Efficient extraction method of luteolin and glucoside derivative thereof in saussurea medusa and application of luteolin and glucoside derivative thereof in resisting rheumatoid arthritis

Abstract

The invention discloses an efficient extraction method of luteolin and glucoside derivatives thereof in saussurea medusa and application of luteolin and glucoside derivatives thereof in resisting rheumatoid arthritis, and belongs to the technical field of natural product chemistry and medicine development. The molecular formulas of the three flavonoid compounds are respectively as follows: C15H10O6 (luteolinin), C21H20O11 (luteolin7-O-glucose) and C21H20O11 (luteolin4 '-O-glucose). The invention further discloses a preparation method of the flavonoid compound. 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, DEPT and other spectrums are adopted for identifying the structure. The flavonoid 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 flavonoid compounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product chemistry and drug development, and particularly relates to a method for efficiently extracting luteolin and its glycoside derivatives from Saussurea maxima and an application thereof in treating rheumatoid arthritis (RA). Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by chronic inflammation of the synovial membrane, which leads to joint pain, swelling, stiffness, and loss of function. In severe cases, it can cause joint deformity and disability. Its etiology is complex and is associated with multiple factors, including genetics, environment, and immune system abnormalities. The core pathological mechanism of RA involves massive infiltration of inflammatory cells in the synovium, the release of inflammatory mediators, and synovial hyperplasia, ultimately leading to the destruction of cartilage and bone tissue. Therefore, finding active drugs that can effectively inhibit the pathological process of RA is of great significance for the prevention and treatment of the disease.

[0003] Bya-rgod-sug-pa, a renowned traditional Tibetan medicine, is widely used in Tibetan medicine to treat a variety of conditions, including head trauma, anthrax, fever and stinging, gynecological disorders, rheumatoid arthritis, and epilepsy. Modern research indicates that Bya-rgod-sug-pa is rich in a variety of bioactive components, including flavonoids, lignans, phenolic acids, and amides. Flavonoids, in particular, have attracted considerable attention for their anti-inflammatory, antibacterial, and anticancer properties. However, reports on the systematic extraction and isolation of these flavonoids from Bya-rgod-sug-pa and their applications are scarce. Therefore, there is an urgent need to develop efficient extraction and isolation methods and explore the potential pharmaceutical applications of these compounds. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention proposes a method for efficiently extracting luteolin and its glycoside derivatives from Saussurea jellyfish and its application in treating rheumatoid arthritis. The flavonoids extracted from Saussurea jellyfish include luteolin (Luteolin, molecular formula: C 15 H 10 O6), luteolin 7-O-glucoside (luteolin-7-O-glucoside, molecular formula: C 21 H 20 O 11 ) and luteolin 4'-O-glucoside (luteolin-4'-O-glucoside, molecular formula: C 21 H 20 O 11), all of which belong to luteolin and its glycosides, which have potential medicinal value in anti-inflammatory and other fields.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] One of the purposes of the present invention is to provide luteolin from Saussurea jellyfish and its glycoside derivatives, the structural formulas of which are shown in Formula 1, Formula 2 and Formula 3:

[0007]

[0008] A second object of the present invention is to provide a method for extracting luteolin and its glycoside derivatives from Saussurea jellyfish, comprising the following steps:

[0009] (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;

[0010] (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;

[0011] (3) The fraction Fr10 obtained in step (2) was loaded onto a Megres C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water to obtain fraction Fr10-3; the fraction Fr10-3 was further loaded onto a Megres C18 preparative column and eluted isocratically with acetonitrile and 0.2% (V / V) formic acid-water to obtain luteolin-7-O-glucoside (compound 2) and luteolin-4'-O-glucoside (compound 3);

[0012] (4) The component Fr5 obtained in step (2) was loaded onto a 200-mesh silica gel column and gradient eluted with a dichloromethane-methanol solvent system to obtain components Fr5-1 to Fr5-11 in sequence;

[0013] (5) The fraction Fr5-5 obtained in step (4) was loaded onto a Dubhe C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water system to obtain fraction Fr5-5-6; the obtained fraction Fr5-5-6 was further loaded onto a Dubhe C18 preparative column and eluted isocratically with acetonitrile and 0.2% (V / V) formic acid-water system to obtain luteolin (compound 1).

