Suaeda salsa small-molecule compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation of traditional Chinese medicine components, relates to a small molecule compound separated and extracted from suaeda glauca, and particularly relates to a flavonoid compound as well as a preparation method and application thereof. According to the invention, 13 small molecule compounds are separated from rhizome of suaeda glauca, and biflavone compounds I-1, I-2 and I-3 and an isoflavone compound II-1 are new compounds reported for the first time at home and abroad; the biflavone compounds I-4 and I-5, the five alkaloid compounds III-1 to III-5 and the isoflavone compound II-3 are obtained by being separated from a suaeda glauca plant for the first time. The five biflavone compounds I <-1 > to I <-5 > and the five alkaloid compounds III <-1 > to III <-5 > prepared by the invention have heart protection activity; the three isoflavone compounds II-1 to II-3 have relatively good activity of inhibiting the growth of tumor cells.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of traditional Chinese medicine ingredients, relates to flavonoids and alkaloid small molecule compounds separated and extracted from Suaeda salsa, and specifically relates to a Suaeda salsa small molecule compound and a preparation method and application thereof. Background Art

[0002] Cardiovascular disease (CVD) and cancer are major diseases that seriously endanger the health and life of the Chinese people. Research data show that CVD and cancer rank first in the proportion of disease deaths among urban and rural residents in my country. Therefore, finding anti-heart failure and anti-tumor drugs with definite efficacy and few side effects is crucial to improving the quality of life of patients.

[0003] The history of the medicinal use of Suaeda glaucu Bunge was first recorded in the early Ming Dynasty's "Jiuhuang Bencao": "The leaves are slightly salty and slightly cold in nature." The Qing Dynasty's "Compendium of Materia Medica" also cited "Yao Xing Kao" to record its efficacy of "clearing heat and eliminating accumulation." Contemporary works such as "Zhonghua Bencao", "Zhongyao Cihai" and "Zhongyao Da Cidian" also detailed its efficacy, but none of them recorded the use of Suaeda glaucu Bunge in the prevention and treatment of cardiovascular and cerebrovascular diseases or tumor inhibition activity, let alone reports on the prevention and treatment of cardiovascular and cerebrovascular diseases or tumor inhibition activity of Suaeda glaucu Bunge extracts.

[0004] At present, the research on Suaeda salsa mainly focuses on its ecological protection and nutritional value. The research on the small molecule chemical components of Suaeda salsa is insufficient, and very few small molecule components have been reported in the literature. Among them, Qiu Pingcai and others from the Jiangsu Institute of Botany, Chinese Academy of Sciences, separated and purified the ethyl acetate part of Suaeda salsa and obtained 10 compounds, which were identified as n-tetradecanoic acid, β-amyrin-n-nonyl ether, β-sitosterol, β-carotene, quercetin, luteolin, luteolin-7-O-β-D-glucoside, isorhamnetin, scopoletin, and stigmasterol. Master Lu Mengdi from Jinzhou Medical University used the roots of Suaeda salsa as the experimental object and isolated 4 chemical components from the extract, which were identified as berberine, (-)-episyringaresinol, scopolamine, and 7S,8R-dihydrodehydroconiferyl alcohol. The antioxidant activity of the four compounds was studied through in vitro antioxidant experiments.

[0005] Chinese patent CN109232756A discloses a method for preparing a non-uniform component of Suaeda polysaccharide, and the obtained non-uniform component of Suaeda polysaccharide can be used to prepare drugs for anti-tumor, immunomodulation or treatment of type 2 diabetes. Patent CN101040711A discloses a new use of a Suaeda-based plant extract in the preparation of health foods with weight loss and lipid-lowering effects. Patent CN118878710A discloses a method for extracting and using Suaeda polysaccharide, and for the first time explores the great potential of Suaeda polysaccharide in cardioprotection, providing a scientific basis for the future clinical application of Suaeda polysaccharide. Patent CN110075137A discloses a method for extracting Suaeda flavonoids from supercritical carbon dioxide, which produces a flavonoid extract, but the specific compound structure is not separated. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and a small molecule compound of Suaeda salsa and a preparation method and application thereof are proposed. 13 small molecule compounds are separated from the rhizomes of Suaeda salsa, among which biflavonoid compounds Ⅰ-1, Ⅰ-2, Ⅰ-3 and isoflavone compound Ⅱ-1 are new compounds reported for the first time at home and abroad; biflavonoid compounds Ⅰ-4 and Ⅰ-5, 5 alkaloid compounds Ⅲ-1 to Ⅲ-5 and isoflavone compound Ⅱ-3 are separated from Suaeda salsa for the first time.

[0007] The technical solution of the present invention is:

[0008] The present invention protects a flavonoid compound, the general structural formula of which is shown in formula (I) or formula (II):

[0009]

[0010] In formula (I), R 1 and R 2 Each of them is independently selected from one or more of hydrogen, hydroxyl and methoxy;

[0011] In formula (II), R 3 and R 4 They are independently selected from one or more of hydrogen, hydroxyl and methoxy.

[0012] Furthermore, when R 1 =H, R 2 =OH, the compound structure is shown in formula (I-1):

[0013]

[0014] When R 1 =H, R 2 =OCH 3 When, the compound structure is shown in formula (I-2):

[0015] When R 1 =OCH 3 , R 2 =OH, the compound structure is shown in formula (I-3):

[0016] When R 1 =OH, R 2 =OH, the compound structure is shown in formula (I-4):

[0017] When R 1 =H, R 2 =H, the compound structure is shown in formula (I-5):

[0018]

[0019] Furthermore, when R 3 =OH, R 4 =OCH 3 When, the compound structure is shown in formula (II-1):

[0020] When R 3 =OCH 3 , R 4 =OCH 3 When, the compound structure is shown in formula (II-2):

[0021]

[0022] When R 3 =OH, R 4 =H, the compound structure is shown in formula (II-3):

[0023]

[0024] The present invention also protects a method for preparing the flavonoid compound described in any one of the above items, comprising the following steps:

[0025] (1) crushing Suaeda salsa, extracting with 95% ethanol under reflux, combining the extracts, and concentrating to obtain Suaeda salsa ethanol extract; extracting the Suaeda salsa ethanol extract with petroleum ether, ethyl acetate, and n-butanol solutions in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, an n-butanol fraction, and a water fraction;

[0026] (2) Using petroleum ether-acetone with a volume ratio of 1:0, 80:1, 50:1, 20:1, 10:1, 6:1, 4:1, 1:1, and 0:1 as the mobile phase, the ethyl acetate fraction sample was gradient eluted using a silica gel column to obtain nine components, Fr.B1 to Fr.B9;

[0027] The Fr.B6 component was eluted using an LH-20 type dextran gel column, and the eluent was methanol. The similar fractions in the obtained Fr.B6.1 to Fr.B6.27 fractions were combined and purified using methanol-water with a volume ratio of 60% to 100%, and compounds II-1 and II-3 were purified from the Fr.B6.25 to Fr.B6.27 fractions;

[0028] The Fr.B8 component was eluted using an LH-20 type dextran gel column, and the eluent was methanol. The similar fractions in the obtained Fr.B8.1 to Fr.B8.65 fractions were combined and purified using methanol-water with a volume ratio of 60% to 100%, and compound II-2 was purified from the Fr.B8.18 to Fr.B8.20 fractions;

[0029] (3) Using ethanol-water with a volume ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 80%, and 100% as the mobile phase, the n-butanol fraction sample was gradient eluted using an HP-20 macroporous adsorption resin column to obtain nine components, Fr.C1 to Fr.C9;

[0030] The Fr.C5 component was eluted using an LH-20 type dextran gel column, with methanol as the eluent, and similar fractions from the obtained Fr.C5.1 to Fr.C5.57 fractions were combined and purified using 30% to 100% methanol-water by volume, and compounds Ⅰ-2 and Ⅰ-3 were purified from the Fr.C5.38 to Fr.C5.40 fractions; compounds Ⅰ-1 and Ⅰ-4 were purified from the Fr.C5.41 to Fr.C5.48 fractions;

[0031] The Fr.C6 component was eluted using an LH-20 type dextran gel column with methanol as the eluent. Similar fractions from the obtained Fr.C6.1 to Fr.C6.52 fractions were combined and purified using methanol-water with a volume ratio of 30% to 100%, and compound I-5 was purified from the Fr.C6.31 to Fr.C6.35 fractions.

