Cichorium glandulosum extract, compound separated from Cichorium glandulosum extract as well as extraction method and application of compound

Abstract

The invention relates to the technical field of medicine research of Cichorium glandulosum, in particular to a Cichorium glandulosum extract, a compound separated from the Cichorium glandulosum extract and an extraction method and application thereof.The Cichorium glandulosum extract comprises Cichorium glandulosum lactone A, Cichorium glandulosum lactone B, Cichorium glandulosum picroside and Cichorium glandulosum. And dissolving the crude extract with a solvent, separating, carrying out gradient elution, carrying out gradient elution on the obtained eluate through ODS column chromatography, and carrying out gradient elution on the obtained eluate through high performance liquid chromatography separation to obtain the cichorium glandulosum extract. According to the invention, the cichorium glandulosum extract and the compound separated from the cichorium glandulosum extract are extracted by taking the whole herb of cichorium glandulosum as a raw material for the first time, and the inhibition effect of the compounds, namely cichorium glandulosum lactone A, cichorium glandulosum lactone B, cichorium glandulosum picroside and cichorium glandulosum, separated from the cichorium glandulosum extract on RAW264.7 cells is disclosed for the first time. Therefore, the compound can be applied to preparation of anti-inflammation or / and inflammation treatment medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of Cichorium glandulosum drug research, and is a Cichorium glandulosum extract, a compound isolated from the Cichorium glandulosum extract, and its extraction method and application. Background Art

[0002] Cichorium glandulosum Boiss. et Huet is a plant of the genus Cichorium in the family Compositae. There are six species of Cichorium plants globally, mainly distributed in Europe, North Africa, Turkey and other places. There are 3 species of Cichorium plants in China, namely Cichorium intybus L., Cichorium endivia L., and Cichorium glandulosum Boiss. et Huet. According to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Cichorii Herba Cichorii Radix is a general term for the dried above-ground parts or roots of the plants Cichorium intybus or Cichorium glandulosum.

[0003] Cichorium glandulosum is a plant of the genus Cichorium in the family Compositae, mainly distributed in the Caucasus, Turkey, Xinjiang of China and other places. In traditional medicine, the above-ground part or root of Cichorium glandulosum is used as medicine, with the effects of clearing the liver and gallbladder, strengthening the stomach and promoting digestion, and diuretic and detumescence, and is used for protecting the liver, anti-tumor, reducing blood sugar and blood lipids, etc.

[0004] The Chinese patent document with the authorization announcement number CN110354116B discloses a preparation method and application of a Cichorium glandulosum extract. The molecular formula of the lignin-like extract of Cichorium glandulosum is C 20 H 24 O6, which can be used as an inflammation lead compound, and this compound has inhibitory effects on NO, TNF-α, and PGE2 produced by LPS-activated RAW264.7 cells.

[0005] The Chinese patent document with the authorization announcement number CN103804334B discloses a method for extracting and separating C 15 H 18 O5 and its uses. The characteristics are as follows: Soak the roots of Cichorium glandulosum with ethanol, collect the percolate by the percolation method, obtain a thick extract with a vacuum distillation device, load the extract onto a silica gel column, an ODS column and a Sephadex LH-20 column, elute, and evaporate the eluate to dryness with a vacuum distillation device to obtain a dry powder, which is the monomer C 15 H 18 O5, and its English name is 11β,13-Dihydrolacurin compound.

[0006] The Chinese patent document with the authorization announcement number CN102351823 discloses a lactucopicrin derivative, its preparation method and uses. This derivative uses lactucopicrin isolated from Cichorium glandulosum Boiss. et Huet as the parent compound, and through structural modification of lactucopicrin, a lactucopicrin derivative is synthesized.

[0007] The Chinese patent document with the publication number CN112939912A discloses a preparation method and anti-inflammatory and hepatoprotective effects of lactucin from Cichorium glandulosum Boiss. et Huet extract. The guaianolide sesquiterpene lactone extract lactucin from Cichorium glandulosum Boiss. et Huet can be used as an inflammation lead compound. Lactucin has inhibitory effects on NO, TNF-α, and IL-6 produced by LPS-activated RAW264.7 cells, and has low cytotoxicity. It can significantly reduce the expression of iNOS and COX-2 proteins, can significantly inhibit the expression of IL-6, iNOS, COX02, and IFN-β genes in RAW264.7 cells, and can reduce the phosphorylation of P44 / 42 and P38MAPK proteins in the MAPK signaling pathway.

[0008] It can be seen that clinically, preparations containing Cichorium glandulosum Boiss. et Huet are mostly used to treat some liver-related diseases: such as Qingre Kasen Granules for protecting the liver and reducing blood pressure; Chicoric Acid Ester Qing Capsules for improving blood sugar, blood lipid, and uric acid metabolism; Hugan Buzure Granules and Yanxiao Dina'er Syrup for treating liver diseases; Compound Muniziqi Granules are mainly used to regulate body fluids and temperament. Modern pharmacological studies have shown that Cichorium glandulosum Boiss. et Huet has various pharmacological activities such as hepatoprotection, hypoglycemic, anti-tumor, lipid-lowering, anti-inflammatory, uric acid-lowering, and antibacterial. According to literature reports, it has various chemical components such as terpenoids, flavonoids, phenylpropanoids, phenolic acids, and sugars, among which sesquiterpene lactone compounds are its characteristic components.

[0009] Therefore, developing and utilizing the compounds extracted from Cichorium glandulosum Boiss. et Huet, further exploring its potential medicinal value, determining and characterizing the structure and physicochemical properties of its compounds, and clarifying the anti-inflammatory activity of the compounds through mouse macrophage model experiments is a very meaningful work. Summary of the Invention

[0010] The present invention provides a Cichorium glandulosum Boiss. et Huet extract, the compounds isolated from the Cichorium glandulosum Boiss. et Huet extract, its extraction method and application, overcoming the deficiencies of the above-mentioned prior art. It first discloses the inhibitory effects of the Cichorium glandulosum Boiss. et Huet extract, the compounds isolated from the Cichorium glandulosum Boiss. et Huet extract, lactucin A, lactucin B, lactucopicrin, and lactucin on RAW264.7 cells, enabling the above compounds to be applied to the preparation of anti-inflammatory or / and anti-inflammatory drugs.

[0011] One of the technical solutions of the present invention is achieved by the following measures: A Cichorium glandulosum Boiss. et Huet extract, including lactucin A, lactucin B, cichorioside, and cichoriin, wherein,

[0012] The structural formula of lactucin A is:

[0013]

[0014] The structural formula of lactucin B is:

[0015]

[0016] The structural formula of cichorioside is:

[0017]

[0018] The structural formula of cichoriin is:

[0019]

[0020] The following is a further optimization or / and improvement of one of the above-mentioned invention technical solutions:

[0021] The above is obtained according to the following steps:

[0022] S1, Mix the required amount of whole Cichorium glandulosum Boiss. et Huet with a solvent, heat and reflux for extraction 2 to 5 times, recover the extractive solution to obtain a crude extract. Among them, 8 mL to 20 mL of solvent is added to each 1 g of whole Cichorium glandulosum Boiss. et Huet, and the solvent is one of pure water, an ethanol solution with a volume fraction of 70% to 95%, and a methanol solution with a volume fraction of 80% to 90%;

[0023] S2, Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate with D101 macroporous adsorption resin and perform a one-time gradient elution to obtain one-time eluates with different polarities. Among them, when performing the one-time gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratio of pure water to ethanol is 100:0 to 5:95, pure water decreases from 100 to 5, and ethanol increases from 0 to 95;

[0024] S3, Subject the one-time eluates with different polarities obtained in step S2 to ODS column chromatography, and perform a two-time gradient elution with a mixed solvent as the mobile phase to obtain the Cichorium glandulosum Boiss. et Huet extract. Among them, the mixed solvent is a mixed solution composed of methanol and pure water or a mixed solution composed of acetonitrile and pure water. The volume ratio of methanol to pure water is 2:8 to 9:1, methanol increases from 2 to 9, pure water decreases from 8 to 1, and the volume ratio of acetonitrile to pure water is 1:9 to 8:2, acetonitrile increases from 1 to 8, and pure water decreases from 9 to 2.

