Amino acid sesquiterpene lactone compound as well as preparation method and application thereof

Abstract

The invention discloses an amino acid sesquiterpene lactone compound 11beta, 13-dihydroxy-13-prolyl-lactucin, which has anti-inflammatory activity, expands the source of anti-inflammatory drugs, and further provides a further research on active monomer components in cichorium intybus.

Description

Technical Field

[0001] The 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 from Europe, India, South Africa, North America and other regions, and is now widely cultivated around the world. It has a variety of 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 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 away heat and detoxifying, promoting digestion, and diuresis and reducing swelling. It is commonly used by the people to treat symptoms such as damp-heat jaundice, stomach pain and loss of appetite, and nephritis edema. Modern science has further revealed that chicory also has a variety of pharmacological activities such as hypoglycemic, anti-inflammatory, anti-tumor, antioxidant and anti-hyperuricemia.

[0003] So far, more than 100 compounds have been isolated from chicory, including sesquiterpene lactones, flavonoids, coumarins, chlorogenic acid derivatives, triterpenes, steroids, polysaccharides, etc. Among them, sesquiterpene lactones are the most abundant and give chicory its unique bitter taste. There are more than 60 sesquiterpene lactone components reported in chicory, mainly with three types of parent core structures: guaiacane type, germarane type and eucalyptane type. 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: taking dried chicory roots, extracting with ethanol solution, removing polysaccharide components from the extract by passing through a macroporous resin, and then purifying through a polyamide resin column, a silica gel column chromatography column, a reverse phase chromatography column chromatography, and a preparative high performance liquid chromatography column in sequence 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 roots and soaking them in 80% ethanol solution for extraction or reflux extraction, filtering and combining the extracts, and concentrating them under reduced pressure at 50°C to obtain an alcohol-free crude extract; the crude extract is suspended in water, loaded onto a D101 macroporous resin, adsorbed for several hours, and after the solvent is naturally drained, eluted with 4-5 column volumes of pure water to remove the polysaccharide components in the sample, and then eluted the adsorbed sample with 90-95% ethanol, 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, and after adsorption, eluted with 4-5 column volumes of pure water, collecting the water elution fraction, and concentrating to obtain a total sesquiterpene lactone mixture. The total sesquiterpene lactone mixture is segmented by column chromatography on a 100-200 mesh silica gel column, and gradient eluted with a solvent system of 3 column volumes of dichloromethane:methanol in a volume ratio of 1:0, 50:1, 20:1, 10:1, 5:1, 1:1, and 1:2, and is divided into 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 in a volume ratio of 0:1, 2:8, 5:5, and 1:0 as the eluent, and the eluent S7-1 obtained with the solvent ratio of 0:1 and 2:8 is combined; S7-1 is purified by preparative liquid phase, and isocratic elution is performed with 9% acetonitrile / water 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 a drug with anti-inflammatory activity.

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

[0012] The present invention separates and identifies a new amino acid sesquiterpene lactone 11β,13-dihydro-13-prolyl-lactucin from chicory. The anti-inflammatory activity of the compound is evaluated, and the results show that the compound has significant NO generation inhibition activity, and its active 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 The compound 1 of the present invention 1 H NMR spectrum;

[0016] Figure 3 The 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 The 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] Fig. 9 is the infrared spectrum of compound 1 of the present invention;

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

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

[0025] Fig.12 is the calculated ECD and experimental ECD of compound 1 of the present invention;

[0026] Fig.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] Fig.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 Nanjing Zhongshan Botanical Garden in June 2022 and identified by researcher Ren Bingru from the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The plant specimen (No. 20220601) is stored in the herbarium of Nanjing Zhongshan Botanical Garden. The samples were washed, cut into small pieces and dried in an oven at 45°C for later use.

[0033] 2. Instruments and Equipment

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

[0035] 3. Chemical reagents

[0036] Deuterated pyridine (pyridine-d5) was purchased from Shanghai Adamas Reagent Co., Ltd.; chromatographic 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 reverse filler (Merck, Germany); liquid preparation was carried out on Shimadzu LC-6AD high performance liquid chromatograph (equipped with SPD-20A UV detector) in Japan, and the preparative column was YMC-Pack ODS-AQ (S-5μm, 12nm, 250×20.0mm). Cell lines were purchased from Shanghai Cell Collection, Chinese Academy of Sciences, DMEM culture medium and fetal bovine serum were purchased from Hyclone; Griess reagent, LPS, MTS, L-NMMA were purchased from Sigma, and ELISA detection kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0037] 4. Extraction and separation methods

[0038] The dried chicory root is soaked and extracted in 80% ethanol solution for 3 times, each time for 7 days (it can also be refluxed, add 6-10 times the volume of 80% ethanol to the sample, and reflux under slight boiling for 2-3 times, each time for 2 hours), filter and combine the extracts, and concentrate under reduced pressure at 50°C until there is no alcohol. The crude extract is suspended with an appropriate amount of water, loaded onto D101 macroporous resin, and adsorbed for several hours. After the solvent is naturally drained, it is first eluted with 4-5 column volumes of pure water to remove the polysaccharide components in the sample. Then the adsorbed sample is eluted with 90-95% ethanol. Concentrate under reduced pressure on a rotary evaporator. The above-obtained sample is vortexed with water again, loaded onto polyamide resin (30-60 mesh), and after adsorption, it is eluted with 4-5 column volumes of pure water. The water elution fraction is collected and concentrated to obtain the total sesquiterpene lactone Fr.S.