[0032] Furthermore, according to the HPLC-UV detection results, similar fractions in the eluted fractions were combined and then purified on a C18 high pressure preparative chromatography column.

[0033] The present invention also protects an alkaloid compound obtained by the above preparation method, wherein the alkaloid compound is extracted from a n-butanol fraction, and a HP-20 macroporous adsorption resin column is used to perform gradient elution on the n-butanol fraction sample using ethanol-water with a volume ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 80%, and 100% as a mobile phase to obtain nine components of Fr.C1 to Fr.C9;

[0034] The Fr.C2 component was eluted using an LH-20 type dextran gel column, and the eluent was a 20% methanol-water solution to obtain Fr.C2.1 to Fr.C2.40 fractions. According to the HPLC-UV detection results, similar fractions were combined into a bottle and purified on a C18 type high pressure preparative chromatography column using 30% to 100% methanol-water by volume to purify compounds III-1 and III-2 from Fr.C2.16 to Fr.C2.20 fractions;

[0035] The Fr.C3 component was eluted using an LH-20 type dextran gel column, and the eluent was pure methanol to obtain Fr.C3.1 to Fr.C3.23 fractions. According to the HPLC-UV detection results, similar fractions were combined into a bottle and purified on a C18 type high pressure preparative chromatographic column using 30% to 100% methanol-water by volume, and compound III-4 was purified from the Fr.C3.15 to Fr.C3.18 fractions; compound III-5 was purified from the Fr.C3.19 to Fr.C3.23 fractions;

[0036] The Fr.C6 component was eluted using an LH-20 type dextran gel column, and the eluent was pure methanol to obtain Fr.Fr.C6.1 to Fr.C6.52 fractions. According to the HPLC-UV detection results, similar fractions were combined into a bottle and purified on a C18 type high pressure preparative chromatography column using 30% to 100% methanol-water by volume, and compound III-3 was purified from Fr.C6.19 to Fr.C6.23 fractions;

[0037]

[0038] The present invention protects the use of the flavonoid compounds in preparing drugs for treating cardiovascular diseases or improving heart damage.

[0039] The present invention protects the use of the flavonoid compounds in the preparation of anti-tumor drugs.

[0040] The present invention protects the use of the alkaloid compounds in preparing drugs for treating cardiovascular diseases or improving heart damage.

[0041] A large number of literatures show that the zebrafish model has unique advantages in screening cardioprotective active ingredients. The zebrafish heart is highly similar to the human heart in structure, function, signaling pathways and ion channels; the lipid composition and metabolism are basically the same as those of humans; and the embryos and their larvae are almost transparent, the cardiovascular system is visible, and the heartbeat, angiogenesis and vascular lesions can be observed in vivo. Based on this, the present invention uses the zebrafish model to screen small molecule compounds extracted and separated from Suaeda salsa, and evaluates its effect on improving heart failure caused by verapamil, providing a scientific basis for its clinical application.

[0042] Beneficial effects of the present invention:

[0043] The five biflavonoid compounds Ⅰ-1, Ⅰ-2, Ⅰ-3, Ⅰ-4 and Ⅰ-5 and the five alkaloid compounds Ⅲ-1, Ⅲ-2, Ⅲ-3, Ⅲ-4 and Ⅲ-5 prepared by the present invention can improve the zebrafish heart damage induced by verapamil hydrochloride at a concentration of 50 μM, indicating that the compounds have anti-cardiac damage activity, that is, have cardioprotective activity.

[0044] The three isoflavone compounds II-1, II-2 and II-3 prepared by the present invention showed toxicity to zebrafish at a concentration of 50 μM, and therefore, their LC50 values ​​were tested, which were 9.41, 39.59 and 24.47 μM, respectively. Based on the above toxicity, it is speculated that compounds II-1, II-2 and II-3 will also have a strong effect on tumor cells. Therefore, at concentrations of 30 and 50 μM, the inhibitory effects of these three compounds on HCT116 cells were detected, and it was found that these three compounds did have good activity in inhibiting the growth of tumor cells, among which compound II-1 had a substantially equivalent effect to the positive drug 5-fluorouracil. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a flow chart for the preparation of the compound;

[0046] Figure 2 is the HR-ESI-MS spectrum of compound Ⅰ-1 in CH3OH solution;

[0047] Figure 3 Compound Ⅰ-1 in DMSO-d 6 1HNMR spectrum in solution (600 MHz);

[0048] Figure 4 Compound Ⅰ-1 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0049] Figure 5 Compound Ⅰ-1 in DMSO-d 6HSQC spectrum in solution (600 MHz);

[0050] Figure 6 Compound Ⅰ-1 in DMSO-d 6 HMBC spectrum in solution (600 MHz);

[0051] Figure 7 Compound Ⅰ-2 in CH 3 HR-ESI-MS spectra in OH solution;

[0052] Figure 8 Compound Ⅰ-2 in DMSO-d 6 In solution 1 HNMR spectrum (600 MHz);

[0053] Fig. 9 Compound Ⅰ-2 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0054] Fig.10 Compound Ⅰ-2 in DMSO-d 6 HSQC spectrum in solution (600 MHz);

[0055] Fig.11 Compound Ⅰ-2 in DMSO-d 6 HMBC spectrum in solution (600 MHz);

[0056] Fig.12 Compound Ⅰ-3 in CH 3 HR-ESI-MS spectra in OH solution;

[0057] Fig.13 Compound Ⅰ-3 in DMSO-d 6 In solution 1 HNMR spectrum (600 MHz);

[0058] Fig.14 Compound Ⅰ-3 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0059] Fig.15 Compound Ⅰ-3 in DMSO-d 6 HSQC spectrum in solution (600 MHz);

[0060] Fig.16 Compound Ⅰ-3 in DMSO-d 6 HMBC spectrum in solution (600 MHz);

[0061] Fig.17 Compound Ⅰ-4 in CH 3 HR-ESI-MS spectra in OH solution;

[0062] Fig.18 Compound Ⅰ-4 in DMSO-d 6 In solution 1 HNMR spectrum (600 MHz);

[0063] Fig.19 Compound Ⅰ-4 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0064] Fig. 20 Compound Ⅰ-4 in DMSO-d 6 HSQC spectrum in solution (600 MHz);

[0065] Fig.21 Compound Ⅰ-4 in DMSO-d 6 HMBC spectrum in solution (600 MHz);

[0066] Fig. 22 Compound Ⅰ-5 in CH 3 HR-ESI-MS spectra in OH solution;

[0067] Fig.23 Compound Ⅰ-5 in DMSO-d 6 In solution 1 HNMR spectrum (600 MHz);

[0068] Fig.24 Compound Ⅰ-5 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0069] Fig.25 Compound II-1 in CH 3 HR-ESI-MS spectra in OH solution;

[0070] Fig.26 Compound II-1 in DMSO-d 6 In solution 1 HNMR spectrum (600 MHz);

[0071] Fig. 27 Compound II-1 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0072] Fig.28 Compound II-1 in DMSO-d 6 HSQC spectrum in solution (600 MHz);

[0073] Fig.29 Compound II-1 in DMSO-d 6 HMBC spectrum in solution (600 MHz);

[0074] Fig.30 Compound II-2 in CH 3 HR-ESI-MS spectra in OH solution;

[0075] Fig.31 Compound II-2 in DMSO-d 6 In solution 1 HNMR spectrum (600 MHz);