[0025] The second technical solution of the present invention is achieved by the following measures: An extraction method of Cichorium glandulosum extract is carried out according to the following steps:

[0026] S1, Mix the required amount of whole Cichorium glandulosum herb with a solvent, heat and reflux for extraction 2 to 5 times, recover the extract to obtain a crude extract. Among them, 8 mL to 20 mL of the solvent is added to every 1 g of the whole Cichorium glandulosum herb, and the solvent is one of pure water, an ethanol solution with a volume fraction of 70% to 95%, and a methanol solution with a volume fraction of 80% to 90%.

[0027] S2, Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate with D101 macroporous adsorption resin and perform a one-time gradient elution to obtain one-time eluates with different polarities. Among them, when performing the one-time gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratio of pure water to ethanol is 100:0 to 5:95, the pure water decreases from 100 to 5, and the ethanol increases from 0 to 95.

[0028] S3, Subject the one-time eluates with different polarities obtained in step S2 to ODS column chromatography, and perform a two-time gradient elution with a mixed solvent as the mobile phase to obtain the Cichorium glandulosum extract. Among them, the mixed solvent is a mixed solution composed of methanol and pure water or a mixed solution composed of acetonitrile and pure water. The volume ratio of methanol to pure water is 2:8 to 9:1, the methanol increases from 2 to 9, and the pure water decreases from 8 to 1. The volume ratio of acetonitrile to pure water is 1:9 to 8:2, the acetonitrile increases from 1 to 8, and the pure water decreases from 9 to 2.

[0029] The third technical solution of the present invention is achieved by the following measures: A compound isolated from the Cichorium glandulosum extract is cichograndatin A, and its structural formula is:

[0030]

[0031] It is obtained by extraction according to the following steps:

[0032] The Cichorium glandulosum extract obtained in step S3 is separated by high performance liquid chromatography, and a three-time gradient elution is performed with a mixed solvent as the mobile phase to obtain cichograndatin A.

[0033] The fourth technical solution of the present invention is achieved by the following measures: A compound isolated from the Cichorium glandulosum extract is cichograndatin B, and its structural formula is:

[0034]

[0035] It is obtained by extraction according to the following steps:

[0036] The Cichorium glandulosum extract obtained in step S3 is separated by high performance liquid chromatography, and a three-time gradient elution is performed with a mixed solvent as the mobile phase to obtain cichograndatin B.

[0037] The fifth technical solution of the present invention is achieved by the following measures: A compound isolated from the extract of Cichorium glandulosum Boiss. et Huet, which is cichorioside, and its structural formula is:

[0038]

[0039] It is obtained by extraction according to the following steps:

[0040] The extract of Cichorium glandulosum Boiss. et Huet obtained in step S3 is separated by high performance liquid chromatography, and three gradient elutions are carried out with a mixed solvent as the mobile phase to obtain cichorioside.

[0041] The sixth technical solution of the present invention is achieved by the following measures: A compound isolated from the extract of Cichorium glandulosum Boiss. et Huet, which is cichoriin, and its structural formula is:

[0042]

[0043] It is obtained by extraction according to the following steps:

[0044] The extract of Cichorium glandulosum Boiss. et Huet obtained in step S3 is separated by high performance liquid chromatography, and three gradient elutions are carried out with a mixed solvent as the mobile phase to obtain cichoriin.

[0045] The seventh technical solution of the present invention is achieved by the following measures: A method for extracting a compound isolated from the extract of Cichorium glandulosum Boiss. et Huet, characterized in that:

[0046] The extraction method of lactucin A is carried out according to the following steps:

[0047] The extract of Cichorium glandulosum Boiss. et Huet obtained in step S3 is separated by high performance liquid chromatography, and three gradient elutions are carried out with a mixed solvent as the mobile phase to obtain lactucin A;

[0048] The extraction method of lactucin B is carried out according to the following steps:

[0049] The extract of Cichorium glandulosum Boiss. et Huet obtained in step S3 is separated by high performance liquid chromatography, and three gradient elutions are carried out with a mixed solvent as the mobile phase to obtain lactucin B;

[0050] The extraction method of cichorioside is carried out according to the following steps:

[0051] The extract of Cichorium glandulosum Boiss. et Huet obtained in step S3 is separated by high performance liquid chromatography, and three gradient elutions are carried out with a mixed solvent as the mobile phase to obtain cichorioside;

[0052] The extraction method of cichoriin is carried out according to the following steps:

[0053] The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is performed three times with a mixed solvent as the mobile phase to obtain chicoric acid.

[0054] The eighth technical solution of the present invention is achieved by the following measures: An application of chicory extract in the preparation of anti-inflammatory and / or anti-inflammatory drugs.

[0055] The ninth technical solution of the present invention is achieved by the following measures: An application of compound lactucin A or / and lactucin B or / and int chicoric acid or / and chicoric acid separated from chicory extract in the preparation of anti-inflammatory and / or anti-inflammatory drugs.

[0056] The present invention first extracts chicory extract and compounds separated from chicory extract from the whole chicory herb, and first discloses the inhibitory effects of compounds lactucin A, lactucin B, int chicoric acid, and chicoric acid separated from chicory extract on RAW264.7 cells, enabling the above compounds to be applied to the preparation of anti-inflammatory and / or anti-inflammatory drugs. Detailed implementation mode

[0057] The present invention is not limited by the following embodiments, and specific implementation modes can be determined according to the technical solutions of the present invention and actual situations. All chemical reagents and chemical supplies mentioned in the present invention are well-known and commonly used chemical reagents and chemical supplies in the prior art unless otherwise specified.

[0058] The present invention will be further described below in conjunction with embodiments:

[0059] Embodiment 1: The chicory extract includes lactucin A, lactucin B, int chicoric acid, and chicoric acid, wherein

[0060] The structural formula of lactucin A is:

[0061]

[0062] The structural formula of lactucin B is:

[0063]

[0064] The structural formula of int chicoric acid is:

[0065]

[0066] The structural formula of chicoric acid is:

[0067]

[0068] Embodiment 2: As an optimization of the above embodiment, it is obtained according to the following steps:

[0069] S1. Mix the required amount of whole Cichorium glandulosum Boiss. et Huet herb with a solvent, heat and reflux for extraction 2 to 5 times, and recover the extract to obtain a crude extract. Among them, 8 mL to 20 mL of solvent is added to every 1 g of whole Cichorium glandulosum Boiss. et Huet herb, and the solvent is one of pure water, an ethanol solution with a volume fraction of 70% to 95%, and a methanol solution with a volume fraction of 80% to 90%.

[0070] S2. Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate with D101 macroporous adsorption resin, and perform a one-time gradient elution to obtain eluates with different polarities for the first time. Among them, when performing the one-time gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratio of pure water to ethanol is 100:0 to 5:95, pure water decreases from 100 to 5, and ethanol increases from 0 to 95.

[0071] S3. Subject the eluates with different polarities for the first time obtained in step S2 to ODS column chromatography, and perform a two-time gradient elution with a mixed solvent as the mobile phase to obtain the Cichorium glandulosum Boiss. et Huet extract. Among them, the mixed solvent is a mixed solution composed of methanol and pure water or a mixed solution composed of acetonitrile and pure water. The volume ratio of methanol to pure water is 2:8 to 9:1, methanol increases from 2 to 9, and pure water decreases from 8 to 1. The volume ratio of acetonitrile to pure water is 1:9 to 8:2, acetonitrile increases from 1 to 8, and pure water decreases from 9 to 2.

[0072] Example 3: The compound isolated from the Cichorium glandulosum Boiss. et Huet extract is lactucin A, and its structural formula is:

[0073]

[0074] It is obtained by extraction according to the following steps:

[0075] S1. Mix 1 kg of whole Cichorium glandulosum Boiss. et Huet herb with 10 L of pure water, heat and reflux for extraction 3 times, and recover the extract under reduced pressure to obtain a crude extract.

[0076] S2. Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate with D101 macroporous adsorption resin, and perform a one-time gradient elution to obtain eluates with different polarities for the first time. Among them, when performing the one-time gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratios of pure water to ethanol are successively 100:0, 70:30, 50:50, 30:70, 5:95, pure water decreases from 100 to 5, and ethanol increases from 0 to 95.