[0014] Furthermore, in step (1),

[0015] The volume concentration of the ethanol is 50-95%;

[0016] The mass ratio of the saussurea jellyfish to the ethanol is 1:(8-10);

[0017] The reflux extraction was performed 3 times, and the time for each reflux extraction was 12 h.

[0018] Furthermore, in step (2), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solvent is 100:0 to 0:100.

[0019] 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.

[0020] Furthermore, in step (3), the gradient elution process is divided into two gradient elutions, and the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution, and the volume ratios of acetonitrile and 0.2% (V / V) formic acid-water solution are 15:85 and 35:65, respectively;

[0021] During the isocratic elution process, the eluents are chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution.

[0022] Furthermore, in step (4), during the gradient elution process, the volume ratios of dichloromethane and methanol are 100:0, 25:1, 12:1, 5:1 and 0:100, respectively.

[0023] Furthermore, in step (5), the gradient elution process is divided into four gradient elutions, and the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution.

[0024] Furthermore, in step (5), during the isocratic elution, the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution.

[0025] The third object of the present invention is to provide a use of luteolin and its glycoside derivatives in Saussurea jellyfish in the preparation of anti-rheumatoid arthritis drugs.

[0026] Compared with the prior art, the present invention has the following advantages and technical effects:

[0027] (1) The luteolin and its two derived glycoside compounds extracted from Saussurea jellyfish and the extraction method thereof in the present invention are not described in the prior art; the extraction method of the present invention is simple and rapid, and the isolated compounds are of high purity and large yield.

[0028] (2) The luteolin and its two derivative glycoside compounds in the jellyfish Saussurea jellyfish of the present invention can be used as precursors for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research, and can be used to prepare drugs with anti-RA effects, and have broad application development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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:

[0030] Figure 1 The compound 1 in DMSO-d6 in Example 1 1 H NMR spectrum;

[0031] Figure 2 The compound 1 in DMSO-d6 in Example 1 13 C NMR spectrum;

[0032] Figure 3 is the (+)-ESI-MS spectrum of compound 2 in Example 1;

[0033] Figure 4 is the (-)-ESI-MS spectrum of compound 2 in Example 1;

[0034] Figure 5 The compound 2 in Example 1 in methanol-d4 1 HNMR spectrum;

[0035] Figure 6 The compound 2 in Example 1 in methanol-d4 13 C NMR spectrum;

[0036] Figure 7 is the DEPT spectrum of compound 2 in Example 1;

[0037] Figure 8 is the HSQC spectrum of compound 2 in Example 1;

[0038] Figure 9 The compound 3 in Example 1 in methanol-d4 1 HNMR spectrum;

[0039] Figure 10 The compound 3 in Example 1 in methanol-d4 13 C NMR spectrum. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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 exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] The embodiment of the present invention provides a method for extracting luteolin and its glycoside derivatives from Saussurea jellyfish, comprising the following steps:

[0046] (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;

[0047] (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;

[0048] (3) The fraction Fr10 obtained in step (2) was loaded onto a Megres C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water to obtain fraction Fr10-3; the fraction Fr10-3 was further loaded onto a Megres C18 preparative column and eluted isocratically with acetonitrile and 0.2% (V / V) formic acid-water to obtain luteolin-7-O-glucoside and luteolin-4'-O-glucoside;

[0049] (4) The component Fr5 obtained in step (2) was loaded onto a 200-mesh silica gel column and gradient eluted with a dichloromethane-methanol solvent system to obtain components Fr5-1 to Fr5-11 in sequence;

[0050] (5) The fraction Fr5-5 obtained in step (4) was loaded onto a Dubhe C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water system to obtain fraction Fr5-5-6; the obtained fraction Fr5-5-6 was further loaded onto a Dubhe C18 preparative column and isocratically eluted with an acetonitrile / 0.2% formic acid-water system to obtain luteolin.

