Preparation and application of phenylethanoid glycoside compound with anti-inflammatory activity in philippine flemingia root

Abstract

The invention belongs to the technical field of medicines, and provides two phenylethanoid glycoside compounds with anti-inflammatory activity in philippine flemingia root as well as a preparation method and application of the two phenylethanoid glycoside compounds. In particular to two novel phenylethanoid glycoside compounds extracted and separated from philippine flemingia root, such as a compound 1 (xylanchoside A) and a compound 2 (xylanchoside B) as shown in structural formulas. The compounds 1-2 have an inhibition effect on cell factors IL-6 and TNF-alpha secreted by LPS-induced macrophages RAW264.7, can form a pharmaceutical composition with a pharmaceutical carrier, and are used for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] The invention belongs to the field of Chinese medicine chemistry and pharmaceutical technology, and specifically relates to two novel phenylethanol glycoside compounds and a preparation method thereof and application thereof in the preparation of anti-inflammatory drugs. Background Art

[0002] Qianjinzhui is the dried tuber of Xylanche himalaica Hook.f.et Thoms., a plant of the Orobanchaceae family. It often parasitizes on the roots of plants of the genus Rhododendron and is mainly distributed in Yunnan, Qinghai, Tibet, Sichuan and Hubei. It is excavated in summer and autumn, and the spherical tubers are used as medicine after air-drying or sun-drying. It has the effects of dispelling wind and activating collaterals, regulating qi and strengthening the stomach. It is used for rheumatic joint pain, irregular menstruation, stomachache, abdominal distension, hernia and cough. Some ethnic minorities in Yunnan also use Qianjinzhui to treat diseases such as schistosomiasis, mumps and aconite poisoning. Qianjinzhui is a medicinal plant unique to Yunnan, and the research on its chemical composition and biological activity is very weak. In order to explore the medicinal value of Xylanchoside chinensis, the present invention takes the active part of Xylanchoside chinensis as the research object, uses the extraction and separation method of natural drug chemistry to conduct a systematic material basis study, and separates and purifies two new phenylethanol glycoside compounds, compound 1 (xylanchoside A) and compound 2 (xylanchoside B), and screens out the two compounds, which have inhibitory effects on cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can be used for the preparation of anti-inflammatory drugs. So far, there is no report on compound 1 (xylanchoside A) and compound 2 (xylanchoside B) in the prior art, nor on the pharmaceutical composition thereof as an active ingredient, nor on the application of the pharmaceutical composition thereof in the preparation of anti-inflammatory drugs. Summary of the invention

[0003] The purpose of the present invention is to provide a preparation method, a pharmaceutical composition and application of two new compounds 1 (xylanchoside A) and 2 (xylanchoside B) with medicinal value. In order to explore the drug value of Xylanchoside A, the present invention separated and purified two unpublished new phenylethanol glycoside compounds 1 and 2 from Xylanchoside A in the process of studying the material basis of the active site; Compounds 1 and 2 have an inhibitory effect on the cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can be combined with a pharmaceutical carrier to form a pharmaceutical composition for the preparation of anti-inflammatory drugs.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides two novel phenylethanoid glycoside compounds, compound 1 (xylanchoside A) and compound 2 (xylanchoside B), whose structures are shown below:

[0006]

[0007] The present invention also provides a method for preparing the novel phenylethanoid glycoside compounds 1-2 in the aforementioned Qianjinzhui, comprising the following steps:

[0008] (1) Crush the dried rhizome of the Chinese angelica root into coarse powder, add water and boil twice, adding 10 times the amount of water each time, boil for 1.5 hours, combine the decoctions, filter, and concentrate the filtrate under reduced pressure at 60-70°C to a relative density of 1.1 (60°C). Cool, add 95% ethanol to make the alcohol content reach 70%, let stand for 24 hours, filter, and concentrate the filtrate under reduced pressure to an extract with a relative density of 1.1-1.2 (60°C).

[0009] (2) The extract was mixed with silica gel and separated by silica gel column chromatography. Gradient elution was performed using chloroform-methanol in a ratio of 15:1→1:1 as the eluent. Each fraction was collected separately. The same fractions were combined under TLC monitoring to obtain 11 component segments Fr1 to Fr11.

