Two sesquiterpenoids in ligularia nakai and application thereof

Abstract

According to the invention, the structural formulas of ligularia nakai are shown as a formula I and a formula II, the ligularia nakai sesquiterpenoids 1 and 2 are extracted from ligularia nakai, and anti-inflammatory activity experiments find that the compound 1 and the compound 2 have anti-inflammatory activity; the compound provided by the invention provides a lead compound for developing an anti-inflammatory preparation, and is beneficial to developing and utilizing plant medicinal resources.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and relates to two erimofenane-type sesquiterpenoid compounds obtained from Ligularia natans and applications thereof in preparing anti-inflammatory drugs. Background Art

[0002] Inflammatory response is a common clinical pathological process. When inflammatory factors act on the body, they will cause damage to tissue cells, while inducing the body's disease resistance to increase, remove inflammatory factors, and repair damaged tissues. The regulation and treatment of inflammatory responses are of great significance to human health. Macrophages are one of the most important cells that regulate the body's inflammatory response. RAW 264.7 (mouse macrophages) cells induced by LPS stimulation can form an inflammatory model. After applying drugs or test compounds, the enzyme-linked immunosorbent assay (ELISA) can be used to detect the effect of drugs or test compounds on inflammatory cells, thereby affecting the content of NO released by cells, to evaluate the in vitro anti-inflammatory activity of drugs or test compounds.

[0003] At present, the widely used anti-inflammatory drugs in the market are relatively few from plants, and the most common ones are non-steroidal anti-inflammatory drugs from the synthetic industry, such as aspirin, indomethacin, ibuprofen, acetaminophen, etc. Ligularia cymbulifera Hand.-Mazz.) is a perennial herbaceous plant of the genus Ligularia in the Asteraceae family. It is widely distributed in the Hengduan Mountains in northwest Yunnan and southwest to northwest Sichuan. It has been recorded in Tibetan medical literature that its young leaves and seedlings are used to treat sores, gallbladder heat and other symptoms, and have the effects of relieving pain, anti-inflammatory and pus removal. Among the plants of the genus Ligularia, some sesquiterpenoid compounds have good biological activities such as antioxidant, anti-inflammatory, antibacterial, anti-cancer and gastric damage protection. The erimofenane-type sesquiterpenoid compounds 1 and 2 prepared from Ligularia natans and their use as anti-inflammatory drugs have not been reported. Summary of the invention

[0004] The present invention aims to provide two new compounds, namely, erimofenane-type sesquiterpenoid compounds 1 and 2, which are isolated from Ligularia natans (Ligularia natans) of the Asteraceae family. Ligularia cymbulifera Hand.-Mazz.), whose chemical structures are shown in Formula I and Formula II:

[0005] Another object of the present invention is to use the above compounds 1 and 2 in the preparation of anti-inflammatory drugs. The anti-inflammatory drugs prepared from the compounds of the present invention can be common tablets, capsules, powders, ointments, solutions, emulsions, injections, etc., and various pharmaceutical excipients can be added to the drug dosage form as needed.

[0006] The advantages and technical effects of the present invention are that the raw materials used for extraction are abundant and easy to obtain, the preparation method is easy to operate, the invented compounds are two new erimofenane-type sesquiterpenoid compounds obtained from the ethyl acetate extraction part of the 95% ethanol extract of the whole plant of Ligularia natans, and the identified new sesquiterpenoid compounds 1 and 2 are proved to have a relatively significant inhibitory activity on the content of inflammatory mediator NO produced by RAW 264.7 cells through anti-inflammatory experiments, and their half inhibitory concentration (IC50) is 2.17~2.13 %. 50 ) values ​​were 11.87 μM and 15.02 μM, respectively. The compounds of this invention provide lead compounds for new anti-inflammatory drugs and expand the medicinal and commercial value of Ligularia natans. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The molecular structural formula diagrams of Compound 1 and Compound 2 of the present invention are shown in FIG.

[0008] Figure 2 The key to the compounds 1 and 2 of the present invention 1 H- 1 H COSY spectrum.

[0009] Figure 3 It is the key HMBC spectrum of compound 1 and compound 2 of the present invention.

[0010] Figure 4 ROESY spectra of Compound 1 and Compound 2 of the present invention.

[0011] Figure 5 is the key HMBC of compound 1 of the present invention, 1 H- 1 Schematic diagram of H COSY and ROESY related signals.

[0012] Figure 6 is the key HMBC of compound 2 of the present invention, 1 H- 1 Schematic diagram of H COSY and ROESY related signals.