[0051] In some optional embodiments of the present invention, in step (1), the volume concentration of the ethanol is 50-95%. 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%.

[0052] 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.

[0053] 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.

[0054] In the following preferred 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. 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.

[0055] In the following preferred embodiments of the present invention, in step (3), the gradient elution process is divided into two gradient elutions (the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution, and the volume ratios of acetonitrile and 0.2% (V / V) formic acid-water solution are 15:85 and 35:65).

[0056] In the following preferred embodiments of the present invention, in step (3), during the isocratic elution process, the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution, and the volume ratio of acetonitrile to 0.2% (V / V) formic acid-water solution is 20:80.

[0057] In the following preferred embodiments of the present invention, in step (4), during the gradient elution process, the volume ratios of dichloromethane and methanol are 100:0, 25:1, 12:1, 5:1 and 0:100, respectively.

[0058] In the following preferred embodiments of the present invention, in step (5), the gradient elution process is divided into four gradient elutions (the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution, and the 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)).

[0059] In the following preferred embodiments of the present invention, in step (5), during the isocratic elution process, the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution, and the volume ratio of acetonitrile and 0.2% (V / V) formic acid-water solution is 27:73).

[0060] The above method can be used to extract luteolin and its glycoside derivatives, specifically including luteolin, luteolin 7-O-glucoside, and luteolin 4'-O-glucoside, with the structural formulas shown in Formula 1, Formula 2, and Formula 3:

[0061]

[0062] The luteolin and its glycoside derivatives in the Saussurea jellyfish can be used in the preparation of anti-rheumatoid arthritis drugs.

[0063] 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.

[0064] 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).

[0065] The technical solution of the present invention is further illustrated by the following examples.

[0066] Example 1

[0067] A method for extracting luteolin and its glycoside derivatives from Saussurea jellyfish comprises the following steps:

[0068] (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;

[0069] (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;

[0070] (3) applying the n-butanol fraction obtained in step (2) to a 200-mesh silica gel column and performing gradient elution using a dichloromethane-methanol solvent system at a dichloromethane-methanol volume ratio of 100:0, 10:1, 5:1, 2:1, and 0:100, respectively. The fractions were combined based on the results of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis to obtain 13 components (Fr1-Fr13, i.e., Fr1, Fr2, Fr3, Fr4, Fr5, Fr6, Fr7, Fr8, Fr9, Fr10, Fr11, Fr12, and Fr13);

[0071] (4) The Fr10 component obtained in step (2) was loaded onto a Megres C18 preparative column (10×250 mm, 5 μm) and eluted in two gradients (eluents were acetonitrile and 0.2% (V / V) formic acid-water solution, with the volume ratio of acetonitrile to 0.2% (V / V) formic acid-water solution being 15:85 and 35:65) at a flow rate of 4.0 mL / min. Fr10-3 was obtained in 41 to 43 min. The obtained Fr10-3 was further loaded onto a Megres C18 preparative column (10×250 mm, 5 μm). An sC18 preparative column (10×250 mm, 5 μm) was used with acetonitrile and 0.2% (v / v) formic acid-water system, and eluted isocratically with 20% acetonitrile (eluent: acetonitrile and 0.2% (v / v) formic acid-water, with a volume ratio of acetonitrile to 0.2% (v / v) formic acid-water of 20:80) at a flow rate of 4.0 mL / min. Fr10-3-1 (luteolin 7-O-glucoside, 7.8 mg) and Fr10-3-2 (luteolin 4′-O-glucoside, 2.0 mg) were obtained at 16 min and 26.5 min, respectively.