[0077] S3. The primary eluates with different polarities obtained in step S2 are fractionated through an ODS column chromatography with a flow rate of (pure water: ethanol) from 100:0 to 50:50, and a secondary gradient elution is performed using a mixed solution composed of methanol and pure water as the mobile phase to obtain the extract of Cichorium glandulosum Boiss. et Huet. Among them, the volume ratios of methanol to pure water are successively 1:9, 3:7, 5:5, 7:3, 9:1, with methanol increasing from 1 to 9 and pure water decreasing from 9 to 1;

[0078] S4. The extract of Cichorium glandulosum Boiss. et Huet. obtained in step S3 is fractionated through high performance liquid chromatography (HPLC - UV) with a flow rate of (methanol: pure water) from 3:7 to 5:5, and a tertiary gradient elution is performed using a mixed solvent as the mobile phase, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile: pure water (15:85), t R is 43 min, and lactucin A (yield is 0.00015‰) is obtained.

[0079] Example 4: The compound isolated from the extract of Cichorium glandulosum Boiss. et Huet. is lactucin B, and its structural formula is:

[0080]

[0081] It is obtained by extraction according to the following steps:

[0082] S1. 1 kg of the whole herb of Cichorium glandulosum Boiss. et Huet. is mixed with 10 L of pure water, heated and refluxed for extraction three times, and the extract is recovered under reduced pressure to obtain a crude extract;

[0083] S2. The crude extract is dissolved in a solvent, and macroporous adsorption resin is taken for sample mixing and drying. After separation by D101 macroporous adsorption resin, a primary gradient elution is performed to obtain primary eluates with different polarities. Among them, when performing the primary gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratios of pure water to ethanol are successively 100:0, 70:30, 50:50, 30:70, 5:95, with water decreasing from 100 to 5 and ethanol increasing from 0 to 95;

[0084] S3. The primary eluates with different polarities obtained in step S2 are fractionated through an ODS column chromatography with a flow rate of (pure water: ethanol) from 100:0 to 50:50, and a secondary gradient elution is performed using a mixed solution composed of methanol and pure water as the mobile phase to obtain the extract of Cichorium glandulosum Boiss. et Huet. Among them, the volume ratios of methanol to pure water are successively 1:9, 3:7, 5:5, 7:3, 9:1, with methanol increasing from 1 to 9 and pure water decreasing from 9 to 1;

[0085] S4. The extract of Cichorium glandulosum Boiss. et Huet. obtained in step S3 is fractionated through high performance liquid chromatography (HPLC - UV) with a flow rate of (methanol: pure water) from 3:7 to 5:5, and a tertiary gradient elution is performed using a mixed solvent as the mobile phase, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile: pure water (28:72), tR It was 35 min, and ethyl lactucin was obtained (the yield was 0.00032‰).

[0086] Example 5: The compound isolated from the extract of Cichorium glandulosum Boiss. et Huet is cichorioside, and its structural formula is:

[0087]

[0088] It was obtained by extraction according to the following steps:

[0089] S1, Mix 1 kg of the whole herb of Cichorium glandulosum Boiss. et Huet with 10 L of pure water, heat and reflux for extraction 3 times, and recover the extract under reduced pressure to obtain a crude extract;

[0090] S2, Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate it with D101 macroporous adsorption resin and perform a primary gradient elution to obtain primary eluates with different polarities. Among them, when performing the primary gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratio of pure water to ethanol is successively 100:0, 70:30, 50:50, 30:70, 5:95, and pure water decreases from 100 to 5, while ethanol increases from 0 to 95;

[0091] S3, Subject the primary eluates with different polarities obtained in step S2 to ODS column chromatography with a flow fraction of (pure water: ethanol) from 100:0 to 50:50, and perform a secondary gradient elution with a mixed solution composed of methanol and pure water as the mobile phase to obtain the extract of Cichorium glandulosum Boiss. et Huet. Among them, the volume ratio of methanol to pure water is successively 1:9, 3:7, 5:5, 7:3, 9:1, methanol increases from 1 to 9, and pure water decreases from 9 to 1;

[0092] S4, Subject the extract of Cichorium glandulosum Boiss. et Huet obtained in step S3 to high-performance liquid chromatography separation with a flow fraction of (methanol: pure water) from 1:9 to 3:7, detect at 210 nm, the flow rate is 3 mL / min, and the mobile phase is acetonitrile: pure water (8:92), t R It was 21 min, and cichorioside was obtained (the yield was 0.00018‰).

[0093] Example 6: The compound isolated from the extract of Cichorium glandulosum Boiss. et Huet is cichoriin, and its structural formula is:

[0094]

[0095] It was obtained by extraction according to the following steps:

[0096] S1, Mix 1 kg of the whole herb of Cichorium glandulosum Boiss. et Huet with 10 L of pure water, heat and reflux for extraction 3 times, and recover the extract under reduced pressure to obtain a crude extract;

[0097] S2. Dissolve the crude extract in a solvent, mix it with macroporous adsorption resin and dry it. After separation by D101 macroporous adsorption resin, perform a one-step gradient elution to obtain eluates with different polarities. During the one-step gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratios of pure water to ethanol are successively 100:0, 70:30, 50:50, 30:70, 5:95, with pure water decreasing from 100 to 5 and ethanol increasing from 0 to 95.

[0098] S3. Subject the eluates with different polarities obtained in step S2 to ODS column chromatography with a flow fraction of (pure water:ethanol) from 100:0 to 50:50, and perform a two-step gradient elution using a mixed solution composed of methanol and pure water as the mobile phase to obtain the chicory extract. Among them, the volume ratios of methanol to pure water are successively 1:9, 3:7, 5:5, 7:3, 9:1, with methanol increasing from 1 to 9 and pure water decreasing from 9 to 1.

[0099] S4. Subject the chicory extract obtained in step S3 to high-performance liquid chromatography separation with a flow fraction of (methanol:pure water) from 3:7 to 6:4, detect at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile:pure water (38:62), R at t = 41 min to obtain chicoryin (yield: 0.00005‰).

[0100] Example 7: Application of the chicory extract in the preparation of anti-inflammatory drugs.

[0101] Example 8: Application of the chicory extract in the preparation of drugs for treating inflammation.

[0102] Example 9: Application of the compound chicory lactone A isolated from the chicory extract in the preparation of anti-inflammatory and / or drugs for treating inflammation.

[0103] Example 10: Application of the compound chicory lactone A isolated from the chicory extract in the preparation of drugs for treating inflammation.

[0104] Example 11: Application of the compound chicory lactone B isolated from the chicory extract in the preparation of anti-inflammatory drugs.

[0105] Example 12: Application of the compound chicory lactone B isolated from the chicory extract in the preparation of drugs for treating inflammation.

[0106] Example 13: Application of the compound chicory glucoside isolated from the chicory extract in the preparation of anti-inflammatory drugs.

[0107] Example 14: Application of the compound chicory glucoside isolated from the chicory extract in the preparation of drugs for treating inflammation.

[0108] Example 15: Application of the compound lactucin isolated from the extract of Cichorium glandulosum Boiss. et Huet in the preparation of anti-inflammatory drugs.

[0109] Example 16: Application of the compound lactucin isolated from the extract of Cichorium glandulosum Boiss. et Huet in the preparation of drugs for treating inflammation.

[0110] The structures of the compounds lactucin A, lactucin B, lactucopicrin, and lactucin isolated from the extract of Cichorium glandulosum Boiss. et Huet obtained in Examples 3 to 6 were identified by their physicochemical properties and spectral data.

[0111] I. The structure identification data of the compound lactucin A are as follows:

[0112] Yellow oil (methanol).

[0113] [α] 2 D 0 +192.4 (c 0.50, MeOH). HR-ESI-MS gave the quasi-molecular ion peak m / z 277.1080 [M+H] + : (calcd. 277.1076 for C 15 H 17 O5), suggesting its molecular formula is C 15 H 16 O5, and the degree of unsaturation is 8.

[0114] 1 The 1H-NMR (600 MHz, CDCl3) spectrum gave two sets of double bond hydrogen signals: δ H 6.02 (1H, d, J = 2.7 Hz, H-6), 6.29 (1H, s, H-3); two sets of methylene hydrogen signals: δ H 4.44 (1H, td, J = 10.5, 3.6 Hz, H-8), 3.18 (1H, dd, J = 10.1, 2.7 Hz, H-7); two sets of methylene hydrogen signals: δ H 3.01 (1H, dd, J = 18.7, 10.8 Hz, H-9a), 3.10 (1H, dd, J = 18.8, 3.6 Hz, H-9b), 4.61 (1H, d, J = 17.1 Hz, H-15a), 4.67 (1H, d, J = 17.5 Hz, H-15b); two sets of methyl hydrogen signals: δ H 1.39 (3H, s, C H 3-13), 2.45 (3H, s, C H 3-14).

