An amino acid sesquiterpene lactone compound and its preparation method and application

Abstract

The present invention discloses an amino acid sesquiterpene lactone compound 11β,13-dihydro-13-prolyl-lactucin, which has anti-inflammatory activity, expands the source of anti-inflammatory drugs, and takes a step further in the study of active monomer components in chicory.

Description

Technical Field

[0001] The present invention relates to a new amino acid sesquiterpene lactone compound with anti-inflammatory activity. Background Art

[0002] Chicory (Cichorium intybus L), also known as blue daisy and chicory, belongs to the genus Cichorium of the Asteraceae family. It is an annual or perennial herb with both medicinal and edible value. Chicory originated in Europe, India, South Africa, North America and other regions, and is now widely cultivated around the world. It has diverse applications and can be used in medicine, forage, vegetable salad making, coffee substitutes and industrial inulin extraction. The above-ground parts, roots and seeds of chicory all have medicinal value and are included in the "Pharmacopoeia of the People's Republic of China", with a long history of medicinal use. It is bitter and cold in nature, and has the functions of clearing heat and detoxifying, promoting digestion, and promoting diuresis and reducing swelling. It is commonly used by the people to treat symptoms such as damp-heat jaundice, stomach pain and poor appetite, and edema caused by nephritis. Modern science has further revealed that chicory also has multiple pharmacological activities such as hypoglycemic, anti-inflammatory, anti-tumor, antioxidant and anti-hyperuricemia.

[0003] To date, over 100 compounds have been isolated from chicory, primarily including sesquiterpene lactones, flavonoids, coumarins, chlorogenic acid derivatives, triterpenes, steroids, and polysaccharides. Sesquiterpene lactones are the most abundant and contribute to chicory's distinctive bitterness. Over 60 sesquiterpene lactones have been reported in chicory, primarily consisting of guaiacane, germarane, and eudesmanane nuclei. Summary of the Invention

[0004] The purpose of the present invention is to further study chicory, obtain monomer compounds with active ingredients therefrom, improve the utilization of natural drug resources, and expand the source of anti-inflammatory active substances.

[0005] In order to achieve the above object, the present invention provides a new amino acid sesquiterpene lactone compound, the chemical structure of which is as follows:

[0006]

[0007] The present invention also provides a method for preparing the amino acid sesquiterpene lactone compound. The amino acid sesquiterpene lactone compound is obtained by separation and purification from chicory roots.

[0008] Furthermore, the amino acid sesquiterpene lactone compound of the present invention is prepared by the following method: dried chicory root is extracted with an ethanol solution, the extract is passed through a macroporous resin to remove polysaccharide components, and then purified in sequence through a polyamide resin column, a silica gel column chromatography column, a reverse phase chromatography column, and a preparative high performance liquid chromatography column to obtain the amino acid sesquiterpene lactone compound.

[0009] More specifically, the amino acid sesquiterpene lactone compound is prepared by the following method: taking dried chicory root, soaking and extracting it in 80% ethanol solution or refluxing extraction, filtering and combining the extracts, and concentrating under reduced pressure at 50°C to obtain an alcohol-free crude extract; the crude extract is suspended in water, loaded onto D101 macroporous resin, adsorbed for several hours, and after the solvent naturally drains away, eluted with 4-5 column volumes of pure water to remove the polysaccharide components in the sample, and then eluted with 90-95% ethanol to remove the adsorbed sample, and concentrated under reduced pressure on a rotary evaporator; the concentrated sample is suspended in water again, loaded onto a 30-60 mesh polyamide resin column, adsorbed, and eluted with 4-5 column volumes of pure water, the water elution fraction is collected, and concentrated to obtain a total sesquiterpene lactone mixture. The total sesquiterpene lactone mixture is fractionated by column chromatography on a 100-200 mesh silica gel column, and gradient eluted with a solvent system of dichloromethane:methanol at a volume ratio of 1:0, 50:1, 20:1, 10:1, 5:1, 1:1, and 1:2 in sequence to obtain 7 segments Fr.S1-Fr.S7; Fr.S7 is then chromatographed on an RP-18 reverse phase column, and gradient eluted with 2 column volumes of methanol:water at a volume ratio of 0:1, 2:8, 5:5, and 1:0 in sequence to obtain the eluate S7-1 obtained with solvent ratios of 0:1 and 2:8; S7-1 is purified by preparative liquid phase purification, and isocratic elution with 9% acetonitrile / water isocratic to obtain the amino acid sesquiterpene lactone compound.

