Leech characteristic pteridine compound as well as preparation method and application thereof

Abstract

The invention discloses a pteridine compound or a salt thereof. The pteridine compound is a compound as shown in a formula I in the specification. One characteristic pteridine compound in the leech is subjected to enrichment and preparation of a reference substance in a column chromatography mode, purity detection is performed through HPLC, and the structure is identified by adopting mass spectrum and nuclear magnetic data and is SZ-1. The compound is a leech characteristic chemical component and is a symbolic component for distinguishing leeches, leeches and related adulterants of leeches and leeches, platelet aggregation experiment results show that SZ-1 has the effect of promoting platelet aggregation, and a candidate standard substance is provided for different original varieties and authenticity identification of leech medicinal materials. No related reports for separating and identifying the SZ-1 from the leeches involved in the invention are found at present. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a characteristic pteridine compound of leech, a preparation method thereof, and an application thereof. Background Art

[0002] Traditional Chinese medicine leech is derived from the dried bodies of animals of the genus Hirudo in the family Hirudinidae, Whitmania pigra Whitman or Whitmania acranulata Whitman, and has the effects of promoting blood circulation to remove blood stasis and dispelling stasis to relieve masses. Modern pharmacological studies have shown that leech has various effects such as anticoagulation, antithrombosis, anti-atherosclerosis, antiplatelet aggregation, antitumor, anti-inflammatory, improving hemorheology, and protecting against cerebral ischemia-reperfusion injury.

[0003] Currently, commercially available traditional Chinese medicine leeches are mostly Whitmania pigra Whitman, Hirudo nipponica Whitman is relatively rare, and Whitmania acranulata Whitman is hardly seen. Although the three are all legal original sources of leech medicinal materials, the anticoagulant activity of Hirudo nipponica Whitman is 100 times higher than that of Whitmania pigra Whitman. Therefore, effectively distinguishing Hirudo nipponica Whitman from Whitmania pigra Whitman is beneficial to ensuring the safety of medication. At present, there is little research on the differential components among different original varieties of leech and their adulterants, and there is still a blank in the report of the characteristic components of leech.

[0004] A characteristic pteridine compound of leech, a preparation method thereof, and an application thereof according to the present invention 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, and the pteridine compound is a compound of formula I:

[0006]

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

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

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

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

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

[0012] Further, the adjuvant is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, complexing agent, flavoring agent, sustained release agent, retention aid, lubricant, dispersant, plasticizer, light shielding agent, and antioxidant.

[0013] Further, the dosage form of the pharmaceutical composition is tablet, dripping pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

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

[0015] Further, the method for preparing 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, rotary evaporation and concentration to obtain a leech extract, and adding a second solvent to the leech extract for dissolution to obtain the leech extract.

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

[0017] Further, the volume percentage concentration of the ethanol is 90% to 100%, such as about 95%.

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

[0019] Further, the power of the ultrasonic extraction is 200 to 300 W, such as about 250 W.

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

[0021] Further, the time of the ultrasonic extraction is 15 to 45 min, such as 20 to 40 min, such as about 30 min.

[0022] Further, the number of times of the ultrasonic extraction is 1 to 5 times, such as 3 times.

[0023] Further, the rotation speed of the centrifugation is 10000 to 15000 r / min, such as about 12000 r / min.

[0024] Further, the time of the centrifugation is 5 to 20 min, such as 5 to 15 min, such as about 10 min.

[0025] Further, the end point of the rotary evaporation and concentration is concentration to dryness.

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

[0027] Further, the volume percentage concentration of the ethanol is 60% - 80%, such as about 70%.

[0028] Further, the volume of the second solvent is 5 - 20 mL, such as about 10 mL.

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

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

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

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

[0033] Further, the volume ratio of the leech extract solution to the volume of the column packing is about 1:about 15.

[0034] Further, the elution flow rate is 1 - 5 mL / min, such as about 3 mL / min.

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

[0036] Further, the volume percentage concentration of the methanol is 10% - 30%, such as about 20%.

[0037] Further, the volume ratio of the third solvent to the column volume of the MCI chromatography column is 2 - 4, such as about 3.

[0038] Further, the method of gel column chromatography 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 a gel column, eluting with the fourth solvent, collecting the eluate, concentrating, and drying to obtain a second dried product.

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

[0040] Further, the volume percentage concentration of the methanol is 40% - 60%, such as about 50%.

