A compound, a preparation method thereof, and an application in an aphrodisiac active product

Abstract

The present invention discloses a compound extracted from Epimedium sagittatum, its preparation method and application. The preparation method comprises the following steps: processing, drying, and ethanol extraction of Epimedium sagittatum to obtain a crude extract; adsorbing the crude extract with macroporous resin and eluting successively with ethanol of different concentrations; performing solvent extraction and separation to obtain a dichloromethane part and an ethyl acetate part; separating the ethyl acetate part by normal-phase solid-phase extraction, using eluents of dichloromethane and methanol in different ratios successively in the normal-phase solid-phase extraction, and separating the eluted liquid by reversed-phase solid-phase extraction. In the reversed-phase solid-phase extraction, gradient elution is performed with an aqueous methanol solution, the eluents are collected successively from low to high methanol concentration, the obtained fractions are subjected to reversed-phase separation, the eluents are collected, and concentrated and dehydrated to obtain the product.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extracts, and particularly to a compound extracted from Epimedium sagittatum, a preparation method thereof, and an application thereof. Background Art

[0002] Epimedium sagittatum Maxim. is a plant of the genus Epimedium in the family Berberidaceae, and is the main medicinal species of the traditional Chinese medicine Epimedium in China. It is widely distributed in Shaanxi, Sichuan, Hubei, Hunan, Anhui, Jiangxi, Guangdong, Fujian, Zhejiang, Guizhou and other places. Epimedium is a traditional Chinese medicine for tonifying the kidney and strengthening yang, which can strengthen tendons and bones, dispel wind and dampness. In recent years, it has been used to treat various diseases such as coronary heart disease, hypertension and sexual neurasthenia, and has certain curative effects. It has become one of the hotspots in the research and development of medicinal plants.

[0003] At present, erectile dysfunction increasingly plagues many middle-aged men. Some first-line drugs such as sildenafil and vardenafil can treat erectile dysfunction by selectively inhibiting phosphodiesterase, resulting in penile tissue dilation and increased blood flow. However, the mechanism of Epimedium processed products in treating erectile dysfunction has not been systematically studied, especially whether it contains active inhibitors of phosphodiesterase.

[0004] Therefore, it is necessary to study and isolate the components of Epimedium to obtain beneficial compounds for use. Summary of the Invention

[0005] The purpose of the present invention is to provide a compound obtained by extracting, chromatographically separating and extracting Epimedium sagittatum after treatment, and a preparation method thereof.

[0006] The present invention solves its technical problems by adopting the following technical solutions.

[0007] The present invention provides a compound, and its structural formula is:

[0008]

[0009] The present invention also provides a preparation method of the above compound, which includes the following steps:

[0010] (1) Alcohol-extracting the powder of Epimedium sagittatum processed products to obtain an extract, filtering and drying the extract, dissolving it in water, filtering with a membrane, and drying to obtain a crude extract;

[0011] (2) Adsorbing the crude extract with macroporous resin, and eluting it with ethanol of different concentrations in turn to obtain a 20% ethanol elution fraction, a 40% ethanol elution fraction, a 60% ethanol elution fraction, an 80% ethanol elution fraction and a 95% ethanol elution fraction;

[0012] (3) The 60% ethanol elution fraction was separated by solvent extraction to obtain a dichloromethane fraction and an ethyl acetate fraction;

[0013] (4) Eluents were prepared in the ratios of dichloromethane:methanol being 25:1, 15:1, and 5:1 respectively. The ethyl acetate fraction was separated by normal-phase solid-phase extraction. In the normal-phase solid-phase extraction, eluents with different ratios of dichloromethane and methanol were used successively to obtain the 25:1 fraction, 15:1 fraction, 5:1 fraction, and pure methanol fraction in turn;

[0014] (5) The 5:1 fraction was separated by reversed-phase solid-phase extraction. In the reversed-phase solid-phase extraction, gradient elution was carried out successively using mobile phase methanol aqueous solutions with concentrations of 20%, 40%, 60%, and 80%. The eluents eluted with 60% and 80% methanol aqueous solutions were collected;

[0015] (6) The obtained eluents were subjected to reversed-phase separation, and the eluents were collected and concentrated and dehydrated to obtain the product; the mobile phase used in the reversed-phase separation preparation was an aqueous solution of methanol.

