Coumarin-lignan glycoside compound separated from opium graveolens and anti-RA application of coumarin-lignan glycoside compound
By isolating and purifying neocoumarin lignan glycosides from Tibetan celery, the problem of the lack of anti-rheumatoid arthritis drugs in the prior art has been solved, and significant anti-RA effects have been achieved.
Patent Information
- Application Number
- CN202511463560.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-14
- Publication Date
- 2026-01-23
AI Technical Summary
Existing technologies have failed to effectively utilize the resources of Tibetan celery to develop its active ingredients for combating rheumatoid arthritis, and there is a lack of effective drugs to combat rheumatoid arthritis.
A novel coumarin lignan glycoside compound was extracted and purified from Tibetan celery using a bioactivity-guided separation method. The compound was then separated using multi-step chromatographic and extraction techniques, and its anti-RA effect was verified by in vitro cell experiments.
A novel coumarin lignan glycoside compound was successfully isolated, exhibiting significant anti-rheumatoid arthritis activity. It can effectively inhibit LPS-induced NO production and has the potential to be used in the preparation of anti-RA drugs.
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Figure CN121378367A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of medicine, and relates to a new coumarin lignan glycoside compound and a medical use thereof, in particular to a new coumarin lignan glycoside compound isolated from Cymopterus tibeticus, and a preparation method of the compound and a use of the compound in preparing an anti-rheumatoid arthritis (RA) drug. BACKGROUND
[0002] Cymopterus tibeticus is a perennial herb of the Umbelliferae family, also known as Dujiaodanggui or Yedanggui. It is warm in nature and pungent, sweet, bitter and slightly bitter in taste, and has the effects of dispelling wind and dampness, dispelling cold and relieving pain in clinical practice. Modern studies have found that Cymopterus tibeticus contains a variety of chemical components, mainly including coumarins, flavonoids, lignans, terpenes and other chemical components. Modern pharmacological studies have shown that Cymopterus tibeticus has a variety of biological activities, mainly including anti-inflammatory, antioxidant, anti-fatigue and other effects. Therefore, it is necessary to further develop the potential medicinal value of Cymopterus tibeticus. SUMMARY
[0003] The purpose of the present application is to take Cymopterus tibeticus as the research object, and to separate and purify the anti-RA active part of Cymopterus tibeticus by using a bioactivity-guided separation method, so as to obtain a new coumarin lignan glycoside compound. At the same time, the present application uses in vitro cell experiments to evaluate and analyze the effect of the coumarin lignan glycoside compound on rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA), and further verifies that it has an anti-RA effect. So far, there is no related literature or patent reported at home and abroad.
[0004] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:
[0005] A new coumarin lignan glycoside compound (denoted as compound I) with a structure as shown in formula I:
[0006]
[0007] The molecular formula of compound I is C 36 H 32 O 17 .
[0008] The name of compound I is 5-hydroxy-8-luteosyl-7'-benzo-4'-hydroxy-10', 15'-diketone indolizine-8'-dihydropyranyl coumarin.
[0009] A preparation method of the coumarin lignan glycoside compound, comprising the following steps:
[0010] Step (1), crushing dry Cynoglossum zangianum, soaking in 75-95% ethanol for 22-26 hours, then heating to reflux extraction for 3-5 times, each time for 4-6 hours, combining the filtrate to obtain Cynoglossum zangianum total extract;
[0011] Step (2), concentrating and drying the Cynoglossum zangianum total extract under reduced pressure to obtain dry Cynoglossum zangianum total extract;
[0012] Step (3), uniformly suspending the Cynoglossum zangianum total extract in distilled water, sequentially extracting with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract, respectively;
[0013] Step (4), loading the n-butanol extract onto a macroporous adsorption resin column, sequentially gradient eluting with 15% ethanol, 30% ethanol, 50% ethanol, 70% ethanol, and 100% ethanol to obtain 5 main fractions: main fraction A, main fraction B, main fraction C, main fraction D, and main fraction E;
[0014] Step (5), separating main fraction A on a medium pressure column, the medium pressure column being YMC-Pack ODS-A column (250×50mm, 5μm), mobile phase: 10-20% methanol, absorption wavelength: 200-220nm, flow rate: 15-20mL / min, to obtain 4 sub-fractions: sub-fraction A-1, sub-fraction A-2, sub-fraction A-3, and sub-fraction A-4;
[0015] Step (6), loading sub-fraction A-3 onto a Sephadex LH-20 column, eluting with 80-100% methanol to obtain 3 fine fractions: fine fraction A-3-1, fine fraction A-3-2, and fine fraction A-3-3;
[0016] Step (7), separating fine fraction A-3-2 by preparative liquid chromatography, the conditions of preparative liquid chromatography being: YMC-Pack ODS-A column (250×20mm, 5μm), mobile phase: 10-20% methanol, absorption wavelength: 200-220nm, flow rate: 2-4mL / min, to obtain compound I.
