with the fourth solvent, collecting the eluate, concentrating it, and drying it to obtain a second dried product.

[0039] Furthermore, the fourth solvent is an alcohol, such as methanol.

[0040] Furthermore, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%.

[0041] Furthermore, the concentration of the first dried product in the first sample solution is 50 to 400 mg / mL, for example, 100 to 300 mg / mL.

[0042] Furthermore, the gel column is a Sephadex LH-20 gel column.

[0043] Furthermore, the ratio of the height (H) to the diameter (d) of the gel column is about 15:about 1.

[0044] Furthermore, the elution flow rate is 10-15 s / drop, for example, about 12 s / drop.

[0045] Furthermore, the silica gel column chromatography method includes: dissolving the second dried object in the fourth solvent to obtain a second sample solution, adding blank silica gel to the second sample solution, stirring evenly, evaporating to dryness, and grinding to obtain a second sample; adding the second sample to a silica gel column for gradient elution, collecting the eluate, concentrating, and drying to obtain a third dried object.

[0046] Furthermore, the concentration of the second dried product in the second sample solution is 50 to 200 mg / mL, for example, 80 to 120 mg / mL.

[0047] Furthermore, the mass / mass (g / g) ratio of the blank silica gel to the second dried product is 1-5, for example, about 4.

[0048] Furthermore, the evaporation to dryness is carried out under water bath conditions.

[0049] Furthermore, the temperature of the water bath is 50-70°C, for example, about 60°C.

[0050] Furthermore, the particle size of the silica gel is 200-300 meshes.

[0051] Furthermore, the ratio of the height (H) to the diameter (d) of the silica gel column is about 8:about 1.

[0052] Furthermore, the gradient elution was performed according to the following procedure: (a) dichloromethane:methanol (100:1), approximately 2 column volumes; (b) dichloromethane:methanol (10:1), approximately 2 column volumes; (c) dichloromethane:methanol (1:1), approximately 2 column volumes; and (d) methanol, approximately 2 column volumes.

[0053] Furthermore, the flow rate of the gradient elution is 10-20 mL / min, for example, about 15 mL / min.

[0054] Furthermore, the third dried product is dissolved in the fifth solvent to obtain a third sample solution, and the third sample solution is separated and purified by high performance liquid chromatography, and the corresponding fractions are collected and dried to obtain the pteridine compound.

[0055] Furthermore, the fifth solvent is an alcohol, such as methanol.

[0056] Furthermore, the concentration of the third dried product in the third sample solution is 1-20 mg / mL, such as 5-15 mg / mL, such as about 10 mg / mL.

[0057] Furthermore, the high performance liquid chromatograph is an Alltech 426 semi-preparative liquid chromatograph.

[0058] Furthermore, the chromatographic conditions of the high performance liquid chromatograph are as follows: using a YMC-Pack ODS-A chromatographic column, isocratic elution with acetonitrile:water (23:77) as the mobile phase, a flow rate of 1-5 mL / min, a detection wavelength of 200-300 nm, and an injection volume of 40-60 μL.

[0059] Furthermore, the specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, and particle size 10 μm.

[0060] Furthermore, the flow rate is about 3 mL / min.

[0061] Furthermore, the detection wavelength is 244 nm.

[0062] Furthermore, the injection volume is about 50 μL.

[0063] Furthermore, the drying is freeze-drying.

[0064] According to another aspect of the present invention, there is provided a use of the above-mentioned pteridine compound or its salt, or the above-mentioned pharmaceutical composition in the preparation of a medicament for promoting hemostasis.

[0065] According to another aspect of the present invention, there is provided a use of the above-mentioned pteridine compound or its salt as a characteristic chemical component of leeches for distinguishing leeches from leeches and their closely related hybrids.

[0066] According to another aspect of the present invention, there is provided a use of the above preparation method for preparing a standard substance.

[0067] Beneficial effects of the present invention:

[0068] The present invention enriched and prepared a reference substance for a characteristic pteridine compound from leeches by column chromatography. The purity was tested by HPLC, and the structure was identified using mass spectrometry and nuclear magnetic resonance data. This compound is SZ-1, a characteristic chemical component of leeches and a hallmark component for distinguishing leeches from leeches and their closely related counterfeits. Platelet aggregation experiments showed that SZ-1 promotes platelet aggregation, providing a candidate standard substance for distinguishing different leech origin varieties and their authenticity. Currently, there are no reports on the isolation and identification of SZ-1 from leeches, as described in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0070] Figure 1 This is a thin-layer diagram of separation and purification by MCI column chromatography. The numbers 1 to 10 represent fractions 1 to 10 during the MCI column chromatography process. Fraction 1 represents the fraction where SZ-1 is located, and the blue-green fluorescent spot in fraction 1 is SZ-1.

[0071] Figure 2 This is a thin layer chromatography image of the separation and purification process using Sephadex LH-20 column chromatography. Numbers 1 to 5 represent fractions 1 to 5 during the gel column chromatography process, while fraction 2 represents the fraction where SZ-1 is located. The blue-green fluorescent spot in fraction 2 is SZ-1.

[0072] Figure 3 This is a thin layer chromatography image of silica gel column chromatography separation and purification. The numbers 1 to 7 represent fractions 1 to 7 during silica gel column chromatography, fraction 4 represents the fraction where SZ-1 is located, and the blue-green fluorescent spot in fraction 4 is SZ-1.

[0073] Figure 4 This is a HPLC preparative chromatogram, in which the peak with a retention time of 15.8 min represents SZ-1.