[0010] (3) Fr8 was taken as a fraction and subjected to MCI column chromatography with a gradient elution of 10% → 40% methanol-water as the eluent. The fractions were collected separately and the same fractions were combined under TLC monitoring to obtain 13 fraction groups Fr8-1 to Fr8-13.

[0011] (4) Fraction group Fr8-13 was separated by Sephadex LH20 gel chromatography column and eluted with methanol to purify compound 1.

[0012] (5) Take the fraction Fr7, and perform gradient elution on an MCI column with 10% → 70% methanol-water as the eluent. Collect the fractions separately, and combine the same fractions under TLC monitoring to obtain 10 fraction groups Fr7.1 to Fr7.10.

[0013] (6) Fraction group Fr7.3 was mixed with silica gel and separated by silica gel column chromatography. Gradient elution was performed with dichloromethane-methanol-water (7:2.5:1→6:4:1) as the eluent. The fractions were collected separately and the same fractions were combined under TLC monitoring to obtain 8 fraction groups Fr7.3.1 to Fr7.3.8.

[0014] (7) Take fraction Fr7.3.5 and pass it through RP 18 The mixture was separated by a reverse phase chromatography column and gradient eluted with 15%→70% methanol solution. The same fractions were combined by TLC detection to obtain 9 subfraction groups Fr7.3.5.1~Fr7.3.5.9.

[0015] (8) The subfraction Fr7.3.5.5 was separated by silica gel column chromatography using 7:2.5:1 dichloromethane-methanol-water as the eluent. After TLC detection, the same fractions were combined to obtain four subfractions Fr7.3.5.5.1 to Fr7.3.5.4.

[0016] (9) The subfraction Fr7.3.5.5.4 was separated by Sephadex LH20 gel chromatography column and eluted with methanol to obtain compound 2.

[0017] In the preparation method, the hook used is Xylanche himalaica Hook.f.et Thoms. of the Orobanchaceae family.

[0018] The present invention provides a pharmaceutical composition and its preparation, which comprises the anti-inflammatory active phenylethanoid glycoside compound in the above-mentioned pounding weight or one or more of the pharmaceutically acceptable salts of the compound, as well as pharmaceutically acceptable carriers and excipients; and also comprises one or a combination of pharmaceutically acceptable carriers, excipients, and diluents. The pharmaceutical composition preparation is divided into an oral pharmaceutical composition or an injectable pharmaceutical composition according to the administration route. The present invention has no special limitation on the dosage form, and the dosage form well known in the art can be selected, including but not limited to tablets, capsule preparations, granules, sprays, sustained-release tablets, oral liquids, powders, granules or injectable preparations.

[0019] The present invention also provides the use of the phenylethanoid glycoside compound with anti-inflammatory activity in the said Qianjinzhui or the pharmaceutically acceptable salt of the compound or the said pharmaceutical composition in the preparation of anti-inflammatory drugs.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The present invention provides two new compounds - novel phenylethanol glycoside compounds, compound 1 (xylanchoside A) and compound 2 (xylanchoside B).

[0022] 2. The present invention provides a method for preparing novel phenylethanol glycoside compounds 1-2, which has readily available raw materials and is easy to operate.

[0023] 3. The present invention provides novel phenylethanoid glycoside compounds 1-2 which have an inhibitory effect on the cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can be combined with a pharmaceutical carrier to form a pharmaceutical composition for the preparation of an anti-inflammatory drug.

[0024] The present invention further enriches the chemical substance basis of the active site of Psoralea corylifolia, lays the foundation for the subsequent relevant biological activity tests of the monomer compounds obtained, provides active lead compounds for the development of new drugs, and also provides a theoretical basis for the research and development of the drugability of Psoralea corylifolia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 are the structural formulas of compound 1 and compound 2;

[0026] Figure 2 For compound 1 1 H- 1 H COSY and HMBC are related;

[0027] Figure 3 For compound 2 1 H- 1 H COSY and HMBC are related;

[0028] Figure 4 The effect of compound 1 and compound 2 on cell viability;

[0029] Figure 5 The effects of compounds 1 and 2 on the cytokines IL-1β, IL-6 and TNF-α in LPS-induced macrophage RAW264.7. DETAILED DESCRIPTION

[0030] In order to better understand the essence of the present invention, the essential content of the present invention is further illustrated below in conjunction with embodiments, but the present invention is not limited thereto.

