Lignan compound, preparation method and application thereof

Abstract

The application discloses a lignan compound and a preparation method and application thereof, and relates to the technical field of medicines. The application specifically relates to four lignan small-molecule compounds with the same planar structure, which are separated from amber, and include two pairs of enantiomers (+)-6 / (-)-6 and (+)-7 / (-)-7. The compound structures are as follows: In a renal fibrosis model experiment, the lignan compound provided by the application can inhibit the expression of proteins related to renal fibrosis, such as alphaSMA protein, type I collagen protein and fibronectin, in NRK-52E cells induced by transforming growth factor beta 1, which indicates that the lignan compound provided by the application has the use of preparing medicines for preventing and treating renal fibrosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a lignan compound, its preparation method, and its application. Background Technology

[0002] Amber, a transparent fossilized organism, is the fossilized resin of plants such as conifers, pine trees, and araucaria. The resin dripped and was buried underground for millions of years, fossilizing under pressure and heat. Some amber contains small insects such as bees, making it exceptionally beautiful. Most amber is formed from the fossilized resin of pine trees, hence it is also known as "pine resin fossil." Amber production is mainly concentrated in Russia, Myanmar, and the provinces of Henan, Liaoning, and Yunnan in China. The main chemical components of amber are resin and volatile oils. Volatile oils include monoterpenes, sesquiterpenes, diterpenes, and aromatic compounds. The earliest records of amber's medicinal use date back to ancient times. Early medicine only used ingredients obtainable from nature: plants, animals, and minerals. As a traditional Chinese medicine, amber has been widely used to treat various diseases since ancient times. In ancient books, amber is described as calming the mind, promoting hydration, and eliminating blood stasis, among other benefits. Several studies have shown that amber has antibacterial, anti-inflammatory, and anti-allergic effects.

[0003] Renal fibrosis is a common pathological manifestation of various chronic kidney diseases (CKD) progressing to end-stage renal disease (ESRD). To date, no specific drug has been found to reverse renal fibrosis. Clinically, interventions to slow the progression of renal fibrosis are key to protecting kidney function. Effective interventions can maximally slow the progression of renal fibrosis and delay renal replacement therapy. Currently, drugs used to treat renal fibrosis mainly include hormones, immunosuppressants, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists (ARBs) for antihypertensive purposes, and various traditional Chinese medicines with kidney-protective effects.

[0004] Further exploration of the effects of components in amber on renal fibrosis will help develop new applications for small molecule compounds in amber. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide two racemic lignan-based small molecule compounds, their preparation methods, and applications. The aim is to provide a novel small molecule compound that prevents and treats renal fibrosis by inhibiting the expression of proteins related to renal fibrosis, such as αSMA protein, type I collagen, and fibronectin, in NRK-52E cells induced by transforming growth factor-β1 (TGF-β1). The details are as follows:

[0006] On one hand, the present invention provides a lignan compound or a salt thereof, having the following structure:

[0007]

[0008] Specifically, the lignan compounds include two compounds, 6 and 7, named Succignans A (compound 6) and Succignans B (compound 7), which are pale yellow oils and contain enantiomers. After further separation, four stereoisomers are obtained, specifically including two pairs of enantiomers (+)-6 / (-)-6 and (+)-7 / (-)-7.

[0009] The structural identification results of the compounds obtained in this invention are as follows:

[0010] High-resolution electrospray ionization mass spectrometry (HRESIMS) of compound 6 showed: 621.2076 [M + Na]. + (Calculated value 621.2095) and high-resolution electrospray ionization mass spectrometry (HRESIMS) of compound 7 showed: 621.2074 [M+Na] + (Calculated value is 621.2095), compounds 6 and 7 with C 35 H 34 O9 has the same molecular formula and is a pale yellow oil.

[0011] It should be noted that any process that uses molecular weight as a clue to trace and obtain the lignan compounds described in this invention is within the scope of protection of this invention.

[0012] Specifically, the lignan compounds are extracted and isolated from amber or obtained through artificial synthesis.