[0076] Fig.32 Compound II-2 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0077] Fig.33 Compound II-3 in CH 3 HR-ESI-MS spectra in OH solution;

[0078] Fig.34 Compound II-3 in DMSO-d 6 In solution 1 HNMR spectrum (600 MHz);

[0079] Fig.35 Compound II-3 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0080] Fig.36 Compound Ⅲ-1 in CH 3 HR-ESI-MS spectra in OH solution;

[0081] Fig.37 Compound III-1 in DMSO-d 6 In solution 1 H NMR spectrum (600 MHz);

[0082] Fig.38 Compound III-1 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0083] Fig.39 Compound III-1 in DMSO-d 6 HSQC spectrum in solution (600 MHz);

[0084] Fig.40 Compound III-1 in DMSO-d 6 HMBC spectrum in solution (600 MHz);

[0085] Fig.41 Compound Ⅲ-2 in CH 3 HR-ESI-MS spectra in OH solution;

[0086] Fig.42 Compound III-2 in DMSO-d 6 In solution 1 H NMR spectrum (600 MHz);

[0087] Fig.43 Compound III-2 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0088] Fig.44 Compound Ⅲ-3 in CH 3 HR-ESI-MS spectra in OH solution;

[0089] Fig.45 Compound III-3 in DMSO-d 6 In solution 1 H NMR spectrum (600 MHz);

[0090] Fig.46 Compound III-3 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0091] Fig.47 Compound Ⅲ-4 in CH 3 HR-ESI-MS spectra in OH solution;

[0092] Fig.48 Compound III-4 in DMSO-d 6 In solution 1 H NMR spectrum (600 MHz);

[0093] Fig.49 Compound III-4 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0094] Fig.50 Compound Ⅲ-5 in CH 3 HR-ESI-MS spectra in OH solution;

[0095] Fig.51 Compound III-5 in DMSO-d 6 In solution 1 H NMR spectrum (600 MHz);

[0096] Fig.52 Compound III-5 in DMSO-d 6 In solution 13 C NMR spectrum (125 MHz);

[0097] Fig.53 The anti-heart failure activities of 5 biflavonoid compounds and 5 alkaloid compounds; (A) Typical new reports of edema and venous congestion in each group of zebrafish; (B) Statistical graph of pericardial edema area; (C) Statistical graph of venous congestion area;

[0098] Fig.54 is the lethal concentration curve of compound II-1 to normal zebrafish;

[0099] Fig.55 is the lethal concentration curve of compound II-2 to normal zebrafish;

[0100] Fig.56 is the lethal concentration curve of compound II-3 to normal zebrafish;

[0101] Fig.57 The growth inhibitory activity of compounds II-1-II-3 on HCT-116 cells. DETAILED DESCRIPTION

[0102] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, 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.

[0103] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0104] Example 1

[0105] like Figure 1As shown, this embodiment provides a method for extracting and separating active ingredients from Suaeda salsa, specifically a method for preparing 13 small molecule compounds, including 5 biflavonoid compounds, 3 isoflavone compounds and 5 alkaloid compounds, and the steps are as follows:

[0106] Weigh 30 kg of dried rhizomes of Suaeda salsa, crush them, and extract them with 95% ethanol reflux for 3 times, each time for 2 hours. Combine the extracts, and recover the solvent under reduced pressure to obtain 8 kg of Suaeda salsa ethanol extract. The extract is placed in 30 L of distilled water and fully dispersed, and extracted with petroleum ether, ethyl acetate and n-butanol solution in turn to obtain petroleum ether fraction (Fr.A, 420 g), ethyl acetate fraction (Fr.B, 680 g), n-butanol fraction (Fr.C, 560 g) and water fraction (Fr.D, 340 g).

[0107] 680g of ethyl acetate fraction sample was separated on a silica gel column, and gradient elution was performed using petroleum ether-acetone as the mobile phase, and the volume ratios of the mobile phases were 1:0, 80:1, 50:1, 20:1, 10:1, 6:1, 4:1, 1:1, and 0:1, respectively. After the eluate was concentrated under reduced pressure, Fr.B1 fraction (15.7g), Fr.B2 fraction (14.5g), Fr.B3 fraction (16.8g), Fr.B4 fraction (36.7g), Fr.B5 fraction (33.6g), Fr.B6 fraction (37.3g), Fr.B7 fraction (41.5g), Fr.B8 fraction (44.3g), and Fr.B9 fraction (38.6g) were obtained, respectively.

[0108] Fr.B6 fraction (37.3 g) was eluted on a LH-20 type dextran gel column (85 cm × 5 cm), and the eluent was a methanol solution, with 50 mL per bottle, and a total of 27 bottles of fractions (Fr.B6.1 to Fr.B6.27) were obtained. According to the HPLC-UV detection results, similar fractions were combined and purified on a C18 type high pressure preparative chromatography column [methanol / water (60% to 100%, v / v)]. Among them, Fr.B6.25 to Fr.B6.27 sub-fractions were eluted by gradient elution to obtain compounds II-1 (8.4 mg) and II-3 (12.6 mg).

[0109] The Fr.B8 fraction was eluted on a LH-20 type dextran gel column (85cm×5cm), and the eluent was a pure methanol solution, with 50mL per bottle, and a total of 65 bottles of fractions (Fr.B8.1~Fr.B8.65) were obtained. According to the HPLC-UV detection results, similar fractions were combined and purified on a C18 type high pressure preparative chromatography column [methanol / water (60%~100%, v / v)]. Among them, the Fr.B8.18~Fr.B8.20 sub-fractions were purified to obtain compound II-2 (18.6mg).

[0110] 560g of the n-butanol fraction was separated on a HP-20 macroporous adsorption resin column and gradient elution was performed using ethanol-water as the mobile phase. The volume ratios of the mobile phases were: 0%, 10%, 20%, 30%, 40%, 50%, 60%, 80%, and 100%, respectively. After the eluate was concentrated under reduced pressure, the following were obtained: Fr.C1 fraction (38.6g), Fr.C2 fraction (8.5g), Fr.C3 fraction (4.8g), Fr.C4 fraction (6.7g), Fr.C5 fraction (11.8g), Fr.C6 fraction (20.7g), Fr.C7 fraction (31.4g), Fr.C8 fraction (12.8g), and Fr.C9 fraction (40.4g).

[0111] The Fr.C2 fraction (8.5 g) was eluted on a LH-20 type dextran gel column (85 cm × 5 cm) with a 20% methanol-water solution as the eluent, 1 bottle of 50 mL each, and a total of 40 bottles of fractions (Fr.C2.1 to Fr.C2.40) were obtained. According to the HPLC-UV detection results, similar fractions were combined and purified on a C18 type high pressure preparative chromatography column [methanol / water (30% to 100%, v / v)]. Among them, the Fr.C2.16 to Fr.C2.20 sub-fractions were eluted by gradient elution to obtain compounds III-1 (25.6 mg) and III-2 (24.8 mg).

[0112] The Fr.C3 site was eluted on a LH-20 type dextran gel column (85cm×5cm), and the eluent was a pure methanol solution, with 50mL per bottle, and a total of 23 bottles of fractions (Fr.C3.1~Fr.C3.23) were obtained. According to the HPLC-UV detection results, similar fractions were combined and purified on a C18 type high pressure preparative chromatographic column [methanol / water (30%~100%, v / v)]. Among them, Fr.C3.15~Fr.C3.18 sub-sites were eluted by gradient elution to obtain compound III-4 (17.6mg); Fr.C3.19~Fr.C3.23 sub-sites were eluted by gradient elution to obtain compound III-5 (26mg).