[0041] Furthermore, the concentration of the first dried substance in the first sample solution is 50 - 400 mg / mL, for example, 100 - 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 flow rate of the elution is 10 - 15 s / drop, for example, about 12 s / drop.

[0045] Furthermore, the method of silica gel column chromatography includes: dissolving the second dried substance with the fourth solvent to obtain a second sample solution, adding blank silica gel to the second sample solution, stirring evenly, evaporating to dryness, 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 substance.

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

[0047] Furthermore, the mass / mass (g / g) ratio of the blank silica gel to the second dried substance 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 mesh.

[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 is carried out according to the following procedure: (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.

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

[0054] Furthermore, dissolve the third dried substance with the fifth solvent to obtain a third sample solution, separate and purify the third sample solution by a high performance liquid chromatograph, collect the corresponding fractions, and dry to obtain the pteridine compound.

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

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

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

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

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

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

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

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

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

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

[0065] According to another aspect of the present invention, there is provided the 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 adulterants.

[0066] According to another aspect of the present invention, there is provided the use of the above-mentioned preparation method for preparing reference substances.

[0067] Advantages of the present invention:

[0068] The present invention enriches and prepares a reference substance for one characteristic pteridine compound in Hirudo by column chromatography, detects its purity by HPLC, and identifies its structure using mass spectrometry and nuclear magnetic resonance data, which is SZ-1. This compound is a characteristic chemical component of Hirudo, a marker component for distinguishing Hirudo from Whitmania pigra and its closely related adulterants. The results of platelet aggregation experiments show that SZ-1 has the effect of promoting platelet aggregation, providing a candidate reference substance for the identification of different original varieties and authenticity of Hirudo medicinal materials. Currently, there is no relevant report on the isolation and identification of SZ-1 from Hirudo involved in the present invention. Description of the Drawings

[0069] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0070] Figure 1 It is the thin layer chromatogram of MCI column chromatography separation and purification. Among them, the numbers 1 to 10 represent fractions 1 to 10 in the process of MCI column chromatography. 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 It is the thin layer chromatogram of gel column chromatography (Sephadex LH-20) separation and purification. Among them, the numbers 1 to 5 represent fractions 1 to 5 in the process of gel column chromatography. Fraction 2 represents the fraction where SZ-1 is located, and the blue-green fluorescent spot in fraction 2 is SZ-1.

[0072] Figure 3 It is the thin layer chromatogram of silica gel column chromatography separation and purification. Among them, the numbers 1 to 7 represent fractions 1 to 7 in the process of 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 It is the high performance liquid chromatography preparative chromatogram. Among them, the chromatographic peak with a retention time of 15.8 min represents SZ-1.

[0074] Figure 5 It is the HPLC chromatogram of SZ-1. Among them, the chromatographic peak with a retention time of 6.9 min represents SZ-1.

[0075] Figure 6 It is the structural schematic diagram of SZ-1.

[0076] Figure 7 It is the HR-ESI-MS diagram of SZ-1.

[0077] Figure 8 It is the IR spectrum of SZ-1.

[0078] Figure 9 It is the UV spectrum of SZ-1.

[0079] Figure 10 It is for SZ-1 1 1H NMR spectrum.

[0080] Figure 11 It is for SZ-1 13 13C NMR spectrum.

[0081] Figure 12 It is for SZ-1 1 H- 1 H COSY spectrum.

[0082] Figure 13 It is the HSQC spectrum of SZ-1.

[0083] Figure 14 It is the HMBC spectrum of SZ-1.

[0084] Figure 15 It is the thin layer chromatography for SZ-1 to identify different batches of leeches and bloodsuckers. Detailed implementation manners

[0085] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0086] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the art in the field of the present invention or the field where the term is applied. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the implementation of the present invention, the preferred methods, conditions, substances or materials are described herein.

[0087] The present invention is expected to cover all alternatives, variations and equivalents, which may be included in the existing field of invention as defined by the claims. Those skilled in the art will recognize many methods and substances similar to or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is by no means limited to the description of methods and substances.

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

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

[0090] As described in the background art section, a characteristic pteridine compound of leech and its preparation method and application of the present invention have not been reported in the prior art. To solve the above problems, the present invention provides a pteridine compound or a salt thereof, and the pteridine compound is a compound of 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 of the family Hirudinidae.

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

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

[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, light shielding agent and antioxidant.

