Three diaryl butyrolactone lignan compounds in saussurea medusa as well as extraction and separation method and application of three diaryl butyrolactone lignan compounds

Abstract

The invention discloses three diaryl butyrolactone lignan compounds in saussurea medusa as well as an extraction and separation method and application of the diaryl butyrolactone lignan compounds, and belongs to the technical field of extraction and separation of traditional Chinese medicines. The molecular formulas of the three lignan compounds are respectively as follows: C27H34O11 (arctiin), C21H24O6 (arctigenin) and C42H46O12 (arctigenin), and the molecular formulas of the three lignan compounds are respectively as follows: C27H34O11 (arctigenin), C21H24O6 (arctigenin) and C42H46O12 ( According to the invention, ethanol extraction, extraction with different polar solvents, silica gel column chromatography and high performance liquid chromatography are adopted for separation, purification and preparation, and ESIMS, 1H-NMR, 13C-NMR, HSQC and other spectrums are adopted for identifying the structure. The diaryl butyrolactone lignan compounds with important medicinal values are efficiently separated from saussurea medusa, and a new source and a material basis are provided for further research and application of the diaryl butyrolactone lignan compounds.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction and separation, and in particular relates to three diarylbutyrolactone-type lignan compounds in Saussurea medusa Maxim., as well as extraction and separation methods and applications thereof. Background Art

[0002] Rheumatoid arthritis (RA) is one of the autoimmune diseases with the highest disability rate in the world. Its treatment faces clinical difficulties such as large drug side effects and poor long-term efficacy. Its clinical characteristics are mainly manifested as persistent synovial inflammatory response, which in turn leads to joint dysfunction and progressive structural damage. The pathogenesis of the disease involves the interaction of multiple factors, including genetic susceptibility, environmental inducers, and immune regulation disorders. From a pathological point of view, the characteristic changes of RA include inflammatory cell infiltration of synovial tissue, excessive secretion of proinflammatory cytokines, and abnormal proliferation of synovial fibroblasts. These pathological changes ultimately lead to articular cartilage erosion and bone destruction. Given the disabling characteristics of RA, the development of therapeutic drugs with disease-modifying effects has important clinical value.

[0003] As an important medicinal plant in traditional Tibetan medicine, Snow Lotus (S. medusa) has a long history of use in the treatment of various diseases. Modern phytochemical research has confirmed that this plant is rich in a variety of pharmacologically active secondary metabolites, primarily including lignans, alkaloids, flavonoids, and terpenoids. These active ingredients exhibit diverse biological activities, such as free radical scavenging, inflammation suppression, radiation protection, and tumor cytotoxicity. Notably, studies have revealed that its lignan components can significantly reduce the expression of proinflammatory cytokines by regulating inflammatory signaling pathways such as NF-κB, thus showing potential application in the treatment of RA.

[0004] In view of this, systematic isolation and identification of lignan components of Saussurea jellyfish and research on their anti-RA mechanism of action will not only help expand the modern application value of traditional medicinal plants, but also provide an important source of candidate molecules for the research and development of innovative anti-RA drugs, and provide new research directions and possibilities for the development of new anti-rheumatoid arthritis drugs. Summary of the Invention

[0005] In response to the above technical problems, the present invention proposes three diarylbutyrolactone-type lignan compounds in Saussurea jellyfish and their extraction and separation methods and applications.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the purposes of the present invention is to provide three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish, with structural formulas as shown in Formula 1, Formula 2 and Formula 3:

[0008]

[0009] A second object of the present invention is to provide a method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish, comprising the following steps:

[0010] (1) extracting Saussurea jellyfish with ethanol reflux, concentrating the extract to obtain a medicinal solution, resuspending the medicinal solution in water, and extracting with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction;

[0011] (2) The n-butanol fraction obtained in step (1) was loaded onto a 200-mesh silica gel column and gradient eluted using a dichloromethane-methanol solvent system to collect the fractions. The similar fractions were combined by thin layer chromatography (TLC) combined with high performance liquid chromatography (HPLC) analysis to obtain fractions Fr1 to Fr13 in sequence;

