Application and preparation method of rhizoma polygonati compound for protecting activity of alpha-glucosidase

Abstract

The invention discloses application and a preparation method of polygonatum kingianum compounds for protecting activity of alpha-glucosidase, the polygonatum kingianum compounds comprise a compound 1 (the molecular formula is C39H62O15), a compound 2 (the molecular formula is C47H72O21) and a compound 3 (the molecular formula is C47H72O21), and the invention relates to the technical field of GAA abnormal expression disease related drugs. The activity of alpha-glucosidase (GAA) can be protected, so that the alpha-glucosidase (GAA) can promote the utilization of glucose in cell respiration in vivo, and the alpha-glucosidase GAA has a good auxiliary curative effect on patients with difficult miscellaneous diseases with abnormal expression of GAA.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs related to GAA abnormal expression diseases, and specifically relates to an application of a polygonatum compound for protecting α-glucosidase activity and a preparation method thereof. Background Art

[0002] α-Glucosidase (GAA) is an enzyme that hydrolyzes glycogen and maltose into glucose. Its normal expression in the body can promote the utilization of glucose in cell respiration. For diabetic patients, the overexpression of the physiological function of GAA will cause hyperglycemia, which will further deteriorate the condition of diabetic patients and accelerate the development of the disease. However, the decrease or deficiency of GAA activity will lead to the deposition of glycogen in lysosomes of skeletal muscle, cardiac muscle and smooth muscle cells due to the inability to be degraded, resulting in cell metabolic disorders, abnormal functions, cell damage and organ damage, and causing a series of clinical manifestations, known as type II glycogen storage disease, also known as Pompe disease. The common abnormality found in the semen examination of infertile men is semen abnormality. Neutral α-glucosidase in seminal plasma is derived from the epididymis, which is a specific enzyme and a marker enzyme of the epididymis. The change of the marker indirectly reflects the functional change of the epididymis, and at the same time directly affects some parameters of sperm, thus affecting male fertility. The level of neutral α-glucosidase activity in male seminal plasma is correlated with the severity of male infertility. The lower the enzyme level, the more serious the condition. In addition, a continuously low expression of GAA activity has been diagnosed in patients with Duchenne muscular dystrophy.

[0003] Traditional Chinese medicine has unique advantages in preventing and treating difficult and complicated diseases. The theory of traditional Chinese medicine points out that: "The spleen is the foundation of acquired constitution and the source of qi and blood production; the kidney is the foundation of congenital constitution, storing primordial yin and yang." The occurrence and development of diseases are closely related to the spleen and kidney. Diabetes belongs to the category of "consumptive thirst". The root cause of the disease is weak spleen and kidney. The method of strengthening the spleen and tonifying the kidney can be used to achieve the purpose of strengthening the healthy qi and consolidating the root. Type II glycogen storage disease and Duchenne muscular dystrophy both belong to the category of "flaccidity disease". If the spleen and stomach are weak, the source of qi, blood and body fluid will be insufficient, and the limb tendons and vessels will not be nourished; if the liver and kidney are deficient, the essence and blood will be insufficient, and the tendons and vessels will lose nourishment, and it will become flaccid over time. The treatment principle should be to strengthen the spleen and tonify the kidney to treat the root cause. The kidney is the root of qi production, and the spleen and stomach are the source of qi production; strengthening the spleen and replenishing qi to nourish the muscle tendons and vessels, and tonifying the kidney is the key, which runs through the whole process. Oligospermia belongs to the category of "infertility". The kidney can store the essence of the human body. The male reproductive function can only function normally under the action of the essence qi in the kidney. Just as stated in "Shennong Ben Cao Jing Shu", "If a man has a weak kidney, his essence will be exhausted and he will have no children." The strength of the spleen and stomach's transportation and transformation function will also directly affect the abundance and deficiency of the kidney essence. Therefore, tonifying the kidney and strengthening the spleen, with sufficient qi and blood and sufficient kidney essence, "the congenital promotes the acquired, and the acquired nourishes the congenital", so as to treat the disease by seeking the root cause. Treating the spleen and kidney simultaneously has obvious curative effects on improving male semen parameters. In addition, the balance of yin and yang is the core thinking of traditional Chinese medicine in grasping diseases. Therefore, it is feasible to find substances that regulate α-glucosidase activity from traditional Chinese medicines with the efficacy of "strengthening the spleen and tonifying the kidney". Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides an application of a polygonatum compound for protecting the activity of α-glucosidase and its preparation method, and solves the problem of low α-glucosidase activity and difficulty in regulation during the prevention and treatment of intractable diseases.