[0013] Figure 7 The experimental and calculated ECD spectrum fitting diagrams of compound 1 and compound 2 of the present invention. DETAILED DESCRIPTION

[0014] The present invention is further described in detail below by way of examples, but the content of the present invention is not limited thereto. The methods in the present examples are all operated according to conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified.

[0015] Example 1: Extraction and separation of erimofenane-type sesquiterpenoid compounds (1) The dried whole herb of Ligularia natans (7.5 kg) was crushed and soaked in 95% ethanol. The mixture was allowed to stand for 8 hours and the extraction was repeated 4-5 times. The combined extracts were concentrated under reduced pressure to obtain 590.0 g of total extract. (2) Add pure water to the extract and stir thoroughly to ensure full contact. Then, add equal volumes of petroleum ether and ethyl acetate solvents to the total extract water suspension in turn, let stand for 12 hours, and repeat the operation three times to obtain an extract. (3) The three extracts were combined and concentrated under reduced pressure to obtain 189.5 g of petroleum ether extract, 103.7 g of ethyl acetate extract and 67.4 g of n-butanol extract; (4) The ethyl acetate portion was subjected to silica gel column chromatography and gradient eluted with petroleum ether-ethyl acetate solvents in a volume ratio of 10:0, 8:2, 6:4, 4:6, 2:8, and 0:10 to obtain 8 portions, Fr.A-Fr.H; (5) Fr.B (10.7 g) was chromatographed on an MCI column (the mobile phase was a methanol-water solution with a volume fraction of 50%-100%) to obtain Fr.B-1 to Fr.B-5. Fr.B-5 (2.1 g) was chromatographed on a silica gel column [the mobile phase was V (petroleum ether): V (ethyl acetate) = 8:1] to obtain compound 1 (1.2 mg); (6) Fr.D (5.1 g) was subjected to MCI column chromatography (mobile phase volume fraction of 30%-60% methanol-water solution) to obtain Fr.D-1 to Fr.D-6. Fr.D-5 (1.2 g) was subjected to semi-preparative liquid chromatography (mobile phase volume fraction of 38% acetonitrile-water solution, retention time 20 min) to obtain compound 2 (5.0 mg). ; The identification results are as follows: Compound 1: White solid, easily soluble in chloroform and dichloromethane; has UV absorption at wavelengths of 210 nm and 254 nm, specific rotation :−15.83°. TLC was developed with petroleum ether:ethyl acetate = 6:1 solvent, R f The value is about 0.5, and the color developing agent does not develop color after the mixed solution of H2SO4-EtOH with a volume fraction of 10%. The quasi-molecular ion peak [M+Na] was obtained by HR-ESI-MS experiment. + for m / z 381.1316 (calculated value is 381.1309), combined with the information of NMR spectrum, its molecular formula is determined to be C 20 H 22 O6, the calculated unsaturation is 10.13 C NMR and DEPT showed that compound 1 had 20 carbon signals and 4 methyl signals. d C [16.0 (C-5′), 20.0 (C-4′), 26.2 (C-14), 9.2 (C-13)], 2 methylene signals d C [27.3 (C-2), 26.9 (C-1)], 6 methine d C [38.7 (C-10), 49.6 (C-4), 68.4 (C-3), 75.8 (C-6), 109.4 (C-9), 140.2 (C-3′)], 8 quaternary carbon signals including: 4 olefinic carbon signals d C [137.8 (C-8), 146.8 (C-7), 129.4 (C-11), 127.0 (C-2′)], 3 carbonyl carbon signals d C [170.0 (C-12), d C [174.5 (C-15), 166.7 (C-1′)] and 1 SP 3 Hybridized quaternary carbon d C [41.1 (C- 5)]. After removing the above unsaturated groups, which account for 6 unsaturations, there are still 4 unsaturations, and it is speculated that the compound has a tetracyclic structure. According to the HSQC spectrum analysis, 1 The protons shown by H-NMR are clearly assigned to their corresponding carbons: d H 1.24 (H-1a) / 26.9 (C-1), d H 1.82 (H-1b) / 26.9 (C-1), d H 1.74 (H-2a) / 27.4 (C-2), d H 2.44 (H-2a) / 27.4 (C-2), d H 5.56 (H-3) / 68.5 (C-3), d H 3.17 (H-4) / 39.6 (C-4), d H 4.96 (H-6) / 75.8 (C-6), d H 5.78 (H-9) / 109.5 (C-9), d H 2.61 (H-10) / 38.8 (C-10), d H 2.08 (H-13) / 9.3 (C-13), 1.35 (H-14) / 26.5 (C-14), d H 6.16 (H-3′) / 140.3 (C-3′), d H 2.03 (H-4′) / 16.1 (C-4′), d H 1.91 (H-5′) / 20.9 (C-5′). 1 H- 1 H COSY spectrum shows: H-1 ( d H 1.24, 1.82) and H-2 ( d H 1.74, 2.44), H-2 ( d H 1.74, 2.44) and H-3 ( d H 5.56), H-3 ( d H 5.56) and H-4 ( d H 3.17) and H-9 ( d H 5.78) and H-10 ( d H 2.61) has a related signal, indicating that C-1 ( d C 26.9)、C-2 ( d C 27.4)、C-3 ( d C 68.5) and C-4 ( d C 49.6) directly connected; H-3′ ( d H 6.16) and H-4′ ( d H 2.03) have related signals, indicating that C-3′ ( d C140.3) and C-4′ ( d C 16.1) are directly connected. According to the HMBC spectrum: H-1 ( d H 1.24, 1.82) have related signals with C-2, C-5, C-3, H-9( d H 5.78) has related signals with C-10, C-8, C-7, H-14 ( d H 1.35) There are related signals with C-10, C-4 and C-6, combined with 1 H- 1 H COSY spectrum shows that compound 1 has a decahydronaphthalene structure similar to compound 2, and C-14 is connected to C-5. H-13 ( d H 2.08) has related signals with C-11, C-8, C-7, and C-9. C-11 and C-7 are alkenyl carbons, indicating that C-13 is connected to alkenyl C-11. H-4 ( d H 3.17) has related signals with C-10, C-3, and C-14, among which C-6 is a methylene carbon connected to oxygen, and C-15 is a carbonyl carbon, indicating the existence of a five-membered lactone ring A and a decahydronaphthalene structure connected thereto. Based on the above information, it is inferred that compound 1 is an erimofenane-type sesquiterpene structure; H-5′ ( d H 1.91) has related signals with C-2′, C-3′, and C-1′, H-4′( d H 2.03) There are related signals with C-2′ and C-3′, C-1′ is the carbonyl carbon, and C-3 is the methyl carbon, from which the substituent and substitution position can be inferred.