[0012] (3) Components Fr4 and Fr5 obtained in step (2) were applied to a 200-mesh silica gel column, and gradient eluted with a dichloromethane-methanol solvent system to obtain components Fr4-1 to Fr4-9 and Fr5-1 to Fr5-11, respectively;

[0013] (4) The components Fr4-3 and Fr5-5 obtained in step (3) were loaded onto Dubhe C18 preparative columns, and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water to obtain compound C of formula 2 in Fr4-3. 21 H 24 O6 (arctigenin) and compound C as described in formula 3 42 H 46 O 12 (diarctigenin), Fr5-5-1 to Fr5-5-6 were obtained in Fr5-5;

[0014] (5) The component Fr5-5-5 obtained in step (4) was loaded onto a Megres C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water system to obtain compound C of formula 1 in Fr5-5-5. 27 H 34 O 11 (arctiin).

[0015] Furthermore, in step (1),

[0016] The volume concentration of the ethanol is 50-95%;

[0017] The mass ratio of the saussurea jellyfish to the ethanol is 1:(8-10);

[0018] The reflux extraction was performed 3 times, and the time for each reflux extraction was 12 h.

[0019] Furthermore, in step (2), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent is 100:0 to 0:100.

[0020] Furthermore, the volume ratios of dichloromethane to methanol during the gradient elution process are 100:0, 10:1, 5:1, 2:1 and 0:100, respectively.

[0021] Furthermore, in step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent is 100:0 to 0:100.

[0022] Furthermore, when gradient elution is performed on the component Fr4, the volume ratios of dichloromethane to methanol are 100:0, 100:1, 50:1, 10:1, 2:1 and 0:100, respectively;

[0023] When the component Fr5 is subjected to gradient elution, the volume ratios of dichloromethane to methanol are 100:0, 25:1, 12:1, 5:1 and 0:100, respectively.

[0024] Furthermore, in step (4):

[0025] When the component Fr4-3 was subjected to gradient elution using a chromatography acetonitrile and 0.2% (V / V) formic acid-water system, three gradient elutions were performed (the volume ratios of acetonitrile and 0.2% (V / V) formic acid-water system were 38:62, 50:50, and 40:60, respectively);

[0026] When the component Fr5-5 is gradient eluted using chromatographic acetonitrile and 0.2% (V / V) formic acid-water system, four gradient elutions are performed (acetonitrile volume concentration is 6-10% (0-12 min), 10-30% (12-35 min), 30-22% (35-36 min) and 22% isocratic (36-60 min)).

[0027] Furthermore, in step (5), when gradient elution of the component Fr5-5-5 is performed using chromatographic acetonitrile and 0.2% (V / V) formic acid-water system, two gradient elutions are performed (the volume ratios of acetonitrile and 0.2% (V / V) formic acid-water system are 27:73 and 40:60, respectively).

[0028] The third object of the present invention is to provide a use of three diarylbutyrolactone-type lignan compounds in Saussurea jellyfish in the preparation of anti-rheumatoid arthritis drugs.

[0029] Compared with the prior art, the present invention has the following advantages and technical effects:

[0030] (1) The diarylbutyrolactone-type lignan compounds extracted from Saussurea jellyfish and the extraction method thereof in the present invention are not described in the prior art; the extraction and separation method of the present invention is simple and rapid, and the separated compounds are of high purity and large yield.

[0031] (2) The three diarylbutyrolactone-type lignan compounds from the present invention can be used as precursors for the synthesis of other compounds, as raw materials for new drug development and pharmacological activity research, and for the preparation of drugs with anti-RA effects. The diarylbutyrolactone-type lignan compounds and derivatives of the present invention can be applied to the development of new natural product traditional Chinese medicines and have broad application and development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 The concentration of compound 1 in CD3OD in Example 1 1 H NMR spectrum;

[0034] Figure 2 The concentration of compound 1 in CD3OD in Example 1 13 C NMR spectrum;

[0035] Figure 3 is the HSQC spectrum of compound 1 in Example 1 in CD3OD;