[0031] In conjunction with the embodiments of the present invention, the technical solutions of the present invention are clearly and completely described. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1: Preparation of Compound 1 (xylanchoside A) and Compound 2 (xylanchoside B)

[0033] (1) Medicinal material crushing

[0034] 17 kg of Qianjinzhui medicinal materials were crushed into coarse powder by a CSJ-60 coarse crusher.

[0035] (2) Preparation of active sites

[0036] The coarse powder of Qianjinzhui was decocted twice with water, adding 10 times the amount of water (170L) each time, and decocted for 1.5 hours. The decoction was filtered through a 200-mesh filter, and the water decoction was combined. The filtrate was concentrated under reduced pressure at 60-70℃ to a relative density of 1.1 (60℃), cooled, and 95% ethanol was added to make the alcohol content reach 70%, allowed to stand for 24 hours, filtered, and the filtrate was concentrated under reduced pressure to 6.5 kg of extract with a relative density of 1.1-1.2 (60℃), with a yield of 38.2%.

[0037] (3) Segmentation of silica gel chromatography column

[0038] Take 2 kg of the extract of the Psoralea corylifolia in step 2, mix it with silica gel (100-200 mesh), separate it by silica gel (100-300 mesh) column chromatography, and use 15:1→1:1 dichloromethane-methanol or chloroform-methanol as the eluent for gradient elution. Collect each fraction separately, and combine the same fractions under TLC monitoring to obtain 11 component segments, which are named Fr1~Fr11 respectively.

[0039] (4) Isolation and purification of compound 1

[0040] 200 g of the extract of the component segment Fr8 obtained in step 3 was taken and subjected to MCI column chromatography with 10%→40% (v / v) methanol-water as the eluent for gradient elution. The fractions were collected separately and the same fractions were combined under TLC monitoring to obtain 13 fraction groups, which were named Fr8-1 to Fr8-13 respectively. 8.23 ​​g of the extract of the fraction group Fr8-13 was taken and separated by Sephadex LH20 gel chromatography column and eluted with methanol to purify and obtain compound 1 (50 mg).

[0041] (5) Isolation and purification of compound 2

[0042] 324.51 g of the extract of the component segment Fr7 obtained in step 3 was taken, and gradient eluted with 10%→70% methanol-water as the eluent by MCI column chromatography, and each fraction was collected separately. The same fractions were combined by TLC monitoring to obtain 10 fraction groups, which were named Fr7.1~Fr7.10 respectively; 200 g of the extract of the fraction group Fr7.3 was taken, mixed with silica gel (100~200 mesh), and separated by silica gel (100~300 mesh) column chromatography, and gradient eluted with 7:2.5:1→6:4:1 (v / v / v) of dichloromethane-methanol-water as the eluent, and each fraction was collected separately. The same fractions were combined by TLC monitoring to obtain 8 fraction groups, which were named Fr7.3.1~Fr7.3.8 respectively; 400 g of the extract of the fraction group Fr7.3.5 was taken, and the sample was mixed with silica gel (100~200 mesh), and the sample was separated by silica gel (100~300 mesh) column chromatography, and the gradient elution was carried out with 7:2.5:1→6:4:1 (v / v / v) of dichloromethane-methanol-water as the eluent. The fractions were collected separately, and the same fractions were combined by TLC monitoring to obtain 8 fraction groups, which were named Fr7.3.1~Fr7.3.8 respectively. g, separated by RP18 reverse phase chromatography column, gradient eluted with 15%→70% methanol solution, TLC detection and combined the same fractions to obtain 9 sub-fraction groups, named Fr7.3.5.1~Fr7.3.5.9; 11 g of sub-fraction group Fr7.3.5.5 extract was mixed with silica gel (100-200 mesh), separated by silica gel (100-300 mesh) column chromatography, eluted with 7:2.5:1 (v / v / v) dichloromethane-methanol-water as eluent, TLC detection and combined the same fractions to obtain 4 secondary sub-fraction groups, named Fr7.3.5.5.1~Fr7.3.5.4; secondary sub-fraction group Fr7.3.5.5.4 (210 mg) was separated by Sephadex LH20 gel chromatography column, eluted with methanol, and purified to obtain compound 2 (24.1 mg).