[0016] ROESY spectrum shows: H-6 ( d H 4.99) and H-14 ( d H 1.35), H-6 ( d H 4.99) and H-4 ( d H 3.17), H-3 ( d H 5.56) and H-4 ( d H 3.17), H-14 ( d H 1.35) and H-4 ( d H 3.17), H-14 ( d H 1.35) and H-10 ( d H 2.61) There are related signals, indicating that H-3, H-4, H-6, H-10, and H-14 are in the same orientation. Based on the above information, the relative configuration of compound 1 was determined.

[0017] by （ 3R*, 4S*, 5S*, 6R*, 10R*)-1 is the calculation molecular model. The ECD curve obtained by ECD calculation is consistent with the experimental ECD result trend of compound 1. It can be determined that the absolute configuration of compound 1 is calculated 3R, 4S, 5S, 6R, 10R.

[0018] Compound 2: White solid, easily soluble in chloroform, dichloromethane and methanol; has UV absorption at wavelengths of 210 nm and 254 nm, and specific rotation :−80.07°. TLC was developed in petroleum ether:ethyl acetate = 8:1 solvent, R f The value is about 0.6. The color developing agent does not develop color after the mixed solution of H2SO4-EtOH with a volume fraction of 10%. The quasi-molecular ion peak [M+Na] was obtained by HR-ESI-MS experiment. + for m / z 327.1208 (calculated value is 327.1203) Its molecular formula is C 17 H 20 O5, calculated unsaturation is 8. 13 C NMR and DEPT showed that compound 2 had 17 carbon signals: 3 methyl signals d C [14.6 (C-2′), 8.8(C-13), 28.3 (C-14)], 4 methylene signals d C [21.0 (C-2), 9.0 (C-1′), 21.7 (C-1), 38.4 (C-9)], 3 methine signals d C [75.2 (C-6), 137.6 (C-3), 38.5 (C-10)], 7 quaternary carbon signals including: 3 olefinic carbon signals d C [130.4 (C-4), 149.3 (C-7), 132.4 (C-11)], 2 carbonyl carbon signals d C[170.5(C-12), 168.8(C-15)], 1 oxygen-linked quaternary carbon signal d C [106.2(C-8)] and 1 SP 3 Hybridized quaternary carbon d C [41.2 (C-5)]. After removing the above unsaturated groups accounting for 4 unsaturations, there are still 4 unsaturations, and it is speculated that compound 2 has a tetracyclic structural fragment.