[0036] Figure 4 is the (+)-ESI-MS spectrum of compound 2 in Example 1;

[0037] Figure 5 The concentration of compound 2 in CD3OD in Example 1 1 HNMR spectrum;

[0038] Figure 6 The concentration of compound 2 in CD3OD in Example 1 13 C NMR spectrum;

[0039] Figure 7 HSQC spectrum of compound 2 in Example 1 in CD3OD;

[0040] Figure 8is the (+)-ESI-MS spectrum of compound 3 in Example 1;

[0041] Figure 9 is the (-)-ESI-MS spectrum of compound 3 in Example 1;

[0042] Figure 10 is the concentration of compound 3 in CD3OD in Example 1 1 HNMR spectrum;

[0043] Figure 11 is the concentration of compound 3 in CD3OD in Example 1 13 C NMR spectrum. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and materials related to the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] The embodiment of the present invention provides a method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish, comprising the following steps:

[0050] (1) extracting Saussurea jellyfish with ethanol reflux, concentrating the extract to obtain a medicinal solution, resuspending the medicinal solution in water, and extracting with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction;

[0051] (2) The n-butanol fraction obtained in step (1) was loaded onto a 200-mesh silica gel column and gradient eluted using a dichloromethane-methanol solvent system to collect the fractions. The similar fractions were combined by thin layer chromatography (TLC) combined with high performance liquid chromatography (HPLC) analysis to obtain fractions Fr1 to Fr13 in sequence;

[0052] (3) Components Fr4 and Fr5 obtained in step (2) were applied to a 200-mesh silica gel column, and gradient eluted with a dichloromethane-methanol solvent system to obtain components Fr4-1 to Fr4-9 and Fr5-1 to Fr5-11, respectively;

[0053] (4) The components Fr4-3 and Fr5-5 obtained in step (3) were loaded onto Dubhe C18 preparative columns, and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water to obtain C in Fr4-3. 21 H 24 O6 (arctigenin) and C 42 H 46 O 12 (diarctigenin), Fr5-5-1 to Fr5-5-6 were obtained in Fr5-5;

[0054] (5) The component Fr5-5-5 obtained in step (4) was loaded onto a Megres C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water system to obtain C in Fr5-5-5. 27 H 34 O 11 (arctiin).

[0055] In some optional embodiments of the present invention, in step (1), the volume concentration of the ethanol is 50-95%. As an example, in the following preferred embodiments of the present invention, the volume concentration of the ethanol is 95%.

[0056] In some optional embodiments of the present invention, in step (1), the mass ratio of the jellyfish lotus to the ethanol is 1: (8-10). As an example, in the following preferred embodiments of the present invention, the mass ratio of the jellyfish lotus to the ethanol is 1:10.

[0057] In the following preferred embodiment of the present invention, in step (1), the reflux extraction is performed three times, and the time for each reflux extraction is 12 hours.

[0058] In some optional embodiments of the present invention, in step (2), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent is 100:0 to 0:100. In the gradient elution process, the volume ratio of dichloromethane to methanol is 100:0, 10:1, 5:1, 2:1 and 0:100, respectively.

[0059] In the following preferred embodiments of the present invention, in step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent is 100:0 to 0:100. When gradient elution is performed on the component Fr4, the volume ratios of dichloromethane to methanol are 100:0, 100:1, 50:1, 10:1, 2:1, and 0:100, respectively; and when gradient elution is performed on the component Fr5, the volume ratios of dichloromethane to methanol are 100:0, 25:1, 12:1, 5:1, and 0:100, respectively.

[0060] In the following preferred embodiments of the present invention, in step (4), when gradient elution of the component Fr4-3 is performed using a chromatographic acetonitrile and 0.2% (V / V) formic acid-water system, three gradient elutions are performed (the volume ratios of acetonitrile and 0.2% (V / V) formic acid-water system are 38:62, 50:50 and 40:60, respectively); when gradient elution of the component Fr5-5 is performed using a chromatographic acetonitrile and 0.2% (V / V) formic acid-water system, four gradient elutions are performed (the volume concentration of acetonitrile is 6-10% (0-12 min), 10-30% (12-35 min), 30-22% (35-36 min) and 22% isocratic (36-60 min)).