[0013] On the other hand, the present invention also provides a method for preparing the lignan compounds or their salts, comprising the following steps:

[0014] ① Extract amber by reflux with dichloromethane at room temperature, combine the extracts and concentrate to obtain crude extract;

[0015] ② The crude extract was subjected to gradient elution with water and 85-100 v% methanol / isopropanol mixed solvent on an MCI CHP 20P column to obtain 9 components Fr.1-Fr.9;

[0016] Fr.6 was eluted with a gradient of water and 88-100 v% methanol / isopropanol mixed solvent to obtain 7 components Fr.6.1-Fr.6.7;

[0017] Fr.6.6 was subjected to gradient elution chromatography on a silica gel column using a dichloromethane / methanol system to obtain 14 fractions: Fr.6.6.1-Fr.6.6.9 and Fr.6.6.A-Fr.6.6.E.

[0018] Fr.6.6.5 was separated by reversed-phase chromatography using an RP-18 column with 86-100 v% methanol / water solution to obtain seven components Fr.6.6.5.1-Fr.6.6.5.7;

[0019] ③ Using acetonitrile-water as the mobile phase, Fr6.6.5.7 was further purified by semi-preparative HPLC to obtain compounds 6 and 7; compounds 6 and 7 were chirally resolved to obtain compounds (+)-6 / (-)-6 and (+)-7 / (-)-7.

[0020] The further technical solution is as follows: in step ①, the ratio of amber to dichloromethane is 1kg:3-8L; reflux extraction is performed 2-3 times, with each extraction lasting 8-36 hours.

[0021] A further technical solution is that, in step ②, the volume ratio of methanol to isopropanol in the methanol / isopropanol mixed solvent is 8:1-20:1; and the volume ratio of dichloromethane to methanol in the dichloromethane / methanol system is 100:1-5:1.

[0022] A further technical solution is that, in step ③, the mobile phase is a 60-85% acetonitrile aqueous solution.

[0023] In another aspect, the present invention provides the use of the lignan compounds or their salts in the preparation of a drug that inhibits the expression of αSMA protein, type I collagen and fibronectin associated with renal fibrosis in transforming growth factor-β1-induced NRK-52E cells.

[0024] In another aspect, the present invention provides the use of the lignan compounds or their salts in the preparation of drugs for the treatment and prevention of renal fibrosis.

[0025] In another aspect, the present invention provides a pharmaceutical composition for treating and preventing renal fibrosis, the pharmaceutical composition comprising lignan compounds or salts thereof as described above.

[0026] Specifically, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient. That is, pharmaceutical compositions containing the lignan compounds of the present invention as active ingredients or their salts, and conventional pharmaceutical excipients or carriers are also included in the present invention.

[0027] The pharmaceutical composition of the present invention uses the lignan compounds 6 and 7 as active ingredients. It does not exclude the possibility of changes in the formulation system and administration method, derivatives of the above compounds after simple chemical modification, pharmaceutical salts, multiple compounds, and multiple degradation agents used together.

[0028] Specifically, the pharmaceutically acceptable salts provided by this invention can be: sodium salts, potassium salts, ammonium salts, amino acid salts, lactates, hydrochlorides, phosphates, acetates, malates, citrates, or aspartates, etc. This invention does not specifically limit the pharmaceutical salts.

[0029] In this invention, the lignan compounds 6 and 7 of this invention can be formulated as active ingredients in a non-toxic, inert, and pharmaceutically acceptable carrier medium; the formulated drug can be administered via conventional routes, including but not limited to oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or local administration.

[0030] When the pharmaceutical composition of the present invention is in the form of a drug for oral administration, it contains a safe and effective amount of the lignan compounds 6 and 7 of the present invention, as well as a pharmaceutically acceptable carrier and / or excipient. The drug for oral administration can be formulated into commonly used dosage forms such as tablets, pills, powders, granules, capsules, emulsions, syrups, ointments, and suppositories. In the present invention, no specific limitation is made on the carrier and / or excipient.

[0031] The pharmaceutical composition of the present invention can also be formulated into an injection, which can be prepared in an aseptic environment with water for injection, physiological saline, or glucose solution. The above-mentioned injection can be prepared by conventional methods.

[0032] Compared with the prior art, the technical effects achieved by the present invention include:

[0033] This invention provides two lignan-based racemic compounds, their preparation methods, and applications. Two lignan-based racemic compounds, named Succignans A (compound 6) and Succignans B (compound 7), were extracted and isolated from amber using dichloromethane. This is the first time novel compounds have been isolated and identified from amber, and this invention also discloses for the first time a method for extracting and isolating new compounds Succignans A and Succignans B from amber. Structural identification confirmed that these small molecule compounds are novel racemic lignan compounds, which exhibit certain effects in anti-renal fibrosis.