[0016] In a preferred embodiment of the present invention, for alcohol extraction, alcohol 8 - 12 times the volume of the powder of the processed Epimedium sagittatum was added and extracted for 1.5 h, and the extraction was carried out 1 to 3 times to obtain an extract. The extract was filtered and dried, dissolved in water, filtered using a membrane, and dried to obtain a crude extract.

[0017] In a preferred embodiment of the present invention, the mesh number of the silica gel used in the normal-phase solid-phase extraction is 200 - 300 mesh.

[0018] In a preferred embodiment of the present invention, the specification of the silica gel used in the reversed-phase solid-phase extraction is 100 - 150 mesh.

[0019] In a preferred embodiment of the present invention, the silica gel used in the normal-phase and reversed-phase solid-phase extractions is spherical silica gel or amorphous silica gel.

[0020] In a preferred embodiment of the present invention, the separation time of the normal-phase solid-phase extraction in step (4) is 30 - 40 min.

[0021] In a preferred embodiment of the present invention, the separation time of the reversed-phase solid-phase extraction in step (5) is 20 - 35 min.

[0022] In a preferred embodiment of the present invention, the reverse separation time in step (6) is 14 - 20 min.

[0023] In a preferred embodiment of the present invention, the packing used in the normal-phase solid-phase extraction is silica gel SiO 2 packing.

[0024] In a preferred embodiment of the present invention, the packing used in the reversed-phase solid-phase extraction and the reversed-phase separation preparation is C18 packing.

[0025] In a preferred embodiment of the present invention, in step (6), the volume fraction of methanol in the aqueous solution of methanol is 60-70%.

[0026] Another object of the present invention is to provide the use of the above compound in the preparation of virility drugs or foods.

[0027] The present invention further provides the use of the compound in the preparation of antioxidant drugs, foods or cosmetics.

[0028] The present invention further provides the use of the compound in the preparation of drugs or foods with turning positive activity.

[0029] The beneficial effects of the compound, its preparation method and application in the embodiments of the present invention are as follows: The compound isolated by the present invention has a relatively high purity, reaching more than 60%; at the same time, PDE5A inhibitory activity detection was carried out, and the result found that the IC 50 value is 1.86 μM, indicating its potential therapeutic effect on erectile dysfunction. In addition, the specific process steps adopted in this application can greatly reduce the pollution of the elution equipment, extend the service life and reduce the generation of impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 Structural formula diagram of the compound provided in the embodiment of the present invention;

[0032] Figure 2 Main HMBC spectrum diagram of the compound provided in the embodiment of the present invention;

[0033] Figure 3 IC of the compound provided in the embodiment of the present invention for PDE5A1 inhibition 50 Curve graph. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0035] The compounds of the embodiments of the present invention, their preparation methods and their applications will be specifically described below.

[0036] As Figure 1 shown, the compounds provided by the present invention have the following structural formula,

[0037]

[0038] The Chinese name of this compound is Sagittatoside I, and the English name is Sagittatoside I.

[0039] The present invention also provides a method for preparing the above-mentioned compound, including the following steps:

[0040] (1) The powder of the processed product of Epimedium sagittatum is extracted with alcohol to obtain an extract. After the extract is filtered and dried, it is dissolved in water and filtered through a membrane, and then dried to obtain a crude extract;

[0041] (2) The crude extract is adsorbed by macroporous resin and eluted successively with ethanol of different concentrations to obtain a 20% ethanol elution fraction, a 40% ethanol elution fraction, a 60% ethanol elution fraction, an 80% ethanol elution fraction and a 95% ethanol elution fraction;

[0042] (3) The 60% ethanol elution fraction is separated by solvent extraction to obtain a dichloromethane fraction and an ethyl acetate fraction;