[0010] The present invention also provides the use of the amino acid sesquiterpene lactone compound in preparing drugs with anti-inflammatory activity.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] The present invention isolates and identifies a new amino acid sesquiterpene lactone 11β,13-dihydro-13-prolyl-lactucin from chicory. The anti-inflammatory activity of the compound was evaluated, and the results showed that the compound has significant NO production inhibition activity, and its IC 50 The value was better than that of the positive control L-NMMA. In addition, the compound could significantly inhibit the expression of inflammatory cytokines TNF-α, IL-6 and COX-2, showing good anti-inflammatory activity.

[0013] The present invention not only makes further progress in the research of active ingredients of chicory, but also broadens the sources of natural medicines with anti-inflammatory active ingredients, laying a theoretical foundation for further resource utilization of chicory. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the structural formula of the amino acid sesquiterpene lactone compound of the present invention (hereinafter referred to as Compound 1);

[0015] Figure 2 Compound 1 of the present invention 1 H NMR spectrum;

[0016] Figure 3 Compound 1 of the present invention 13 C NMR spectrum;

[0017] Figure 4 is the HSQC spectrum of compound 1 of the present invention;

[0018] Figure 5 is the HMBC spectrum of compound 1 of the present invention;

[0019] Figure 6 Compound 1 of the present invention 1 H- 1 H COSY spectrum;

[0020] Figure 7 is the ROESY spectrum of compound 1 of the present invention;

[0021] Figure 8 is the HR-ESI-MS mass spectrum of compound 1 of the present invention;

[0022] Figure 9 is the infrared spectrum of compound 1 of the present invention;

[0023] Figure 10 is the UV spectrum of compound 1 of the present invention;

[0024] Figure 11 The key to compound 1 of the present invention 1 H- 1 H COSY, HMBC and ROESY are related;

[0025] Figure 12 are the calculated ECD and experimental ECD of compound 1 of the present invention;

[0026] Figure 13 The inhibitory effect of compound 1 of the present invention on the expression of inflammatory factors TNF-α, IL-6 and COX-2 (*p<0.05, **p<0.01, n=5.)

[0027] Figure 13 In the table, control is the blank control group, model is the model group, and 1 is the compound 1 drug group. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to specific embodiments.

[0029] Example 1

[0030] Preparation of Amino Acid Sesquiterpene Lactone 11β,13-dihydro-13-prolyl-lactucin

[0031] 1. Experimental Materials

[0032] Fresh chicory roots were collected from the Nanjing Sun Yat-sen Botanical Garden in June 2022 and identified by Researcher Ren Bingru of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The plant specimen (accession number 20220601) is stored in the Nanjing Sun Yat-sen Botanical Garden Herbarium. The samples were washed, cut into small pieces, and oven-dried at 45°C until ready for use.

[0033] 2. Instruments and equipment

[0034] The nuclear magnetic resonance (NMR) spectra (1D & 2D NMR) of the compounds were measured on a Bruker DRX-600 NMR spectrometer; TMS was used as the internal standard, and the chemical shift (δ) unit was ppm, and the coupling constant (J) unit was Hertz (Hz); the infrared (IR) spectra were measured on a Bio-Rad FTS-135 infrared spectrometer; the ultraviolet (UV) data were measured on a Shimadzu UV2401 spectrometer; the optical rotation spectra (α) were measured on a JASCO P-1020 polarimeter in Tokyo, Japan; the electron circular dichroism (ECD) spectra were measured on a Chirascan circular dichroism spectrometer; the high-resolution mass spectra were measured on an API QSTARPular-1 mass spectrometer; and the absorbance was measured on a Thermo Fisher Scientific Multiscan FC microplate reader.