[0034] Specifically, in renal fibrosis model experiments, compounds Succignans A and Succignans B can inhibit the expression of renal fibrosis-related proteins such as αSMA protein, type I collagen, and fibronectin in NRK-52E cells induced by transforming growth factor-β1 (TGF-β1). This indicates that compounds Succignans A and Succignans B provided by this invention have the potential use in preparing drugs for the prevention and treatment of renal fibrosis. Therefore, the novel compounds Succignans A and Succignans B, which were isolated from amber for the first time in this invention, have a novel use in preparing drugs for the prevention and treatment of renal fibrosis. Attached Figure Description

[0035] Figure 1 The image shows the hydrogen nuclear magnetic resonance spectrum of Succignans A, a compound obtained in Example 1 of this invention.

[0036] Figure 2 This is the carbon NMR spectrum of Succignans A, a compound obtained in Example 1 of this invention.

[0037] Figure 3 The image shows the hydrogen nuclear magnetic resonance spectrum of Succignans B, a compound obtained in Example 1 of this invention.

[0038] Figure 4 This is the carbon NMR spectrum of Succignans B, a compound obtained in Example 1 of this invention.

[0039] Figure 5 This demonstrates in Example 2 of the present invention that compounds Succignans A and Succignans B did not show significant cytotoxicity at 20 μM.

[0040] Figure 6 This is the experimental result of compounds Succignans A and Succignans B in Example 2 of the present invention in inhibiting the expression of proteins related to renal fibrosis, such as αSMA protein, type I collagen and fibronectin, in NRK-52E cells induced by transforming growth factor-β1 (TGF-β1). Detailed Implementation

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] It should be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0043] Example 1

[0044] This invention provides a lignan compound or a salt thereof, having the following structure:

[0045]

[0046] The lignan compounds specifically include two compounds, 6 and 7, named Succignans A (compound 6) and Succignans B (compound 7).

[0047] This embodiment also provides a method for preparing the above-mentioned compound, including the following steps:

[0048] S100, providing amber raw materials (29kg);

[0049] S200. The amber raw material was pulverized and extracted twice with dichloromethane (150L, ​​24 hours). The extracts were combined and concentrated to obtain crude extract (5.1kg).

[0050] S300. The crude extract was subjected to a first column chromatography separation using an MCI CHP 20P column, with gradient elution using water and a mixed solvent of 85-100 v% methanol / isopropanol = 10:1, to obtain 9 fractions (Fr.1-Fr.9).

[0051] S400. The Fr.6 (299.8g) was subjected to a second column chromatography separation using an MCI CHP 20P column. Gradient elution was performed with water and a mixed solvent of 88-100v% methanol / isopropanol = 15:1 to obtain 7 fractions, namely (Fr.6.1-Fr.6.7).

[0052] S500, the Fr.6.6 (67.4 g) was subjected to a third column chromatography separation, and eluted on a silica gel column with a gradient of dichloromethane / methanol system (100:1-5:1) to obtain 14 fractions, namely (Fr.6.6.1-Fr.6.6.9 and Fr.6.6.A-Fr.6.6.E);

[0053] S600, the Fr.6.6.5 (8.0 g) was subjected to a fourth column chromatography separation, purified by reverse-phase RP-18 column with 86-100 v% methanol / water, and eluted to obtain 7 fractions (Fr.6.6.5.1-Fr.6.6.5.7);

[0054] S700. The Fr6.6.5.7 (541.0 mg) was purified by semi-preparative HPLC (mobile phase: acetonitrile / water 80 v%, flow rate: 3.0 mL / min, to obtain compound 6 (15.5 mg, tR = 18.2 min) and compound 7 (27.1 mg, tR = 16.5 min).

[0055] S800, racemic compounds 6 and 7 were given their enantiomers by chiral high performance liquid chromatography: (-)-6 (4.8 mg, tR = 23.0 min) and (+)-6 (4.6 mg, tR = 25.0 min) (mobile phase: acetonitrile / water 60 v%, flow rate: 1.0 mL / min); (-)7 (3.9 mg, tR = 21.3 min) and (+)7 (3.7 mg, tR = 23.4 min) (mobile phase: acetonitrile / water 60 v%, flow rate: 1.0 mL / min).