[0115] 1313C-NMR (150 MHz, CDCl3) gave 15 carbon signals of the guaiane-type sesquiterpene lactone nucleus, including 3 sets of double bond carbon signals: δ C 128.8 (C-1), 129.7 (C-3), 168.0 (C-4), 138.8 (C-5), 118.4 (C-6), 149.1 (C-10); 2 sets of methine carbon signals: δ C 54.6 (C-7), 76.3 (C-8); 2 sets of methylene carbon signals: δ C 45.4 (C-9), 59.1 (C-15); 2 sets of methyl carbon signals: δ C 18.9 (C-13), 21.7 (C-14); 2 sets of carbonyl carbon signals: δ C 197.0 (C-2), 180.1 (C-12) and 1 quaternary carbon signal: δ C 76.7 (C-11).

[0116] In the HMBC spectrum, it was observed that δ H 6.29 (H-3) correlated with δ C 128.8 (C-1), 138.8 (C-5), 168.0 (C-4), 59.1 (C-15), 197.0 (C-2), δ H 6.02 (H-6) correlated with δ C 168.0 (C-4), 128.8 (C-1), 76.3 (C-8), δ H 3.18 (H-7) correlated with δ C 45.4 (C-9), 18.9 ( C H3-13), δ H 3.01 (H-9a), 3.10 (H-9b) correlated with δ C 149.1 (C-10), 128.8 (C-1), confirming the tricyclic ring of the compound; δ H 4.61 (H-15a), 4.67 (H-15b) correlated with δ C 168.0 (C-4), 129.7 (C-3), confirming the substitution position of the hydroxymethyl group at C-4 in the compound; δ H 1.39 (C H 3-13) correlated with δ C 54.6 (C-7), 76.3 (C-8), 180.1 (C-12), δ H 2.45 (C H 3-14) correlated with δ CIt is related to 128.8 (C-1), 149.1 (C-10), and 45.4 (C-9), confirming that the substitution positions of methyl groups in the compound are at C-11 and C-10.

[0117] In the NOESY spectrum, δ H 4.44 (H-8) is more strongly correlated with δ H 1.39 (C H 3-13) than δ H 3.18 (H-7) is correlated with δ H 1.39 (C H 3-13), suggesting that H-8 and CH3-13 are in the β orientation.

[0118] When J H-7,8 > 8.5 Hz, H-7,8 is trans; when J H-7,8 < 8.5 Hz, H-7,8 is cis. The J H-7,8 of compound lactucin A is 10.1 Hz, suggesting that H-7,8 in compound lactucin A is trans. The relative configuration of the chiral center is speculated to be 7S*, 8S*, 11R*.

[0119] By comparing the measured electronic circular dichroism spectrum (ECD) and the calculated ECD data, the absolute configuration of compound lactucin A was determined to be 7S, 8S, 11R. It is a new compound not reported in the literature, named mongolicumin G in English and lactucin A in Chinese.

[0120] II. The structure identification data of compound lactucin B are as follows:

[0121] Yellow oil (MeOH). [α] 2 D 0 +14 (c 0.10, MeOH). HR-ESI-MS gave the quasi-molecular ion peak m / z 247.1331 [M + H] + : (calcd. 247.1334 for C 15 H 19 O3), and its molecular formula was speculated to be C 15 H 18 O3, with an unsaturation degree of 7.

[0122] 1 1H-NMR (600 MHz, CD3OD) gave two sets of double-bond hydrogen signals: δ H 6.24 (1H, dd, J = 4.5, 1.8 Hz, H-6), 6.14 (1H, s, H-3); five sets of methine hydrogen signals: δ H3.18 (1H, t-like, J = 2.4 Hz, H-1), 3.28 (1H, m, H-7), 4.43 (1H, td, J = 10.8, 3.3 Hz, H-8), 2.61 (1H, m, H-10), 2.88 (1H, p, J = 7.8 Hz, H-11); one set of methylene hydrogen signals: δ H 2.03 (1H, td, J = 12.1, 4.0 Hz, H-9a), 2.46 (1H, dt, J = 12.9, 3.6 Hz, H-9b); three sets of methyl hydrogen signals: δ H 1.39 (3H, d, J = 7.7 Hz, H-13), 0.75 (3H, d, J = 7.2 Hz, H-14), 2.25 (3H, t, J = 1.0 Hz, H-15).

[0123] 13 C-NMR (150 MHz, CD3OD) gives 15 carbon signals of the guaianolide sesquiterpene lactone nucleus, including two sets of double bond carbon signals: δ C 133.1 (C-3), 172.0 (C-4), 146.9 (C-5), 121.4 (C-6); five sets of methine carbon signals: δ C 54.2 (C-1), 48.1 (C-7), 78.4 (C-8), 30.9 (C-10), 40.1 (C-11); one set of methylene carbon signals: δ C 42.0 (C-9); three sets of methyl carbon signals: δ C 11.8 (C-13), 13.1 (C-14), 13.9 (C-15); two sets of carbonyl carbon signals: δ C 208.9 (C-2), 181.6 (C-12).

[0124] In the HMBC spectrum, it can be observed that δ H 6.24 (H-6) correlates with δ C 54.2 (C-1), 40.1 (C-11), 48.1 (C-7), 78.4 (C-8), 172.0 (C-4), δ H 6.14 (H-3) correlates with δ C 54.2 (C-1), 146.9 (C-5), 172.0 (C-4), 208.9 (C-2), δ H 3.28 (H-7) correlates with δ C 54.2 (C-1), δ H 2.46 (H-9b), 2.03 (H-9a) correlate with δ C 48.1 (C-7), 54.2 (C-1), δH 2.88 (H-11) is related to δ C 78.4 (C-8), confirming the tricyclic ring of the compound; δ H 2.25 (C H 3-15) is related to δ C 133.1 (C-3), 172.0 (C-4), 146.9 (C-5), δ H 1.39 (C H 3-13) is related to δ C 40.1 (C-11), 48.1 (C-7), 181.6 (C-12), δ H 0.75 (C H 3-14) is related to δ C 30.9 (C-10), 42.0 (C-9), 54.2 (C-1), confirming that the substitution positions of methyl groups in the compound are at C-4, C-10, and C-11 positions.

[0125] In the NOESY spectrum, δ H 2.03 (H-9a) is related to δ H 3.18 (H-1), δ H 3.28 (H-7), indicating that H-9a, H-1, and H-7 are in the β orientation; δ H 2.46 (H-9b) is related to δ H 0.75 (C H 3-14), δ H 4.43 (H-8), δ H 4.43 (H-8) is related to δ H 1.39 (C H 3-13), indicating that H-9b, C H 3-14, H-8, C H 3-13 are in the α orientation. The relative configuration of the chiral centers is speculated to be 1S*, 7R*, 8S*, 10S*, 11S*.

[0126] By comparing the measured electronic circular dichroism spectrum (ECD) and the calculated ECD data, the absolute configuration of the compound hypochoerisin B was determined to be 1S, 7R, 8S, 10S, 11S. It is a new compound not reported in the literature, named hypochoeros O in English and hypochoerisin B in Chinese.

[0127] III. The structural identification data of the compound hypochoerisin A are as follows:

[0128] Yellow oil (MeOH). HR-ESI-MS gave the quasi-molecular ion peak m / z 421.1507 [M-H]- :(calcd. 421.1499 for C 21 H 25 O9), the molecular formula is speculated to be C 21 H 26 O9, and the degree of unsaturation is 9.

[0129] 1 1H-NMR (600 MHz, CD3OD) gives two sets of double bond hydrogen signals: δ H 6.10 (1H, d, J = 2.8 Hz, H-6), 6.43 (1H, s, H-3); three sets of methine hydrogen signals: δ H 4.63 (1H, td, J = 10.6, 3.5 Hz, H-8), 3.38 (1H, m, H-7), 2.95 (1H, p, J = 7.8 Hz, H-11); two sets of methylene hydrogen signals: δ H 3.13 (1H, dd, J = 18.9, 3.5 Hz, H-9a), 3.01 (1H, dd, J = 18.9, 10.9 Hz, H-9b), 4.99 (1H, dd, J = 16.1, 1.3 Hz, H-15a), 4.67 (1H, dd, J = 16.1, 1.1 Hz, H-15b); two sets of methyl hydrogen signals: δ H 1.30 (3H, d, J = 7.7 Hz, C H 3-13), 2.44 (3H, s, C H 3-14); in addition, there is also a set of hydrogen signals of sugar, among which δ H 4.39 (1H, d, J = 7.8 Hz, H-1′) is the anomeric hydrogen signal of sugar.