[0074] Figure 5 The HPLC spectrum of SZ-1 is shown in FIG. The chromatographic peak with a retention time of 6.9 min represents SZ-1.

[0075] Figure 6 This is a schematic diagram of the structure of SZ-1.

[0076] Figure 7 HR-ESI-MS spectrum of SZ-1.

[0077] Figure 8 This is the IR graph of SZ-1.

[0078] Figure 9 This is the UV map of SZ-1.

[0079] Figure 10 For SZ-1 1 H NMR spectrum.

[0080] Figure 11 For SZ-1 13 C NMR spectrum.

[0081] Figure 12 For SZ-1 1 H- 1 H COSY plot.

[0082] Figure 13 This is the HSQC diagram of SZ-1.

[0083] Figure 14 This is the HMBC diagram of SZ-1.

[0084] Figure 15 This is the thin layer chromatogram of SZ-1 used to identify different batches of leeches and leeches. DETAILED DESCRIPTION

[0085] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0086] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those skilled in the art in the field of the present invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0087] The present invention is intended to encompass all alternatives, modifications, and equivalents that may come within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0088] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0089] In the present invention, the term "comprising" is synonymous with "including." As used herein, the terms "comprises," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0090] As described in the background technology section, the prior art does not report the pteridine compound characteristic of leeches, its preparation method, and its application. To address the above problems, the present invention provides a pteridine compound or a salt thereof, wherein the pteridine compound is a compound of the following formula I:

[0091]

[0092] In a preferred embodiment, the pteridine compound is derived from the dried body of leech.

[0093] In a preferred embodiment, the leech is Hirudo nipponica Whitman.

[0094] In a preferred embodiment, the detection conditions of the pteridine compounds are: using an LC-20A high performance liquid chromatograph, using a Kromasil (250×4.5 mm, 5 μm) chromatographic column; 10% acetonitrile isocratic elution for 40 min; a flow rate of 1 mL / min; and wavelengths of 244 nm and 254 nm.

[0095] According to another aspect of the present invention, a pharmaceutical composition comprising the above-mentioned pteridine compound or a salt thereof is provided.

[0096] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0097] In a preferred embodiment, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention aid, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0098] In a preferred embodiment, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

[0099] According to another aspect of the present invention, a method for preparing the above-mentioned pteridine compound is provided, which comprises: subjecting a leech extract to MCI column chromatography, gel column chromatography, silica gel column chromatography, and high performance liquid chromatography in sequence to obtain the pteridine compound.

[0100] The method of the present invention is a compound preparation and separation method with simple process, good reproducibility, high purity of the separated compound, and good industrial application prospects, so as to solve the problem of lack of relevant preparation and separation methods in the prior art.

[0101] In a preferred embodiment, the preparation method of the leech extract comprises: weighing an appropriate amount of leech powder, adding a first solvent for ultrasonic extraction, filtering, centrifuging the filtrate, taking the supernatant, spin-drying and concentrating to obtain a leech extract, and adding a second solvent to the leech extract for dissolution to obtain the leech extract.

[0102] In a preferred embodiment, the first solvent is an alcohol, such as ethanol.

[0103] In the present invention, when volume percentage concentration, ratio, mass, power, frequency, time, number of times, rotation speed, volume, flow rate, concentration, temperature, particle size, wavelength, or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "90% to 100%" is disclosed, the described range should be interpreted to include the range of "90% to 98%", "90% to 96%", "90% to 94%", "90% to 92%", "92% to 100%", "92% to 98%", "92% to 96%", "92% to 94%", "94% to 100%", "94% to 98%", "94% to 96%", "96% to 100%", "96% to 98%", "98% to 100%", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range.

[0104] In a preferred embodiment, the volume percentage concentration of the ethanol is 90% to 100%, such as about 95%.

[0105] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 95%" includes ±5% of 95%, or from 90.25% to 99.75%.

[0106] In a preferred embodiment, the mass / volume (g / mL) ratio of the leech powder to the first solvent is 0.01-1, such as 0.06-0.2, such as about 0.1.

[0107] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.

[0108] In a preferred embodiment, the power of the ultrasonic extraction is 200-300W, for example, about 250W.

[0109] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5.

[0110] In a preferred embodiment, the frequency of the ultrasonic extraction is 30 to 60 kHz, such as 30 to 50 kHz, such as about 40 kHz.

[0111] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0112] In a preferred embodiment, the ultrasonic extraction time is 15 to 45 minutes, such as 20 to 40 minutes, such as about 30 minutes.

[0113] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes ±5% of 30, or from 28.5 to 31.5.

[0114] In a preferred embodiment, the ultrasonic extraction is performed 1 to 5 times, for example 3 times.

[0115] In a preferred embodiment, the centrifugal rotation speed is 10000-15000 r / min, for example, about 12000 r / min.

[0116] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12,000" includes ±5% of 12,000, or from 11,400 to 12,600.

[0117] In a preferred embodiment, the centrifugation time is 5 to 20 min, such as 5 to 15 min, such as about 10 min.

[0118] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0119] In a preferred embodiment, the endpoint of the spin-drying concentration is concentration to dryness.

[0120] In a preferred embodiment, the second solvent is an alcohol, such as ethanol.

[0121] In a preferred embodiment, the volume percentage concentration of the ethanol is 60% to 80%, such as about 70%.

[0122] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 70%" includes ±5% of 70%, or from 66.5% to 73.5%.