[0056] The structures of compounds 6 and 7 prepared above were identified, and the results are as follows:

[0057]

[0058] Compound 6, a pale yellow oil, is named Succignans A. HRESIMS m / z 621.2076 [M+Na] + (calcd for C 35 H 34 O9Na,621.2095); 1H NMR(600MHz,CD3OD)δ8.08(dd,J=8.3,1.4,H-2″′,6″′,2H),7.84(dd,J=8.3,1.4,H-2′,6′,2H),7.56(t,H-4″′,1H),7.49(t,J=7.4,H-4′,1H),7.45(t,H-3″′,5″′,2H),7.36(t,H-3′,5′,2H),6.97(d,J=1.6,H-2″,1H),6.91(dd,J=8.1,1.6,H-6″,1H),6.89(d,J=8.1,H-5″,1H),6.85(d,J=1.1,H-2,1H),6.84(dd,J=8.1,1.1,H-6,1H),6.75(d,J=8.1,H-5,1H),6.63(dd,J=15.8,1.3,H-7,1H),6.25(dt,J=15.8,6.5,H-8,1H),4.96(dd,J=6.5,1.3,H-9,2H),4.65(overlap,H-9″,8″,3H),4.48(m,H-7″,H),3.85(s,OCH3,3H),3.70(s,OCH3,3H),3.31(s,OCH3,3H); 13 C NMR(150MHz,CD3OD)δ131.3(s,C-1),110.1(s,C-2),146.7(s,C-3),148.2(s,C-4),118.7(s,C-5),119.9(s,C-6),134.3(s,C-7),122.0(s,C-8),65.7(s,C-9),130.0(s,C-1′),129.7(s,C-2′,6′),128.3(s,C-3′,5′),133.0(s,C-4′),166.5(s,C-7′),130.0(s,C-1″),109.9(s,C-2″),151.0(s,C-3″),145.6(s,C-4″),114.1(s,C-5″),121.0(s,C-6″),82.9(s,C-7″),82.4(s,C-8″),64.5(s,C-9″),130.3(s,C-1″′),129.7(s,C-2″′,6″′),128.5(s,C-3″′,5″′),133.1(s,C-4″′),166.6(s,C-7″′),56.0(3-OCH3),55.8(3″-OCH3),57.3(7″-OCH3)。

[0059] Compound 7, a pale yellow oil, is named Succignans B. HRESIMS m / z 621.2076 [M+Na] + (calcd for C 35 H 34 O9Na,621.2095); 1 H NMR (600MHz, CD3OD) δ8.08(dd,J=8.3,1.4,H-2″′,6″′,2H),7.85(dd,J=8.3,1 .4,H-2′,6′,2H),7.57(t,H-4″′,1H),7.50(t,J=7.4,H-4′,1H),7.45(t,J=7. 7,H-3″′,5″′,2H),7.37(t,J=7.7,H-3′,5′,2H),6.98(d,J=8.1,H-5,1H),6.9 6(d,J=1.1,H-2″,1H),6.90(dd,J=8.1,1.1,H-6,1H),6.89(d,J=8.1,H-5″,1H ),6.88(dd,overlap,H-2,6″,2H),6.66(dd,J=15.7,1.3,H-7,1H),6.27(dt,J =15.7,6.5,H-8,1H),4.96(dd,J=6.5,1.3,H-9,2H),4.65(td,J=6.0,3.9,H-8 ″,1H),4.51(d,J=6.0,H-7″,H),4.43(dd,J=11.8,3.9,Ha-9″,H),4.32(dd,J= 11.8,6.0,Hb-9″,H),3.82(s,OCH3,3H),3.74(s,OCH3,3H),3.32(s,OCH3,3H); 13C NMR(150MHz,CD3OD)δ131.1(s,C-1),110.0(s,C-2),146.9(s,C-3),148.7(s,C-4),118.3(s,C-5),120.0(s,C-6),134.4(s,C-7),121.9 (s,C-8),65.8(s,C-9),129.6(s,C-1′),129.7(s,C-2′,6′),128.4(s,C-3′,5′),133.1(s,C-4′),166.3(s,C-7′),130.3(s,C-1″),109. 8(s,C-2″),150.9(s,C-3″),145.8(s,C-4″),114.3(s,C-5″),120.8(s,C-6″),83.6(s,C-7″),82.2(s,C-8″),64.5(s,C-9″),129.9(s,C -1″′),129.7(s,C-2″′,6″′),128.5(s,C-3″′,5″′),133.1(s,C-4″′),166.6(s,C-7″′),56.0(3-OCH3),55.8(3″-OCH3),57.3(7″-OCH3).