[0005] To achieve the above object, the present invention is realized through the following technical solutions: An application of a polygonatum compound for protecting the activity of α-glucosidase and its preparation method, the polygonatum compound includes compound 1 (molecular formula: C 39 H 62 O 15 ), compound 2 (molecular formula: C 47 H 72 O 21 ), and compound 3 (molecular formula: C 47 H 72 O 21 ).

[0006] Preferably, the preparation method includes extraction, extraction, separation and purification;

[0007] The extraction: Take the dried rhizome of polygonatum, heat and reflux with 85-95% ethanol for 1-3 times, 1-4 hours each time, filter, combine the filtrates, recover ethanol to obtain an extract, and then heat and reflux the residue with 50%-70% ethanol for 1-2 times, 1-2 hours each time, filter, combine the filtrates, and obtain an extract in the same way;

[0008] The extraction: Disperse the extract in water, and extract it successively with equal volumes of petroleum ether, chloroform, ethyl acetate, and n-butanol. The extract is extracted 2-3 times, and the extraction solutions are combined, and the solvents are recovered to obtain each extraction part;

[0009] The separation: Combine the n-butanol layer extraction parts, dissolve appropriately, load the column with chloroform, load the sample by dry method, and elute with a chloroform-methanol system gradient (volume ratio 100:0-0:100), 1000 ml for each fraction, at least 15 fractions for each gradient, a total of 261 fractions;

[0010] Purification: Analyzed by liquid chromatograph, similar fractions were combined and divided into groups A - T in total. Group N was eluted with a methanol - water gradient (0% - 100%) using an ODS open column chromatograph. The fractions eluted with 60% - 80% methanol were taken and purified by semi - preparative liquid chromatograph, and compounds 1 - 3 were separated using acetonitrile - water (30% - 45%) as the mobile phase. Polygonatum sibiricum: It is the dried rhizome of Polygonatum sibiricum, sweet in taste and neutral in nature. It acts on the spleen, lung and kidney meridians. It has the effects of invigorating the spleen and kidney, replenishing qi and nourishing yin, and moistening the lungs. It is used for internal heat diabetes, spleen - stomach qi deficiency, fatigue, stomach yin deficiency, dry mouth and less food intake, dry cough due to lung deficiency, chronic cough with hemoptysis, insufficiency of essence and blood, weakness of the waist and knees, and premature whitening of hair. Polygonatum sibiricum was first recorded in "Shennong Ben Cao Jing" and got its name because "it obtains the essence of Kun soil", and it is a traditional classic Chinese medicine with both medicinal and edible properties. "Ben Cao Cheng Ya Ban Jie" records that Polygonatum sibiricum "supplements the body of earth and fills the functions of earth", "Zheng Lei Ben Cao" records that it "tonifies deficiency and replenishes essence", and "Dian Nan Ben Cao" records that it "strengthens the primordial yang". Therefore, it is feasible to discover Polygonatum sibiricum compounds with the activity of protecting α - glucosidase from Polygonatum sibiricum with the effect of invigorating the spleen and kidney.

[0011] Beneficial effects

[0012] The present invention provides an application of a Polygonatum sibiricum compound for protecting α - glucosidase activity and its preparation method, having the following beneficial effects:

[0013] 1. Compounds 1 - 3 are extracted from the dried rhizome of Polygonatum sibiricum, which can protect the activity of α - glucosidase (GAA), enabling it to promote the utilization of glucose in cell respiration in vivo, and having a good adjuvant therapeutic effect on patients with difficult and complicated diseases of abnormal GAA expression.

[0014] 2. High separation and purification efficiency, and compounds 1 - 3 can be obtained simultaneously under one separation and purification condition. Brief description of the drawings

[0015] Figure 1 It is the structure of compound 1 of the present invention.