[0017] In step (1), the dry Cynoglossum zangianum is crushed to 60-90 mesh.
[0018] The dry Cynoglossum zangianum is dry root of Cynoglossum zangianum.
[0019] In step (2), the temperature for concentrating and drying under reduced pressure is 45-55°C, and the pressure is vacuum.
[0020] In step (3), the volume ratio of the Cynoglossum zangianum total extract to distilled water is 1:1.5.
[0021] The anti-RA activity of different extraction parts is screened by the bioactivity-guided separation method, and the n-butanol extraction part is determined as the anti-AR active part.
[0022] In step (4), the macroporous adsorption resin is D101 macroporous adsorption resin.
[0023] The anti-RA activity of the five main fractions is screened, and the result shows that the main fraction A has significant anti-AR activity.
[0024] In step (5), preferably, the medium-pressure column is YMC-Pack ODS-A column (250*50mm, 5um), the mobile phase is 15% methanol, the absorption wavelength is 210nm, and the flow rate is 18mL / min.
[0025] In step (6), preferably, Sephadex LH-20 column is used for the sub-fraction A-3, and 90% methanol is used for elution.
[0026] In step (7), preferably, the preparation liquid chromatography is prepared under the following conditions: YMC-Pack ODS-A column (250*20mm, 5um), the mobile phase is 15% methanol, the absorption wavelength is 210nm, and the flow rate is 3mL / min.
[0027] Another object of the present application is to provide the coumarin lignan glycoside compound in the preparation of a drug for treating rheumatoid arthritis.
[0028] Another object of the present application is to provide a pharmaceutical composition, wherein the coumarin lignan glycoside compound is used as a main active ingredient, and the pharmaceutical composition is prepared into a pharmaceutically acceptable dosage form together with a pharmaceutically acceptable excipient.
[0029] The dosage form is tablet, capsule, granule, pill, oral liquid, suspension, and patch.
[0030] The present application has the following beneficial effects:
[0031] The present application selects the Tibetan medicine, i.e., Tibetan Cnicus lactifolius as a research object, the plant resource is rich, the raw material is easy to obtain, and has obvious regional characteristics.
[0032] The present application adopts 75-95% ethanol to heat reflux extract the Tibetan Cnicus lactifolius, uses the bioactivity tracking separation method to directionally separate and purify the anti-RA active part of the Tibetan Cnicus lactifolius, and separates a new coumarin lignan glycoside compound from the Tibetan Cnicus lactifolius.
[0033] The coumarin lignan glycoside compound has novel and unique structure, the preparation method is simple and easy to operate, has significant anti-RA activity, and can be prepared into anti-RA medicine. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 This is a flowchart illustrating the preparation process of compound I of the present invention.
[0035] Figure 2 This is a coupling correlation diagram of compound I of the present invention. Detailed Implementation
[0036] The following specific embodiments further illustrate the substantive content of the present invention, but do not limit the scope of protection of the present invention.
[0037] Example 1
[0038] like Figure 1 As shown, a novel coumarin lignan glycoside compound isolated from Tibetan celery is prepared by the following method:
[0039] Step (1): Crush 20.0 kg of dried roots of Tibetan celery to 70 mesh, soak in 85% ethanol for 24 hours, and then heat and reflux to extract 4 times, 5 hours each time. Combine the filtrates to obtain the total extract of Tibetan celery.
[0040] The total extract of Tibetan celery obtained in steps (2) and (1) was concentrated and dried under vacuum at 55°C to obtain dried total extract of Tibetan celery (4585.5 g).
[0041] Step (3): The total extract of Tibetan celery and distilled water were uniformly suspended in distilled water at a volume ratio of 1:1.5. The extract was then extracted with petroleum ether, ethyl acetate and n-butanol in sequence to obtain petroleum ether extract (674.6 g), ethyl acetate extract (110.2 g) and n-butanol extract (358.4 g) respectively.