[0098] In a preferred embodiment, the dosage form of the pharmaceutical composition is tablet, dripping pill, capsule, powder, injection, film, lozenge, granule or oral liquid.

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

[0100] The method of the invention is a compound preparation and separation method with simple process, good reproducibility, high purity of separated compounds and 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 to dissolve it 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 value or parameter is 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 as including 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 the 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 30-60 kHz, such as 30-50 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, such as 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 12000" includes ±5% of 12000, or from 11400 to 12600.

[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, such as 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 the 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 filler is about 1:about 15.

[0132] In the present invention, "about" refers to a value within the 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 1-5 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, such as 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 the 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%, such as 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, such as 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, such as 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 with the fourth solvent to obtain a second sample solution, adding blank silica gel to the second sample solution, stirring evenly, evaporating to dryness, 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 under water bath conditions.

[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 meshes.

[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 the 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 procedure: (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 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 1-5 mL / min, a detection wavelength of 200-300 nm, and an injection volume of 40-60 μL.

[0170] In a preferred embodiment, the specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, 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 drug 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 only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

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

[0182] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features 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 iS10FTIR 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 Pulisheng Instrument Co., Ltd.); PocH100 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 150g of leech powder, add 10 times the amount of 95% ethanol, ultrasonically treat (power 250W, frequency 40KHz) for 30min, filter, repeat 3 times, combine the filtrate, centrifuge at 12000r / min for 10min to obtain the supernatant, and then use a rotary evaporator to concentrate and remove the solvent to constant weight to obtain 5.7385g 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 it into the treated MCI CHP20 (the ratio of height (H) to diameter (d) is about 10: about 1), the flow rate is about 3mL / min, and 3BV (3 times column volume) of 20% methanol is used for elution, and the 20% methanol elution fractions are collected, and each fraction is subjected to thin layer examination. The thin layer development conditions are chloroform: methanol = 8:1. After development under this developing agent ratio, 365nm ultraviolet light is irradiated, and it can be found that the Rf value is about 0.3 and there are obvious blue-green fluorescent spots, as shown in the following example. Figure 1 As shown, among the fractions 1 to 10, the enriched fraction 1 (containing SZ-1) was concentrated and dried to obtain 1.3566 g of the first dried product.

[0192] 2.1.3 Gel column separation and enrichment

[0193] Weigh all of 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 12 s / 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 and irradiation with a 365nm ultraviolet lamp, it can be found that there are obvious blue-green fluorescent spots with an Rf value of about 0.3, as shown in the following example. Figure 2 As shown, among the fractions 1 to 5, the enriched fraction 2 (containing SZ-1) was 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 material, dissolve it in 50% methanol to prepare a second sample solution with a concentration of 80 mg / mL to 120 mg / mL, mix the mass of the second dried material contained in the second sample solution and the mass of the blank silica gel in a ratio of 1:4, evaporate and grind to fine powder in a 60°C water bath to obtain a 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 examination. The thin layer development conditions are dichloromethane: methanol = 5:1. After development under this developing agent ratio, 365nm ultraviolet lamp is irradiated, and it can be found that there are 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; 244 nm wavelength). The HPLC preparative chromatogram of the sample is shown in 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 test

[0199] The HPLC chromatogram of the sample was obtained by using an LC-20A high performance liquid chromatograph according to the 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 N 4 O 5 S 2, the unsaturation is 9.

[0202] exist 1 H NMR (DMSO-d 6 , 600MHz) spectrum, there are two active hydrogen signal peaks [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 of an oxygen-containing methylene group [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 C NMR (DMSO-d 6 , 150MHz) spectrum, there are 12 carbon signals, combined with HSQC spectrum analysis, [δ C 161.9(C-2),δ C 153.7 (C-4)] are two keto 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 signals of 2 hydroxyl-substituted methine carbons and methylene carbons 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)], these hydrogen and carbon signals are assigned in Table 1. The above information is similar to the compound (R)-hirudonucleodisulfide B in the literature, which is speculated to be a pteridine derivative. The main difference between them is that the nitrogen atom at the 3rd position of this compound is substituted by a methyl group, and the sulfur atom at the 11th position is a sulfinyl group. The chemical shifts of the two methyl groups substituted at the 3rd and 11th positions [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 crossover signals H-13 / H2-14 in the H COSY spectrum showed the presence of C13-C14 (vicinal diol) structure fragments. The related signals of H-10 / C-2 / C-4, H-12 / C-6, H-13 / C-6, and H-14 / C-7 in the HMBC spectrum verified the methyl substitution of N-3, the sulfinyl substitution of C-6, and the vicinal diol substitution of C-7, respectively.