[0016] Figure 2 It is the structure of compound 2 of the present invention.

[0017] Figure 3 It is the structure of compound 3 of the present invention.

[0018] Figure 4 It is the 1 1H - NMR (600 MHz, Pyridine - d5) spectrum of compound 1 of the present invention.

[0019] Figure 5 It is the 13 13C - NMR (150 MHz, Pyridine - d5) spectrum of compound 1 of the present invention.

[0020] Figure 6 The HSQC spectrum of Compound 1 of the present invention.

[0021] Figure 7 The HMBC spectrum of Compound 1 of the present invention.

[0022] Figure 8 For Compound 2 of the present invention 1 1H-NMR (600 MHz, Pyridine-d5) spectrum.

[0023] Figure 9 For Compound 2 of the present invention 13 13C-NMR (150 MHz, Pyridine-d5) spectrum.

[0024] Figure 10 The HSQC spectrum of Compound 2 of the present invention.

[0025] Figure 11 The HMBC spectrum of Compound 2 of the present invention.

[0026] Figure 12 For Compound 3 of the present invention 1 1H-NMR (600 MHz, Pyridine-d5) spectrum.

[0027] Figure 13 For Compound 3 of the present invention 13 13C-NMR (150 MHz, Pyridine-d5) spectrum.

[0028] Figure 14 The HSQC spectrum of Compound 3 of the present invention.

[0029] Figure 15 The HMBC spectrum of Compound 3 of the present invention. Detailed implementation manners

[0030] 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 only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Please refer to Figures 1-15 , the present invention provides a technical solution: an application of a polygonatum compound for protecting α-glucosidase activity and its preparation method. The polygonatum compound includes Compound 1 (molecular formula: C 39 H 62 O 15 ), Compound 2 (molecular formula: C 47 H72 O 21 ) and compound 3 (molecular formula: C 47 H 72 O 21 ).

[0032] Furthermore, the preparation method includes extraction, extraction, separation and purification;

[0033] For the said extraction: Take the dried rhizome of Polygonatum sibiricum, extract it by heating under reflux with 85% - 95% ethanol for 1 - 3 times, 1 - 4 h each time, filter, combine the filtrates, recover the ethanol to obtain an extract, then extract the medicinal residues by heating under reflux with 50% - 70% ethanol for 1 - 2 times, 1 - 2 h each time, filter, combine the filtrates, and obtain an extract in the same way;

[0034] For the said extraction: Disperse the extract in water, extract it successively with equal volumes of petroleum ether, chloroform, ethyl acetate, and n - butanol, extract the extract 2 - 3 times, combine the extraction solutions, and recover the solvent to obtain each extraction part;

[0035] For the said separation: Combine the extraction parts of the n - butanol layer, dissolve appropriately, load the column with chloroform, load the sample by dry method, elute with a chloroform - methanol system gradient (volume ratio 100:0 - 0:100), each fraction is 1000 ml, at least 15 fractions for each gradient, a total of 261 fractions;

[0036] For the said purification: Analyze by a liquid chromatograph, combine the similar fractions, divide them into groups A - T in total, group N is eluted with a methanol - water gradient (0% - 100%) using an ODS open column chromatograph, take the fractions eluted with 60% - 80% methanol, and purify them with a semi - preparative liquid chromatograph, and separate compounds 1 - 3 using acetonitrile - water (30% - 45%) as the mobile phase.

[0037] Perform structure identification on compounds 1 - 3:

[0038] Compound 1

[0039] is a light yellowish - white powdery substance, soluble in pyridine. High - resolution mass spectrometry gives the quasi - molecular ion peak m / z793.3965 [M + Na], and combined with the 1H - NMR spectrum and 13C - NMR spectrum, determine the molecular formula of this compound as C 39 H 62 O 15In the 1H-NMR (600 MHz, Pyridine-d5) spectrum, a vinylic proton signal appears in the low-field region: δH 5.51 (1H, brd, J = 5.5 Hz, H-6), and two anomeric proton signals of sugar moieties: δH 5.26 (1H, d, J = 7.9 Hz, H-1”), 4.91 (1H, d, J = 7.7 Hz, H-1'); four methyl proton signals appear in the high-field region: δH 1.15 (3H, d, J = 7.0 Hz, H-21), 1.14 (3H, s, H-19), 1.07 (3H, s, H-18), 0.71 (3H, d, J = 6.0 Hz, H-27). These four methyl proton signals are characteristic signals of the methyl protons at positions 18, 19, 21, and 27 of steroidal saponin compounds, suggesting that Compound 1 is a steroidal saponin compound; the 13C-NMR (150 MHz, Pyridine-d5) spectrum gives 39 carbon signals, a set of olefinic carbon signals: δC 139.3 (C-5), 125.2 (C-6); a set of galactose carbon signals: δC 103.1 (C-1'), 79.9 (C-4'), 75.5 (C-5'), 75.3 (C-3'), 73.5 (C-2'), 61.0 (C-6'); a set of glucose carbon signals: δC 107.1 (C-1”), 78.8 (C-3”), 78.6 (C-5”), 76.40 (C-2”), 72.4 (C-4”), 63.2 (C-6”); five oxygenated carbon signals: δC 111.9 (C-22), 81.7 (C-16), 77.9 (C-1), 75.1 (C-3), 66.1 (C-26), four methyl carbon signals: δ 17.0 (C-27), 16.9 (C-18), 14.8 (C-21), C 13.8 (C-19); further in its HMBC spectrum, δH 5.51 (H-6) has long-range correlations with δC 43.7 (C-10), 39.8 (C-4), 32.9 (C-8), indicating that the olefinic bond is located at positions 5 and 6; δH 4.91 (H-1') has a long-range correlation with δC 75.5 (C-3), indicating that galactose is located at position 3; δH 5.26 (H-1”) has a long-range correlation with δC 79.9 (C-4'), indicating that glucose is linked to the 4'-position of galactose. Therefore, the structure of the compound can be determined accordingly and named as polygonatum grandifolium saponin.

[0040] Compound 2

[0041] is a pale white powder and is readily soluble in pyridine. High-resolution mass spectrometry gives the quasi-molecular ion peak m / z 995.4442 [M+Na]. Combining with the 1H-NMR spectrum and 13C-NMR spectrum, the molecular formula of this compound is determined to be C 47 H 72 O 21In the 1H-NMR (600 MHz, Pyridine-d5) spectrum, a vinylic proton signal is given in the low-field region: δH 5.33 (1H, brd, J = 5.5 Hz, H-6), and anomeric proton signals of three sugar moieties: δH 5.35 (1H, d, J = 7.7 Hz, H-1'''), δH 5.07 (1H, d, J = 7.9 Hz, H-1''), 4.85 (1H, d, J = 7.7 Hz, H-1'); four methyl proton signals are given in the high-field region: δH 1.40 (3H, d, J = 7.0 Hz, H-21), 1.27 (3H, s, H-18), 0.90 (3H, s, H-19), 0.73 (3H, d, J = 6.0 Hz, H-27). These four methyl proton signals are characteristic signals of the methyl protons at positions 18, 19, 21, and 27 of steroidal saponin compounds. It is speculated that compound 1 is a steroidal saponin compound. The methyl proton signal of the acetyl group, δH 2.15 (3H, s), can also be seen; the 13C-NMR (150 MHz, Pyridine-d5) spectrum gives 47 carbon signals.Among them, there are two carbonyl carbon signals: δC 212.2 (C-12) and the carbonyl carbon signal of the acetyl group δC 170.5; a set of olefinic carbon signals: δC 140.6 (C-5), 121.6 (C-6); a set of galactose carbon signals: δC 101.1 (C-1'), 80.2 (C-4'), 75.6 (C-5'), 74.8 (C-2'), 72.8 (C-3'), 60.4 (C-6'); two sets of glucose carbon signals: δC 105.1 (C-1''), 83.8 (C-2''), 78.5 (C-3''), 78.5 (C-5''), 71.6 (C-4''), 63.0 (C-6'') and δC 105.8 (C-1'''), 78.5 (C-5'''), 77.7 (C-3'''), 76.4 (C-2'''), 70.6 (C-4'''), 62.2 (C-6'''); 4 oxygen-linked carbon signals: δC 111.8 (C-22), 80.3 (C-16), 78.8 (C-3), 66.1 (C-26), 5 methyl carbon signals: δC 18.8 (C-19), 16.9 (C-27), 16.1 (C-18), 13.6 (C-21) and the methyl carbon signal of the acetyl group δC 21.3; further in its HMBC spectrum, δH 5.33 (H-6) has long-range correlations with δC 39.2 (C-4), C 37.5 (C-10), 30.8 (C-8), indicating that the olefinic bond is located at positions 5 and 6; δH 3.67 (H-3) has a long-range correlation with δC 101.0 (C-1'), indicating that galactose is located at the 3-position of the parent nucleus; δH 5.07 (H-1'') has a long-range correlation with δC 80.2 (C-4'), indicating that the first glucose is linked to the 4'-position of galactose; δH 5.35 (H-1''') has a long-range correlation with δC 83.8 (C-2''), indicating that the second glucose is linked to the 2''-position of the first glucose. Also, the methyl proton signals δH 2.15 and δH 5.64 (H-2') of the acetyl group have long-range correlations with its carbonyl carbon signal δC 170.5, indicating that the acetyl group is located at the 2'-position of galactose. Therefore, the structure of the compound can be determined accordingly and named 2'-acetoxy-kingianoside G.