[0035] 3. Chemical reagents

[0036] Deuterated pyridine (pyridine-d5) was purchased from Shanghai Adamas Reagent Co., Ltd.; chromatography-grade acetonitrile was purchased from Tiandi Reagent Company, USA. Column chromatography materials included D101 macroporous resin (Cangzhou, China), polyamide resin (30–60 mesh, Sinopharm Shanghai Trial), 100–200 mesh silica gel (Qingdao Ocean Chemical Co., Ltd.), and RP-18 reversed phase packing (Merck, Germany). Liquid chromatography was performed on a Shimadzu LC-6AD high-performance liquid chromatograph equipped with an SPD-20A UV detector, using a YMC-Pack ODS-AQ preparative column (5 μm, 12 nm, 250 × 20.0 mm). Cell lines were purchased from the Shanghai Cell Collection, Chinese Academy of Sciences. DMEM medium and fetal bovine serum were purchased from Hyclone. Griess reagent, LPS, MTS, and L-NMMA were purchased from Sigma. ELISA kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0037] 4. Extraction and separation methods

[0038] Dried chicory roots are soaked and extracted in 80% ethanol three times for seven days each time. (Reflux extraction can also be performed by adding 6-10 volumes of 80% ethanol to the sample and gently boiling it under reflux for two to three times, each for two hours.) The extracts are filtered, combined, and concentrated under reduced pressure at 50°C until alcohol-free. The crude extract is suspended in an appropriate amount of water and applied to D101 macroporous resin for adsorption for several hours. After the solvent has naturally drained, the sample is eluted with 4-5 column volumes of pure water to remove polysaccharides. The adsorbed sample is then eluted with 90-95% ethanol. The sample is then concentrated under reduced pressure on a rotary evaporator. The resulting sample is again swirled with water and applied to a polyamide resin (30-60 mesh). After adsorption, it is eluted with 4-5 column volumes of pure water. The water-eluted fraction is collected and concentrated to obtain the total sesquiterpene lactone Fr.S.

[0039] The obtained chicory total sesquiterpene lactone Fr.S was fractionated by column chromatography on a silica gel column (100-200 mesh) and gradient eluted with a solvent system of dichloromethane:methanol (v / v = 1:0, 50:1, 20:1, 10:1, 5:1, 1:1, 1:2) with 3 column volumes, dividing it into 7 fractions Fr.S1-Fr.S7. Fr.S7 was then chromatographed on an RP-18 reverse phase column with a gradient elution of methanol:water (v / v = 0:1, 2:8, 5:5, 1:0) with 2 column volumes. The eluents S7-1 obtained with solvent ratios of 0:1 and 2:8 were combined. Sample S7-1 was then purified by preparative liquid chromatography using isocratic elution with 9% acetonitrile / water to obtain compound 11β,13-dihydro-13-prolyl-lactucin (10.5 mg). The structural formula is as follows: Figure 1 shown.

[0040] 5. Structural identification of compound 11β,13-dihydro-13-prolyl-lactucin

[0041] 5.1 Physicochemical properties

[0042] 11β,13-dihydro-13-prolyl-lactucin, yellow powder, [α] D 25 -12.5(c 0.1,CH3OH),UVλ max (methanol)258nm; IR:ν max 3432,1740,1630cm -1 . 1H NMR(600MHz,pyridine-d5): δ6.97(1H,brs,H-3),3.68(1H,m,overlap,H-5),3.60(1H,m,overlap,H-6),2.48(1H,m,H-7),3.96(1H,m, H-8),2.79(1H,dd,J=13.7,10.7Hz,H-9a),2.58(1H,dd,J=13.7,2.2Hz,H-9b),3.23(1H,m,H-11),3.08(1H,dd,J=13.1,10.0Hz,H-13), 3.63(1H,overlap,H-13),2.43(3H,s,H-14),5.29(1H,brd,J=18.5Hz,H-15a),4.72(1H,brd,J=18.5Hz,H-15b),3.65(2H,overlap,H-1 ”),2.14(2H,dd,J=10.0,6.6Hz,H-2”),1.68(1H,m,H-3”a),1.84(1H,m,H-3”b),3.18(1H,m,H-4”a),2.71(1H,q,J=8.5,8.0Hz,H-4”b); 13 C NMR (150MHz, pyridine-d5): δ132.8(C-1),194.8(C-2),133.0(C-3),175.2(C-4),48.5(C-5),80.8(C-6),62.3(C-7),67.5(C-8),47.1(C-9),14 7.2(C-10),46.4(C-11),174.8(C-12),55.8(C-13),20.9(C-14),62.5(C -15),66.7(C-1”),30.2(C-2”),23.8(C-3”),54.5(C-4”),176.4(C-5”).