[0043] (4) The ethyl acetate fraction is separated by normal-phase solid-phase extraction. In the normal-phase solid-phase extraction, eluents of different ratios of dichloromethane and methanol are used successively to obtain a 25:1 fraction, a 15:1 fraction, a 5:1 fraction and a pure methanol fraction; among them, the mesh number of the silica gel used in the normal-phase solid-phase extraction is 200-300 mesh; preferably, the silica gel used in the normal-phase solid-phase extraction is spherical silica gel or amorphous silica gel; more preferably, the chromatography separation time in the normal-phase solid-phase extraction is 30-40 min;

[0044] (5) The 5:1 fraction is separated by reverse-phase solid-phase extraction. In the reverse-phase solid-phase extraction, gradient elution is carried out successively with mobile phase methanol aqueous solutions with concentrations of 20%, 40%, 60% and 80%, and the eluents eluted with 60% and 80% methanol aqueous solutions are collected; among them, the mesh number of the silica gel used in the reverse-phase solid-phase extraction is 150-200 mesh; preferably, the silica gel used in the reverse-phase solid-phase extraction is spherical silica gel or amorphous silica gel; more preferably, the chromatography separation time in the reverse-phase solid-phase extraction is 20-35 min; preferably, each concentration is eluted twice, and the eluent is collected starting from the eluent after the second 60% methanol elution.

[0045] (6) The eluate collected is subjected to reverse-phase separation and preparation using high-pressure liquid phase. The eluate containing the target compound is collected, concentrated and dehydrated to obtain the product. The mobile phase used in the reverse-phase separation and preparation is an aqueous solution of methanol. Preferably, the time for reverse-phase separation is 14 - 20 min; more preferably, the volume fraction of methanol in the aqueous solution of methanol is 60 - 70%; further preferably, the packing material used in the reverse-phase solid-phase extraction and reverse-phase separation and preparation is C18 packing material.

[0046] In the present invention, Epimedium sagittatum is subjected to six pre-treatments including processing, alcohol extraction, macroporous resin adsorption, solvent extraction, normal-phase solid-phase extraction, and rough separation by reverse-phase solid-phase extraction, and finally the target compound is separated and purified by using the method of repeated reverse-phase preparative chromatography.

[0047] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0048] Example 1

[0049] In this example, it is prepared by the following method Figure 1 the shown compound:

[0050] (1) The processed product of Epimedium sagittatum is pulverized into powder and dried, and extracted with ethanol with a volume fraction of 50% which is 10 times its volume for 1.5 h. The extractions are carried out twice and the extracts are combined. The extract is filtered, evaporated to dryness, dissolved in ultrapure water, filtered through a 5000 DA ultrafiltration membrane, concentrated and dried to obtain the crude extract.

[0051] (2) The crude extract is adsorbed by macroporous resin D101 and eluted successively with ethanol of different concentrations to obtain the 20% part, 40% part, 60% part, 80% part and 95% part.

[0052] (3) The 60% part is separated by solvent extraction to obtain the dichloromethane part and the ethyl acetate part.

[0053] (4) The ethyl acetate part is separated by SiO 2 normal-phase solid-phase extraction. In the normal-phase solid-phase extraction, eluents of dichloromethane and methanol with different ratios are used successively to obtain the 25:1 part, 15:1 part, 5:1 part and pure methanol part. Among them, the mesh number of the silica gel used in the normal-phase solid-phase extraction is 200 mesh, the silica gel used in the normal-phase solid-phase extraction is spherical silica gel, and the time for chromatographic separation in the normal-phase solid-phase extraction is 30 min.

[0054] (5) Separate the 5:1 fraction by C18ME-HPLC reversed-phase solid-phase extraction. The mobile phase used in the reversed-phase solid-phase extraction is an aqueous solution of methanol, and the methanol concentrations are 20%, 40%, 60%, and 80% in sequence. Each concentration is eluted twice, and the eluents eluted with 60% and 80% aqueous methanol solutions are collected. Among them, the mesh number of the silica gel used in the reversed-phase solid-phase extraction is 200 mesh, the silica gel used in the reversed-phase solid-phase extraction is spherical silica gel, and the time for chromatographic separation in the reversed-phase solid-phase extraction is 20 min;

[0055] (6) Prepare by reversed-phase separation of the 60% fraction using high-performance liquid chromatography. Collect the eluent containing the target compound, and concentrate and dehydrate to obtain the compound of the above structural formula. The mobile phase used in the reversed-phase separation preparation is an aqueous solution of methanol. The time for reversed-phase separation is 14 min, and the volume fraction of methanol in the aqueous solution of methanol is 66%.