[0042] The activity of different extraction fractions was screened by using a bioactivity-guided separation method, and the n-butanol extraction fraction was identified as the anti-AR active fraction.
[0043] Step (4): The anti-AR active fraction (i.e., the n-butanol extract fraction) obtained in step (3) is loaded onto a D101 macroporous adsorption resin column and eluted sequentially with 15% ethanol → 30% ethanol → 50% ethanol → 70% ethanol → 100% ethanol to obtain 5 main fractions, which are respectively labeled as main fraction A (42.6 g), main fraction B (62.5 g), main fraction C (84.8 g), main fraction D (49.6 g), and main fraction E (30.7 g).
[0044] Step (5), the main fraction A was separated by YMC-Pack ODS-A column (length 250 x inner diameter 50 mm, particle size 5 μm), mobile phase: 15% methanol, absorption wavelength: 210 nm, flow rate: 18 mL / min, to obtain four sub-fractions, which were recorded as sub-fraction A-1 (6.3 g), sub-fraction A-2 (12.6 g), sub-fraction A-3 (10.3 g), sub-fraction A-4 (6.5 g), respectively;
[0045] Step (6), the sub-fraction A-3 obtained in step (5) was separated by Sephadex LH-20 column, eluted with 90% methanol, to obtain three fine fractions, which were recorded as fine fraction A-3-1 (1.9 g), fine fraction A-3-2 (3.3 g), fine fraction A-3-3 (2.6 g), respectively;
[0046] Step (7), the fine fraction A-3-2 obtained in step (6) was separated by preparative liquid chromatography, using YMC-Pack ODS-A column (250 x 20 mm, 5 μm), mobile phase: 15% methanol, absorption wavelength: 210 nm, flow rate: 3 mL / min, through multiple preparations and purifications, finally compound I (9.06 mg) was obtained.
[0047] Compound I was a light yellow solid, HR-ESI-MS m / z 759.1535 [M+Na] + suggesting that the molecular formula was C 36 H 32 O 17 (calcd for C 36 H 32 O 17 Na, 759.1537). The IR v max :3437, 1693, 1620, 1347 cm -1 ; UV (MeOH) λ max :220, 260, 285, 356 nm, according to the IR and UV spectra of the compound, it was preliminarily inferred that the compound was a coumarin-pyranolignan compound. The 1 H NMR (600 MHz, MeOD-d4) and 13 C NMR (150 MHz, MeOD-d4) data of compound I are shown in Table 1. In the 1 H NMR and 13 C NMR spectra of compound I, there was a group of typical coumarin characteristic signals: δ H6.43 (IH, d, J = 9.5 Hz, H-3), 7.74 (IH, d, J = 9.5 Hz, H-4) and δc 162.5 (C-2), 117.1 (C-3), 145.7 (C-4), again confirming that the compound belongs to the coumarinolignan class. In addition, in the 1 H NMR and 13 CNMR in the middle field spectrum, there is a set of typical signals characteristic of the rutinosyl group: δ H 5.78 (IH, d, J = 7.8 Hz, H-1"), 3.82 (IH, m, H-2"), 3.61 (IH, m, H-3"), 3.39 (IH, m, H-4"), 4.01 (IH, m, H-5"), 3.51 (IH, dd, J = 14.4, 7.2 Hz, H-6"a), 3.96 (IH, J = 7.2, 3.6 Hz, H-6"b) and δ C 101.1 (C-1"), 72.0 (C-2"), 69.3 (C-3"), 67.7 (C-4"), 70.2 (C-5"), 64.2 (C-6"); 4.98 (IH, d, J = 1.8 Hz, H-1"'), 3.38 (IH, m, H-2"'), 3.32 (IH, m, H-3"'), 3.28 (IH, m, H-4"'), 3.79 (IH, m, H-5"'), 1.21 (IH, d, J = 7.2 Hz, H-6"') and δ C 99.3 (C-1"'), 71.6 (C-2"'), 68.9 (C-3"'), 72.7 (C-4"'), 64.8 (C-5"'), 15.0 (C-6"') (Table 1). From the HMBC correlation diagram ( Figure 2 ): H-1" and C-8 / 3" correlation, H-5" and C-3" correlation, H-1"' and C-6" / 3"' correlation, H-6"' and C-4"' correlation, it was determined that the rutinosyl group is attached at the C-8 position. In addition, in the 1 H NMR and 13 C NMR in the aromatic region, there is a symmetrical benzophenone 4'-hydroxy-10', 15'-dione indolizine fragment: δ H 7.25 (2H, d, J = 6.5 Hz, H-11' / 14'), 7.41 (2H, d, J = 6.5 Hz, H-12' / 13') and δ C130.2 (C-1'), 133.4 (C-2' / 6'), 128.6 (C-3' / 5'), 156.1 (C-4'), 132.0 (C-11' / 14'), 142.3 (C-12' / 13'), 198.7 (C-10' / 15'), according to HMBC correlation: H-8' / 9' and C-1' correlation, H-11' and C-2' correlation, H-12' and C-10' correlation, H-13' and C-15' correlation, H-14' and C-6' correlation, it was determined that the group was connected at the C-7' position.