[0205] In addition, the characteristic signal peaks in the IR spectrum such as amino (3239cm -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 - 14 Sure.

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

[0207] Position <![CDATA[δ H > <![CDATA[δ C > Position <![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[N 3 -CH 3 > 3.26 27.8 6 - 128.8 <![CDATA[-CH 3 > 3.15 40.2 7 - 153.2 13 - OH 6.27, brs - 8a - 141.6 14 - OH 5.11, brs -

[0208] The above research results show that leeches contain a component different from other leeches, which is SZ-1, which proves the particularity of leech composition. The entire preparation and separation process is environmentally friendly, simple and easy to operate, so leeches can be used as the raw material for the separation 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 fasted but not watered for 12 h before blood collection from the abdominal aorta), placed in a 3.8% sodium citrate anticoagulation blood collection tube, and rotated 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, aspirate the supernatant, and obtain platelet-poor plasma (PPP) for later use. After counting platelets with PRP, adjust the platelet count to 400,000 / μL with PPP.

[0212] 2.4.2 Solution preparation

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

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

[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 fasted but not watered for 12 h before blood collection from the abdominal aorta), placed in a 3.8% sodium citrate anticoagulation blood collection tube, and rotated 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, aspirate the supernatant, and obtain platelet-poor plasma (PPP) for later use. After counting platelets with PRP, adjust the platelet count to 400,000 / μL with PPP.

[0217] 2.4.4 Determination of in vitro platelet aggregation induction

[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, add 270 μL of PPP to the zeroed test cups, and then add 30 μL of saline (without a stirrer); add 270 μL of PRP to each test cup in the blank group, and then add 30 μL of saline (with a stirrer); add 300 μL of SZ-1 containing a final concentration of 50 μM PRP to each test cup in the sample group (with a stirrer), and incubate in the incubation chamber for 4 min.

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

[0222] (5) Take out the zeroing test cup and put it into the PRP sample group test cup. After the light flux value stabilizes, press the corresponding channel number key. Pipette 6μl of ADP (adenosine diphosphate) inducer into the bottom of the cup, immediately press the corresponding channel key, and 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 by using an in vitro platelet aggregation model. The results are shown in Table 2. At a dose of 50 μM, SZ-1 has a certain activity in promoting platelet aggregation.

[0225] Table 2 Effect of SZ-1 on in vitro induced 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 agent ratio, 365nm ultraviolet light was used to illuminate the sample, and an obvious blue-green fluorescent spot with an Rf value of about 0.3 was found. Four batches of leeches contained 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 promising application prospects. It has good social and economic benefits, will play a positive role in promoting the economic development of the leech industry, and will 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 used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content 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 its salt according to claim 1, characterized in that The pteridine compound is derived from the dried body of leech; Preferably, the leech is a leech of the family Hirudinidae (Hirudo nipponica Whitman); Preferably, 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; flow rate 1 mL / min; wavelengths of 244 nm and 254 nm.

3. A pharmaceutical composition comprising the pteridine compound or a salt thereof according to claim 1 or 2.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention agent, lubricant, dispersant, plasticizer, opacifier and antioxidant; More preferably, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

5. A method for preparing a pteridine compound according to claim 1 or 2, characterized in that: The preparation method comprises: subjecting the 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.