[0061] In the following preferred embodiments of the present invention, in step (5), when the component Fr5-5-5 is gradient eluted using chromatographic acetonitrile and 0.2% (V / V) formic acid-water system, two gradient elutions are performed (the volume ratios of acetonitrile and 0.2% (V / V) formic acid-water system are 27:73 and 40:60, respectively).

[0062] The above method can be used to extract C 21 H 24 O6 (arctigenin), C 42 H 46 O 12 (diarctigenin) and C 27 H 34 O 11 (arctiin), whose structural formulas are shown in Formula 1, Formula 2 and Formula 3 respectively:

[0063]

[0064] The three diarylbutyrolactone-type lignan compounds in the Saussurea jellyfish can be used in the preparation of anti-rheumatoid arthritis drugs.

[0065] In the application examples of the present invention, HFLS-RA human rheumatoid arthritis fibroblast-like synoviocytes were purchased from Guangzhou Jinio Biotechnology Co., Ltd. DMEM high-glucose cell culture medium was purchased from Shanghai Dathill Biotechnology Co., Ltd. Cell freezing solution, fetal bovine serum, and PBS buffer were purchased from Guangzhou Hucheng Technology Co., Ltd. Penicillin-streptomycin sulfate and trypsin were purchased from Wuhan Boster Bioengineering Co., Ltd. DMSO was purchased from Tianjin Best Chemical Co., Ltd. MTT cell proliferation and toxicity assay kits were purchased from Wuhan Boster Bioengineering Co., Ltd. PeproTech human TNF-α dry powder was purchased from Thermo Fisher Scientific Inc.

[0066] The main instruments used in the application examples of the present invention include: an AG204 microbalance (Mettler Toledo, Switzerland); a DragonLab pipette and a DM0412E centrifuge (Dalong Xingchuang Laboratory Instrument Co., Ltd.) and a Reaserch pipette (Eppendorf, Germany); a Model 680 microplate reader (Bio-Rad, USA); an HF90 CO2 cell culture incubator (Shanghai Likang Biomedical Technology Holdings Co., Ltd.); a Hirayama HVE-50 high-pressure sterilizer (Hirayama Manufacturing Co., Ltd., Japan); a Shangjing BHC-1600IIB2 biological safety cabinet (Shaoxing Shangyu Aike Instrument Equipment Co., Ltd.); and an Olympus CKX41 inverted imaging microscope (Olympus Corporation, Japan).

[0067] The technical solution of the present invention is further illustrated by the following examples.

[0068] Example 1

[0069] The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish comprises the following steps:

[0070] (1) 15 kg of dried medicinal materials of Saussurea jellyfish were taken, 150 kg of 95% by volume ethanol was added, and reflux extraction was performed at 70° C. for 3 times, each time for 12 h. The extract was then filtered, concentrated, and ethanol was recovered under reduced pressure to obtain 800 g of extract;

[0071] (2) The extract prepared in step (1) was resuspended in water and extracted with 4 L of petroleum ether, ethyl acetate, and n-butanol five times, respectively, to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction, wherein the mass of the n-butanol fraction was 200.7 g;

[0072] (3) applying the n-butanol fraction obtained in step (2) to a 200-mesh silica gel column and using a dichloromethane-methanol solvent system to perform gradient elution at a dichloromethane to methanol volume ratio of 100:0, 10:1, 5:1, 2:1, and 0:100, respectively. The fractions were combined according to the results of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis to obtain 13 components (Fr1 to Fr13, i.e., Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10, Fr11, Fr12, and Fr13);