[0043] Example 2 Structural identification of new compounds

[0044] (1) Structural identification of compound 1

[0045]

[0046] xylanchoside A

[0047] Molecular formula: C 37 H 48 O 21

[0048] Appearance: Light yellow oil

[0049] HRESI-MS m / z: 851.2585 [M+Na] + (Calculated value: C 37 H 48 O 21 Na, 851.2580)

[0050] Compound 1, light yellow oily substance, according to positive ion HRESI-MS m / z 851.2585 (calculated value 851.2580 [M+Na] + )and 13 C NMR data confirmed the molecular formula to be C 37 H 48 O 21 Combined with HSQC, the 1 H and 13 C NMR (Table 2) showed three typical sugar end group signals [4.47 (d, J = 8.1 Hz, H-1″), 4.80 (dd, J = 7.8,6.5 Hz, H-1″′), 4.57 (d, J = 7.8 Hz, H-1″′′); δ C 101.83 (C-1″), 102.50 (C-1″′), 105.58 (C-1″′′)] and the characteristic signal of rhamnose at position 6 [1.31 (d, J = 6.2 Hz, H-6″′, δ C 18.16 (C-6″′)], 1 acetyl [δ H 1.97 (d, J = 4.2 Hz); δ C 20.86, 171.25], two groups of aromatic signals of ABX spin system [δ H 6.62 (d, J = 1.9 Hz, H-2), 6.65 (d, J = 8.0 Hz, H-5), 6.50 (dd, J =8.0, 1.9 Hz, H-6), δ H 7.05 (d, J = 1.9 Hz, H-2′), 6.79 (t, J = 8.3 Hz, H-5′), 6.92 (dd, J = 8.3, 1.9 Hz, H-6′); δ C 117.19 (C-2), 116.27 (C-5), 121.16 (C-6), 115.03 (C-2′), 116.53 (C-5′), 123.14 (C-6′)], a group of methylene signals [δ H 2.68 (dd, J =10.9, 6.1 Hz, H-7; δ C 36.29 (C-7)], a group of oxymethylene signals [δ H 3.99 (dd, J = 6.1, 3.5Hz, H-8a), 3.61 (m, H-8b), δ C71.84 (C-8)], a group of trans-olefin signals [6.30 (d, J = 15.9 Hz, H-7′), 7.57 (d, J = 15.9 Hz, H-8′); δ C 114.73 (C-7′), δ C 147.72 (C-8′)] and one keto carbonyl group [δ C 169.05 (C-9′)]. Based on the above information, it is speculated that compound 1 is a typical phenylethanol glycoside compound. 1 H and 13 The C NMR data were carefully compared. The only difference was that 80.21 (C-3″) and 62.14 (C-6″) in Monoacetylrossicaside A shifted downfield to 82.14 (C-3″) and 64.25 (C-6″), suggesting that the position of the caffeoyl group may have changed. In the HMBC spectrum, H-6″ (δ H 4.36, 4.50) is related to C-9′(169.05), indicating that the caffeoyl group is connected to glucose C-6″, confirming the above speculation. In summary, the structure of compound 1 is a new compound that has not been reported in the literature and is named xylanchoside A. 1 H NMR and 13 C NMR (DEPT) data are shown in Table 1.

[0051]

[0052] (2) Structural data of compound 2.

[0053]

[0054] Xylanchoside B

[0055] Molecular formula: C 29 H 44 O 18

[0056] Appearance: White solid

[0057] HRESI-MS m / z: 703.2414 [M+Na] + , (calculated value: C 20 H 44 O 18 Na, 703.2420)