[0123] In a preferred embodiment, the volume of the second solvent is 5-20 mL, for example, about 10 mL.

[0124] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0125] In a preferred embodiment, the mass / volume (g / mL) ratio of the leech extract to the second solvent is 0.1-1, such as about 0.5.

[0126] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0127] In a preferred embodiment, the MCI column chromatography method comprises: adding the leech extract to a pretreated MCI chromatography column, eluting with a third solvent, collecting the eluate, concentrating, and drying to obtain a first dried product.

[0128] In a preferred embodiment, the MCI chromatography column is an MCI CHP20 chromatography column.

[0129] In a preferred embodiment, the ratio of the height (H) to the diameter (d) of the MCI CHP20 chromatography column is about 10:about 1.

[0130] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0131] In a preferred embodiment, the ratio of the volume of the leech extract to the volume of the column packing is about 1:about 15.

[0132] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0133] In a preferred embodiment, the elution flow rate is 1 to 5 mL / min, such as about 3 mL / min.

[0134] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0135] In a preferred embodiment, the third solvent is an alcohol, such as methanol.

[0136] In a preferred embodiment, the volume percentage concentration of the methanol is 10% to 30%, for example, about 20%.

[0137] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 20%" includes ±5% of 20%, or from 19% to 21%.

[0138] In a preferred embodiment, the ratio of the volume of the third solvent to the column volume of the MCI chromatography column is 2-4, for example, about 3.

[0139] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0140] In a preferred embodiment, the gel column chromatography method comprises: adding a fourth solvent to the first dried product to dissolve it to obtain a first sample solution, adding the first sample solution to a gel column, eluting with the fourth solvent, collecting the eluate, concentrating it, and drying it to obtain a second dried product.

[0141] In a preferred embodiment, the fourth solvent is an alcohol, such as methanol.

[0142] In a preferred embodiment, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%.

[0143] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, "about 50%" includes ±5% of 50%, or from 47.5% to 52.5%.

[0144] In a preferred embodiment, the concentration of the first dried product in the first sample solution is 50-400 mg / mL, for example, 100-300 mg / mL.

[0145] In a preferred embodiment, the gel column is a Sephadex LH-20 gel column.

[0146] In a preferred embodiment, the ratio of the height (H) to the diameter (d) of the gel column is about 15:about 1.

[0147] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0148] In a preferred embodiment, the elution flow rate is 10-15 s / drop, for example, about 12 s / drop.

[0149] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12" includes ±5% of 12, or from 11.4 to 12.6.

[0150] In a preferred embodiment, the silica gel column chromatography method comprises: dissolving the second dried product in the fourth solvent to obtain a second sample solution, adding blank silica gel to the second sample solution, stirring evenly, evaporating to dryness, and grinding to obtain a second sample; adding the second sample to a silica gel column for gradient elution, collecting the eluate, concentrating, and drying to obtain a third dried product.

[0151] In a preferred embodiment, the concentration of the second dried product in the second sample solution is 50 to 200 mg / mL, such as 80 to 120 mg / mL.

[0152] In a preferred embodiment, the mass / mass (g / g) ratio of the blank silica gel to the second dried product is 1-5, for example, about 4.

[0153] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0154] In a preferred embodiment, the evaporation to dryness is carried out in a water bath.

[0155] In a preferred embodiment, the temperature of the water bath is 50-70°C, such as about 60°C.

[0156] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0157] In a preferred embodiment, the particle size of the silica gel is 200-300 mesh.

[0158] In a preferred embodiment, the ratio of the silica gel column height (H) to the diameter (d) is about 8: about 1.

[0159] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 8" includes ±5% of 8, or from 7.6 to 8.4; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0160] In a preferred embodiment, the gradient elution is performed according to the following program: (a) dichloromethane:methanol (100:1), about 2 column volumes; (b) dichloromethane:methanol (10:1), about 2 column volumes; (c) dichloromethane:methanol (1:1), about 2 column volumes; and (d) methanol, about 2 column volumes.

[0161] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.

[0162] In a preferred embodiment, the flow rate of the gradient elution is 10-20 mL / min, such as about 15 mL / min.

[0163] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0164] In a preferred embodiment, the third dried product is dissolved in the fifth solvent to obtain a third sample solution, and the third sample solution is separated and purified by high performance liquid chromatography, and the corresponding fractions are collected and dried to obtain the pteridine compound.

[0165] In a preferred embodiment, the fifth solvent is an alcohol, such as methanol.

[0166] In a preferred embodiment, the concentration of the third dried product in the third sample solution is 1-20 mg / mL, such as 5-15 mg / mL, such as about 10 mg / mL.

[0167] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0168] In a preferred embodiment, the high performance liquid chromatograph is an Alltech 426 semi-preparative liquid chromatograph.

[0169] In a preferred embodiment, the chromatographic conditions of the high performance liquid chromatograph are: using a YMC-Pack ODS-A chromatographic column, isocratic elution with acetonitrile: water (23:77) as the mobile phase, a flow rate of 1 to 5 mL / min, a detection wavelength of 200 to 300 nm, and an injection volume of 40 to 60 μL.

[0170] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, and particle size 10 μm.

[0171] In a preferred embodiment, the flow rate is about 3 mL / min.

[0172] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes ±5% of 3, or from 2.85 to 3.15.

[0173] In a preferred embodiment, the detection wavelength is 244 nm.

[0174] In a preferred embodiment, the injection volume is about 50 μL.

[0175] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5.