[0060] The proton NMR spectrum of compound Succignans A (compound 6) is shown below. Figure 1 As shown, the carbon spectrum is as follows Figure 2 As shown. The 1H NMR spectrum of compound Succignans B (compound 7) is shown below. Figure 3 As shown, the carbon spectrum is as follows Figure 4 As shown.

[0061] Example 2

[0062] The DNA damage repair efficiency of compounds Succignans A (compound 6) and Succignans B (compound 7) was tested.

[0063] In this embodiment, the cell culture method is as follows: normal rat kidney proximal tubular epithelial cells (NRK-52E) (Shanghai Cell Bank of Chinese Academy of Sciences, China) were cultured in high glucose DMEM (C11995500BT, Gibco, USA; 10% fetal bovine serum, Gibco, USA, 2094468CP) medium at 37°C in a humid environment containing 5% carbon dioxide, with the addition of 100 U / mL penicillin and 100 μg / mL streptomycin.

[0064] The cell viability assay method is as follows: NRK-52E cells (5×10⁻⁶) were used to determine cell viability. 4Cells were seeded at 100 cells / mL into 96-well plates containing complete DMEM medium and incubated for 24 h. Then, cells were treated with different concentrations of compound 6, compound 7, or DMSO (control) for 48 h. Cell counting kit-8 (CCK-8, Beyotime, Shanghai) was then added to each well, and the plates were incubated at 37°C for 1 h. The absorbance of each well was measured at 450 nm using a microplate reader (BioTek, USA).

[0065] The Western blot analysis method was as follows: NRK-52E cells were blotted at 2 mL / well (5 × 10⁻⁶ mcg). 4 Cells were seeded at 100 cells / mL into 6-well plates and incubated overnight. After 6 hours of starvation, cells were treated with different concentrations of Compound 6, Compound 7, or DMSO in DMEM containing 4% FBS for 48 hours. Additionally, recombinant TGF-β1 (5 ng / mL) was added to each plate to induce cell fibrosis. Total protein was extracted from the cell lines using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) containing a protease mixture (Roche, Germany). Protein samples were then quantified using a BCA assay (Thermo Fisher Scientific, USA). Western blot analysis was performed as previously described. The primary antibodies used in this study were as follows: anti-fibronectin (dilution 1:1000; ab 268020; Abcam), anti-collagen 1 (dilution 1:1000; ab 270993; Abcam), anti-αSMA (dilution 1:1000; A2547; Sigma), and anti-GAPDH (dilution 1:2000; sc 365062; Santa Cruz).

[0066] All data were analyzed using IBM SPSS Statistics 22 and expressed as mean ± SEM. One-way ANOVA was used for inter-group comparisons, followed by the Student-Newman-Kuels test. P < 0.05 was considered statistically significant. Detailed statistical tests are illustrated in the legends for each experiment.

[0067] Excessive extracellular matrix (ECM) deposition is a constant feature of chronic kidney disease (CKD). Furthermore, extensive research has described TGF-β1 as the most important pathogenic factor associated with glomerular and tubulointerstitial fibrosis. Therefore, in this study, we induced fibrosis using TGF-β1-stimulated NRK-52E cells, followed by the detection of ECM proteins such as fibronectin, collagen type I, and α-SMA to assess the severity of fibrosis.

[0068] CCK-8 assays were performed to rule out the possibility that the compound's biological effects were caused by cytotoxicity. Results were as follows... Figure 5 The results showed that these compounds at concentrations of 20 μM and below had no significant cytotoxicity. Subsequent dose-response studies indicated that their antifibrotic activity appeared to be more pronounced with increasing concentrations of compounds (-)-6 and (+)-6 (see results). Figure 6 The effects of compounds (-)-7 and (+)-7 on fibronectin expression did not show a dose-response relationship, but they were able to inhibit the expression of α-SMA, type I collagen, and fibronectin at lower concentrations (see results). Figure 6 EH).