[0113] The Fr.C5 fraction was eluted on a LH-20 Sephadex column (85 cm × 5 cm), and the eluent was pure methanol solution. Each 50 mL was collected in one bottle, and a total of 57 fractions (Fr.C5.1 - Fr.C5.57) were obtained. According to the HPLC-UV detection results, the similar fractions were combined into one bottle and then purified on a C18 high-pressure preparative chromatography column [methanol / water (30% - 100%, v / v)]. Among them, after gradient elution of the sub-fractions Fr.C5.38 - Fr.C5.40, compounds Ⅰ-2 (8.6 mg) and Ⅰ-3 (26.3 mg) were obtained; after gradient elution of the sub-fractions Fr.C5.41 - Fr.C5.48, compounds Ⅰ-1 (21.6 mg) and Ⅰ-4 (26.3 mg) were obtained.

[0114] The Fr.C6 fraction was eluted on a LH-20 Sephadex column (85 cm × 5 cm), and the eluent was pure methanol solution. Each 50 mL was collected in one bottle, and a total of 52 fractions (Fr.C6.1 - Fr.C6.52) were obtained. According to the HPLC-UV detection results, the similar fractions were combined into one bottle and then purified on a C18 high-pressure preparative chromatography column [methanol / water (30% - 100%, v / v)]. Among them, after gradient elution of the sub-fractions Fr.C6.19 - Fr.C6.23, compound Ⅲ-3 (12.4 mg) was obtained, and after gradient elution of the sub-fractions Fr.C6.31 - Fr.C6.35, compound Ⅰ-5 (11.4 mg) was obtained.

[0115] The above samples were concentrated under reduced pressure at 40 °C and freeze-dried for later use.

[0116] Example 2

[0117] The structures of the above compounds were characterized and analyzed by nuclear magnetic resonance spectroscopy ( 1 1H-NMR, 13 13C-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) or two-dimensional nuclear magnetic resonance spectroscopy (HSQC, HMBC).

[0118] Compound Ⅰ-1: Yellow amorphous powder, with the molecular formula C 55 18 60 20 31 O

[0119] As Figure 2-6 shown, the spectral information of compound Ⅰ-1 is as follows. Among them, HR-ESI-MS m / z 1215.3210 [M + H] + (theoretical value 1217.3197).

[0120] 1 1H-NMR (600 MHz, DMSO-d6 )δ:12.63(1H,s,5-OH),10.61(1H,s,7-OH),9.66

[0121] (1H,s,3′-OH),9.14(1H,s,4′-OH),7.74(1H,d,J=1.8Hz,H-2′),7.62(1H,dd,J=8.4,1.8Hz,H-6′),6.83(1H,d,J=8.4Hz,H-5′),6.17(1H,s,H-6),5.39(1H,d,J=7.6Hz,H-1″),4.43(1H,d,J=1.2Hz,H-1″′),4.15(2H,s,H-11),3.72(1H,overlap,H-6″a),3.47(1H,m,H-2″′),3.34(1H,m,H-3″′),3.32(1H,m,H-6″b),3.31(1H,m,H-5″′),3.25(1H,m,H-3″),3.19(1H,m,H-2″),3.12(1H,m,H-4″′),3.10(1H,m,H-4″),1.02(3H,d,J=6.2Hz,H-6″′),12.60(1H,s,5a-OH),10.55(1H,s,7a-OH),10.10(1H,s,4a′-OH),8.05(1H,d,J=8.4Hz,H-2a′),8.05(1H,d,J=8.4Hz,H-6a′),6.85(1H,d,J=8.4Hz,H-5a′),6.20(1H,s,H-6a),5.37(1H,d,J=7.6Hz,H-1a″),4.43(1H,d,J=1.2Hz,H-1a″′),3.72(1H,overlap,H-6a″a),3.47(1H,m,H-2a″′),3.34(1H,m,H-3a″′),3.32(1H,m,H-6a″b),3.31(1H,m,H-5a″′),3.25(1H,m,H-3a″),3.19(1H,m,H-2a″),3.12(1H,m,H-4a″′),3.10(1H,m,H-4a″),1.03(3H,d,J=6.2Hz,H-6a″′)。

[0122] 13 C-NMR(DMSO-d 6 ,600MHz)δ:156.34(C-2),132.97(C-3),177.78(C-4),158.86

[0123] (C-5), 98.29 (C-6), 162.15 (C-7), 104.84 (C-8), 153.90 (C-9), 103.65 (C-10), 16.45

[0124] (C-11), 121.75(C-1′), 116.51(C-2′), 144.69(C-3′), 148.37(C-4′), 115,12(C-5′), 121.61

[0125] (C-6′), 101.50(C-1″), 74.10(C-2″), 76.31(C-3″), 69.76(C-4″), 75.78(C-5″), 66.88

[0126] (C-6″), 100.72(C-1″′), 70.29(C-2″′), 70.52(C-3″′), 71.81(C-4″′), 68.29(C-5″′), 17.77

[0127] (C-6″′), 156.59(C-2a), 133.09(C-3a), 177.81(C-4a), 158.88(C-5a), 98.35(C-6a), 162.18(C-7a), 104.86(C-8a), 154.00(C-9a), 103.82(C-10a), 121.81(C-1a′), 130.87

[0128] (C-2a′), 115.12(C-3a′), 159.90(C-4a′), 115,12(C-5a′), 130.87(C-6a′), 101.60(C-1a″), 74.21(C-2a″), 76.40(C-3a″), 69.85(C-4a ″), 75.92(C-5a″), 66.97(C-6a″), 100.75(C-1a″′), 70.31(C-2a″′), 70.58(C-3a″′), 71.82(C-4a″′), 68.32(C-5a″′), 17.78(C-6a″′).

[0129] Compound Ⅰ-2: Yellow amorphous powder, molecular formula is C 56 H 62 O 31 .

[0130] like Figure 7-11 The following is the spectrum information of compound Ⅰ-2. Among them, HR-ESI-MS m / z 1231.3256[M+H] +(Theoretical value: 1231.3353).

[0131] 1 H-NMR (600 MHz, DMSO-d 6 ) δ: 12.61 (1H, s, 5-OH), 10.63 (1H, s, 7-OH), 9.75 (1H, s, 4′-OH), 7.98 (1H, d, J = 1.8 Hz, H-2′), 7.58 (1H, dd, J = 8.4, 2.4 Hz, H-6′), 6.88 (1H, overlap, H-5′), 6.21 (1H, overlap, H-6), 5.37 (1H, d, J = 7.6 Hz, H-1″), 4.42 (1H, d, J = 1.2 Hz, H-1″′), 4.14 (2H, s, H-11), 3.79 (3H, s, 3′-OCH 3 ), 3.72 (1H, overlap, H-6″a), 3.47 (1H, m, H-2″′), 3.33 (1H, m, H-3″′), 3.32 (1H, m, H-6″b), 3.31 (1H, m, H-5″′), 3.25 (1H, m, H-3″), 3.19 (1H, m, H-2″), 3.12 (1H, m, H-4″′), 3.10 (1H, m, H-4″), 1.00 (3H, d, J = 6.2 Hz, H-6″′), 12.62 (1H, s, 5a-OH), 10.65 (1H, s, 7a-OH), 10.10 (1H, s, 4a′-OH), 8.05 (1H, d, J = 8.4 Hz, H-2a′), 8.05 (1H, d, J = 8.4 Hz, H-6a′), 6.86 (1H, d, J = 8.4 Hz, H-5a′), 6.21 (1H, s, H-6a), 5.37 (1H, d, J = 7.6 Hz, H-1a″), 4.46 (1H, d, J = 1.2 Hz, H-1a″′), 3.72 (1H, t, J = 10.8 Hz, H-6a″a), 3.47 (1H, m, H-2a″′), 3.33 (1H, m, H-3a″′), 3.32 (1H, m, H-6a″b), 3.31 (1H, m, H-5a″′), 3.25 (1H, m, H-3a″), 3.19 (1H, m, H-2a″), 3.12 (1H, m, H-4a″′), 3.10 (1H, m, H-4a″), 1.00 (3H, d, J = 6.2 Hz, H-6a″′).