[0039] The obtained chicory total sesquiterpene lactone Fr.S was segmented by column chromatography on a silica gel column (100-200 mesh), and gradient eluted with a solvent system of 3 column volumes of dichloromethane:methanol (v / v=1:0, 50:1, 20:1, 10:1, 5:1, 1:1, 1:2), and divided into 7 segments Fr.S1-Fr.S7, wherein Fr.S7 was further chromatographed on an RP-18 reverse phase column, and gradient eluted with 2 column volumes of methanol:water (v / v=0:1, 2:8, 5:5, 1:0) as the eluent, and the eluent S7-1 obtained with the solvent ratio of 0:1 and 2:8 was combined. Subsequently, sample S7-1 was purified by preparative liquid phase, and prepared by 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 can be identified by high resolution mass spectrometry (HR-ESI-MS). 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 the molecular formula is C 20 H 25 NO7, unsaturation is 9; infrared signal (such as Fig. 9 shown) is shown in ν max 3432, 1630 and 1740cm -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; Fig.10 As shown, the UV absorption λ max Around 258 nm indicates that there may be an unsaturated ketone structure in the molecule.

[0048] Combination 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) 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 C-7 / C-11 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 identified as proline groups by two-dimensional COSY, HSQC and HMBC analysis. 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 inferred that the proline group was attached to the C-13 position (e.g. Fig.11 As shown). Therefore, 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. Fig.11 As shown, in the ROESY spectrum, H-6, H-8, and H-11 are correlated, 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. Fig.11 The absolute configuration of compound 1 was determined by calculating the ECD. Fig.12 The cotton curve of compound 1 is consistent with the 5S, 6R, 7R, 8S, 11R, 1'R configuration, and thus 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 the cardiovascular system. When immune cells are stimulated by microbial endotoxins, inflammatory mediators, etc., a large amount of induced nitric oxide synthase (iNOS) will be 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, and 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 inoculated into 96-well plates and stimulated with 1 μg / ml LPS. The test compound (final concentration 50 μM) was added at the same time. A drug-free group and an L-NMMA-positive drug group were set as controls. After overnight cell culture, the culture medium was taken to detect NO production and the absorbance was measured at 570 nm. MTS was added to the remaining culture medium to detect cell viability to exclude the toxic effects of the compound 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 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, and its 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 generation 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] c Positive control.

[0062] At the same time, compared with the blank control group and the model group, the compound 1 of the present invention can effectively inhibit the expression of inflammatory factors TNF-α, IL-6 and COX-2. Fig.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 separated and purified from chicory roots.

3. The preparation method according to claim 2, characterized in that: The amino acid sesquiterpene lactone compound is prepared by the following method: taking dried chicory roots, extracting with ethanol solution, removing polysaccharide components from the extract by passing through a macroporous resin, and then purifying through a polyamide resin column, a silica gel column chromatography column, a reverse phase chromatography column chromatography, and a preparative high performance liquid chromatography column in sequence to obtain the amino acid sesquiterpene lactone compound.

4. The preparation method according to claim 3, characterized in that: The amino acid sesquiterpene lactone compound is prepared by the following method: taking dried chicory roots and soaking them in 80% ethanol solution for extraction or reflux extraction, filtering and combining the extracts, and concentrating them under reduced pressure at 50° C. to obtain an alcohol-free crude extract; adding water to the crude extract for suspension, loading the crude extract onto D101 macroporous resin, adsorbing for several hours, and after the solvent naturally drains away, eluting the sample 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 suspended in water again and loaded onto a 30-60 mesh polyamide resin column. After adsorption, it was eluted with 4-5 column volumes of pure water, and the water elution fractions were collected and concentrated to obtain a total sesquiterpene lactone mixture. The total sesquiterpene lactone mixture was segmented by column chromatography on a 100-200 mesh silica gel column, and was gradient eluted with a solvent system of 3 column volumes of dichloromethane:methanol in a volume ratio of 1:0, 50:1, 20:1, 10:1, 5:1, 1:1, and 1:2 in sequence, and was divided into 7 segments Fr. S1 - Fr. S7, take Fr. S7 and then chromatograph on an RP-18 reverse phase column, use 2 times the column volume of methanol: water as the eluent in the volume ratio of 0:1, 2:8, 5:5, 1:0 in sequence for gradient elution, and combine the eluent S7-1 obtained with the solvent ratio of 0:1 and 2:8; S7-1 is purified by preparative liquid phase, prepared by isocratic elution with 9% acetonitrile / water, to obtain the amino acid sesquiterpene lactone compound.

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

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