[0043] The NMR data of compound 11β,13-dihydro-13-prolyl-lactucin (hereinafter referred to as compound 1) are shown in the following table:

[0044] Table 1. NMR data of compound 1 1 H (600MHz) and 13 C(150MHz)(δin ppm,J in Hz,pyridine-d5)

[0045]

[0046] 5.2 Structure Identification

[0047] Compound 1 is a yellow powder, which was analyzed by high resolution mass spectrometry HR-ESI-MS (e.g. Figure 8 The quasi-molecular ion peak was determined to be 392.1697 (calcd for 392.1704 [M+H] + ), combined with 13 C NMR indicates that its molecular formula is C 20 H 25 NO7, unsaturation is 9; infrared signal (such as Figure 9 shown) is shown in ν max 3432, 1630 and 1740 cm -1 There is strong absorption at , indicating that there may be hydroxyl, γ-lactone, α, β-unsaturated ester and α, β-unsaturated ketone structures in the molecule; Figure 10 As shown, the UV absorption λ max Around 258 nm indicates that there may be an unsaturated ketone structure in the molecule.

[0048] Combine Figures 2 to 7 ,from 1 H and 13 C NMR and HSQC spectra can infer that there is a CH3 group (δ C 20.9), a free carboxyl group (δ C 176.4) and an α,β-unsaturated ketone structure (δ C 194.8), CO(δ C 174.8). Based on the HSQC and COSY correlation, it can be inferred that the two fragments C(9)-C(8)-C(7)-C(6)-C(5), C(7)-C(11)-C(13) and C(1')-C(2')-C(3')-C(4' are connected. H 3.68) and C-1(δ C 132.8), C-4(δ C 175.2), H-8(δ H 3.96) and C-10(δ C 147.2) are related, and H-9 (δ H 2.79) and C-14 (δ C 20.9), H-13(δ H 3.08) and C-12(δ C 174.8), etc., and it is speculated that the compound has a similar core structure to that of compound 11β,13-dihydro-lactucin, except that the chemical shifts at the C-7 / C-11 positions are different, and the C-13 position changes from CH3 to CH2 (δ C55.8) signal. In addition, the five additional carbon signals (δ C 54.5, 30.2, 23.8, 66.7, 174.8) were analyzed by two-dimensional COSY, HSQC and HMBC spectra and identified as proline groups. Finally, based on the long-range correlations between H-13 and C-1' and C-4', H-4' and C-13, and H-1' and C-13 in HMBC, it was speculated that the proline group was attached to the C-13 position (e.g. Figure 11 As shown). Thus, the planar structure of compound 1 was identified as 11β,13-dihydro-13-polyl-lactucin, which is a new compound.

[0049] The relative configuration of compound 1 was determined by ROESY spectrum. Figure 11 As shown, H-6, H-8, and H-11 are correlated in the ROESY spectrum, while H-5, H-7, and H-13 are correlated. It is speculated that H-6, 8, and 11 are on the same side, and H-5, 7, and 13 are on the same side (e.g. Figure 11 The absolute configuration of compound 1 was determined by calculating ECD. After comparing the experimental ECD with the calculated ECD (as shown in Figure 12 As shown), the cotton curve of compound 1 is consistent with the 5S, 6R, 7R, 8S, 11R, 1'R configuration, and the final structure of compound 1 is identified, as shown in FIG. Figure 1 shown.