[0130] 13 13C-NMR (150 MHz, CD3OD) gives 15 carbon signals of the guaianolide sesquiterpene lactone nucleus, including three sets of double bond carbon signals: δ C 128.5 (C-1), 130.5 (C-3), 164.2 (C-4), 138.2 (C-5), 121.0 (C-6), 149.4 (C-10); three sets of methine carbon signals: δ C 47.3 (C-7), 79.5 (C-8), 40.6 (C-11); two sets of methylene carbon signals: δ C 45.5 (C-9), 65.6 (C-15); two sets of methyl carbon signals: δ C 11.6 (C-13), 21.7 (C-14); two sets of carbonyl carbon signals: δ C 197.0 (C-2), 181.3 (C-12); one set of carbon signals on glucose: δC 104.0 (C-1′), 75.1 (C-2′), 78.0 (C-3′), 71.7 (C-4′), 78.2 (C-5′), 62.8 (C-6′).

[0131] In the HMBC spectrum, it can be observed that δ H 6.43 (H-3) correlates with δ C 65.6 (C-15), 128.5 (C-1), 138.2 (C-5), 164.2 (C-4), 197.0 (C-2), δ H 6.10 (H-6) correlates with δ C 164.2 (C-4), 128.5 (C-1), 79.5 (C-8), 40.6 (C-11), δ H 3.38 (H-7) correlates with δ C 138.2 (C-5), δ H 3.13 (H-9a), 3.01 (H-9b) correlate with δ C 149.4 (C-10), 128.5 (C-1), δ H 2.95 (H-11) correlates with δ C 79.5 (C-8), confirming the tricyclic ring of the compound; δ H 1.30 (C H 3-13) correlates with δ C 47.3 (C-7), 40.6 (C-11), 181.3 (C-12), δ H 2.44 (C H 3-14) correlates with δ C 128.5 (C-1), 149.4 (C-10), 45.5 (C-9), confirming the substitution positions of the methyl groups in the compound at C-11 and C-10; δ H 4.99 (H-15a) correlates with δ C 130.5 (C-3), 164.2 (C-4), 104.0 (C-1′) to determine the sugar linkage position.

[0132] In the NOESY spectrum, δ H 4.63 (H-8) correlates with δ H 3.13 (H-9a), 1.30 (C H 3-13); δ H 3.38 (H-7) correlates with δ H 3.01 (H-9b), δ HRelated to 2.95(H-11), suggesting H-7,8-trans in the compound, indicating that H-8,CH3-13 is in the β-orientation, and H-7,H-11 are in the α-orientation. The relative configuration of the chiral centers is speculated to be 7R*,8S*,11S*. The sugar in compound 3 was determined to be D-glucose by enzymatic hydrolysis with snailase.

[0133] By comparing the measured electronic circular dichroism spectrum (ECD) and the calculated ECD data, the absolute configuration of the aglycone of compound hypochoeroside was determined to be 7R,8S,11S. After literature search, it was found to be a new compound not reported before, named hypochoeroside Q in English and hypochoeroside in Chinese.

[0134] Among them, the 1H-NMR and 13C-NMR data assignments of compounds lactucin A, lactucin B, and hypochoeroside are shown in Table 1.

[0135] Table 1

[0136] IV. The structure identification data of compound lactucin are as follows:

[0137] Yellow oil (MeOH). HR-ESI-MS gave the quasi-molecular ion peak m / z 347.1497 [M+H] + : (calcd. 347.1495 for C 19 H 23 O6), suggesting its molecular formula is C 19 H 22 O6, and the degree of unsaturation is 9.

[0138] 1 The 1H-NMR (600 MHz, CDCl3) spectrum gave a set of AA′BB′ coupled aromatic hydrogen signals: δ H 6.78 (2H, d, J = 8.3 Hz, H-3″,5″), δ H 7.16 (2H, d, J = 8.3 Hz, H-2″,6″); a set of meta-coupled aromatic hydrogen signals: δ H 6.33 (2H, s, H-2′,6′); four sets of methylene hydrogen signals: δ H 1.90 (2H, m, H2-2), 2.54 (2H, t, J = 7.5 Hz, H2-3), 3.55 (2H, s, H2-5), 4.10 (2H, t, J = 6.4 Hz, H2-1); two sets of methoxy hydrogen signals: δ H 3.84 (6H, s, 3′,5′-OC H 3).

[0139] 13 The 13C-NMR (150 MHz, CDCl3) spectrum gave 19 carbon signals, including one set of meta-coupled aromatic carbon signals: δ C 132.3 (C-1′), 105.1 (C-2′, 6′), 147.1 (C-3′, 5′), 133.0 (C-4′); one set of AA′BB′-coupled aromatic carbon signals: δ C 126.5 (C-1″), 130.6 (C-2″, 6″), 115.6 (C-3″, 5″), 154.8 (C-4″); four sets of methylene carbon signals: δ C 30.6 (C-2), 32.4 (C-3), 40.8 (C-5), 64.1 (C-1); two sets of methoxy carbon signals: δ C 56.4 (3′,5′-O C CH3); one carbonyl carbon signal: δ C 172.1 (C-4).

[0140] In the HMBC spectrum, it was observed that δ H 6.33 (H-2′, 6′) was correlated with δ C 133.0 (C-4′), 32.4 (C-3), δ H 6.78 (H-3″, 5″) was correlated with δ C 115.6 (C-3″, 5″), 154.8 (C-4″), 126.5 (C-1″), δ H 7.16 (H-2″, 6″) was correlated with δ C 130.6 (C-2″, 6″), 154.8 (C-4″), 40.8 (C-5), demonstrating the presence of two benzene ring systems in the structure; δ H 3.84 (3′,5′-OCH H 3) was correlated with δ C 147.1 (C-3′, 5′) to determine the attachment position of the methoxy group on the benzene ring; δ H 3.55 (H-5) was correlated with δ C 126.5 (C-1″), 130.6 (C-2″, 6″), 172.1 (C-4), δ H 2.54 (H-3) was correlated with δ C 105.1 (C-2′, 6′), 64.1 (C-1), δ H 1.90 (H-2) was correlated with δ C 132.3 (C-1′), δ H 4.10 (H-1) was correlated with δ CIt is related to 32.4 (C-3) and 172.1 (C-4), determining the connection position of the two benzene ring systems.

[0141] Through literature search, it is a new compound not reported before. Its English name is 3-(4'-hydroxy-3',5'-dimethoxyphenyl)propyl 5-(4”-hydroxyphenyl)acetate, and its Chinese name is lactucopicrin.

[0142] Among them, the NMR data attribution of the hydrogen spectrum and carbon spectrum of the compound lactucopicrin is shown in Table 2.

[0143] Table 2

[0144] Name <![CDATA[δ H |(J in Hz)]]> <![CDATA[δ c > No. <![CDATA[δ H (J in Hz)]]> <![CDATA[δ c > 1 4.10(t，6.4) 64.1 5' - 147.1 2 1.90(m) 30.6 6’ 6.33(s) 105.1 3 2.54(t，7.5) 32.4 1” - 126.5 4 - 172.1 2” 7.16(d，8.3) 130.1 5 3.55(s) 40.8 3” 6.78(d，8.3) 115.6 1' - 132.3 4” - 154.8 2' 6.33(s) 105.1 5” 6.78(d，8.3) 115.6 3 - 147.1 6” 7.16(d，8.3) 130.1 4' - 133.0 <![CDATA[3', 5'-OCH3]]> 3.84(s) 56.4

[0145] Example 17: The compound isolated from the lactuca tatarica extract is lactucin A. The extraction method is the same as that of Example 3 in steps S1 and S2, and the differences are as follows:

[0146] S3, subjecting the primary eluate with different polarities obtained in step S2 to ODS column chromatography with a flow fraction of (pure water: ethanol) from 85:15 to 30:70, and performing secondary gradient elution with a mixed solution composed of methanol and pure water as the mobile phase to obtain the lactuca tatarica extract. Among them, the volume ratios of methanol and pure water are successively 2:8, 4:6, 6:4, 8:2, and 9:1;

[0147] S4, subjecting the lactuca tatarica extract obtained in step S3 to separation by high performance liquid chromatography (HPLC-UV) with a flow fraction of (methanol: pure water) from 4:6 to 6:4, performing tertiary gradient elution with a mixed solvent as the mobile phase, detecting at 210 nm, with a flow rate of 3 mL / min, and the mobile phase being acetonitrile: pure water (22:78), t R is 39 min, to obtain lactucin A (the yield is 0.00013‰).