[0058] Compound 2, white solid, was identified by positive ion HR-ESI-MS (m / z 703.2414 [M+Na]+ , calculated value: 703.2420) to determine the molecular formula as C 29 H 44 O 18 , the unsaturation is 8. 1 H and 13 C NMR spectrum (Table 2) shows a typical ABX spin system [δ H 6.67 (d, J = 2.1 Hz, H-3), 6.63 (dd, J = 2.1, 8.2 Hz, H-5), 6.80 (d, J = 8.2 Hz, H-6); δ C 117.1 (C-3), 121.2 (C-5), 112.8 (C-6) ], 1 oxymethyl [δ H 3.81(s); δ C 56.5 (2-OCH3)], 1 acetyl group [δ H 1.97 (s); δ C 20.9, 171.6], a group of methylene signals [δ H 2.70 (m, H-7; δ C 36.3 (C-7)], a group of oxymethylene signals [δ H 3.63 (m, H-8a), 4.05 (m, H-8b), δ C 71.5 (C-8)], 3 terminal sugar signals [δ H 4.44 (d, J = 8.1 Hz, H-1'), 4.78 (d, J =1.6 Hz, H-1"), 4.56 (d, J = 7.8 Hz, H-1‴); δ C 101.7 (C-1'), 102.6(C-1"), 105.6(C-1‴)]. From the above information, it can be seen that compound 2 is a typical phenylethanol glycoside compound. HMBC spectrum ( Figure 3 ), H-1' and C-8 (δ C 71.5) and H-1" and C-3' (δ C 82.7), H-1‴ and C-4" (δ C 83.3), indicating that the sugar chain is connected at C-8, rhamnose is connected at C-3', and glucose is connected at C-4". In addition, H-1' and H-2' [δ H 4.75 (m)] and δ C171.6 correlation, indicating that the acetyl group is connected to the C-2' position. The coupling constant J >7.0 Hz determined that glucose was in the β configuration; in addition, rhamnose was determined to be in the α configuration by comparing the C-3 and C-5 chemical shifts of α-L-Rha and β-L-Rha. In summary, the structure of compound 2 was determined to be a new phenylethanol glycoside compound and was named xylanchoside B. 1 H NMR and 13 C NMR (DEPT) data are shown in Table 2.

[0059]

[0060] Example 3: In vitro anti-inflammatory activity of compounds 1-2 in macrophage RAW264.7

[0061] (1) CCK8 assay to detect the effects of compounds 1-2 on cell survival rate

[0062] RAW264.7 cells were routinely cultured in DMEM medium. When the cells grew to 80% confluence and the cell morphology was normal under microscope, the cells were plated at 2×10 5 The cells were inoculated at a density of 100 μL / well in a 96-well plate. After overnight culture in a carbon dioxide incubator, the supernatant in the wells was discarded, and different concentrations of compound 1 and compound 2 sample solutions (200, 100, 50, 25, 12.5 μg / mL) were added to treat the cells, 100 μL per well, 6 wells for each concentration, and the blank control group was added with the same volume of DMEM and cultured in a carbon dioxide incubator. After 24 hours, 10 μL of CCK-8 reagent solution was added to each well. After 4 hours of culture in a carbon dioxide incubator, the absorbance was detected at a wavelength of 450nm with an enzyme reader to calculate the cell survival rate. The results are shown in Figure 4 As shown: The maximum non-toxic dose of compound 1 and compound 2 to RAW264.7 cells is 50 μg / mL.

[0063] (2) ELISA test to detect the effects of compounds 1-2 on LPS-induced inflammatory indicators of macrophage RAW264.7

[0064] RAW264.7 cells were routinely cultured in DMEM medium. When the cells grew to 80% confluence and the cell morphology was normal under microscope, the cells were plated at 2×10 5Cells were inoculated at a density of 100 μg / mL in a 6-well plate, 2 mL / well, and the supernatant in the wells was discarded after overnight culture in a carbon dioxide incubator. Compounds 1 and 2 were added as high- and low-dose groups at a concentration of 50 and 25 μg / mL (diluted by DMEM, and the concentration was confirmed by the results of the cytotoxicity test). Lipopolysaccharide (LPS) was added for modeling at a concentration of 200 ng / ml, 1 mL per well, and 3 wells for each concentration. The blank control group was added with the same volume of DMEM, and the model control group was added with a DMEM solution containing the same concentration of LPS, and cultured in a carbon dioxide incubator. After 24 hours, the cell supernatant was aspirated and IL-1β, IL-6, and TNF-α were detected using a kit. The results are shown in Figure 5 As shown: Combined with the comprehensive effects on the three inflammatory mediators, compounds 1 to 2 can reduce the concentrations of IL-1β, IL-6 and TNF-α in the supernatant of RAW264.7 cells induced by LPS to varying degrees, suggesting that they have anti-inflammatory effects.