[0132] 13 C-NMR (DMSO-d 6, 600MHz) δ: 156.25(C-2), 132.73(C-3), 177.76(C-4), 158.63(C-5), 98.22(C-6), 162.08(C-7), 104.70(C-8), 153.99(C-9), 103.69(C-10), 16.37(C-11), 121.27(C-1′), 113.19(C-2′), 146.91(C-3′), 149.34(C-4′), 115. 01(C-5′), 122.34(C-6′), 101.36(C-1″), 74.17(C-2″), 76.30(C-3″), 69.72(C-4″), 75.76(C-5″), 66.74(C-6″ ), 100.72(C-1″′), 70.23(C-2″′), 70.54(C-3″′), 71.77(C-4″′), 68.28(C-5″′), 17.74(C-6″′), 55.50(3′-OCH 3 ), 156.67(C-2a), 132.95(C-3a), 177.76(C-4a), 158.91(C-5a), 98.30(C-6a), 162.29(C-7a), 104.77(C-8a) , 154.01(C-9a), 103.74(C-10a), 121.41(C-1a′), 130.91(C-2a′), 115.11(C-3a′), 159.89(C-4a′), 115,11(C -5a′), 130.91(C-6a′), 101.54(C-1a″), 74.28(C-2a″), 76.33(C-3a″), 69.88(C-4a″), 75.95(C-5a″), 66.77( C-6a″), 100.88(C-1a″′), 70.29(C-2a″′), 70.54(C-3a″′), 71.77(C-4a″′), 68.33(C-5a″′), 17.74(C-6a″′).

[0133] Compound I-3: Yellow amorphous powder, molecular formula C 56 H 62 O 32 .

[0134] like Figure 12-16 The following is the spectrum information of compound Ⅰ-3. Among them, HR-ESI-MS m / z 1247.3296[M+H] + (Theoretical value 1247.3302).

[0135] 1H-NMR(600MHz,DMSO-d 6 )δ:12.63(1H,s,5-OH),10.59(1H,s,7-OH),9.13(1H,s,4′-OH),8.00(1H,d,J=1.8Hz,H-2′),7.66(1H,overlap,H-6′),6.83(1H,d,J=8.4Hz,H-5′),6.17(1H,s,H-6),5.51(1H,d,J=7.6Hz,H-1″),4.42(1H,d,J=1.2Hz,H-1″′),4.16(2H,s,H-11),3.80(3H,s,3′-OCH 3 ),3.73(1H,overlap,H-6″a),3.47(1H,m,H-2″′),3.34(1H,m,H-3″′),3.32(1H,m,H-6″b),3.31(1H,m,H-5″′),3.25(1H,m,H-3″),3.19(1H,m,H-2″),3.12(1H,m,H-4″′),3.10(1H,m,H-4″),1.02(3H,d,J=6.2Hz,H-6″′),12.65(1H,s,5a-OH),10.61(1H,s,7a-OH),9.67(1H,s,4a′-OH),7.75(1H,d,J=1.8Hz,H-2a′),7.65(1H,overlap,H-6a′),6.88(1H,d,J=8.4Hz,H-5a′),6.19(1H,s,H-6a),5.40(1H,d,J=7.6Hz,H-1a″),4.46(1H,d,J=1.2Hz,H-1a″′),3.73(1H,overlap,H-6a″a),3.47(1H,m,H-2a″′),3.34(1H,m,H-3a″′),3.32(1H,m,H-6a″b),3.31(1H,m,H-5a″′),3.25(1H,m,H-3a″),3.19(1H,m,H-2a″),3.12(1H,m,H-4a″′),3.10(1H,m,H-4a″),1.04(3H,d,J=6.2Hz,H-6a″′)。

[0136] 13 C-NMR(DMSO-d 6 ,600MHz)δ:156.37(C-2),132.72(C-3),177.79(C-4),158.93

[0137] (C-5), 98.37(C-6), 162.41(C-7), 104.73(C-8), 153.95(C-9), 103.67(C-10), 16.45

[0138] (C-11), 121.63(C-1′), 113.17(C-2′), 146.94(C-3′), 149.36(C-4′), 115.29(C-5′), 122.36

[0139] (C-6′), 101.39(C-1″), 74.34(C-2″), 76.45(C-3″), 69.94(C-4″), 75.97(C-5″), 66.77

[0140] (C-6″), 100.89(C-1″′), 70.28(C-2″′), 70.55(C-3″′), 71.82(C-4″′), 68.29(C-5″′), 17.76

[0141] (C-6″′), 55.51(3′-OCH 3 ), 156.21(C-2a), 133.06(C-3a), 177.79(C-4a), 158.89(C-5a), 98.27(C-6a), 162.21(C-7a), 104.85(C-8a), 153.93(C-9a) , 103.66(C-10a), 121.69(C-1a′), 116.56(C-2a′), 144.76(C-3a′), 148.44(C-4a′), 115,20(C-5a′), 121.47(C-6a′), 101.46

[0142] (C-1a″), 74.13(C-2a″), 76.39(C-3a″), 69.88(C-4a″), 75.94(C-5a″), 66.91(C-6a″), 1 00.71(C-1a″′), 70.36(C-2a″′), 70.60(C-3a″′), 71.86(C-4a″′), 68.37(C-5a″′), 17.76

[0143] (C-6a″′).

[0144] Compound I-4: Yellow amorphous powder, molecular formula: C 55 H 60 O 32 .

[0145] like Figure 17-21The following is the spectrum information of compound Ⅰ-4. Among them, HR-ESI-MS m / z 1231.3003[M+H] + (Theoretical value 1231.3353).

[0146] 1 H-NMR (600 MHz, DMSO-d 6 )δ: 12.63 (2H, s, 5-OH, 5a-OH), 10.54 (2H, s, 7-OH, 7a-OH), 9.65 (2H, s, 3′-OH, 3a′-OH), 9.16 (2H, s, 4′-OH, 4a′-OH), 7.76 (2H, d, J=1 .8Hz, H-2′, 2a′), 7.68 (2H, dd, J=8.4, 1.8Hz, H-6′, 6a′), 6.83 (2H, d, J=8.4Hz, H-5′, 5a′), 6.17 (2H, s, H-6, 6a), 5.41 (2H, d, J=7.6H z, H-1″, 1a″), 4.43 (2H, d, J = 1.2Hz, H-1″′, 1a″′), 4.18 (2H, s, H-11), 3.74 (2H, d, J = 10.4Hz, H-6″a, 6a″a), 3.47 (2H, m, H-2″′, 2a″′) , 3.35 (2H, m, H-3″′, 3a″′), 3.32 (2H, m, H-6″b, 6a″b), 3.31 (2H, m, H-5″′, 5a″′), 3.25 (2H, m, H-3″, 3a″), 3.20 (2H, m, H-2″, 2a″), 3.12

[0147] (2H, m, H-4″′, 4a″′), 3.11 (2H, m, H-4″, 4a″), 1.04 (6H, d, J = 6.2Hz, H-6″′, 6a″’).

[0148] 13 C-NMR (DMSO-d 6, 600MHz) δ: 156.2 (C-2, 2a), 133.3 (C-3, 3a), 177.8 (C-4, 4a), 158.9 (C-5, 5a), 98.3 (C-6), 162.3 (C-7), 104.8(C-8,8a), 153.9(C-9,9a), 103.6(C-10,10a), 16.5(C-11), 121.7(C-1′, 1a′), 116.5(C-2′, 2a′), 1 44.8(C-3′, 3a′), 148.5(C-4′, 4a′), 115.2(C-5′, 5a′), 121.6(C-6′, 6a′), 101.5(C-1″, 1a″), 74.1(C-2″ , 2a″), 76.4(C-3″, 3a″), 69.9(C-4″, 4a″), 75.9(C-5″, 5a″), 67.0(C-6″, 6a″), 100.7(C-1″′, 1a″′), 70.3

[0149] (C-2″′, 2a″′), 70.6(C-3″′, 3a″′), 71.8(C-4″′, 4a″′), 68.3(C-5″′, 5a″′), 17.8(C-6″′, 6a″′)

[0150] Compound Ⅰ-5: Yellow amorphous powder, molecular formula C 55 H 60 O 30 .