[0148] Example 18: The compound isolated from the lactuca tatarica extract is lactucin B. The extraction method is the same as that of Example 17 in steps S1, S2, and S3, and the differences are as follows:

[0149] S4, subjecting the lactuca tatarica extract obtained in step S3 to separation by high performance liquid chromatography (HPLC-UV) with a flow fraction of (methanol: pure water) from 4:6 to 6:4, performing tertiary gradient elution with a mixed solvent as the mobile phase, detecting at 210 nm, with a flow rate of 3 mL / min, and the mobile phase being acetonitrile: pure water (25:75), t R is 41 min, to obtain lactucin B (the yield is 0.00028‰).

[0150] Example 19: The compound isolated from the Cichorium glandulosum extract is cichoriin. The extraction method is the same as that in Example 17 in steps S1, S2, and S3, with the difference being:

[0151] S4. The Cichorium glandulosum extract obtained in step S3 is separated by high-performance liquid chromatography with a fraction of (methanol: pure water) from 2:8 to 6:4. Detection is carried out at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (11:89), and t R is 35 min, obtaining cichoriin (yield: 0.00011‰).

[0152] Example 20: The compound isolated from the Cichorium glandulosum extract is cichorioside. The extraction method is the same as that in Example 17 in steps S1, S2, and S3, with the difference being:

[0153] S4. The Cichorium glandulosum extract obtained in step S3 is separated by high-performance liquid chromatography with a fraction of (methanol: pure water) from 4:6 to 8:2. Detection is carried out at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (35:65), and t R is 35 min, obtaining cichorioside (yield: 0.00009‰).

[0154] The structural identification methods of the compounds cicholactone A, cicholactone B, cichoriin, and cichorioside isolated from the Cichorium glandulosum extracts obtained in Examples 17 to 20 are the same as those in Examples 16 to 19.

[0155] Example 21: The compound isolated from the Cichorium glandulosum extract is cicholactone A, which is obtained by extraction according to the following steps:

[0156] S1. Mix 1.5 kg of the whole Cichorium glandulosum plant with 15 L of ethanol with a volume fraction of 80%, heat and reflux for extraction 3 times, and recover the extract under reduced pressure to obtain a crude extract;

[0157] S2. Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate with D101 macroporous adsorption resin, and perform a one-step gradient elution to obtain eluates with different polarities in one step. Among them, during the one-step gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratios of pure water and ethanol are successively 100:0, 80:20, 60:40, 40:60, 20:80, 5:95;

[0158] S3. The eluates with different polarities in one step obtained in step S2 are separated by ODS column chromatography with a fraction of (pure water: ethanol) from 80:20 to 20:80, and a mixed solution composed of methanol and pure water is used as the mobile phase for a two-step gradient elution to obtain the Cichorium glandulosum extract. Among them, the volume ratios of methanol and pure water are successively 1:9, 3:7, 5:5, 7:3, 8:2;

[0159] S4. The chicory extract obtained in step S3 is fractionated with (acetonitrile: pure water) at a ratio of 3:7 to 7:3 by high performance liquid chromatography (HPLC-UV), and three gradient elutions are performed with the mixed solvent as the mobile phase. Detection is carried out at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (19:81), and t R is 42 min, and chicory lactone A (yield 0.00020‰) is obtained.

[0160] Example 22: The compound isolated from the chicory extract is chicory lactone B. The extraction method is the same as that in Example 21 for steps S1, S2 and S3, except that:

[0161] S4. The chicory extract obtained in step S3 is fractionated with (acetonitrile: pure water) at a ratio of 3:7 to 7:3 by high performance liquid chromatography (HPLC-UV), and three gradient elutions are performed with the mixed solvent as the mobile phase. Detection is carried out at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (25:75), and t R is 35 min, and chicory lactone B (yield 0.00018‰) is obtained.

[0162] Example 23: The compound isolated from the chicory extract is chicory picroside. The extraction method is the same as that in Example 21 for steps S1, S2 and S3, except that:

[0163] S4. The chicory extract obtained in step S3 is fractionated with (acetonitrile: pure water) at a ratio of 1:9 to 3:7 by high performance liquid chromatography (HPLC-UV), and three gradient elutions are performed with the mixed solvent as the mobile phase. Detection is carried out at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (8:82), and t R is 22 min, and chicory picroside (yield 0.00008‰) is obtained.

[0164] Example 24: The compound isolated from the chicory extract is chicoryin. The extraction method is the same as that in Example 21 for steps S1, S2 and S3, except that:

[0165] S4. The chicory extract obtained in step S3 is fractionated with (acetonitrile: pure water) at a ratio of 3:7 to 7:3 by high performance liquid chromatography (HPLC-UV), and three gradient elutions are performed with the mixed solvent as the mobile phase. Detection is carried out at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (37:63), and t R is 35 min, and chicoryin (yield 0.00018‰) is obtained.

[0166] The structural identification methods of the compounds lactucin A, lactucin B, cichorioside, and lactucopicrin isolated from the chicory extract obtained in Examples 21 to 24 are the same as those in Examples 16 to 19.

[0167] Example 25: The compound isolated from the chicory extract is lactucin A, which is obtained by extraction according to the following steps:

[0168] S1, Mix 1 kg of whole chicory herb with 10 L of ethanol with a volume fraction of 95%, heat and reflux for extraction three times, and recover the extract under reduced pressure to obtain a crude extract;

[0169] S2, Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate it with D101 macroporous adsorption resin, and perform a one-time gradient elution to obtain eluates with different polarities at one time. Among them, a mixed solution composed of pure water and ethanol is used for elution during the one-time gradient elution, and the volume ratios of pure water and ethanol are 100:0, 70:30, 50:50, 30:70, and 5:95 in sequence;

[0170] S3, Subject the eluates with different polarities obtained in step S2 to ODS column chromatography with a flow fraction of (pure water: ethanol) from 100:0 to 30:70, and perform a two-time gradient elution with a mixed solution of methanol and pure water as the mobile phase to obtain a chicory extract. Among them, the volume ratios of methanol and pure water are 1:9, 2:8, 4:6, 6:4, and 8:2 in sequence;

[0171] S4, Subject the chicory extract obtained in step S3 to separation by high-performance liquid chromatography (HPLC-UV) with a flow fraction of (acetonitrile: pure water) from 2:8 to 6:4, perform a three-time gradient elution with a mixed solvent as the mobile phase, detect at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (15:85), t R is 40 min, and lactucin A is obtained (the yield is 0.00018‰).

[0172] Example 26: The compound isolated from the chicory extract is lactucin B. Compared with Example 25, steps S1, S2, and S3 are the same, and the difference lies in:

[0173] S4, Subject the chicory extract obtained in step S3 to separation by high-performance liquid chromatography (HPLC-UV) with a flow fraction of (acetonitrile: pure water) from 2:8 to 6:4, perform a three-time gradient elution with a mixed solvent as the mobile phase, detect at 210 nm, the flow rate is 3 mL / min, the mobile phase is acetonitrile: pure water (25:75), t R is 35 min, and lactucin B is obtained (the yield is 0.00018‰).

[0174] Example 27: The compound isolated from the Cichorium glandulosum Boiss. et Huet extract is cichoriin. The extraction method is the same as that of Example 25 in steps S1, S2, and S3, except that:

[0175] S4. The Cichorium glandulosum Boiss. et Huet extract obtained in step S3 is fractionated with (acetonitrile: pure water) from 1:9 to 4:6 and separated by high performance liquid chromatography (HPLC-UV). It is eluted three times with a gradient using a mixed solvent as the mobile phase, detected at 210 nm, with a flow rate of 3 mL / min. The mobile phase is acetonitrile: pure water at 6:94, and t R is 18 min, obtaining cichoriin (yield: 0.00009‰).