[0048] According to the spectral data and HMBC of compound I, 1 H- 1 H COSY coupling correlation information Figure 2 ), SciFinder search found that compound I was a new coumarin lignan glycoside compound.
[0049] Table 1. Compound I 1 H NMR, 13 C NMR and HMBC correlation data
[0050]
[0051] Note: 1 H NMR (600MHz, MeOD-d4), 13 C NMR (150MHz, MeOD-d4).
[0052] Example 2
[0053] Determination and evaluation of the anti-RA activity of compound I
[0054] 1 Experimental materials, reagents and instruments
[0055] Rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA), compound I, FM-2 medium (fibroblast culture medium-2), CCK-8 kit, NO kit, Griess Reagent I and II, PBS buffer, LPS (lipopolysaccharide), cell counter, trypsin, inverted biological microscope, table centrifuge, microplate reader, etc.
[0056] 2 Experimental method
[0057] 2.1 Determination of cell viability
[0058] First, when the rheumatoid arthritis fibroblast-like synoviocytes proliferate to 80%, remove the culture medium in the original culture bottle, add 2 mL PBS to rinse twice, then add 1 mL trypsin, digest for 0.5-1 min, and observe the cell edge under a microscope. When the cell edge begins to shrink and the adhesion is loose, stop the digestion by adding 2 mL culture medium, and then gently blow the cells with a pipette to detach and disperse the cell layer. Transfer the cells to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend with 1 mL FM-2 medium, count using a cell counter, dilute, and inoculate 1x10 4
[0059] Cell survival rate = [(experimental well-blank well) / (control well-blank well)]x100%
[0060] The CCK-8 method was used to verify the effect of compound I on the activity and proliferation of HFLS-RA cells. The results are shown in Table 2, which shows that compound I has no significant effect on the cell activity of HFLS-RA at a dose of less than 40 μM, but has significant cytotoxicity at a concentration of 80-320 μM. Therefore, compound I at a concentration of 10, 20, and 40 μM was selected for subsequent experiments.
[0061] Table 2. Effect of compound I on HFLS-RA cell proliferation (mean ± SD, n = 3)
[0062]
[0063]
[0064] Note: *P <0.05, **P <0.01.
[0065] 2.2 Effect of compound I on LPS-induced NO production by HFLS-RA cells (mean ± SD, n = 3)
[0066] First, when the rheumatoid arthritis fibroblast-like synoviocytes proliferate to 80%, remove the culture medium in the original culture bottle, add 2 mL PBS to moisten and wash 2 times, then add 1 mL trypsin, digest for 0.5-1 min, observe under a microscope, when the cell edge begins to shrink and the adhesion is loose, add 2 mL culture medium to stop the digestion, then gently blow the cells with a pipette, blow off and scatter the cell layer, and then transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 1 mL FM-2 culture medium to resuspend, count using a cell counter, dilute, and inoculate 10,000 cells per hole into a 96-well plate, 3 replicate wells per group, culture for 24 h, discard the supernatant, and divide into a blank group, a model group, and different concentrations of compound I treatment groups. The compound I treatment groups: add FM-2 culture medium containing different concentrations (10, 20, 40 μM) of compound I (first prepare a compound I stock solution with DMSO, and then dilute with FM-2 culture medium) and culture for 24 h, then add LPS (5450 μg / mL) and continue to culture for 24 h. The blank group: only add FM-2 culture medium for culture. The model group: only add FM-2 culture medium containing LPS (5450 μg / mL) for culture. Aspirate 50 μL of the culture supernatant, then add 50 μL of Griess Reagent I and II in sequence, and measure the absorbance at 540 nm.