[0072] (5) The Fr5 component obtained in step (2) was loaded onto a 200-mesh silica gel column and gradient eluted using 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 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 purified by using DubheC 18 preparative column (20 × 250 mm, 5 μm), four gradient elutions (eluent: chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution, acetonitrile volume concentration: 6-10% (0-12 min), 10-30% (12-35 min), 30-22% (35-36 min) and 22% isocratic (36-60 min)), flow rate: 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 Fr5-5-6 subcomponent was added to Dubhe C18 preparative column (20×250 mm, 5 μm) was isocratically eluted with 27% volume concentration of acetonitrile (the eluent was chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution, and the volume ratio of acetonitrile and 0.2% (V / V) formic acid-water solution was 27:73) at a flow rate of 18.0 mL / min to obtain Fr5-5-6-1 (luteolin, 17.5 mg) in 20 min.

[0073] Using ESIMS, 1 H-NMR, 13 The structures of the three 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 10 As shown. Figures 1-2 is the spectrum of compound 1; Figures 3 to 8 is the spectrum of compound 2, Figures 9-10 is the spectrum of compound 3. 1 H-NMR and 13 C-NMR and 13 The C-NMR data of compound 2 were obtained by measuring in DMSO-d6. 1 The H-NMR data and the data of compound 3 were obtained in methanol-d4.

[0074] Luteolin (Compound 1): Light yellow to yellow-brown powder; 1 H NMR (600MHz, DMSO-d6) δ: 12.99 (s, 1H, 5-OH), 7.42 (d, J = 6.5Hz, 2H, H-5', 6' ),6.91(s,1H,H-2'),6.67(s,1H,H-3),6.45(s,1H,H-8),6.20(s,1H,H-6); 13 C NMR (150MHz, DMSO-d6) δ: 181.63(C-4), 164.45(C-7), 163.89(C-2), 161.50(C-5), 157.33(C-9), 149.87(C-4'), 145.83(C- 3'),121.43(C-1'),118.98(C-6'),116.04(C-5'),113.34(C-2'),103.61(C-10),102.82(C-3),98.93(C-6),93.91(C-8).

[0075] Luteolin 7-O-glucoside (Compound 2): Off-white to brown crystals; ESI-MS: m / z 449.11 ([M+H] + ), 447.10([MH] - ); 11H NMR (600 MHz, Methanol-d4) δ: 7.47 (1H, d, J = 8.0 Hz, H-6'), 7.41 (1H, s, H-2'), 6.91 (s, 1H, H-5'), 6.79 (s, 1H, H-3), 6.74 (s, 1H, H-8), 6.46 - 6.43 (m, 1H, H-6), 5.08 (d, J = 7.5 Hz, 1H, H-1”), 3.71 (d, J = 10.9 Hz, 1H), 3.52 - 3.47 (m, 1H), 3.45 (d, J = 9.2 Hz, 1H), 3.28 (dd, J = 20.1, 7.8 Hz, 3H), 3.18 (d, J = 8.8 Hz, 1H); 13 13C NMR (150 MHz, DMSO-d6) δ: 181.83 (C-4), 164.44 (C-7), 162.90 (C-2), 160.83 (C-5), 156.93 (C-9), 149.97 (C-4'), 145.73 (C-3'), 121.25 (C-1'), 119.17 (C-6'), 115.93 (C-5'), 113.48 (C-2'), 105.31 (C-10), 103.15 (C-3), 99.84 (C-1”), 99.48 (C-6), 94.72 (C-8), 77.12 (C-5”), 76.28 (C-3”), 73.03 (C-2”), 69.45 (C-4”), 60.52 (C-6”).