[0176] Example 28: The compound isolated from the Cichorium glandulosum Boiss. et Huet extract is cichorioside. The extraction method is the same as that of Example 21 in steps S1, S2, and S3, except that:

[0177] S4. The Cichorium glandulosum Boiss. et Huet extract obtained in step S3 is fractionated with (acetonitrile: pure water) from 2:8 to 6:4 and separated by high performance liquid chromatography (HPLC-UV). It is eluted three times with a gradient using a mixed solvent as the mobile phase, detected at 210 nm, with a flow rate of 3 mL / min. The mobile phase is acetonitrile: pure water (35:65), and t R is 32 min, obtaining cichorioside (yield: 0.00020‰).

[0178] The structural identification methods of the compounds cicholactone A, cicholactone B, cichoriin, and cichorioside isolated from the Cichorium glandulosum Boiss. et Huet extracts obtained in Examples 25 to 28 are the same as those in Examples 16 to 19.

[0179] Example 29: The compound isolated from the Cichorium glandulosum Boiss. et Huet extract is cicholactone A, which is obtained by extraction according to the following steps:

[0180] S1. Mix 1.5 kg of the whole Cichorium glandulosum Boiss. et Huet plant with 15 L of ethanol with a volume fraction of 80%, heat and reflux for extraction three times, and recover the extract under reduced pressure to obtain a crude extract;

[0181] S2. Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate it with D101 macroporous adsorption resin and perform a single gradient elution to obtain eluates with different polarities in the first step. Among them, when performing the single gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratios of pure water to ethanol are successively 95:5, 75:25, 50:50, 25:75, and 5:95;

[0182] S3. Subject the first eluates with different polarities obtained in step S2 to ODS column chromatography with a fraction of (pure water: ethanol) from 80:20 to 20:80, and perform secondary gradient elution using a mixed solution composed of methanol and pure water as the mobile phase to obtain the extract of Cichorium glandulosum Boiss. & Huet. Among them, the volume ratios of methanol to pure water are successively 3:7, 5:5, 7:3, 8:2, and 9:1.

[0183] S4. Subject the extract of Cichorium glandulosum Boiss. & Huet. obtained in step S3 to separation by high performance liquid chromatography (HPLC-UV) with a fraction of (methanol: pure water) from 3:7 to 8:2, perform tertiary gradient elution using a mixed solvent as the mobile phase, detect at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile: pure water (13:87), t R is 52 min to obtain lactucin A (yield is 0.00011‰).

[0184] Example 30: The compound isolated from the extract of Cichorium glandulosum Boiss. & Huet. is lactucin B. Compared with Example 21 in the extraction method, steps S1, S2, and S3 are the same, the difference is:

[0185] S4. Subject the extract of Cichorium glandulosum Boiss. & Huet. obtained in step S3 to separation by high performance liquid chromatography (HPLC-UV) with a fraction of (methanol: pure water) from 3:7 to 8:2, perform tertiary gradient elution using a mixed solvent as the mobile phase, detect at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile: pure water (21:79), t R is 31 min to obtain lactucin B (yield is 0.00021‰).

[0186] Example 31: The compound isolated from the extract of Cichorium glandulosum Boiss. & Huet. is cichoriin. Compared with Example 21 in the extraction method, steps S1, S2, and S3 are the same, the difference is:

[0187] S4. Subject the extract of Cichorium glandulosum Boiss. & Huet. obtained in step S3 to separation by high performance liquid chromatography (HPLC-UV) with a fraction of (methanol: pure water) from 3:7 to 5:5, perform tertiary gradient elution using a mixed solvent as the mobile phase, detect at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile: pure water 10:90, t R is 33 min to obtain cichoriin (yield is 0.00008‰).

[0188] Example 32: The compound isolated from the extract of Cichorium glandulosum Boiss. & Huet. is cichorioside. Compared with Example 21 in the extraction method, steps S1, S2, and S3 are the same, the difference is:

[0189] S4. The chicory extract obtained in step S3 is separated by high performance liquid chromatography (HPLC-UV) with a fraction of (methanol: pure water) from 3:7 to 8:2. Three gradient elutions are performed with the mixed solvent as the mobile phase, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile: pure water (31:69), t R is 28 min, and chicoryin (yield is 0.00025‰) is obtained.

[0190] The structural identification methods of the compounds chicory lactone A, chicory lactone B, chicory bitter glycoside, and chicoryin isolated from the chicory extracts obtained in Examples 29 to 32 are the same as those in Examples 16 to 19.

[0191] The anti-inflammatory activities of the compounds chicory lactone A, chicory lactone B, chicory bitter glycoside, and chicoryin isolated from the chicory extracts obtained in Examples 16 to 32 are tested.

[0192] (1) Experimental principle: The abnormal activation of macrophages is an important link in the inflammatory response and is closely related to the occurrence and development of various inflammatory diseases. Therefore, inhibiting the excessive activation of macrophages may become an important target for drug development. LPS can activate macrophages to release inflammatory factors such as NO, TNF-α, IL-6, and IL-1β. In this experiment, an in vitro model of abnormally activated RAW264.7 mouse macrophages induced by LPS is established, and the anti-inflammatory activities of the compounds chicory lactone A, chicory lactone B, chicory bitter glycoside, and chicoryin are evaluated using the NO release amount as an index.

[0193] (2) Experimental method:

[0194] ① Culture of RAW264.7 macrophages

[0195] All glassware and metal instruments (culture flasks, pipettes, solution bottles, etc.) used in the experiment are autoclaved at 121 °C for 30 minutes to completely remove contaminated LPS. Using DMEM medium as the basis, a cell culture solution containing 10% fetal bovine serum (FBS) is prepared. RAW264.7 cells are cultured and passaged at a concentration of about 2.0×10 5 cells / mL under the conditions of 37 °C and 5% CO2. When the cells on the bottom of the culture flask cover 70% to 80% of the area, the adherent cells are digested with trypsin and passaged. The resuscitated RAW264.7 cells cryopreserved at -80 °C are used as the starting cells for the experiment, and cells of the 3rd to 8th generations are selected for the experiment.

[0196] ② Method for preparing the drug

[0197] All the compounds to be tested were dissolved in DMSO to prepare a stock solution (100 mM) and stored at -20°C. Before use, they were diluted with DMEM culture medium to 100 μM, 30 μM, 10 μM, and 1 μM successively. The final concentration of DMSO was <1‰.

[0198] ③ Detection of the inhibitory effect of the compound on LPS-activated RAW264.7 cells by the Griess method

[0199] RAW264.7 cells in the logarithmic growth phase were adjusted to a cell density of 2.0×10 5 cells / mL with fresh DMEM culture medium containing 10% fetal bovine serum, seeded in 96-well plates at 100 μL / well, and cultured at 37°C and 5% CO2 for 24 hours until the cells adhered. Then the medium was replaced with serum-free fresh medium, and the compound (at concentrations of 100 μM, 30 μM, 10 μM, and 1 μM) was added for co-treatment with LPS. The final concentration of LPS was 100 ng / mL. A blank control group (without LPS and compound) was set. The final concentration of LPS in each drug treatment group was 100 ng / mL. After the cells were treated with the drug for another 24 hours, the supernatant was collected, and the NO content in the supernatant was detected by the Griess colorimetric method.

[0200] ④ Detection of the effect of the compound on the cell viability of microglia by the MTT method

[0201] RAW264.7 cells in the logarithmic growth phase were adjusted to a cell density of 2.0×10 5 cells / mL with fresh DMEM medium containing 10% fetal bovine serum, seeded in 96-well plates at 100 μL / well, and cultured in an incubator at 37°C and 5% CO2. After the cells adhered and were cultured for 24 hours, the medium was replaced with fresh medium, and drug treatment was carried out simultaneously. The compound doses of 100 μM, 30 μM, 10 μM, and 1 μM were co-treated with LPS. A blank control was set at the same time. The final concentration of LPS in each drug treatment group was 100 ng / mL. After the cells were treated with the drug for another 24 hours, MTT solution (10 μL / well) was added to the cell culture medium, and the cells were incubated with 0.25 mg / mL MTT at 37°C for 3 hours. Then the culture medium was aspirated, and 150 μL of DMSO solution was added to measure the optical density OD value. Data processing was performed using the enzyme-linked immunosorbent assay (ELISA) reader software. The average OD value of 3 wells for each sample was calculated, and the cell viability (cell viability, CV%) was calculated using the average value according to the following formula.