[0067] The experimental results are shown in Table 3, which shows that compound I can effectively scavenge NO free radicals, and the scavenging ability increases with the increase of the concentration. Compound I (10, 20, 40 μM) inhibits the production of NO in HFLS-RA induced by LPS in a dose-dependent manner. Therefore, compound I has anti-RA activity and can be used for preparing a medicine for treating rheumatoid arthritis.
[0068] Table 3. Effect of compound I on the production of NO in LPS-induced HFLS-RA cells (mean ± SD, n = 3)
[0069]
[0070] Note: *P < 0.05, **P < 0.01.
Claims
1. A coumarin pterocarpan glycoside compound having a structure as shown in Formula I:
2. The preparation method of the coumarin-lignan glycoside compound according to claim 1, characterized in that: Comprising the following steps: Step (1), crushing dry Cibotium barometz, soaking in 75-95% ethanol for 22-26 hours, then heating to reflux extraction for 3-5 times, each time for 4-6 hours, combining the filtrate to obtain total extract of Cibotium barometz; Step (2), drying the total extract of Cibotium barometz by vacuum concentration to obtain total extract of dry Cibotium barometz; Step (3), uniformly suspending the total extract of Cibotium barometz in distilled water, then sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract, respectively; Step (4), loading the n-butanol extract onto a macroporous adsorption resin column, then sequentially eluting with 15% ethanol, 30% ethanol, 50% ethanol, 70% ethanol and 100% ethanol to obtain five main fractions: main fraction A, main fraction B, main fraction C, main fraction D and main fraction E; Step (5), separating main fraction A on a medium pressure column, wherein the medium pressure column is YMC-Pack ODS-A column (250×50mm, 5μm), the mobile phase is 10-20% methanol, the absorption wavelength is 200-220nm, and the flow rate is 15-20mL / min, to obtain four sub-fractions: sub-fraction A-1, sub-fraction A-2, sub-fraction A-3 and sub-fraction A-4; Step (6), loading sub-fraction A-3 onto a Sephadex LH-20 column, eluting with 80-100% methanol to obtain three fine fractions: fine fraction A-3-1, fine fraction A-3-2 and fine fraction A-3-3; Step (7), separating fine fraction A-3-2 by preparative liquid chromatography, wherein the preparative liquid chromatography is performed on YMC-Pack ODS-A column (250×20mm, 5μm) with the mobile phase of 10-20% methanol, the absorption wavelength of 200-220nm and the flow rate of 2-4mL / min, to obtain compound I.
3. The method for preparing coumarin lignan glycosides according to claim 2, characterized in that: In step (1), the dry Cibotium barometz is crushed to 60-90 mesh.
4. The method for preparing coumarin lignan glycosides according to claim 2, characterized in that: In step (2), the drying temperature of vacuum concentration is 45-55℃.
5. The method for preparing coumarin lignan glycosides according to claim 2, characterized in that: In step (4), the macroporous adsorption resin is D101 macroporous adsorption resin.
6. The method for preparing coumarin lignan glycosides according to claim 2, characterized in that: In step (5), the medium pressure column is YMC-Pack ODS-A column (250×50mm, 5μm) with the mobile phase of 15% methanol, the absorption wavelength of 210nm and the flow rate of 18mL / min.
7. The method for preparing coumarin lignan glycosides according to claim 2, characterized in that: According to the preparation method of the coumarin pterocarpan glycoside compound of claim 2, in step (6), sub-fraction A-3 is loaded onto a Sephadex LH-20 column and eluted with 90% methanol.
8. The method for preparing coumarin lignan glycosides according to claim 2, characterized in that: In step (7), the preparative liquid chromatography is performed on YMC-Pack ODS-A column (250×20mm, 5μm) with the mobile phase of 15% methanol, the absorption wavelength of 210nm and the flow rate of 3mL / min.
9. The coumarin pterocarpan glycoside compound of claim 1 for use in the preparation of a medicament for treating rheumatoid arthritis.
10. A pharmaceutical composition, characterized by: The pharmaceutical composition is prepared by using the coumarin lignan glycoside compound of claim 1 as the main active ingredient and pharmaceutically acceptable adjuvants.