[0151] like Figure 22-24 The following is the spectrum information of compound Ⅰ-5. Among them, HR-ESI-MS m / z 1201.3156[M+H] + (Theoretical value 1201.3248). 1 H-NMR (600 MHz, DMSO-d 6 )δ: 12.61 (2H, s, 5-OH, 5a-OH), 10.63

[0152] (2H, s, 7-OH, 7a-OH), 10.10 (2H, s, 4′-OH, 4a′-OH), 8.00 (4H, d, J = 8.6Hz, H-2′, 6 ′, 2a′, 6a′), 6.85 (4H, d, J=8.6Hz, H-3′, 5′, 3a′, 5a′), 6.21 (2H, s, H-6, 6a), 5.36

[0153] (2H, d, J=7.6Hz, H-1″, 1a″), 4.43 (2H, d, J=1.2Hz, H-1″′, 1a″′), 4.14 (2H, s, H-11), 3.71( 2H, d, J = 10.4Hz, H-6″a, 6a″a), 3.48 (2H, m, H-2″′, 2a″′), 3.33 (2H, m, H-3″′, 3a″′), 3.32( 2H, m, H-6″b, 6a″b), 3.31 (2H, m, H-5″′, 5a″′), 3.22 (2H, m, H-3″, 3a″), 3.19 (2H, m, H-2″, 2 a"), 3.12 (2H, m, H-4"', 4a"'), 3.10 (2H, m, H-4", 4a"), 1.01 (6H, d, J = 6.2Hz, H-6"', 6a"').

[0154] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 156.7 (C-2, 2a), 133.0 (C-3, 3a), 177.8 (C-4, 4a), 158.9 (C-5, 5a), 98.4 (C-6), 162.2 (C-7), 104.8(C-8,8a), 154.0(C-9,9a), 103.7(C-10,10a), 16.4(C-11), 121.2(C-1′, 1a′), 130.9(C-2′, 6′, 2a′ , 6a′), 115.1(C-3′, 5′, 3a′, 5a′), 159.9(C-4′, 4a′), 101.6(C-1″, 1a″), 74.2(C-2″, 2a″), 76.3(C-3″, 3 a″), 69.7(C-4″, 4a″), 75.8(C-5″, 5a″), 66.8(C-6″, 6a″), 100.7(C-1″′, 1a″′), 70.3(C-2″′, 2a″′), 70.6

[0155] (C-3″′, 3a″′), 71.8 (C-4″′, 4a″′), 68.3 (C-5″′, 5a″′), 17.8 (C-6″′, 6a″′).

[0156] Compound II-1: Yellow amorphous powder, molecular formula C 17 H 12 O 7 .

[0157] like Figure 25-29 The following is the spectrum information of compound II-1. Among them, HR-ESI-MS m / z 329.0658[M+H] +(Theoretical value 329.0661).

[0158] 1 H-NMR (600 MHz, DMSO-d 6 )δ: 12.70 (1H, s, 5-OH) 10.63 (1H, s, 7-OH), 7.96

[0159] (1H, m, H-3′), 7.78 (1H, m, H-6′), 7.49 (1H, m, H-4′), 7.47 (1H, m, H-5′), 3.86

[0160] (3H, s, 6-OCH 3 ), 3.81(3H, s, 8-OCH 3 ).

[0161] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 165.2(C-2), 96.6(C-3), 178.9(C-4), 149.5(C-5), 132.4(C-6), 143.2(C-7), 128.5(C-8), 149.2(C-9 ), 102.2(C-10), 148.8(C-1′), 122.0(C-2′), 120.7(C-3′), 125.6(C-4′), 125.5(C-5′), 111.8(C-6′), 60.3(6-OCH 3 ), 61.3(8-OCH 3 ).

[0162] Compound II-2: Yellow amorphous powder, molecular formula: C 18 H 14 O 7 .

[0163] like Figure 30-32 The following is the spectrum information of compound II-2. Among them, HR-ESI-MS m / z 343.0822[M+H] + (Theoretical value 343.0818).

[0164] 1 H-NMR (600 MHz, DMSO-d 6 )δ: 10.56 (1H, s, 7-OH), 8.03 (1H, m, H-3′), 7.75 (1H, m, H-6′), 7.47 (1H, m, H-4′), 7.45 (1H, m, H-5′), 3.90 (3H, s, 6-OCH 3 ), 3.84 (3H, s, 5-OCH3 ), 3.83 (3H, s, 8-OCH 3 ).

[0165] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 163.7(C-2), 98.0(C-3), 172.3(C-4), 148.5(C-5), 132.9(C-6), 145.0(C-7), 140.2(C-8), 149.1(C-9 ), 110.4(C-10), 148.6(C-1′), 123.0(C-2′), 120.7(C-3′), 125.2(C-4′), 125.1(C-5′), 111.5(C-6′), 62.0(5-OCH 3 ), 61.3(6-OCH 3 ), 61.1(8-OCH 3 ).

[0166] Compound II-3: Yellow amorphous powder, molecular formula C 16 H 10 O 6 .

[0167] like Figure 33-35 The following is the spectrum information of compound II-3. Among them, HR-ESI-MS m / z 299.0574[M+H] + (Theoretical value 299.0566).

[0168] 1 H-NMR (600 MHz, DMSO-d 6 )δ: 12.92 (1H, s, 5-OH), 10.86 (1H, s, 7-OH), 7.96 (1H, m, H-3′), 7.78 (1H, m, H-6′), 7. 48 (2H, overlap, H-4′), 7.47 (2H, overlap, H-5′), 6.70 (1H, s, H-8), 3.78 (3H, s, 6-OCH 3 ).

[0169] 13 C-NMR (DMSO-d 6, 125MHz) δ: 165.1(C-2), 96.6(C-3), 178.8(C-4), 153.9(C-5), 132.1(C-6), 156.7(C-7), 95.2(C-8), 148.8(C-9) , 103.2(C-10), 149.8(C-1′), 122.0(C-2′), 120.7(C-3′), 125.6(C-4′), 125.5(C-5′), 111.8(C-6′), 60.0(6-OCH 3 ).

[0170] Compound III-1: Off-white amorphous powder, molecular formula: C 15 H 13 NO 2 .

[0171] like Figure 36-40 The following is the spectrum information of compound III-1. Among them, HR-ESI-MS m / z 240.1021[M+H] + (Theoretical value 240.1025).

[0172] 1 H-NMR (600 MHz, DMSO-d 6 )δ: 7.78 (1H, m, H-4′), 7.69 (2H, overlap, H-2′, 6′), 7.68 (2H, overlap, H-3′, 5′), 6.96 (1H, s, H-6), 6.79 (1H, s, H-3), 3.85 (1H, t, J = 8.4Hz, H-8a), 3.03 (1H, t, J = 8.4Hz, H-7a).

[0173] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 171.7(C-2), 130.7(C-2a), 119.5(C3), 144.8(C-4), 155.0(C-5), 115.7(C-6), 118.2 (C-6a), 24.4(C-7), 41.1(C-8), 133.5(C-1′), 130.2(C-2′,6′), 128.9(C-3′,5′), 133.1(C-4′).

[0174] Compound III-2: Off-white amorphous powder, molecular formula: C 16 H 17 NO 2 .

[0175] like Figures 41-43The following is the spectrum information of compound III-2. Among them, HR-ESI-MS m / z 256.1333[M+H] + (Theoretical value 256.1338).

[0176] 1 H-NMR (600 MHz, DMSO-d 6 )δ: 7.37 (2H, overlap, H-3′, 5′), 7.33 (2H, overlap, H-2′, 6′), 7.31 (1H, overlap, H-4′), 6.57 (1H, s, H-6), 6.49 (1H, s, H-3), 3.33 (1H, m, H-8b), 3.27 (1H, m, H-9b), 3.18 (1H, m, H-8a), 3.09 (1H, m, H-9a), 2.87 (1H, m, H-7b), 2.78 (1H, m, H-7a).