[0176] In a preferred embodiment, the drying is freeze-drying.

[0177] According to another aspect of the present invention, there is provided a use of the above-mentioned pteridine compound or its salt, or the above-mentioned pharmaceutical composition in the preparation of a medicament for promoting hemostasis.

[0178] According to another aspect of the present invention, there is provided a use of the above-mentioned pteridine compound or its salt as a characteristic chemical component of leeches for distinguishing leeches from leeches and their closely related hybrids.

[0179] According to another aspect of the present invention, there is provided a use of the above preparation method for preparing a standard substance.

[0180] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or conditions recommended by the manufacturer.

[0181] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0182] The above-mentioned features of the present invention or the features described in the embodiments may be combined in any combination. All features disclosed in this patent specification may be used in any combination, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal, or similar purpose. Therefore, unless otherwise specified, the features disclosed are only general examples of equal or similar features.

[0183] Example

[0184] 1. Experimental Materials

[0185] LC-20A high-performance liquid chromatograph (Shimadzu Corporation, Japan); N-1100EYELA rotary evaporator (Shanghai Airo Instrument Co., Ltd.); Sartorius BSA-124S-CW 1 / 10,000 electronic balance (Sartorius); XSE 105DU 1 / 100,000 balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ-250DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Nicolet iS10 FTIR spectrometer (Thermo Fisher Scientific); Evolution 300 UV-visible spectrophotometer (Thermo Fisher Scientific); Alltech 426 semi-preparative chromatograph equipped with UV2000 detector (Alltech); N1248 intelligent sample grinder (Beijing Hede Technology Co., Ltd.); Xevo G2-S QTOF mass spectrometer (Waters Corporation); CHP20 MCI GEL (Japanese trigone); Sephadex LH-20 type gel (Japanese trigone); (200-300 mesh) silica gel Qingdao Ocean Chemical Plant; silica gel G plate (Qingdao Ocean Chemical Plant); LBY-NJ4 platelet aggregation instrument (Beijing Prism Instrument Co., Ltd.); PocH100 fully automatic blood cell counter (Japan Sysmex Co., Ltd.); adenosine diphosphate (ADP) (Beijing Tailikangxin Technology Co., Ltd.); Ticagrelor was purchased from MedChemexpress Biotechnology Co., Ltd. in the United States; healthy SPF-grade SD rats (male, weighing 240±10g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0006).

[0186] 2. Methods and Results

[0187] 2.1 Extraction and separation

[0188] 2.1.1 Leech Extraction

[0189] Method: Weigh 150 g of leech powder, add 10 times the amount of 95% ethanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 min, filter, repeat 3 times, combine the filtrate, centrifuge at 12000 r / min for 10 min to obtain the supernatant, and then use a rotary evaporator to remove the solvent to constant weight to obtain 5.7385 g of leech extract.

[0190] 2.1.2 MCI column separation and enrichment

[0191] Weigh the above leech extract, add 70% ethanol to dissolve and obtain leech extract (the mass / volume (g / mL) ratio of the leech extract to the 70% ethanol is about 0.5, and the volume ratio of the leech extract to the column filler is about 1: about 15), slowly add the treated MCI CHP20 (the ratio of height (H) to diameter (d) is about 10: about 1), the flow rate is about 3 mL / min, and 3BV (3 times the column volume) of 20% methanol is used for elution. The 20% methanol elution fractions are collected and each fraction is subjected to thin layer chromatography. The thin layer development conditions are chloroform: methanol = 8:1. After development under this developing agent ratio, irradiation with a 365nm ultraviolet lamp can reveal obvious blue-green fluorescent spots with an Rf value of about 0.3, as shown in the following example. Figure 1 As shown, among the 1 to 10 fractions, the enriched fraction 1 (containing SZ-1) was collected and concentrated to dryness to obtain 1.3566 g of the first dried product.

[0192] 2.1.3 Gel column separation and enrichment

[0193] Weigh all the first dried material, dissolve it in 50% methanol, and prepare a first sample solution with a concentration of 100 mg / mL to 300 mg / mL. Add the first sample solution to a gel column (Sephadex LH-20) (the ratio of height (H) to diameter (d) is about 15: about 1); add 50% methanol solvent for isocratic elution with a flow rate of about 12s / drop to obtain eluted fractions, and perform thin layer examination on each fraction. The thin layer development conditions are chloroform: methanol = 8:1. After development under this developing agent ratio, irradiation with a 365nm ultraviolet lamp can reveal obvious blue-green fluorescent spots with an Rf value of about 0.3, as shown in the following example. Figure 2 As shown, among fractions 1 to 5, fraction 2 (containing SZ-1) was enriched and concentrated to dryness to obtain 462.3 mg of the second dried product.

[0194] 2.1.3 Silica gel column separation and enrichment

[0195] Weigh all the second dried matter, dissolve it in 50% methanol, and prepare a second sample solution with a concentration of 80 mg / mL to 120 mg / mL. Stir the mass of the second dried matter contained in the second sample solution and the mass of the blank silica gel in a ratio of 1:4, evaporate and grind into fine powder in a 60°C water bath to obtain the second sample. Add the second sample to a silica gel column (200-300) (the ratio of height (H) to diameter (d) is about 8: about 1); add dichloromethane-methanol solvent for gradient elution, the volume of the organic solvent ratio is 2 times the volume of the silica gel column, the flow rate is about 15 mL / min, and elution fractions are obtained, 1 to 7 fractions, and each fraction is subjected to thin layer inspection. The thin layer development conditions are dichloromethane: methanol = 5:1. After development under this developing agent ratio, irradiation with a 365nm ultraviolet lamp can reveal obvious blue-green fluorescent spots with an Rf value of about 0.6. Specifically, Figure 3 As shown, the enriched fraction 4 (containing SZ-1) was collected and concentrated to dryness to obtain 76.4 mg of the third dried product.