[0056] Example 2

[0057] In this example, the compound shown below is prepared by the following method: Figure 1 The compound shown:

[0058] (1) Grind the processed product of Epimedium sagittatum into powder and dry it. Extract with ethanol with a volume fraction of 50% at 12 times its volume for 1.5 h, extract twice and combine to obtain the extract. The extract is filtered, evaporated to dryness, dissolved in ultrapure water, filtered through a 5000 DA ultrafiltration membrane, concentrated, and dried to obtain the crude extract;

[0059] (2) Adsorb the crude extract with macroporous resin D101 and elute it with ethanol at different concentrations in sequence to obtain the 20% fraction, 40% fraction, 60% fraction, 80% fraction, and 95% fraction;

[0060] (3) Separate the 60% fraction by solvent extraction to obtain the dichloromethane fraction and the ethyl acetate fraction;

[0061] (4) Separate the ethyl acetate fraction by normal-phase solid-phase extraction using SiO 2 Normal-phase solid-phase extraction. In the normal-phase solid-phase extraction, eluents of different ratios of dichloromethane and methanol are used in sequence to obtain the 25:1 fraction, 15:1 fraction, 5:1 fraction, and pure methanol fraction. Among them, the mesh number of the silica gel used in the normal-phase solid-phase extraction is 300 mesh, the silica gel used in the normal-phase solid-phase extraction is spherical silica gel, and the time for chromatographic separation in the normal-phase solid-phase extraction is 40 min;

[0062] (5) The 5:1 portion was separated by C18ME-HPLC reversed-phase solid-phase extraction. The mobile phase used in the reversed-phase solid-phase extraction was an aqueous solution of methanol, and the methanol concentrations were 20%, 40%, 60%, and 80% in sequence. Each concentration was eluted twice, and the eluates obtained by eluting with aqueous solutions of 60% and 80% methanol were collected. Among them, the mesh number of the silica gel used in the reversed-phase solid-phase extraction was 150 mesh, the silica gel used in the reversed-phase solid-phase extraction was spherical silica gel, and the time for chromatographic separation in the reversed-phase solid-phase extraction was 35 min;

[0063] (6) The 60% portion was prepared by reversed-phase separation using high-performance liquid chromatography, and the eluate containing the target compound was collected and concentrated and dehydrated to obtain the compound of the above structural formula; the mobile phase used in the reversed-phase separation preparation was an aqueous solution of methanol, the time for the reversed-phase separation was 20 min, and the volume fraction of methanol in the aqueous solution of methanol was 66%.