[0065] Example 4: New compound pharmaceutical preparation

[0066] (1) 5.0 g of compound 1 or / and compound 2, 5.0 g of microcrystalline cellulose, and 14.7 g of compressible starch were mixed evenly, and an appropriate amount of 2.5% polyethylene glycol was added, mixed evenly, and spherical micropellets were prepared by extrusion and spheronization. The micropellets were put into capsules to prepare capsules;

[0067] (2) Grind compound 1 and / or compound 2 into fine powder, pass through a 100-mesh sieve, take 5.0 g of the powder, mix with 16.0 g of polyethylene oxide and 3.0 g of sodium chloride, pass through a 100-mesh sieve, add 2% povidone (95% ethanol) as a binder to prepare a soft material, pass through a 20-mesh sieve to prepare granules, dry at 40°C, pass through an 18-mesh sieve to prepare granules, press into tablets, and coat with a semi-permeable membrane using cellulose acetate as a skeleton material and polyethylene glycol as a pore-forming agent (10% of the coating solid amount) to prepare sustained-release tablets.

[0068] (3) Compound 1 and / or Compound 2 are taken and prepared into an oral liquid according to a conventional oral liquid preparation method.

[0069] It can be seen from the above examples that the present invention provides two novel phenylethanoid glycoside compounds 1 (xylanchoside A) and compound 2 (xylanchoside B) and preparation methods thereof; compounds 1 and 2 have inhibitory effects on cytokines IL-6 and TNF-α secreted by LPS-induced macrophages RAW264.7, and can be combined with pharmaceutical carriers to form a pharmaceutical composition for the preparation of anti-inflammatory drugs.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A phenylethanol glycoside compound with anti-inflammatory activity in the herb, characterized in that: The compound is compound 1 xylanchoside A or compound 2 xylanchoside B, and its chemical structure is as follows: .

2. A pharmaceutical composition, characterized in that: It comprises one or more of the phenylethanoid glycoside compounds according to claim 1 or their pharmaceutically acceptable salts.

3. A pharmaceutical preparation, characterized in that The invention comprises a therapeutically effective amount of the pharmaceutical composition according to claim 2, and a pharmaceutically acceptable carrier and excipient.

4. The pharmaceutical preparation according to claim 3, characterized in that The pharmaceutically acceptable carrier is selected from one or more of a filler, a disintegrant, a binder and a lubricant.

5. The pharmaceutical preparation according to claim 3, characterized in that The pharmaceutical preparation is a tablet, capsule preparation, granule, spray, sustained-release tablet, oral liquid, powder, granule or injection preparation.

6. Use of the phenylethanol glycoside compound or its pharmaceutically acceptable salt, pharmaceutical composition and preparation thereof having anti-inflammatory activity in the weight according to any one of claims 1 to 3 in the preparation of anti-inflammatory drugs.

7. The method for preparing the phenylethanoid glycoside compounds with anti-inflammatory activity in the weight according to claim 1, characterized in that: The preparation method of the compound comprises the following steps: (1) Take the medicinal material of Qianjinzhui, crush it, add 10 times the amount of water and boil it twice, each time for 1.5 hours, filter it, and concentrate the filtrate under reduced pressure at 60-70°C to a relative density of 1.1, cool it, add 95% ethanol to make the alcohol content reach 70%, let it stand, filter it, and concentrate the filtrate under reduced pressure to obtain an extract; take the extract and perform silica gel column chromatography separation, and use a chloroform-methanol volume ratio v / v of 15:1 to 1:1 as gradient elution to obtain component segments Fr1 to Fr11; (2) Component segment Fr8 was subjected to MCI column chromatography and eluted with a methanol-water gradient to obtain fraction groups Fr8-1 to Fr8-13; fraction group Fr8-13 was purified by Sephadex LH20 gel column chromatography to obtain compound 1; (3) Fraction Fr7 was subjected to silica gel column chromatography and gradient elution with dichloromethane-methanol-water (v / v / v) of 7:2.5:1 to 6:4:1 to obtain fractions Fr7.3.1 to Fr7.3.

8. Fraction Fr7.3.5 was subjected to RP 18 After separation by reverse phase chromatography column, 9 subfraction groups Fr7.3.5.1-Fr7.3.5.9 were obtained by gradient elution with methanol-water; subfraction group Fr7.3.5.5 was purified by silica gel column chromatography and sephadex LH-20 gel column chromatography to obtain compound 2.

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