6. The preparation method according to claim 5, characterized in that: The preparation method of the leech extract comprises: weighing an appropriate amount of leech powder, adding a first solvent to perform ultrasonic extraction, filtering, centrifuging the filtrate, taking the supernatant, spinning and concentrating to obtain a leech extract, adding a second solvent to the leech extract to dissolve it, and obtaining the leech extract; Preferably, the first solvent is an alcohol, such as ethanol; More preferably, the volume percentage concentration of the ethanol is 90% to 100%, such as about 95%; More preferably, the mass / volume (g / mL) ratio of the leech powder to the first solvent is 0.01 to 1, such as 0.06 to 0.2, such as about 0.1; More preferably, the power of the ultrasonic extraction is 200-300W, for example, about 250W; More preferably, the frequency of the ultrasonic extraction is 30 to 60 kHz, such as 30 to 50 kHz, such as about 40 kHz; More preferably, the ultrasonic extraction time is 15 to 45 min, such as 20 to 40 min, such as about 30 min; More preferably, the number of ultrasonic extractions is 1 to 5 times, for example 3 times; Preferably, the centrifugal speed is 10000-15000 r / min, for example, about 12000 r / min; Preferably, the centrifugation time is 5 to 20 min, such as 5 to 15 min, such as about 10 min; Preferably, the end point of the spin-drying concentration is concentration to dryness; Still more preferably, the second solvent is an alcohol, such as ethanol; Particularly preferably, the volume percentage concentration of the ethanol is 60% to 80%, for example, about 70%; Particularly preferably, the volume of the second solvent is 5 to 20 mL, for example about 10 mL; Particularly preferably, the mass / volume (g / mL) ratio of the leech extract to the second solvent is 0.1 to 1, such as about 0.5; Particularly preferably, 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; Particularly preferably, the MCI chromatography column is an MCI CHP20 chromatography column; Particularly preferably, the ratio of the height (H) to the diameter (d) of the MCI CHP20 chromatography column is about 10: about 1; Particularly preferably, the volume ratio of the leech extract to the volume of the column filler is about 1: about 15; Particularly preferably, the elution flow rate is 1 to 5 mL / min, for example about 3 mL / min; Particularly preferably, the third solvent is an alcohol, such as methanol; Particularly preferably, the volume percentage concentration of the methanol is 10% to 30%, for example, about 20%; Particularly preferably, the ratio of the volume of the third solvent to the column volume of the MCI chromatography column is 2 to 4, for example about 3; Particularly preferably, 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 it with the fourth solvent, collecting the eluate, concentrating it, and drying it to obtain a second dried product; Particularly preferably, the fourth solvent is an alcohol, such as methanol; Particularly preferably, the volume percentage concentration of the methanol is 40% to 60%, for example, about 50%; Particularly preferably, 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; Particularly preferably, the gel column is a Sephadex LH-20 gel column; Particularly preferably, the ratio of the height (H) to the diameter (d) of the gel column is about 15: about 1; Particularly preferably, the elution flow rate is 10-15 s / drop, such as about 12 s / drop.

7. The preparation method according to claim 6, characterized in that: The silica gel column chromatography method comprises: dissolving the second dried product with the fourth solvent to obtain a second sample solution, adding blank silica gel to the second sample solution, stirring evenly, evaporating to dryness, 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; Preferably, 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; More preferably, the mass / mass (g / g) ratio of the blank silica gel to the second dried product is 1 to 5, for example, about 4; More preferably, the evaporation to dryness is carried out under water bath conditions; Also preferably, the temperature of the water bath is 50-70°C, for example about 60°C; Preferably, the particle size of the silica gel is 200-300 mesh; Preferably, the ratio of the height (H) to the diameter (d) of the silica gel column is about 8: about 1; More preferably, the gradient elution is performed according to the following procedure: (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; Still more preferably, the flow rate of the gradient elution is 10 to 20 mL / min, for example about 15 mL / min; Particularly preferably, the third dried product is dissolved in the fifth solvent to obtain a third sample solution, the third sample solution is separated and purified by a high performance liquid chromatography, and the corresponding fractions are collected and dried to obtain the pteridine compound; Particularly preferably, the fifth solvent is an alcohol, such as methanol; Particularly preferably, the concentration of the third dried product in the third sample solution is 1 to 20 mg / mL, such as 5 to 15 mg / mL, such as about 10 mg / mL; Particularly preferably, the high performance liquid chromatograph is an Alltech 426 semi-preparative liquid chromatograph; Particularly preferably, 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; Particularly preferably, the specifications of the chromatographic column are: column length 250 mm, inner diameter 10 mm, particle size 10 μm; Particularly preferably, the flow rate is about 3 mL / min; Particularly preferably, the detection wavelength is 244 nm; Particularly preferably, the injection volume is about 50 μL; Particularly preferably, the drying is freeze-drying.

8. Use of the pteridine compound or the salt thereof according to claim 1 or 2, or the pharmaceutical composition according to claim 3 or 4, in the preparation of a medicament for promoting hemostasis.

9. Use of the pteridine compound or salt thereof according to claim 1 or 2 as a characteristic chemical component of leeches for distinguishing leeches from leeches and their closely related hybrids.

10. Use of the preparation method according to claims 5 to 9 for preparing standard substances.

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