[0019] According to HSQC spectrum analysis, 1 The protons shown by H-NMR are clearly assigned to their corresponding carbons: d H 1.58(H-1a) / 21.7 (C-1), d H 2.01 (H-1b) / 21.7 (C-1), d H 2.41 (H-2) / 21.0 (C-2), d H 6.91 (H-3) / 137.6(C-3), d H 5.05 (H-6) / 78.2 (C-6), d H 1.92 (H-9a) / 38.4 (C-9), d H 2.19 (H-9b) / 38.4 (C-9), d H 1.92 (H-10) / 32.5 (C-10), d H 2.01 (H-13) / 8.8 (C-13), d H 1.34 (H-14) / 28.3 (C-14), d H 3.24 (H-1′) / 59.0 (C-1′), d H 1.05(H-2′) / 14.6(C-2′).

[0020] 1 H- 1 H COSY spectrum shows: H-1 ( d H 2.01) and H-2 ( d H2.41), indicating that C-1 ( d C 21.7) directly connected to C-2 ( d C 21.0); H-9 ( d H 2.19) and H-10 ( d H 1.92), indicating that C-9 ( d C 38.4) directly connected to C-10 ( d C 32.5); H-2 ( d H 2.41) and H-3 ( d H 6.91), indicating that C-2 ( d C 21.0) and C-3 ( d C 137.6) directly connected; H-1′ ( d H 3.24) and H-2′ ( d H 1.05), indicating that C-1′ ( d C 59.0) directly connected to C-2′ ( d C 14.6).

[0021] HMBC spectrum shows: H-6 ( d H 5.05) has related signals with C-14, C-5, C-11, C-8, C-7, and C-15. C-6 is connected to oxygen, and C-15 is a carbonyl carbon. Therefore, it is inferred that there is a five-membered lactone ring A in the compound, and the methyl carbon of C-14 is connected to C-5. H-1 ( d H 2.01, 1.58) have related signals with C-2, C-3, C-5, H-14 ( d H 1.34) has related signals with C-10, C-5, C-4 and C-6, H-9 ( d H 1.92, 2.19) with related signals to C-10, C-5 and C-8, H-10 ( d H1.92) has related signals with C-1 and C-8, and it can be inferred that the two six-membered rings A and B are directly connected, and the presence of a decahydronaphthalene skeleton is confirmed. H-13 ( d H 2.01) has related signals with C-11, C-7, C-12, and C-8, and C-12 is a carbonyl carbon, which indicates that the five-membered lactone ring B is connected to the six-membered ring B. Based on the above information, it can be inferred that the compound is an erimofenane-type sesquiterpene structure; H-1′( d H 3.24) has a related signal with C-2′, and C-8 is an oxygen-connected quaternary carbon, from which the structure and substitution position of the substituent are inferred; based on the NMR data reported in the reference, it is determined that compound 2 is different from 8 reported in the literature. β -Ethoxyeremop- hil-3,7(11)-diene-8 α ,12(6 α ,15)-diolide has the same planar structure, but the specific rotations of the two compounds are different (the specific rotations of the compounds published in the literature are : +52°) has a large difference.

[0022] ROESY spectrum shows: H-1′ ( d H 3.24) and H-14 ( d H 1.34), H-14 ( d H 1.34) and H-6 ( d H 5.05) There are related signals, indicating that H-14, H-6, and H-1′ have the same orientation, while there are no related signals between H-10 and H-14, H-6, and H-1′, indicating that H-10 has a different orientation from H-14, H-6, and H-1′, and the relative configuration is opposite to that of the compounds in the literature. Therefore, the relative configuration of compound 2 was determined.

[0023] Combined with the ROESY analysis results, using (5S*, 6R*, 8S*, 10S*) as the calculation molecular model, the curve obtained by ECD calculation is consistent with the ECD result trend of compound 2, and it can be determined that the absolute configuration of compound 2 is calculated as 5 S ,6 R ,8 S ,10 S .

[0024] The above-mentioned compounds 1 and 2 are both new natural organic compounds.