[0069] In summary, compounds 6 and 7 isolated from amber in this invention can limit the expression of α-SMA, collagen type I, and fibronectin in TGF-β1-induced NRK-52E cells, indicating that compounds 6 and 7 have the potential to treat renal fibrosis and can be used to prepare drugs for the prevention and treatment of renal fibrosis.

[0070] Example 3

[0071] This embodiment provides a pharmaceutical composition for preparing drugs to prevent and treat renal fibrosis. The compound obtained in Example 1 is prepared by adding an injection solvent according to conventional methods, followed by fine filtration, filling and sterilization to prepare an injection solution.

[0072] Example 4

[0073] This embodiment provides a pharmaceutical composition for preparing drugs to prevent and treat renal fibrosis. The compound prepared in Example 1 is dissolved in sterile water for injection, filtered through a sterile funnel, dispensed, freeze-dried at low temperature, and then sterilely sealed to obtain a powder for injection.

[0074] Example 5

[0075] This embodiment provides a pharmaceutical composition for preparing drugs to prevent and treat renal fibrosis. The compound obtained in Example 1 can be formulated into tablets or capsules using conventional methods with various pharmaceutical excipients. Using the compound in Example 1 as the active pharmaceutical ingredient, and using several conventional excipients as excipients for preparing the combined drug tablets or capsules, samples containing 10-300 mg of the pharmaceutical ingredient per tablet or capsule are prepared according to conventional methods.

[0076] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0077] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A lignan compound or a salt thereof, characterized in that, It has the following structure:

2. The lignan compound or its salt as described in claim 1, characterized in that, The lignans mentioned are extracted and isolated from amber, or obtained through artificial synthesis.

3. The method for preparing lignan compounds or their salts as described in claim 1, characterized in that, Includes the following steps: ① Extract amber by reflux with dichloromethane at room temperature, combine the extracts and concentrate to obtain crude extract; ② The crude extract was subjected to gradient elution with water and 85-100 v% methanol / isopropanol mixed solvent through an MCI CHP 20P column to obtain 9 components Fr.1-Fr.9; Fr.6 was eluted with a gradient of water and 88-100 v% methanol / isopropanol mixed solvent to obtain 7 components Fr.6.1-Fr.6.7; Fr.6.6 was subjected to gradient elution chromatography on a silica gel column using a dichloromethane / methanol system to obtain 14 fractions: Fr.6.6.1-Fr.6.6.9 and Fr.6.6.A-Fr.6.6.E. Fr.6.6.5 was separated by reversed-phase chromatography using an RP-18 column with 86-100 v% methanol / water solution to obtain seven components Fr.6.6.5.1-Fr.6.6.5.7; ③ Using acetonitrile-water as the mobile phase, Fr6.6.5.7 was further purified by semi-preparative HPLC to obtain compounds 6 and 7; compounds 6 and 7 were chirally resolved to obtain compounds (+)-6 / (-)-6 and (+)-7 / (-)-7.

4. The preparation method according to claim 3, characterized in that, In step ①, the ratio of amber to dichloromethane is 1 kg: 3-8 L; reflux extraction is performed 2-3 times, with each extraction lasting 8-36 hours.

5. The preparation method according to claim 3, characterized in that, In step ②, the volume ratio of methanol to isopropanol in the methanol / isopropanol mixed solvent is 8:1-20:1; and the volume ratio of dichloromethane to methanol in the dichloromethane / methanol system is 100:1-5:

1.

6. The preparation method according to claim 3, characterized in that, In step ③, the mobile phase is a 60-85 v% aqueous solution of acetonitrile.

7. The use of the lignan compound or its salt as described in claim 1 or 2, or the lignan compound prepared by the method described in any one of claims 3-5, in the preparation of a drug that inhibits the expression of αSMA protein, type I collagen and fibronectin related to renal fibrosis in NRK-52E cells induced by transforming growth factor-β1.

8. The use of the lignan compound or its salt as described in claim 1 or 2, or the lignan compound prepared by the preparation method as described in any one of claims 3-5, in the preparation of drugs for treating and preventing renal fibrosis.

9. A pharmaceutical composition for treating and preventing renal fibrosis, characterized in that, Includes the lignan compounds or their salts as described in claim 1 or 2, or lignan compounds prepared by the preparation method as described in any one of claims 3-5.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipient.

Images