[0196] 2.1.4 Preparative liquid chromatography separation, purification and enrichment

[0197] The sample solution was separated, purified and enriched using an Alltech 426 semi-preparative liquid chromatograph under chromatographic conditions (YMC-Pack ODS-A (250×10 mm, 10 μm); 23% isocratic acetonitrile; flow rate 3 mL / min; wavelength 244 nm). The HPLC preparative chromatogram of the sample is shown in FIG. Figure 4 .like Figure 4 As shown, the enriched peak 1 was freeze-dried to obtain a total of 3.7 mg of SZ-1.

[0198] 2.2 Purity Testing

[0199] The HPLC chromatogram of the sample was obtained by using an LC-20A high performance liquid chromatograph under the following chromatographic conditions (Kromasil (250 × 4.5 mm, 5 μm); 10% isocratic acetonitrile isocratic elution for 40 min; flow rate 1 mL / min; wavelengths 244 nm and 254 nm). Figure 5 .like Figure 5 As shown, peak 1 is SZ-1.

[0200] 2.3 Structure identification

[0201] SZ-1 is a green solid. HR-ESI-MS gives a quasi-molecular ion peak of m / z 357.0328 [M+H] + The calculated value is 357.0327, and the inferred molecular formula is C 12 H 12 N4O5S2, degree of unsaturation is 9.

[0202] exist1 In the H NMR (DMSO-d6, 600 MHz) spectrum, two active hydrogen signal peaks were observed [δ H 6.27 (1H, brs), δ H 5.11 (1H, brs)], a hydrogen signal of an oxygen-containing methine [δ H 5.68 (1H, t, J = 5.8 Hz, 13-OH], a hydrogen signal containing an oxymethylene [δ H 3.65 (1H, d, J = 5.7 Hz, 14-OH)], and two methyl characteristic signal peaks [δ H 3.26(3H,s,H-10), 3.15(3H,s,H-12)].

[0203] exist 13 There are 12 carbon signals in the C NMR (DMSO-d6, 150 MHz) spectrum. Combined with HSQC spectrum analysis, [δ C 161.9(C-2), δ C 153.7 (C-4)] are two ketone carbonyl signal peaks, [125.3 (C-4a), 137.5 (C-5a), 128.8 (C-6), 153.2 (C-7), 141.6 (C-8a), δ C 157.7 (C-9a)] are 6 unsaturated carbon signals. The two hydroxyl-substituted methine carbon and methylene carbon signals are [δ C 67.1(C-13), δ C 66.9 (C-14)]. The remaining two carbon signals are attributed to two methyl groups [δ C 27.8 (C-10), 40.2 (C-12)], and these hydrogen and carbon signals are assigned in Table 1. The above information is similar to the compound (R)-hirudonucleodisulfide B in the literature, and it is speculated to be a pteridine derivative. The main difference between them is that the nitrogen atom at position 3 of this compound is substituted by a methyl group, and the sulfur atom at position 11 is a sulfinyl group. The chemical shifts of the two methyl groups substituted at positions 3 and 11 [δ H 3.26 (3H, s, H-10), 3.15 (3H, s, H-12)] also indirectly confirmed this.

[0204] 2D NMR spectroscopy also provided some evidence for the structure of the compound. 1 H- 1The H COSY spectrum's crossover signals H-13 / H2-14 indicate the presence of a C13-C14 (vicinal diol) structure. The HMBC spectrum's signals H-10 / C-2 / C-4, H-12 / C-6, H-13 / C-6, and H-14 / C-7 confirm methyl substitution at N-3, sulfinyl substitution at C-6, and vicinal diol substitution at C-7, respectively.

[0205] In addition, the characteristic signal peaks in the IR spectrum such as amino (3239 cm -1 ), methyl (2921cm - 1 ), carbonyl (1728cm -1 ), carbon-nitrogen double bond (1668cm -1 ), sulfinyl (1594cm -1 ), double bond (1545cm -1 ) is similar to Whitmanine B and (R)-hirudonucleodisulfide B. Therefore, the structure of the compound was determined and named SZ-1. Its structural formula is as follows Figure 6 The structure of SZ-1 is shown in Figures 7 to 14 Sure.

[0206] Table 1 SZ-1 1 H NMR and 13 C NMR data

[0207] Location <![CDATA[δ H ]]> <![CDATA[δ C ]]> Location <![CDATA[δ H ]]> <![CDATA[δ C ]]> 2 - 161.9 9a - 157.7 4 - 153.7 13 5.68,t(5.8) 67.1 4a - 125.3 14 3.65,d(5.7) 66.9 5a - 137.5 <![CDATA[N3-CH3]]> 3.26 27.8 6 - 128.8 <![CDATA[-CH3]]> 3.15 40.2 7 - 153.2 13-OH 6.27,brs - 8a - 141.6 14-OH 5.11,brs -

[0208] These results indicate that leeches contain a unique component, SZ-1, that is distinct from other leech species, demonstrating the unique nature of their composition. The entire preparation and separation process is environmentally friendly, simple, and easy to operate, making leeches suitable as a raw material for the isolation of SZ-1.