[0050] Example 2

[0051] Anti-inflammatory activity test

[0052] Nitric oxide (NO) has a wide range of important biological regulatory functions and plays an important role in inflammation, tumors and cardiovascular systems. When immune cells are stimulated by microbial endotoxins, inflammatory mediators, etc., a large amount of induced nitric oxide synthase (iNOS) is generated to produce NO for immune response. Therefore, inhibiting NO production is a direct indicator of the anti-inflammatory activity of the compound. LPS lipopolysaccharide was used to induce mouse mononuclear macrophage RAW264.7 to produce nitric oxide synthase. The culture medium was aspirated and the absorbance was measured at a wavelength of 570nm by the Griess method to detect nitrite (NO). 2- ) content, which can characterize the cellular NO production.

[0053] RAW264.7 cells were seeded into 96-well plates and stimulated with 1 μg / ml LPS. Test compounds were then added (final concentration 50 μM). A drug-free group and an L-NMMA-positive group served as controls. After overnight culture, the culture medium was collected to assess NO production by measuring absorbance at 570 nm. MTS was added to the remaining culture medium to assess cell viability and exclude potential toxic effects of the compounds on the cells.

[0054] NO production inhibition rate (%) = (OD570nm of non-drug treatment group - OD570nm of sample group) / OD570nm of non-drug treatment group × 100%

[0055] IC 50 (50% inhibitory concentration) was calculated according to the Reed & Muench method.

[0056] Subsequently, the cell culture supernatant in the above step was collected, and the expression levels of inflammatory factors IL-6, TNF-α and COX-2 in the cell supernatant were detected using an ELISA detection kit according to the instructions of the instruction manual.

[0057] In this example, L-monomethylarginine (L-NMMA) was used as a positive control to evaluate the nitric oxide (NO) production inhibitory activity of compound 1 isolated from chicory. The results showed that compound 1 had significant NO production inhibitory activity, with a half inhibitory concentration (IC 50 ) was 20.14 μM, which was more active than the positive control L-NMMA (32.77 μM), as shown in Table 2 below.

[0058] Table 2. NO production inhibitory activity of compound 1a

[0059]

[0060] a Values ​​are expressed as mean ± standard deviation (n = 3); b represents the minimum concentration required to inhibit 50% NO production (μM);

[0061] cPositive control.

[0062] At the same time, compared with the blank control group and the model group, compound 1 of the present invention can effectively inhibit the expression of inflammatory factors TNF-α, IL-6 and COX-2, such as Figure 13 shown.

Claims

1. An amino acid sesquiterpene lactone compound, whose chemical structural formula is as follows: 。 2. The method for preparing the amino acid sesquiterpene lactone compound according to claim 1, characterized in that: The amino acid sesquiterpene lactone compound is prepared by the following method: taking dried chicory roots, soaking or refluxing in an 80% ethanol solution for extraction, filtering and combining the extracts, and concentrating under reduced pressure at 50° C. to obtain an alcohol-free crude extract; suspending the crude extract in water, applying the sample to a D101 macroporous resin, and adsorbing the extract for several hours. After the solvent naturally drains, the extract is first eluted with 4-5 column volumes of pure water to remove polysaccharide components in the sample, and then eluting the adsorbed sample with 90-95% ethanol, and concentrating the sample under reduced pressure on a rotary evaporator; The concentrated sample was resuspended in water and loaded onto a 30-60 mesh polyamide resin column. After adsorption, the sample was eluted with 4-5 column volumes of pure water. The water-eluted fractions were collected and concentrated to obtain a total sesquiterpene lactone mixture. The total sesquiterpene lactone mixture was fractionated by column chromatography on a 100-200 mesh silica gel column using a gradient elution system of dichloromethane:methanol with a volume ratio of 1:0, 50:1, 20:1, 10:1, 5:1, 1:1, and 1:2 (3 column volumes) to divide the fraction into seven fractions, Fr. S1 - Fr. S7, take Fr. S7 and then perform RP-18 reverse phase chromatography column chromatography, using 2 column volumes of methanol: water as eluents in volume ratios of 0:1, 2:8, 5:5, and 1:0 for gradient elution, and combine the eluents S7-1 obtained at solvent ratios of 0:1 and 2:8; S7-1 is purified by preparative liquid phase purification, using 9% acetonitrile / water isocratic elution to obtain the amino acid sesquiterpene lactone compound.

3. Use of the amino acid sesquiterpene lactone compound according to claim 1 in the preparation of drugs with anti-inflammatory activity.

Images