[0177] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 55.1 (C-2), 122.7 (C-2a), 113.6 (C3), 144.0 (C-4), 145.2 (C-5), 115.2 (C-6), 122.3 (C-6a ), 24.2(C-7), 39.0(C-8), 40.1(C-9), 136.1(C-1′), 129.6(C-2′,6′), 128.7(C-3′,5′), 127.1(C-4′).

[0178] Compound III-3: White amorphous powder, molecular formula C 19 H 24 NO 3 + .

[0179] like Figures 44-46 The following is the spectrum information of compound Ⅲ-3. Among them, HR-ESI-MS m / z 315.1836[M+H] + (Theoretical value 315.1829).

[0180] 1 H-NMR (600 MHz, DMSO-d 6)δ: 9.50 (1H, s, 8-OH), 9.40 (1H, s, 4'-OH), 7.09 (2H, d, J = 8.4Hz, H-2', 6'), 7.02 (1H, d, J = 8.4Hz, H-6), 6 .73 (1H, d, J = 8.4Hz, H-5), 6.67 (2H, d, J = 8.4Hz, H-3′, 5′), 4.99 (2H, d, J = 7.2Hz, H-1), 3.81 (3H, s, 7-OCH 3 ), 3.71 (1H, m, H-4a), 3.40 (4H, m, H-3, 9), 3.05 (3H, s, N-CH 3 ), 2.99(3H, s, N-CH 3 ), 2.95 (1H, m, H-4b).

[0181] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 67.9 (C-1), 53.3 (C-3), 22.4 (C-4), 121.2 (C-4a), 119.9 (C-5), 112.2(C-6), 145.8(C-7), 142.5(C-8), 118.9(C-8a), 36.1(C-9), 56.1(-OCH 3 ), 52.6(N-CH 3 ), 50.3(N-CH 3 ), 127.8(C-1′), 130.1(C-2′, 6′), 115.2(C-3′, 5′), 156.0(C-4′).

[0182] Compound III-4: White amorphous powder, molecular formula C 12 H 13 NO 3 .

[0183] like Figures 47-49 The following is the spectrum information of compound III-4. Among them, HR-ESI-MS m / z 288.0974[M+H] + (Theoretical value 288.0977).

[0184] 1 H-NMR (600 MHz, DMSO-d 6)δ: 6.51 (1H, s, H-10), 6.50 (1H, s, H-7), 4.57 (1H, t, J = 8.4Hz, H-10b), 3.96 (1H, ddd, J = 12.4, 5.4, 2.4Hz, H-5), 2.91 (1H, td, J = 12.4 , 5.4, 2.4Hz, H-5), 2.59 (1H, m, H-1), 2.55 (1H, m, H-6), 2.40 (1H, m, H-2), 2.22 (1H, ddd, J=16.2, 9.2, 1.8Hz, H-2), 1.59 (1H, m, H-1).

[0185] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 27.4(C-1), 31.3(C-2), 172.0(C-3), 36.7(C-5), 27.3(C-6), 123.7(C-6a ), 115.4(C-7), 143.9(C-8), 144.1(C-9), 111.6(C-10), 128.4(C-10a), 55.6(C-10b).

[0186] Compound III-5: White amorphous powder, molecular formula C 9 H 9 NO 3 .

[0187] like Figure 50-52 The following is the spectrum information of compound III-5. Among them, HR-ESI-MS m / z 180.0636[M+H] + (Theoretical value 180.0661).

[0188] 1 H-NMR (600 MHz, DMSO-d 6 )δ: 11.06 (1H, s, 6-OH), 9.09 (1H, s, 7-OH), 7.23 (1H, s, H-8), 6.60 (1H, s, H-5), 3.27 (2H, t, J = 6.4Hz, H-3), 2.68 (2H, t, J = 6.4Hz, H-4).

[0189] 13 C-NMR (DMSO-d 6 , 600MHz) δ: 165.0 (C-1), 40.2 (C-3), 27.2 (C-4), 120.6 (C-4a), 113.9 (C-5), 148.8 (C-6), 143.8 (C-7), 114.1 (C-8), 131.3 (C-8a).

[0190] Test Example 1

[0191] Evaluation of the activity of compounds in improving verapamil hydrochloride-induced cardiac injury

[0192] Wild-type AB strain zebrafish larvae at 2 dpf and normal development were selected and placed in 6-well plates, with 30 fish per well. Samples dissolved in DMSO were added to make the final concentration of the drug-treated groups: 50 μmol / mL. Zebrafish experimental water was used as the control group. The 6-well plates after drug addition were placed in a 28.5℃ incubator for 24 hours, and the zebrafish status was observed and recorded, and the mortality rate and deformity rate were counted. The results showed that compared with the blank control group, at a concentration of 50 μmol / mL, isoflavone compounds II-1 to II-3 caused different degrees of deformity or death in zebrafish; while biflavonoid compounds I-1 to I-5 and alkaloid compounds III-1 to III-5 did not cause deformity or death in zebrafish at this concentration, so the cardiac activity of compounds I-1 to I-5 and III-1 to III-5 was evaluated.

[0193] Then, wild-type AB zebrafish of 2 dpf and normal development were selected and placed in a 6-well plate, with 30 fish per well, and each well was fixed to 3 mL with experimental water. Normal group, model group, positive drug group, and drug administration groups of different concentrations were set. The normal group was treated with experimental water only; the model group was treated with 20 μg / mL verapamil hydrochloride for 20 min; the positive drug group was first treated with 8 μg / mL digoxin DMSO solution for 4 h, and then verapamil hydrochloride was added for 20 min; the drug administration group was added with 50 μmol / mL concentration of the above 10 compounds for 4 h; except for the normal group, verapamil hydrochloride was added to each group for 20 min. Ten zebrafish were randomly selected from each group, fixed on a slide with methylcellulose, adjusted to a lateral position to fully expose the atria and ventricles, and recorded under a stereomicroscope with white light (magnification of 60 times). Image J software was used to analyze the pericardial edema area and venous congestion area of ​​zebrafish.

[0194] Test results such as Fig.53As shown in the results, compared with the normal control group, the zebrafish in the model group had obvious pericardial edema and congestion under the yolk sac. The pericardial edema area and congestion area were significantly different from those in the normal group, indicating that verapamil hydrochloride successfully induced heart damage in zebrafish. Compared with the model group, biflavonoid compounds Ⅰ-1 to Ⅰ-5 and alkaloid compounds Ⅲ-1 to Ⅲ-5 had significant improvement effects on the pericardial edema area and venous congestion area of ​​zebrafish induced by verapamil hydrochloride at a concentration of 50μM, and there were statistical differences. Among them, compound Ⅲ-2 reduced the pericardial edema area of ​​zebrafish by 35.73%, which was better than the positive drug digoxin (22.89%) at a concentration of 8μg / mL. Compounds Ⅰ-3 and Ⅰ-4 reduced the venous congestion area of ​​zebrafish by 78.60% and 74.96%, respectively, which was better than the positive drug digoxin (67.73%) at a concentration of 8μg / mL.

[0195] Test Example 2

[0196] Evaluation of the lethal effects of compounds on zebrafish

[0197] Since compounds II-1 to II-3 caused different degrees of deformity or death in zebrafish at a concentration of 50 μM, we tested their LC50 values, which were 9.41, 39.59 and 24.47 μM respectively. The specific test process is as follows:

[0198] First, 2dpf (day post fertilization) zebrafish were selected and placed in a 24-well plate, with 10 fish per well. Compounds 11-13 were set up as a normal control group and 7 drug-treated groups with concentrations of 5, 10, 20, 30, 40, 50, and 70 μM, respectively, with 3 replicates in each group. After incubation for 24 hours, photos were taken and observed, and the deformity rate and mortality rate of zebrafish in each group were counted to determine the half-lethal concentration (LC50) of zebrafish for each compound. The test results are shown in Figure 2. Figure 54-56 As shown, the median lethal concentrations of compounds II-1 to II-3 for zebrafish were 9.41 μM, 39.59 μM, and 24.47 μM, respectively.