[0042] Compound 3

[0043] It is a pale yellowish white powder and is readily soluble in pyridine. High-resolution mass spectrometry gave a quasi-molecular ion peak of m / z 995.4444 [M+Na]. Combining with the 1H-NMR spectrum and 13C-NMR spectrum, the molecular formula of this compound was determined to be C47H72O21. Carefully observing its 1H-NMR (600 MHz, Pyridine-d5) spectrum and 13C-NMR (150 MHz, Pyridine-d5) spectrum, its signals were highly similar to those of compound 2. Combining with its HMBC spectrum, there was a long-range correlation between the methyl proton signals of the acetyl group δH 2.13, 4.77 (H-3') and its carbonyl carbon signal δC 172.8, indicating that the acetyl group was located at the 3'-position of galactose. Therefore, the structure of the compound could be determined accordingly and named 3'-acetyl-kingianoside G.

[0044] Activity test

[0045] Test materials: purified water, α-glucosidase (10 U / mg), PNPG (substrate), anhydrous sodium carbonate, DMSO, PBS (pH 6.8, 2.5 mM).

[0046] Preparation of test solutions

[0047] Preparation of the solution of the compound to be tested: Weigh 1.14 mg, 1.40 mg, and 1.72 mg of compounds 1-3 in turn, dissolve them in DMSO to form a solution of about 4.0 mM, and then serially dilute them to different concentrations in a 2-fold decreasing manner.

[0048] Preparation of the enzyme solution: Weigh 0.50 mg of the enzyme, dissolve it in 1 ml of PBS to form a stock solution, mix well, dispense, store at -20 °C, and dilute it with PBS to a working solution of 0.3 U / ml before use.

[0049] Preparation of the substrate solution: Weigh 11.30 mg of the substrate, dissolve it in PBS to prepare a 2.5 mM solution, mix well, and refrigerate.

[0050] Preparation of the termination solution: Weigh 852.00 mg of anhydrous sodium carbonate, dissolve it in water to form a 200 mM solution, and mix well.

[0051] Activity test method

[0052] Add each test solution in the order shown in the following table in a 96-well plate. After the reaction is completed, measure the absorbance at 405 nm with an enzyme-linked immunosorbent assay reader.

[0053]

[0054] Note: a. When adding DMSO, which accounts for 10% of the reaction system, the enzyme can be inactivated by 30%. Three values are measured for each group, and the RSD of the values within the group ≤ 10%.

[0055] Resurrection rate (%) = [(A sample - A sample blank) - (ADMSO - ADMSO blank)] / [(APBS - APBS blank) - (ADMSO - ADMSO blank)] × 100%.

[0056] Test results

[0057] When the drug concentration was 0.4 mM, the average values of the promotion of the recovery of α-glucosidase activity by Compounds 1 - 3 were 70.0%, 65.0%, and 61.1% respectively.

[0058] The above results indicate that Compounds 1 - 3 have very good rehabilitation benefits for the activity of damaged α-glucosidase and can be used for related diseases with reduced α-glucosidase activity.