[0025] Table 1: Compounds 1 and 2 1 H NMR and13 C NMR data

[0026] Example 2: Inhibitory effect of compounds 1 and 2 on LPS-induced NO release in RAW 264.7 cells The cytotoxicity of different concentrations of the test substances on RAW 264.7 cells was determined by MTT method using compound 1 and compound 2, and the in vitro anti-inflammatory activity of the test substances was determined by Griess method. The specific methods are as follows: (1) Cytotoxicity experiment: Cells in the logarithmic growth phase were cultured at a volume of approximately 1×10 cells / well in 100 μL. 4 Cells were inoculated in 96-well plates and cultured until the cell density reached 60%-70%. After about 24 hours, the experiment could be carried out. The blank group and model group were set as references. The concentrations of the monomer compound in the drug-treated group were 6.25, 12.5, 25, 50, and 100 μM. Three replicates were set for each concentration. The model group and drug-treated group were stimulated with 1 μg / mL of LPS. The blank was supplemented with the same volume of complete medium DMEM. Finally, the amount of DMSO in each well was balanced. The plate was cultured for 24 hours in an incubator. 20 μL of MTT at a concentration of 5 mg / mL was added to each well. The plate was cultured for another 4 hours. The solution in the plate was gently shaken out and inverted on a clean paper. 150 μL of DMSO was added to each well. After being protected from light, it was placed on a shaker with low speed for 15 minutes to fully dissolve the precipitate. The absorbance value was detected at 490 nm on an ELISA reader. Cell survival rate = (experimental well-blank well) / (control well-blank well)×100%.

[0027] (2) Preparation of standard curve: Use complete medium DMEM to dilute the standard NaNO2 (1M) to different concentrations (0, 1, 2, 5, 10, 20, 40, 80, 100 μM), and add 70 μL per well to a clean 96-well plate (avoid bubbles). Set up 3 replicate wells for each concentration. Then, add 50 μL of Griess A to each well, place it on a shaker in the dark for 10 min, then add 50 μL of Griess B to each well, place it on a shaker in the dark for 10 min, and use an enzyme reader to detect the absorbance at a wavelength of 540 nm. Use concentration as the horizontal axis and absorbance as the vertical axis to make a standard curve.

[0028] (3) Inhibitory activity of compounds 1 and 2 on LPS-induced NO production in RAW 264.7 cells: Cells in the logarithmic growth phase were inoculated with 100 μL / well (approximately 1×10 4) were planted in 96-well plates and cultured for 24 h. A blank control group (Control), model group (LPS), positive drug group (LPS+DXM), and experimental group (LPS+different concentrations of compounds) were set up. 50 μL of the supernatant was aspirated and discarded. The experimental group and positive drug group were given DXM and monomer compounds with final concentrations of 6.25, 12.5, 25, 50, and 100 μM, respectively. LPS with a final concentration of 1 μg / mL was added together with the model group. After the blank control reagent was equilibrated, it was filled up to 100 μL with DMEM and cultured in an incubator for 24 h. 70 μL of the supernatant culture solution was aspirated and placed in a new 96-well plate. 50 μL of GriessA was added to each well in turn, and the plates were placed on a shaker for reaction in the dark for 10 min. Then 50 μL of Griess B was added to each well, and the plates were placed on a shaker for reaction in the dark for 10 min. The absorbance value was detected at a wavelength of 540 nm using an enzyme reader, and the NO content was calculated by substituting it into the standard curve equation.

[0029] (4) Data analysis: SPSS statistical software was used for data processing. The data of each group were expressed as mean ± standard deviation. One-way analysis of variance was used to compare the statistical differences among the groups. P < 0.05 was considered statistically significant.

[0030] (5) Results and discussion Table 2: Effects of compounds 1 and 2 at 100 μM on the viability of RAW 264.7 cells

[0031] The results demonstrate that the effects of compounds 1 and 2 on the survival rate of RAW 264.7 cells at a concentration of 100 μM are substantially equivalent to those of dexamethasone, and compounds 1 and 2 have no effect on the normal proliferation and growth of RAW 264.7 cells and do not exhibit cytotoxicity.

[0032] Table 3: Effects of compounds 1 and 2 at 1-100 μM on LPS-induced NO production in RAW 264.7 cells

[0033] Compounds 1 and 2 had significant inhibitory activity on LPS-induced NO production in RAW 264.7 cells, among which compound 1 had significantly better activity than dexamethasone; compounds 1 and 2 could significantly reduce the nitrite concentration in the supernatant of LPS-induced RAW 264.7 cells. As shown in Table 3, compounds 1 and 2 could almost completely inhibit the release of NO, which was equivalent to the effect of dexamethasone; compound 2 had a significant inhibitory effect on the release of NO compared with the dexamethasone group, but its activity was slightly lower than that of dexamethasone.

Claims

1. The erimofenane-type sesquiterpenoid compounds with the structural formulas shown in Formula I and Formula II:

2. Use of the erimofenane-type sesquiterpenoid compound described in claim 1 in the preparation of anti-inflammatory drugs.

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