[0202] Cell viability % = [Average OD value of the sample group / Average OD value of the blank control group] × 100%

[0203] ⑤ Statistical method

[0204] All data were analyzed using the SPSS (19.0) statistical software package. The results were expressed as mean ± standard error. To evaluate the overall differences, one-way ANOVA was used to analyze the homogeneity of variance among group means, and Dunnett's test was combined for inter-group comparison. The Levene test was used for the homogeneity test of variances of multiple samples. When p > 0.05, the variances were homogeneous, and Dunnett's two-sided T test was used to analyze the differences in means among multiple groups. When p < 0.05, the variances were heterogeneous, and Dunnett T3 test was used to analyze the differences in means among multiple groups.

[0205] ⑥IC 50 Calculation method

[0206] Parameters such as each dose and inhibition rate were used for non-linear regression fitting to calculate IC 50 .

[0207] (3) Experimental results:

[0208] The experimental results are shown in Table 3. Table 3 shows the experimental results of the effects of lactucin A (Compound 1), lactucin B (Compound 2), lactucopicrin (Compound 3), and lactucin (Compound 4) on the release of NO from LPS-activated RAW264.7 cells.

[0209] In Table 3, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS-induced group; ### P < 0.001 compared with the control group.

[0210] Table 3

[0211]

[0212] As can be seen from Table 3, the compounds lactucin A (10 μM, 30 μM, 100 μM), lactucin B (1 μM, 10 μM, 30 μM, 100 μM), lactucopicrin (10 μM, 30 μM, 100 μM), and lactucin (1 μM, 10 μM, 30 μM, 100 μM) isolated from the extract of Cichorium glandulosum Boiss. et Huet obtained in Examples 16 to 32 can significantly inhibit the release of NO from LPS-induced RAW264.7 cells.

[0213] In summary, the present invention for the first time uses the whole herb of Cichorium glandulosum Boiss. et Huet as a raw material to extract the extract of Cichorium glandulosum Boiss. et Huet and the compounds isolated from the extract of Cichorium glandulosum Boiss. et Huet, and for the first time discloses the inhibitory effects of the compounds lactucin A, lactucin B, lactucopicrin, and lactucin isolated from the extract of Cichorium glandulosum Boiss. et Huet on RAW264.7 cells, enabling the above compounds to be applied to the preparation of anti-inflammatory or / and anti-inflammatory drugs.

[0214] The above technical features constitute an embodiment of the present invention, which has strong adaptability and implementation effects. Non-essential technical features can be added or subtracted according to actual needs to meet the requirements of different situations.

Claims

1. A Cichorium glandulosum Boiss. et Huet extract, characterized in that It includes lactucopicrin A, lactucopicrin B, cichorioside, and lactucin. Among them, The structural formula of lactucopicrin A is: The structural formula of lactucopicrin B is: The structural formula of cichorioside is: The structural formula of lactucin is:

2. The chicory extract according to claim 1, characterized in that It is obtained according to the following steps: S1. Mix the required amount of whole Cichorium glandulosum Boiss. et Huet with a solvent, heat and reflux for extraction 2 to 5 times, recover the extract to obtain a crude extract. Among them, 8 mL to 20 mL of solvent is added to every 1 g of whole Cichorium glandulosum Boiss. et Huet, and the solvent is one of pure water, an ethanol solution with a volume fraction of 70% to 95%, and a methanol solution with a volume fraction of 80% to 90%. S2. Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate with D101 macroporous adsorption resin and perform a primary gradient elution to obtain primary eluates with different polarities. Among them, when performing the primary gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratio of pure water to ethanol is 100:0 to 5:95, pure water decreases from 100 to 5, and ethanol increases from 0 to 95. S3. Subject the primary eluates with different polarities obtained in step S2 to ODS column chromatography, and perform a secondary gradient elution with a mixed solvent as the mobile phase to obtain a Cichorium glandulosum Boiss. et Huet extract. Among them, the mixed solvent is a mixed solution composed of methanol and pure water or a mixed solution composed of acetonitrile and pure water. The volume ratio of methanol to pure water is 2:8 to 9:1, methanol increases from 2 to 9, and pure water decreases from 8 to 1. The volume ratio of acetonitrile to pure water is 1:9 to 8:2, acetonitrile increases from 1 to 8, and pure water decreases from 9 to 2.

3. A method for extracting the extract of Cichorium glandulosum Boiss. et Huet according to claim 1, characterized in that It is carried out according to the following steps: S1. Mix the required amount of whole Cichorium glandulosum Boiss. et Huet with a solvent, heat and reflux for extraction 2 to 5 times, recover the extract to obtain a crude extract. Among them, 8 mL to 20 mL of solvent is added to every 1 g of whole Cichorium glandulosum Boiss. et Huet, and the solvent is one of pure water, an ethanol solution with a volume fraction of 70% to 95%, and a methanol solution with a volume fraction of 80% to 90%. S2. Dissolve the crude extract with a solvent, take macroporous adsorption resin for sample mixing and drying, separate with D101 macroporous adsorption resin and perform a primary gradient elution to obtain primary eluates with different polarities. Among them, when performing the primary gradient elution, a mixed solution composed of pure water and ethanol is used for elution, and the volume ratio of pure water to ethanol is 100:0 to 5:95, pure water decreases from 100 to 5, and ethanol increases from 0 to 95. S3. Subject the primary eluates with different polarities obtained in step S2 to ODS column chromatography, and perform a secondary gradient elution with a mixed solvent as the mobile phase to obtain a Cichorium glandulosum Boiss. et Huet extract. Among them, the mixed solvent is a mixed solution composed of methanol and pure water or a mixed solution composed of acetonitrile and pure water. The volume ratio of methanol to pure water is 2:8 to 9:1, methanol increases from 2 to 9, and pure water decreases from 8 to 1. The volume ratio of acetonitrile to pure water is 1:9 to 8:2, acetonitrile increases from 1 to 8, and pure water decreases from 9 to 2.

4. A compound isolated from the extract of Cichorium glandulosum Boiss. et Huet according to claim 2, characterized in that It is lactucopicrin A, and its structural formula is: It is extracted according to the following steps: Subject the Cichorium glandulosum Boiss. et Huet extract obtained in step S3 to high performance liquid chromatography separation, and perform a tertiary gradient elution with a mixed solvent as the mobile phase to obtain lactucopicrin A.

5. A compound isolated from the extract of Cichorium glandulosum Boiss. et Huet according to claim 2, characterized in that It is lactucopicrin B, and its structural formula is: It is extracted according to the following steps: The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is carried out three times with a mixed solvent as the mobile phase to obtain lactucin B.

6. A compound isolated from the extract of Cichorium glandulosum Boiss. et Huet according to claim 2, characterized in that It is lactucopicrin, and its structural formula is: It is obtained by extraction according to the following steps: The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is carried out three times with a mixed solvent as the mobile phase to obtain lactucopicrin.

7. A compound isolated from the extract of Cichorium glandulosum Boiss. et Huet according to claim 2, characterized in that It is lactucin, and its structural formula is: It is obtained by extraction according to the following steps: The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is carried out three times with a mixed solvent as the mobile phase to obtain lactucin.

8. A method for extracting a compound isolated from the extract of Cichorium glandulosum Boiss. et Huet according to claim 4 or 5 or 6 or 7, characterized in that : The extraction method of lactucin A is carried out according to the following steps: The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is carried out three times with a mixed solvent as the mobile phase to obtain lactucin A; Or / and, the extraction method of lactucin B is carried out according to the following steps: The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is carried out three times with a mixed solvent as the mobile phase to obtain lactucin B; Or / and, the extraction method of lactucopicrin is carried out according to the following steps: The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is carried out three times with a mixed solvent as the mobile phase to obtain lactucopicrin; Or / and, the extraction method of lactucin is carried out according to the following steps: The chicory extract obtained in step S3 is separated by high performance liquid chromatography, and gradient elution is carried out three times with a mixed solvent as the mobile phase to obtain lactucin.

9. Use of the chicory extract according to claim 1 or 2 in the preparation of anti-inflammatory and / or anti-inflammatory drugs.

10. Use of the compound lactucin A or / and lactucin B or / and lactucopicrin or / and lactucin separated from the chicory extract according to claim 4 or 5 or 6 or 7 in the preparation of anti-inflammatory and / or anti-inflammatory drugs.

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