[0073] (4) The Fr4 component in step (3) was loaded onto a 200-mesh silica gel column and eluted with a dichloromethane-methanol solvent system at a volume ratio of dichloromethane to methanol of 100:0, 100:1, 50:1, 10:1, 2:1 and 0:100, respectively. The fractions were combined according to the results of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis to obtain 9 subcomponents (Fr4-1 to Fr4-9, i.e., Fr4-1, Fr4-2, Fr4-3, Fr4-4, Fr4-5, Fr4-6, Fr4-7, Fr4-8 and Fr4-9); the obtained Fr4-3 subcomponent was loaded onto a Dubhe A C18 preparative column (20×250 mm, 5 μm) was used for chromatography, using a system of acetonitrile and 0.2% (v / v) formic acid-water. Three gradient elutions (volume ratios of 38:62, 50:50, and 40:60) were used at a flow rate of 18.0 mL / min. Fr4-3-2 (445.8 mg, arctigenin) and Fr4-3-4 (251.8 mg, dialctigenin) were obtained in 21 and 40 minutes, respectively.

[0074] (5) The Fr5 component in step (3) was loaded onto a 200-mesh silica gel column and eluted with a dichloromethane-methanol solvent system at a volume ratio of dichloromethane to methanol of 100:0, 25:1, 12:1, 5:1 and 0:100, respectively. The fractions were combined according to the results of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis to obtain 11 subcomponents (Fr5-1 to Fr5-11, i.e., Fr5-1, Fr5-2, Fr5-3, Fr5-4, Fr5-5, Fr5-6, Fr5-7, Fr5-8, Fr5-9, Fr5-10 and Fr5-11); the obtained Fr5-5 subcomponent was loaded onto a Dubhe A C18 preparative column (20×250 mm, 5 μm) was used with chromatographic acetonitrile and 0.2% (V / V) formic acid-water system, with four gradient elutions (acetonitrile volume concentration of 6-10% (0-12 min), 10-30% (12-35 min), 30-22% (35-36 min) and 22% isocratic (36-60 min)) at a flow rate of 18.0 mL / min. Fr5-5-2, Fr5-5-5 and Fr5-5-6 were obtained at 13 min, 39 min and 50 min, respectively. The obtained Fr5-5-5 subcomponent was subjected to Megres chromatography. A C18 preparative column (10×250 mm, 5 μm) was used with chromatographic acetonitrile and 0.2% (V / V) formic acid-water system. Two gradient elutions (volume ratios of 27:73 and 40:60) were performed at a flow rate of 4.0 mL / min to obtain Fr5-5-5-1 (4.9 mg, arctiin) in 13 min.

[0075] Using ESIMS, 1 H-NMR, 13 The structures of the three diarylbutyrolactone lignan compounds extracted and separated in Example 1 were identified by C-NMR, HSQC and other spectral techniques. The results are shown in the attached figure. Figures 1 to 11 As shown. Figures 1 to 3 is the spectrum of compound 1; Figures 4 to 7 is the spectrum of compound 2, Figures 8-11 is the spectrum of compound 3. 1 H-NMR and 13 All C-NMR data were obtained in CD3OD (Methanol-d4).

[0076] Arctiin (Compound 1): Brown-black crystals; molecular formula is C 27 H 34 O 11 . 1H NMR (600MHz, Methanol-d4) δ: 7.06 (d, J=8.2Hz, 1H, H-5), 6.84 (d, J=8.0Hz, 1H, H-5'), 6.77 (d, J=2.0Hz, 1H, H-2), 6.69-6.61 (m, 2H, H-6 ,6'),6.61(s,1H,H-2'),4.21(t,J=8.2Hz,1H,H-9'a),3.96(t,J=8.3Hz,1H,H-9'b),3.81(s,6H,Glu-H),2.56-2.48(m,2H,H-7'a,7'b); 13 C NMR (150MHz, Methanol-d4) δ: 181.39(C-9), 150.69(C-3), 150.50(C-3'), 149.19(C-4), 146.88(C-4' ),134.25(C-1),132.74(C-1'),122.98(C-6'),122.10(C-6),117.88(C-5),114.80(C-5'),113.61(C- 2'),113.07(C-2),102.91(C-1”),78.19(C-5”),77.84(C-3”),74.92(C-2”),72.92(C-9’),71.34(C- 4"),62.50(C-6"),56.67,56.50,56.44(OCH3),47.66(C-8),42.49(C-8'),38.92(C-7'),35.41(C-7).