[0076] Luteolin 4'-O-glucoside (Compound 3): It is a brown powder; 1 1H NMR (600 MHz, Methanol-d4) δ: 7.46 (s, 1H, H-6'), 7.33 (d, J = 6.3 Hz, 1H, H-5'), 6.62 (s, 1H, H-3), 6.47 (s, 1H, H-8), 6.22 (s, 1H, H-6), 4.94 (d, J = 6.6 Hz, 1H, H-1”), 3.93 (d, J = 11.6 Hz, 1H), 3.74 (dd, J = 11.2, 4.5 Hz, 1H), 3.53 (dt, J = 28.0, 8.4 Hz, 3H), 3.43 (t, J = 8.7 Hz, 1H), 3.31 (s, 6H); 13C NMR (150MHz, Methanol-d4) δ: 183.87(C-4), 166.18(C-7), 165.55(C-2), 163.28(C-5 ),159.45(C-9),150.02(C-4'),148.68(C-3'),127.25(C-1'),119.83(C-6'),118.0 0(C-5'),114.88(C-2'),105.43(C-3),105.09(C-10),103.22(C-1”),100.23(C-6), 95.06(C-8),78.49(C-5”),77.55(C-3”),74.78(C-2”),71.29(C-4”),62.42(C-6”).

[0077] Since the above spectra can clearly show the structures of the three compounds, this also proves that the three extracted compounds are of high purity.

[0078] Application Example 1

[0079] The anti-inflammatory activity of luteolin and its glycoside derivatives extracted and separated in Example 1 was tested. The specific experimental steps are as follows:

[0080] (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.

[0081] (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 to calculate cell viability. The results are shown in Table 1.

[0082] Table 1 Cell viability test results

[0083]

[0084] As can be seen from the above table, compounds 1 to 3 showed strong anti-RA activity, among which the anti-inflammatory activity of compound 1 and compound 2 exceeded that of the positive control drug dexamethasone.

[0085] 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. Luteolin and its glycoside derivatives in Saussurea jellyfish, characterized in that: The structural formulas are shown in Formula 1, Formula 2 and Formula 3:

2. The method for extracting luteolin and its glycoside derivatives 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) The fraction Fr10 obtained in step (2) was loaded onto a Megres C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water to obtain fraction Fr10-3; the fraction Fr10-3 was further loaded onto a Megres C18 preparative column and eluted isocratically with acetonitrile and 0.2% (V / V) formic acid-water to obtain luteolin-7-O-glucoside and luteolin-4'-O-glucoside; (4) The component Fr5 obtained in step (2) was loaded onto a 200-mesh silica gel column and gradient eluted with a dichloromethane-methanol solvent system to obtain components Fr5-1 to Fr5-11 in sequence; (5) The fraction Fr5-5 obtained in step (4) was loaded onto a Dubhe C18 preparative column and eluted with a gradient of acetonitrile and 0.2% (V / V) formic acid-water system to obtain fraction Fr5-5-6; the fraction Fr5-5-6 was further loaded onto a Dubhe C18 preparative column and eluted isocratically with acetonitrile and 0.2% (V / V) formic acid-water system to obtain luteolin.

3. The method for extracting luteolin and its glycoside derivatives 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 luteolin and its glycoside derivatives 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 luteolin and its glycoside derivatives 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 luteolin and its glycoside derivatives from Saussurea jellyfish according to claim 2, characterized in that: In step (3), the gradient elution process is divided into two gradient elutions, and the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution; During the isocratic elution process, the eluents are chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution.

7. The method for extracting luteolin and its glycoside derivatives from Saussurea jellyfish according to claim 2, characterized in that: In step (4), during the gradient elution process, the volume ratios of dichloromethane and methanol are 100:0, 25:1, 12:1, 5:1 and 0:100, respectively.

8. The method for extracting luteolin and its glycoside derivatives from Saussurea jellyfish according to claim 2, characterized in that: In step (5), the gradient elution process is divided into four gradient elutions, and the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution.

9. The method for extracting luteolin and its glycoside derivatives from Saussurea jellyfish according to claim 2, characterized in that: In step (5), during the isocratic elution, the eluent is chromatographic acetonitrile and 0.2% (V / V) formic acid-water solution.

10. Use of the luteolin and its glycoside derivatives in Saussurea jellyfish as claimed in claim 1 in the preparation of anti-rheumatoid arthritis drugs.

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