[0209] 2.4 Effect of SZ-1 on in vitro induced platelet aggregation

[0210] 2.4.1 Preparation of platelet-rich plasma

[0211] After the rats were anesthetized, blood was collected from the abdominal aorta (the rats were fasting for 12 hours before blood collection) and placed in a 3.8% sodium citrate anticoagulant blood collection tube at 1200 r·min. -1 Centrifuge for 10 min, separate the supernatant to obtain platelet-rich plasma (PRP); the remaining blood is centrifuged at a speed of 4000 r·min -1 Centrifuge for 10 minutes and aspirate the supernatant to obtain platelet-poor plasma (PPP) for later use. After platelet count using PRP, adjust the platelet count to 400,000 / μL using PPP.

[0212] 2.4.2 Solution preparation

[0213] Preparation of SZ-1: Weigh 1.2 mg of sample and dissolve in 134 μL of ultrapure water to a concentration of 25 mM to prepare a stock solution. Pipette 2 μL of the stock solution and add 998 μL of PRP to a final concentration of 50 μM SZ-1.

[0214] Preparation of Ticagrelor (positive drug): Weigh 4.5 mg of ticagrelor powder and add 172.2 μl of DMSO to a 50 mM concentration to prepare a stock solution. Pipette 4 μl of the stock solution and dilute it with 28 μl of DMSO to obtain a 6.25 mM stock solution. Pipette 4 μl of the stock solution and add 996 μl of PRP to obtain a final ticagrelor concentration of 25 μM.

[0215] 2.4.3 Preparation of platelet-rich plasma

[0216] After the rats were anesthetized, blood was collected from the abdominal aorta (the rats were fasting for 12 hours before blood collection) and placed in a 3.8% sodium citrate anticoagulant blood collection tube at 1200 r·min. -1 Centrifuge for 10 min, separate the supernatant to obtain platelet-rich plasma (PRP); the remaining blood is centrifuged at a speed of 4000 r·min -1 Centrifuge for 10 minutes and aspirate the supernatant to obtain platelet-poor plasma (PPP) for later use. After platelet count using PRP, adjust the platelet count to 400,000 / μL using PPP.

[0217] 2.4.4 Determination of in vitro platelet aggregation

[0218] (1) Preheat the instrument for 30 minutes.

[0219] (2) Set the instrument to TEST mode and press ENT to enter the test interface.

[0220] (3) Add clean test cups to the incubation chamber, adjust the test cup to zero, add 270 μL of PPP, and then add 30 μL of normal saline (without adding a stirrer). For the blank group, add 270 μL of PRP to each test cup, and then add 30 μL of normal saline (with adding a stirrer). For the sample group, add 300 μL of SZ-1 containing a final concentration of 50 μM PRP to each test cup (with adding a stirrer). Incubate in the incubation chamber for 4 minutes.

[0221] (4) Place the zero test cup into the detection channel and adjust to zero.

[0222] (5) Remove the zero-adjusted test cup and place it in the PRP sample test cup. After the light flux value stabilizes, press the corresponding channel number key. Pipette 6 μl of ADP (adenosine diphosphate) inducer into the bottom of the cup and immediately press the corresponding channel key to record the maximum platelet aggregation rate within 5 minutes.

[0223] 2.4.4 Results of SZ-1 on platelet aggregation in vitro

[0224] The activity of SZ-1 was evaluated using an in vitro platelet aggregation model. The results are shown in Table 2. At a dose of 50 μM, SZ-1 had a certain activity in promoting platelet aggregation.

[0225] Table 2 Effects of SZ-1 on in vitro platelet aggregation

[0226] sample Concentration (μM) Maximum aggregation rate (%) P blank - 42.83±4.94 - Ticagrelor 25 0.26±2.26 <0.01 SZ-1 50 56.57±5.50 <0.05

[0227] 3. Thin layer identification

[0228] The thin layer development conditions were chloroform:methanol = 8:1. After development under this developing solvent ratio, irradiation with a 365nm UV lamp revealed a clear blue-green fluorescent spot with an Rf value of around 0.3. Four batches of leeches had this spot, while the other four batches did not. Figure 15 shown.

[0229] The discovery of this characteristic pteridine component in leeches has certain practical significance, and its application prospects are promising. It has good social and economic benefits, will play a positive role in promoting the economic development of the leech industry, and can better serve the public.

[0230] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method of the present invention and its core concept. At the same time, changes or modifications made by those skilled in the art based on the concept of the present invention, the specific implementation methods of the present invention, and the scope of application are all within the scope of protection of the present invention. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A pteridine compound or a salt thereof, characterized in that: The pteridine compound is a compound of the following formula I:

2. The pteridine compound or salt thereof according to claim 1, characterized in that The pteridine compound is derived from the dried body of leech.

3. The pteridine compound or salt thereof according to claim 2, characterized in that The leech is Hirudo nipponica Whitman of the Hirudinidae family.

4. The pteridine compound or salt thereof according to claim 1, characterized in that The detection conditions of the pteridine compounds are: using an LC-20A high performance liquid chromatograph, using a Kromasil chromatographic column; 10% acetonitrile isocratic elution for 40 minutes; a flow rate of 1 mL / min; and wavelengths of 244 nm and 254 nm.

5. The pteridine compound or salt thereof according to claim 4, characterized in that The specifications of the chromatographic column are 250×4.5 mm, 5 μm.