[0077] Arctigenin (Compound 2): Pale yellow-brown translucent crystals; ESI-MS: m / z 373.16 ([M+H] + ), molecular formula is C 21 H 24 O6, its hydrogen spectrum data is: 1 H NMR (600MHz, Methanol-d4) δ: 6.79 (d, J = 8.5 Hz, 1H), 6.70 (d, J = 7.9 Hz, 1H), 6.66 (s, 1H), 6.56 (d, J = 5.0 Hz, 4H), 4.15-4.10 (m, 1 H),3.89(t,J=8.1Hz,1H),2.87(dd,J=13.9,5.2Hz,1H),2.78(dd,J=13.9,7.3Hz,1H),2.62(q,J=7.3Hz,1H),2.54-2.44(m,4H); 13C NMR (150MHz, Methanol-d4) δ: 181.42 (C-9'), 150.39 (C-3), 149.07 (C-3'), 148. 94(C-4),146.35(C-4'),132.78(C-1),130.72(C-1'),123.00(C-6'),122.00(C- 6),116.07(C-5'),113.83(C-2'),113.56(C-2),112.98(C-5),72.82(C-9),56. 43,56.33,56.32(C-OMe),47.68(C-8'),42.43(C-8),38.80(C-7),35.39(C-7').

[0078] Diarctigenin (Compound 3): brownish yellow crystals; ESI-MS: m / z 743.33 ([M+H] + ), 741.45([MH] - ), molecular formula is C 42 H 46 O 12 . 1 H NMR(600MHz, Methanol-d4)δ: 6.74-6.69(m,2H,H-5,6'),6.64-6.61(m,1H,H-6),6. 54(d,J=5.4Hz,2H,H-2,2'),4.17-4.11(m,1H,H-9'),3.87(t,J=8.3Hz,1H,H-9),3. 82(s,2H),3.72(s,2H),3.60(s,2H),3.31(s,1H),2.86(qd,J=14.0,6.0Hz,2H),2.6 4(q,J=6.7,6.1Hz,1H),2.61-2.55(m,1H),2.54(d,J=5.8Hz,1H),2.53-2.46(m,1H); 13CNMR(150MHz,Methanol-d4)δ: 181.49(C-9'),150.31(C-3),149.46(C-3'),148 .99(C-4),143.42(C-4'),132.77(C-1'),130.37(C-1),126.79(C-6'),125.66(C -5'),122.00(C-6),113.50(C-2'),112.94(C-2),112.59(C-5),72.92(C-9),56. 55,56.39,56.27(C-OMe),47.75(C-8'),42.19(C-8),38.78(C-7),35.36(C-7').

[0079] Since the above spectra can clearly show the structures of the three new diarylbutyrolactone-type lignan compounds, this also proves that the three extracted lignan compounds are of high purity.

[0080] Application Example 1

[0081] The three diarylbutyrolactone-type lignan compounds extracted and separated in Example 1 were tested for their anti-inflammatory activity. The specific experimental steps are as follows:

[0082] (1) Cell culture: HFLS-RA cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-antibody in a cell culture incubator at 37°C and 5% carbon dioxide.

[0083] (2) MTT assay for cell viability: HFLS-RA cells in the logarithmic growth phase were cultured at a rate of 2×10 3The cells were inoculated into 96-well plates at a concentration of 100 μL / well and cultured, with 5 replicates per group. After the cells were inoculated into 96-well plates and cultured for 24 hours until they adhered to the wall, the original culture medium of each well was discarded, and then culture medium containing extracts of Saussurea jellyfish from different parts at different concentrations was added to each group. In order to further strengthen the inflammatory response and proliferation of HFLS-RA cells and better simulate the inflammatory environment, the present invention added TNF-α to the cells for intervention. In each group, 110 μL of culture medium was added to the zero-adjustment group, 110 μL of a mixed solution of culture medium and TNF-α was added to the model control group (TNF-α concentration 0.09 ng / mL, the same below), and 110 μL of a mixed solution of drug-containing culture medium and TNF-α was added to the experimental treatment group. The experimental treatment group contained a corresponding compound concentration of 200 μg / mL. To promote drug dissolution, the above-mentioned drug-containing or drug-free culture medium contained DMSO at a concentration of 2.5 parts per thousand. After 24 hours of treatment, the culture medium from each well was aspirated, and 110 μL of DMEM medium containing 9% MTT reagent was added to each well in the dark, followed by incubation at 37°C for 4 hours. Following treatment, the culture medium from each well was aspirated, and 100 μL of DMSO was added. After a further 10 minutes of incubation, the absorbance (A) of each well at 570 nm was measured using a microplate reader, and the cell viability was calculated. The results are shown in Table 1.