[0064] The compound prepared above was subjected to structural identification. It was a light yellow powder, and its molecular formula was speculated to be C - ) from HR-ESI-MS (m / z 821.2662 [M-H] 42 H 46 O 17 . Its ultraviolet spectrum (λmax 210, 247, 255 nm) showed the characteristic absorption of phenyl and carboxyl groups; infrared spectrum analysis showed the presence of OH groups (3361 cm -1 ), C=O groups (1660 cm -1 ) and aromatic rings (1489 cm -1 ); the 13C NMR and DEPT (Table 1) spectra showed 42 carbons, including 15 flavonoid backbone carbons, 12 glycosyl carbons, 9 cardamonoyl carbons, 5 isopentenyl carbons and 1 methoxy carbon. These results indicated that the compound had a flavonoid backbone and side chains. 1 The 1H NMR spectrum showed that a set of A 2 B 2 coupled system protons were at δ H 7.89 (2H, d, J = 8.9 Hz, H-2′ / H-6′) and 7.10 (2H, d, J = 9.0 Hz, H-3′ / H-5′). In addition, δ H 3.89 (3H, s) had a cross peak with C-4 (δ C 163.5) to confirm the position of the methoxy group connected to C-4′. H-14 (δ H 1.64) with C-14 (δ C 25.9), H-15 (δ H 1.71) with C-15 (δ C 18.3), H-12 (δ H 5.22) and (δC 123.6), H-11a (δ H 3.62), H-11b (δ H 3.52) and C-11 (δ C 22.7) showed cross peaks, confirming the presence of an isopentenyl group; the signals of H-11 were correlated with C-7 (δ C 161.5) and C-9 (δ C 155.0), confirming that the isopentenyl group was located at the C-8 position. An aromatic proton at δ H 6.70 (H, s, H-6) had HMQC correlation with C-6 (δ C 99.7), and had HMBC correlations with C-5 (δ C 161.9), C-8 (δ C 110.9) and C-10 (δ C 107.7), confirming its location at the C-6 position and the hydroxyl group was on C-5. 1 The 1H NMR spectrum showed that δ H 5.41 (1H, d, J = 1.7 Hz, H-1") and δ H 0.89 (1H, d, J = 6.2 Hz, H-6") indicated a rhamnoside; the cross peak between H-1″ and C-3 (δ C 136.5) confirmed that the rhamnosyl moiety was attached to the C-3 position. In addition, from δ H 5.08 (1H, d, J = 7.5 Hz, H-1"') and the methylene signals of H-6a"' (δ H 4.28), H-6b"' (δ H 4.58), a glucosyl moiety was inferred. The cross peaks between H-1"' and C-7, and H-6"' and C-5"' (δ C 75.8) indicated that glucose was attached to the C-7 position. The large coupling constant of the vicinal proton signals at δ H 5.08 (1H, d, J = 7.5 Hz, H-1"') indicated that the glucosyl moiety was in the β configuration, and the large coupling constant of the vicinal proton signals at δ H 5.41 (1H, d, J = 1.7 Hz, H-1") indicated that the hydroxysugar moiety was in the α configuration. The absolute configurations of a D-glucosyl and an L-rhamnosyl were determined by GC analysis. From the typical A 2 B 2 coupled aromatic protons δ H 7.42 (2H, d, J = 8.6 Hz, H-5”” / H-9””) and 6.79 (2H, d, J = 8.6 Hz, H-6”” / H-8””) and δ H6.37 (1H, d, J = 16.0 Hz, H-2””) and 7.57 (1H, d, J = 15.9 Hz, H-3””) suggest the presence of a cardamonoyl group, as the large coupling constants indicate the presence of a trans-olefin and an α,β-unsaturated ester group at C-1”” (δc 169.1). The HMBC correlation between H-2" (δ H 4.24) and C-1”” indicates that the p-coumaroyl group is attached to C-2″; the structure and name of the compound can be identified as arrowroot glycoside I based on the above spectral data. The main HMBC spectrum of the obtained compound is as shown in Figure 2 Figure.

[0065] The complete 1H NMR assignment of this compound is as follows:

[0066] (c = 0.001, MeOH); IR (KBr) ν (cm-1): 3289, 1758, 1614, 1585, 1486;

[0067] UV (MeOH) λ max (logε): 272 (4.18), 312 (3.96) nm; IR (KBr) ν max 3361, 2931, 1697, 1558, 1456 cm -1 ; HR-ESI-MS m / z 821.2662 [M-H] - (calcd for C 42 H 45 O 17 , 821.2656);

[0068] The 1H NMR and 13C NMR spectra are shown in Table 1.

[0069] [Table 1. 1H NMR data (600 MHz; in DMSO) (δ ppm)

[0070] and 13C NMR data (150 MHz; in DMSO) (δ ppm)

[0071]

[0072] The antioxidant activity of the prepared compound was tested as follows:

[0073] 1.1 Materials and Reagents

[0074] MgCl 2 , Tris-HCl, cGMP, PDE5A1: Sigma-Aldrich (Merck KGaA, Darmstadt, Germany)

[0075] 1.2 Instruments and Equipment

[0076] HPLC: Agilent Technologies, USA;

[0077] 1.3 Test Methods

[0078] Dilute the compound in the assay buffer containing 100 mM MgCl 2 and 50 mM Tris-HCl (pH 8.0). Take 30 μL of PDE5A1 solution (0.0625 μg / ml) and mix it with 30 μL of the extract at room temperature and incubate for 5 min. Then add 30 μL of cGMP solution (7.5 μg / ml) and react at 35 °C for 90 min. After the reaction, incubate in boiling water at 100 °C for 5 min to inactivate the activity of PDE5A1, and then cool and detect by HPLC. Use the peak area of HPLC to calculate the inhibition rate.