6. A pharmaceutical composition comprising the pteridine compound or a salt thereof according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that The auxiliary materials are selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, retention aids, lubricants, dispersants, plasticizers, opacifiers and antioxidants.

9. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition is tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

10. A method for preparing a pteridine compound according to any one of claims 1 to 5, characterized in that: The preparation method comprises: subjecting an extract of Hirudo nipponica Whitman of the Hirudidae family to MCI column chromatography, gel column chromatography, silica gel column chromatography and high performance liquid chromatography in sequence to obtain the pteridine compound; The MCI column chromatography method comprises: adding the extract of Hirudo nipponica Whitman to a pretreated MCI CHP20 chromatography column, eluting with 10% to 30% alcohol at a flow rate of 1 to 5 mL / min, collecting the eluate, concentrating, and drying to obtain a first dried product; The gel column chromatography method comprises: adding 40% to 60% alcohol to the first dried product to dissolve it to obtain a first sample solution, adding the first sample solution to a Sephadex LH-20 gel column, eluting with the 40% to 60% alcohol at a flow rate of 10 to 15 seconds per drop, collecting the eluate, concentrating it, and drying it to obtain a second dried product; The silica gel column chromatography method includes: dissolving the second dried product in the 40% to 60% alcohol to obtain a second sample solution, adding blank silica gel to the second sample solution, stirring evenly, evaporating to dryness, and grinding to obtain a second sample; adding the second sample to a silica gel column for gradient elution, wherein the gradient elution is performed according to the following procedure: (a) eluting with a dichloromethane:methanol ratio of 100:1 for 1.9 to 2.1 times the column volume; (b) eluting with a dichloromethane:methanol ratio of 10:1 for 1.9 to 2.1 times the column volume; (c) eluting with a dichloromethane:methanol ratio of 1:1 for 1.9 to 2.1 times the column volume; and (d) eluting with methanol for 1.9 to 2.1 times the column volume, the flow rate of the gradient elution is 10 to 20 mL / min, collecting the eluate, concentrating, and drying to obtain a third dried product; The third dried product is dissolved in the alcohol to obtain a third sample solution, and the third sample solution is separated and purified by a high performance liquid chromatography. The chromatographic conditions of the high performance liquid chromatography are as follows: a YMC-Pack ODS-A column is used, and an acetonitrile: water ratio of 23:77 is used as the mobile phase for isocratic elution. The flow rate is 1 to 5 mL / min, the detection wavelength is 200 to 300 nm, the injection volume is 40 to 60 μL, and the corresponding fractions are collected and dried to obtain the pteridine compound.

11. The preparation method according to claim 10, characterized in that: The preparation method of the Hirudo nipponica Whitman extract comprises: weighing an appropriate amount of leech powder, adding a first solvent to perform ultrasonic extraction, filtering, centrifuging the filtrate, taking the supernatant, and concentrating it by spin drying to obtain a leech extract; and adding a second solvent to the leech extract for dissolution to obtain the Hirudo nipponica Whitman extract.

12. The preparation method according to claim 11, characterized in that The first solvent is alcohol.

13. The preparation method according to claim 12, characterized in that The first solvent is ethanol.

14. The preparation method according to claim 13, characterized in that The volume percentage concentration of the ethanol is 90% to 100%.

15. The preparation method according to claim 14, characterized in that The volume percentage concentration of the ethanol is 90.25% to 99.75%.

16. The preparation method according to claim 11, characterized in that The mass / volume ratio of the leech powder to the first solvent is 0.01 to 1, with the unit being g / mL.

17. The preparation method according to claim 16, characterized in that The mass / volume ratio of the leech powder to the first solvent is 0.06 to 0.2, with the unit being g / mL.

18. The preparation method according to claim 17, characterized in that: The mass / volume ratio of the leech powder to the first solvent is 0.095-0.105, with the unit being g / mL.

19. The preparation method according to claim 11, characterized in that The power of the ultrasonic extraction is 200-300W.

20. The preparation method according to claim 19, characterized in that The power of the ultrasonic extraction is 237.5-262.5W.

21. The preparation method according to claim 11, characterized in that The frequency of the ultrasonic extraction is 30 to 60 kHz.

22. The preparation method according to claim 21, characterized in that The frequency of the ultrasonic extraction is 30 to 50 kHz.

23. The preparation method according to claim 22, characterized in that The frequency of the ultrasonic extraction is 38-42 kHz.

24. The preparation method according to claim 11, characterized in that The ultrasonic extraction time is 15 to 45 minutes.

25. The preparation method according to claim 24, characterized in that The ultrasonic extraction time is 20 to 40 minutes.

26. The preparation method according to claim 25, characterized in that The ultrasonic extraction time is 28.5 to 31.5 minutes.

27. The preparation method according to claim 11, characterized in that The ultrasonic extraction is performed 1 to 5 times.

28. The preparation method according to claim 27, characterized in that The ultrasonic extraction was performed 3 times.

29. The preparation method according to claim 11, characterized in that The centrifugal speed is 10000-15000 r / min.

30. The preparation method according to claim 29, characterized in that The centrifugal speed is 11400-12600 r / min.

31. The preparation method according to claim 11, characterized in that The centrifugation time is 5 to 20 minutes.

32. The preparation method according to claim 31, characterized in that The centrifugation time is 5 to 15 minutes.

33. The preparation method according to claim 32, characterized in that The centrifugation time is 9.5 to 10.5 minutes.

34. The preparation method according to claim 11, characterized in that The endpoint of the spin-drying concentration is concentration to dryness.