[0059] Those skilled in the art shall process the materials in this case. For the specific processing process and operation sequence, reference should be made to the following working principle. The detailed connection means are well-known techniques in this field. The following mainly introduces the working principle and process.

[0060] Example: During the preparation process:

[0061] First, take 21.97 kg of the dried rhizome of Polygonatum sibiricum, and extract it three times by heating under reflux with 6, 5, and 5 times the amount (M / M) of 95% ethanol for 3 hours each time. Filter with gauze, combine the filtrates, and concentrate under reduced pressure at 60°C to obtain 3.18 kg of extract. Then, extract the residue with 6 and 5 times the amount (M / M) of 60% ethanol by heating under reflux twice for 2 hours each time. Filter with gauze, combine the filtrates, and concentrate in the same way to obtain 14.05 kg of extract.

[0062] Then, disperse the extract in water and extract it successively with equal volumes of petroleum ether, chloroform, ethyl acetate, and n-butanol. The 95% ethanol extract is extracted three times, and the 60% ethanol extract is extracted twice. Respective extraction liquids are combined, and the solvents are recovered under reduced pressure at 60°C to obtain each extraction part.

[0063] Then, combine the n-butanol layer extraction parts, dissolve appropriately, mix with 535 g of column chromatography silica gel, load the sample onto a column filled with 802.5 g of blank column chromatography silica gel with chloroform, and perform gradient elution with a chloroform - methanol system (volume ratio 100:0.5, 1 - 6, 8, 10, 12, 14, 17, 20, 25, 30, 50, 100; methanol). Each fraction is 1000 ml, and there are at least 15 fractions for each gradient, with a total of 261 fractions.

[0064] Finally, analysis was performed using a liquid chromatograph, and similar fractions were combined, divided into groups A to T in total. Group N (100:14, Fr.169 - 180, 9.98 g) was eluted with a methanol - water gradient (0% - 100%) using an ODS open column chromatograph. The first fraction eluted with 70% methanol (P3 - N - 70 - 1, 467.7 mg) was purified using a semi - preparative liquid chromatograph, and compounds 1 - 3 were separated using acetonitrile - water (37%, tR = 37.5, 18.9, 22.7 min) as the mobile phase.

[0065] It should be noted that in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non - exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising said element.

[0066] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application of a polygonatum compound for protecting the activity of α-glucosidase and a preparation method thereof, characterized in that: The polygonatum compounds include compound 1 (molecular formula: C 39 H 62 O 15 ), compound 2 (molecular formula: C 47 H 72 O 21 ), and compound 3 (molecular formula: C 47 H 72 O 21 ).

2. The preparation method of a polygonatum compound for protecting the activity of α-glucosidase according to claim 1, characterized in that: The preparation method includes extraction, extraction, separation and purification; The extraction: Take the dried rhizome of Polygonatum sibiricum, extract it by heating under reflux with 85-95% ethanol for 1-3 times, each time for 1-4 h, filter, combine the filtrates, recover the ethanol to obtain an extract, and then extract the medicinal residues by heating under reflux with 50%-70% ethanol for 1-2 times, each time for 1-2 h, filter, combine the filtrates, and obtain an extract in the same way; The extraction: Disperse the extract in water, and extract it successively with equal volumes of petroleum ether, chloroform, ethyl acetate, and n-butanol. The extract is extracted 2-3 times, and the extraction liquids are combined, and the solvents are recovered to obtain each extraction part; The separation: Combine the extraction parts of the n-butanol layer, dissolve appropriately, load the column with chloroform, load the sample by dry method, and elute with a chloroform-methanol system gradient (volume ratio 100:0-0:100), each fraction is 1000 ml, and each gradient has at least 15 fractions, with a total of 261 fractions; The purification: Analyze by a liquid chromatograph, combine similar fractions, divide them into groups A-T in total. Group N is eluted with a methanol-water gradient (0%-100%) using an ODS open column chromatograph, and the fractions eluted with 60-80% methanol are taken and purified by a semi-preparative liquid chromatograph, and compounds 1-3 are separated using acetonitrile water (30%-45%) as the mobile phase.

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