[0079] 1.4 Results

[0080] The inhibitory effect of the compound on PDE5A1 is shown as follows Figure 3 shown; the measured IC 50 value is 1.86 μM.

[0081] As described above, within the test concentration range, the IC 50 value of the compound on PDE5A1 is 1.86 μM, which has inhibitory activity on PDE5A and has a significant aphrodisiac effect.

[0082] In summary, the preparation method of the compound provided by the embodiments of the present invention can extract a new compound from Epimedium sagittatum, named icariside I. This compound has a significant inhibitory effect on PDE5A1, indicating its application in the preparation of drugs or foods with aphrodisiac activity.

[0083] The embodiments described above are some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. A compound extracted from Epimedium sagittatum, characterized in that, the structural formula of the compound is:

2. The preparation method of the compound according to claim 1, characterized in that, comprising the following steps: (1) Alcohol extract the powder of the processed product of Epimedium sagittatum to obtain an extract, filter and dry the extract, dissolve it in water, use membrane filtration, and dry to obtain a crude extract; (2) Adsorb the crude extract with macroporous resin, and elute it successively with ethanol of different concentrations to obtain 20% ethanol elution fraction, 40% ethanol elution fraction, 60% ethanol elution fraction, 80% ethanol elution fraction and 95% ethanol elution fraction; (3) Use solvent extraction and separation for the 60% ethanol elution fraction to obtain dichloromethane fraction and ethyl acetate fraction; (4) Prepare eluents according to the ratios of dichloromethane:methanol of 25:1, 15:1, and 5:1 respectively, use normal-phase solid-phase extraction to separate the ethyl acetate fraction, and successively use eluents of different ratios of dichloromethane and methanol in the normal-phase solid-phase extraction to obtain 25:1 fraction, 15:1 fraction, 5:1 fraction and pure methanol fraction in turn; (5) Use reverse-phase solid-phase extraction to separate the 5:1 fraction. In the reverse-phase solid-phase extraction, gradient elution is carried out successively with mobile phase methanol aqueous solutions with concentrations of 20%, 40%, 60%, and 80%, and collect the eluents eluted with 60% and 80% methanol aqueous solutions; (6) Carry out reverse-phase separation on the collected eluents, collect the eluents, and concentrate and dehydrate to obtain; The mobile phase used in the reverse-phase separation preparation is an aqueous solution of methanol.

3. According to the preparation method described in claim 2, characterized in that, The alcohol extraction is to add alcohol with a volume 8 - 12 times that of the powder of the processed product of Epimedium sagittatum, extract for 1.5 h, extract 1 to 3 times to obtain an extract, filter and dry the extract, dissolve it in water, use membrane filtration, and dry to obtain a crude extract.

4. According to the preparation method described in claim 2, characterized in that, In step (4), the mesh number of the silica gel used in the normal-phase solid-phase extraction is 200 - 300 mesh; the separation time of the normal-phase solid-phase extraction is 30 - 40 min.

5. According to the preparation method described in claim 2, characterized in that, In step (5), the specification of the silica gel used in the reverse-phase solid-phase extraction is 100 - 150 mesh; the separation time of the reverse-phase solid-phase extraction is 20 - 35 min.

6. According to the preparation method described in claim 2, characterized in that, The silica gel used in the normal-phase and reverse-phase solid-phase extractions is spherical silica gel or amorphous silica gel.

7. According to the preparation method described in claim 6, characterized in that, The packing material used in normal-phase solid-phase extraction is silica gel SiO 2 packing material.

8. According to the preparation method described in claim 6, characterized in that, The packing materials used in the reverse-phase solid-phase extraction and the reverse-phase separation preparation are C18 packing materials.

9. According to the preparation method described in claim 2, characterized in that, In step (6), the reverse separation time is 14 - 20 min; the volume fraction of methanol in the aqueous solution of methanol is 60 - 70%.

10. The application of the compound according to claim 1 in the preparation of a drug for inhibiting PDE5A1.

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