[0199] Test Example 3

[0200] Evaluation of the inhibitory activity of compounds on HCT116 cell growth

[0201] HCT116 cells were cultured in DMEM containing 10% FBS at 37°C and 5% CO 2cultured in a constant temperature incubator. Compounds Ⅱ-1, Ⅱ-2 and Ⅱ-3 were dissolved in DMSO to prepare a stock solution, which was diluted to the corresponding concentration with culture medium during work. HCT116 in the logarithmic growth phase was taken, and the appropriate cell density was adjusted. The cells were inoculated in a 96-well plate at a density of 2000 cells / well and cultured for 24 hours. A control group, a positive drug group and a drug-treated group were set up, and each group had 3 replicate wells. The entire experimental process was repeated 3 times. The drug-treated group was treated with 30 and 50 μM compounds Ⅱ-1, Ⅱ-2 and Ⅱ-3, the positive drug group was treated with 30 and 50 μM 5-Fluorouracil, and the control group was not treated with drugs. After 24 hours of drug action, the cells were incubated with 0.25 mg / mL MTT at 37°C for 4 hours. After discarding the culture medium, 100 LDMSO was added to each well. After shaking, the optical density OD value was measured at 490 nm using an enzyme reader. Finally, the OD value of the control group was taken as 100%, and the cell survival rate of each group was calculated. The test results are as follows Fig.57 As shown:

[0202] The results showed that the inhibition rates of positive drugs, as well as compounds II-1, II-2 and II-3 at 30 and 50 μM concentrations on HCT116 cells were: (39.27%, 53.87%), (32%, 56.59%), (24.2%, 35.72%), (26.45%, 46.48%). That is, compared with the normal group, compounds II-1, II-2 and II-3 at 30 and 50 μM concentrations all had inhibitory effects on HCT116 cells, and there were statistical differences. Among them, the inhibitory activity of compound II-1 was basically equivalent to that of 5-fluorouracil at the same concentration.

[0203] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A flavonoid compound, characterized in that: The general structural formula of the flavonoid compound is shown in formula (I) or formula (II): In formula (I), R1 and R2 are independently selected from one or more of hydrogen, hydroxyl and methoxy; In formula (II), R3 and R4 are independently selected from one or more of hydrogen, hydroxyl and methoxy.

2. The flavonoid compound according to claim 1, characterized in that When R1=H, R2=OH, the compound structure is as shown in formula (I-1): When R1=H, R2=OCH3, the compound structure is shown in formula (I-2): When R1=OCH3, R2=OH, the compound structure is shown in formula (I-3): When R1=OH, R2=OH, the compound structure is as shown in formula (I-4): When R1=H, R2=H, the compound structure is shown in formula (I-5):

3. The flavonoid compound according to claim 1, characterized in that When R3=OH, R4=OCH3, the compound structure is shown in formula (II-1): When R3=OCH3, R4=OCH3, the compound structure is shown in formula (II-2): When R3=OH, R4=H, the compound structure is shown in formula (II-3):

4. A method for preparing a flavonoid compound according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) crushing Suaeda salsa, extracting with 95% ethanol under reflux, combining the extracts, and concentrating to obtain Suaeda salsa ethanol extract; extracting the Suaeda salsa ethanol extract with petroleum ether, ethyl acetate, and n-butanol solutions in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, an n-butanol fraction, and a water fraction; (2) Using petroleum ether-acetone with a volume ratio of 1:0, 80:1, 50:1, 20:1, 10:1, 6:1, 4:1, 1:1, and 0:1 as the mobile phase, the ethyl acetate fraction sample was gradient eluted using a silica gel column to obtain nine components, Fr.B1 to Fr.B9; The Fr.B6 component was eluted using an LH-20 type dextran gel column, and the eluent was methanol. The similar fractions in the obtained Fr.B6.1 to Fr.B6.27 fractions were combined and purified using methanol-water with a volume ratio of 60% to 100%, and compounds II-1 and II-3 were purified from the Fr.B6.25 to Fr.B6.27 fractions; The Fr.B8 component was eluted using an LH-20 type dextran gel column, and the eluent was methanol. The similar fractions in the obtained Fr.B8.1 to Fr.B8.65 fractions were combined and purified using methanol-water with a volume ratio of 60% to 100%, and compound II-2 was purified from the Fr.B8.18 to Fr.B8.20 fractions; (3) Using ethanol-water with a volume ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 80%, and 100% as the mobile phase, the n-butanol fraction sample was gradient eluted using an HP-20 macroporous adsorption resin column to obtain nine components, Fr.C1 to Fr.C9; The Fr.C5 component was eluted using an LH-20 type dextran gel column, with methanol as the eluent, and similar fractions from the obtained Fr.C5.1 to Fr.C5.57 fractions were combined and purified using 30% to 100% methanol-water by volume, and compounds Ⅰ-2 and Ⅰ-3 were purified from the Fr.C5.38 to Fr.C5.40 fractions; compounds Ⅰ-1 and Ⅰ-4 were purified from the Fr.C5.41 to Fr.C5.48 fractions; The Fr.C6 component was eluted using an LH-20 type dextran gel column with methanol as the eluent. Similar fractions from the obtained Fr.C6.1 to Fr.C6.52 fractions were combined and purified using methanol-water with a volume ratio of 30% to 100%, and compound I-5 was purified from the Fr.C6.31 to Fr.C6.35 fractions.

5. The preparation method according to claim 4, characterized in that: According to the HPLC-UV detection results, similar fractions in the eluted fractions were combined and then purified on a C18 high-pressure preparative chromatographic column.

6. The alkaloid compound obtained by the preparation method according to claim 4, characterized in that: The alkaloid compounds are extracted from the n-butanol part, and the n-butanol part sample is gradient eluted using an HP-20 macroporous adsorption resin column with ethanol-water at a volume ratio of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 80%, and 100% as the mobile phase to obtain nine components of Fr.C1 to Fr.C9; The Fr.C2 component was eluted using an LH-20 type dextran gel column, and the eluent was a 20% methanol-water solution to obtain Fr.C2.1 to Fr.C2.40 fractions. According to the HPLC-UV detection results, similar fractions were combined into a bottle and purified on a C18 type high pressure preparative chromatography column using 30% to 100% methanol-water by volume to purify compounds III-1 and III-2 from Fr.C2.16 to Fr.C2.20 fractions; The Fr.C3 component was eluted using an LH-20 type dextran gel column, and the eluent was pure methanol to obtain Fr.C3.1 to Fr.C3.23 fractions. According to the HPLC-UV detection results, similar fractions were combined into a bottle and purified on a C18 type high pressure preparative chromatographic column using 30% to 100% methanol-water by volume, and compound III-4 was purified from the Fr.C3.15 to Fr.C3.18 fractions; compound III-5 was purified from the Fr.C3.19 to Fr.C3.23 fractions; The Fr.C6 component was eluted using an LH-20 type dextran gel column, and the eluent was pure methanol to obtain Fr.Fr.C6.1 to Fr.C6.52 fractions. According to the HPLC-UV detection results, similar fractions were combined into a bottle and purified on a C18 type high pressure preparative chromatography column using 30% to 100% methanol-water by volume, and compound III-3 was purified from Fr.C6.19 to Fr.C6.23 fractions; 7. Use of the flavonoid compound according to claim 2 in the preparation of drugs for treating cardiovascular diseases or improving heart damage.

8. Use of the flavonoid compound according to claim 3 in the preparation of anti-tumor drugs.

9. Use of the alkaloid compound according to claim 6 in the preparation of a drug for treating cardiovascular diseases or a drug for improving heart damage.

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