[0084] Table 1 Cell viability test results

[0085]

[0086] As can be seen from the above table, compounds 1 to 3 showed strong anti-RA activity, among which the activities of compounds 2 and 3 exceeded that of the positive control drug dexamethasone.

[0087] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish, characterized in that: The structural formulas are shown in Formula 1, Formula 2 and Formula 3:

2. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 1, characterized in that: The following steps are involved: (1) extracting Saussurea jellyfish with ethanol reflux, concentrating the extract to obtain a medicinal solution, resuspending the medicinal solution in water, and extracting with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction; (2) The n-butanol fraction obtained in step (1) was loaded onto a 200-mesh silica gel column and gradient eluted using a dichloromethane-methanol solvent system to collect the fractions. Similar fractions were combined using thin-layer chromatography combined with high-performance liquid chromatography analysis results to obtain fractions Fr1 to Fr13 in sequence; (3) Components Fr4 and Fr5 obtained in step (2) were applied to a 200-mesh silica gel column, and gradient eluted with a dichloromethane-methanol solvent system to obtain components Fr4-1 to Fr4-9 and Fr5-1 to Fr5-11, respectively; (4) The components Fr4-3 and Fr5-5 obtained in step (3) were respectively applied to a Dubhe C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (v / v) formic acid-water system to obtain a compound of formula 2 and a compound of formula 3 in Fr4-3, and obtain Fr5-5-1 to Fr5-5-6 in Fr5-5; (5) The component Fr5-5-5 obtained in step (4) was loaded onto a Megres C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water system to obtain a compound with the structural formula 1 in Fr5-5-5.

3. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 2, characterized in that: In step (1): The volume concentration of the ethanol is 50-95%; The mass ratio of the saussurea jellyfish to the ethanol is 1:(8-10); The reflux extraction was performed 3 times, and the time for each reflux extraction was 12 h.

4. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 2, characterized in that: In step (2), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent is 100:0 to 0:

100.

5. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 4, characterized in that: The volume ratios of dichloromethane to methanol during the gradient elution process are 100:0, 10:1, 5:1, 2:1 and 0:100, respectively.

6. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 2, characterized in that: In step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent is 100:0 to 0:

100.

7. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 5, characterized in that: When gradient elution is performed on the component Fr4, the volume ratios of dichloromethane to methanol are 100:0, 100:1, 50:1, 10:1, 2:1 and 0:100, respectively; When the component Fr5 is subjected to gradient elution, the volume ratios of dichloromethane to methanol are 100:0, 25:1, 12:1, 5:1 and 0:100, respectively.

8. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 2, characterized in that: In step (4): When the component Fr4-3 is subjected to gradient elution using a chromatography acetonitrile and 0.2% (V / V) formic acid-water system, three gradient elutions are performed; When the component Fr5-5 is subjected to gradient elution using a chromatography system of acetonitrile and 0.2% (V / V) formic acid-water, four gradient elutions are performed.

9. The method for extracting and separating three diarylbutyrolactone-type lignan compounds from Saussurea jellyfish according to claim 2, characterized in that: In step (5), when gradient elution of the component Fr5-5-5 is performed using a chromatographic acetonitrile and 0.2% (V / V) formic acid-water system, two gradient elutions are performed.

10. Use of the three diarylbutyrolactone lignan compounds from Saussurea jellyfish as claimed in claim 1 in the preparation of anti-rheumatoid arthritis drugs.

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