35. The preparation method according to claim 11, characterized in that The second solvent is alcohol.

36. The preparation method according to claim 35, characterized in that The second solvent is ethanol.

37. The preparation method according to claim 36, characterized in that The volume percentage concentration of the ethanol is 60% to 80%.

38. The preparation method according to claim 37, characterized in that The volume percentage concentration of the ethanol is 66.5% to 73.5%.

39. The preparation method according to claim 11, characterized in that The volume of the second solvent is 5 to 20 mL.

40. The preparation method according to claim 39, characterized in that The volume of the second solvent is 9.5-10.5 mL.

41. The preparation method according to claim 11, characterized in that The mass / volume ratio of the leech extract to the second solvent is 0.1-1, with the unit being g / mL.

42. The preparation method according to claim 41, characterized in that The mass / volume ratio of the leech extract to the second solvent is 0.475-0.525, with the unit being g / mL.

43. The preparation method according to claim 10, characterized in that The ratio of the height H to the diameter d of the MCI CHP20 chromatography column is (9.5-10.5): (0.95-1.05).

44. The preparation method according to claim 10, characterized in that In the MCI column chromatography method, the volume ratio of the leech Hirudo nipponica Whitman extract to the column filler is (0.95-1.05): (14.25-15.75).

45. The preparation method according to claim 10, characterized in that In the MCI column chromatography method, the elution flow rate is 2.85 to 3.15 mL / min.

46. ​​The preparation method according to claim 10, characterized in that In the MCI column chromatography method, the alcohol is methanol.

47. The preparation method according to claim 46, characterized in that The volume percentage concentration of the methanol is 19% to 21%.

48. The preparation method according to claim 46, characterized in that The ratio of the volume of the methanol to the column volume of the MCI chromatography column is 2-4.

49. The preparation method according to claim 48, characterized in that The ratio of the volume of the methanol to the column volume of the MCI chromatography column is 2.85 to 3.

15.

50. The preparation method according to claim 10, characterized in that In the gel column chromatography method, the alcohol is methanol.

51. The preparation method according to claim 50, characterized in that The volume percentage concentration of the methanol is 47.5% to 52.5%.

52. The preparation method according to claim 10, characterized in that The concentration of the first dried product in the first sample solution is 50 to 400 mg / mL.

53. The preparation method according to claim 52, characterized in that The concentration of the first dried product in the first sample solution is 100 to 300 mg / mL.

54. The preparation method according to claim 10, characterized in that The ratio of the height H to the diameter d of the gel column is (14.25-15.75): (0.95-1.05).

55. The preparation method according to claim 10, characterized in that In the gel column chromatography method, the elution flow rate is 11.4 to 12.6 s / drop.

56. The preparation method according to claim 10, characterized in that The concentration of the second dried product in the second sample solution is 50 to 200 mg / mL.

57. The preparation method according to claim 56, characterized in that The concentration of the second dried product in the second sample solution is 80 to 120 mg / mL.

58. The preparation method according to claim 10, characterized in that The mass / mass ratio of the blank silica gel to the second dried product is 1 to 5, with the unit being g / g.

59. The preparation method according to claim 58, characterized in that The mass / mass ratio of the blank silica gel to the second dried product is 3.8 to 4.2, with the unit being g / g.

60. The preparation method according to claim 10, characterized in that The evaporation to dryness is carried out in a water bath.

61. The preparation method according to claim 60, characterized in that The temperature of the water bath is 50-70°C.

62. The preparation method according to claim 61, characterized in that The temperature of the water bath is 57-63°C.

63. The preparation method according to claim 10, characterized in that The particle size of the silica gel is 200-300 meshes.

64. The preparation method according to claim 10, characterized in that The ratio of the height H to the diameter d of the silica gel column is (7.6-8.4): (0.95-1.05).

65. The preparation method according to claim 10, characterized in that The flow rate of the gradient elution is 14.25-15.75 mL / min.

66. The preparation method according to claim 10, characterized in that In the high performance liquid chromatography preparation, the alcohol is methanol.

67. The preparation method according to claim 10, characterized in that The concentration of the third dried product in the third sample solution is 1 to 20 mg / mL.

68. The preparation method according to claim 67, characterized in that The concentration of the third dried product in the third sample solution is 5 to 15 mg / mL.

69. The preparation method according to claim 68, characterized in that The concentration of the third dried product in the third sample solution is 9.5 to 10.5 mg / mL.

70. The preparation method according to claim 10, characterized in that The high performance liquid chromatograph is an Alltech 426 semi-preparative liquid chromatograph.

71. The preparation method according to claim 10, characterized in that The specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, and particle size 10 μm.

72. The preparation method according to claim 10, characterized in that The flow rate is 2.85-3.15 mL / min.

73. The preparation method according to claim 10, characterized in that In the high performance liquid chromatography preparation, the detection wavelength is 244 nm.

74. The preparation method according to claim 10, characterized in that The injection volume is 47.5 to 52.5 μL.

75. The preparation method according to claim 10, characterized in that In the high performance liquid chromatography preparation, the drying is freeze drying.

76. Use of the pteridine compound or a salt thereof according to any one of claims 1 to 5 in the preparation of a medicament for promoting hemostasis.

77. Use of the pharmaceutical composition according to any one of claims 6 to 9 in the preparation of a medicament for promoting hemostasis.

78. Use of the pteridine compound or salt thereof according to any one of claims 1 to 5 as a characteristic chemical component of leeches for distinguishing leeches from leeches.

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