Flavonoid compound from daphne odora branches and leaves as well as extraction method and application of flavonoid compound

Abstract

The invention belongs to the field of traditional Chinese medicine extraction, and particularly relates to a flavonoid compound from daphne odora branches and leaves, an extraction method of the flavonoid compound and application of the flavonoid compound in preparation of anti-inflammatory drugs. The flavonoid compound is any one of compounds as shown in general formulas (I), (II), (III) and (IV), or an isomer of the compound, or a pharmaceutically acceptable salt of the compound. The structural diversity of active substances in daphne odora is further enriched, on the basis, a foundation is laid for related biological activity tests of subsequently obtained monomeric compounds, an active lead compound is provided for new drug development, and meanwhile, a theoretical basis is provided for deep research and development of daphne odora. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine extraction, and particularly relates to a flavonoid compound from branches and leaves of Daphne koreana and an extraction method thereof and an application of the flavonoid compound in the preparation of anti-inflammatory drugs. Background Art

[0002] As traditional medicinal plants in my country, Daphne plants of the Thymelaeaceae family have about 65 species, which are widely distributed in Shaanxi, Gansu, Ningxia, Qinghai and other places in my country. The plants of this genus are warm in nature and bitter in taste, and have the effects of removing rheumatism, promoting blood circulation and relieving pain. Zushima is the root bark and stem bark of Daphne giraldii Nitsche, a plant of the genus Daphne in the Thymelaeaceae family. It can also be used as medicine for plants of the same genus, such as Daphne tangutica Maxim, Daphne retusa Hemsl., Daphne koreana Nakai. and Edgeworthia chrysantha Lindl.. Daphne giraldii Nitsche is mainly used to treat stomachache, headache, numbness of limbs, rheumatic arthralgia, traumatic injury, arthritis, rheumatoid arthritis and other symptoms. As early as the 1970s, scholars have studied the chemical components of the medicinal plant Daphne oleoides. The compounds isolated from it, such as coumarins, flavonoids, lignans, and diterpenes, have novel structures and diverse biological activities. Therefore, in-depth exploration of active ingredients with medicinal value is of great significance for the research and development of the drug. (Hu Xiaojia, Jin Huizi, Yan Lan, et al. Study on the chemical components of Daphne oleoides (English) [J]. Natural Product Research and Development, 2011, 23(01): 20-24. Wang Mingwei, Li Chengyi, Li Bo. Progress in the pharmacy research of Daphne oleoides [C]; Academic Seminar on Chinese Medicine Identification of Chinese Association of Traditional Chinese Medicine, National Academic Seminar on Chinese Medicine of Chinese Association of Integrated Traditional Chinese and Western Medicine. 2007.) Based on the fact that Daphne oleoides can be used in folk medicine to treat rheumatoid arthritis and lumbago, Erdem et al.

[66] studied the anti-inflammatory activity of compounds isolated from the aerial parts of Olea europaea. The experimental results showed that Daphnetoxin, 1,2-dehydrodaphnetoxin and Daphnetoxin had inhibitory effects on the biosynthesis of interleukin-1 and tumor necrosis factor. Zheng Weifa et al.

[67] established inflammatory mouse and rat models to evaluate the anti-inflammatory activity of different doses of ethanol extract of Daphne genkwa root. The results showed that at a dose of 40 mg / kg, the ethanol extract of Daphne genkwa root could inhibit the increase of vascular permeability and inhibit the rat foot edema induced by Chondrus crispus; at a dose of 30 mg / kg, the ethanol extract of Daphne genkwa root could inhibit rat granulomas and enhance the phagocytosis of the mouse reticuloendothelial system. Further studies have found that the ethanol extract of Daphne genkwa root can also inhibit lipid peroxidation and the production of tumor necrosis factor and interleukin, enhance the activity of superoxide dismutase and catalase, and thus exert an anti-inflammatory effect ( E, Taninaka H, ​​Takaishi Y, et al. In vitro inhibitory effects of Daphne oleoides ssp.oleoides on inflammatory cytokines and activity-guided isolation of active constituents [J]. Cytokine, 2001, 13: 359-364; Zheng Weifa, Wang Li, Shi Feng. Anti-inflammatory activity of ethanol extract of Daphne genkwa root [J]. Chinese Herbal Medicine, 2004, 25: 66-73.). In order to maximize the medicinal value of Daphne oleoides, a systematic component study was conducted on the branches and leaves of Daphne oleoides. Summary of the invention

[0003] The present invention aims to provide a flavonoid compound from branches and leaves of Daphne koreana and an extraction method thereof and an application of the flavonoid compound in the preparation of anti-inflammatory drugs.

[0004] To achieve the above purpose, the present invention adopts the technical solution as follows:

[0005] A flavonoid compound in the branches and leaves of Daphne koreana, wherein the flavonoid compound is any one of the compounds represented by general formula (I), (II), (III), (IV) or an isomer of the compound, or a pharmaceutically acceptable salt of the compound;

[0006]

[0007] The method for preparing flavonoid compounds from branches and leaves of Daphne koreana is to extract and purify the dried branches and leaves of Daphne koreana as raw materials to obtain compounds represented by general formulas (I), (II), (III) and (IV) respectively.

[0008] Further,

[0009] (1) Using the dried branches and leaves of Daphne koreana as raw materials, adding ethanol aqueous solution of 8 to 15 times the mass of the raw materials, reflux extraction 2 to 4 times, each extraction time 2 to 4 hours, combining to obtain extracts, recovering the solvent under reduced pressure, and concentrating to obtain a total extract;

[0010] (2) dispersing the total extract into water of 5 to 10 times its mass, and extracting with petroleum ether and ethyl acetate respectively to obtain a petroleum ether layer, an ethyl acetate layer and a water layer;

[0011] (3) collecting the ethyl acetate extract concentrate and subjecting it to silica gel column chromatography, using dichloromethane-methanol in a volume ratio of 100:0 to 0:1 as an eluent for gradient elution, collecting fractions E1 with a volume ratio of 100:1, E2 with a volume ratio of 50:1, E3 with a volume ratio of 20:1, and E4 with a volume ratio of 10:1;

[0012] (4) The concentrated fraction E2 collected above is separated by silica gel column chromatography using dichloromethane-methanol with a volume ratio of 80:1-1:0 to purify the obtained compound to obtain the compound represented by the general formula (I), the general formula (III) and the general formula (IV);

[0013] (5) The concentrated fraction E3 collected above was further purified by silica gel column chromatography using dichloromethane-methanol in a volume ratio of 30:1-1:0 to obtain the compound represented by general formula (II).

[0014] In the step (4), the concentrated fraction E2 is subjected to silica gel column chromatography using dichloromethane-methanol with a volume ratio of 80:1-1:0, and a fraction with a volume ratio of dichloromethane to methanol of 20:1 is collected, which is recorded as E22; after the fraction E22 is concentrated, it is subjected to MCI chromatography using an aqueous solution containing 80%-100% methanol as an eluent, and the fraction eluted with the 80% methanol aqueous solution is collected, which is recorded as E221; after the fraction E221 is concentrated, it is subjected to ODS column chromatography, and gradient elution is performed using methanol-water with a volume ratio of 2:8, 4:6, 6:4, 7:3, and 1:0 as an eluent, and a fraction with a volume ratio of 4:6 is collected, which is recorded as E2212; the fraction E2212 is concentrated and subjected to preparative HPLC chromatography using methanol-water with a volume ratio of 30:70 as a mobile phase, and purified to obtain the general formula (I) (t R =23.8min), general formula (III) (t R =31.2min)、(Ⅳ)(t R =39.0min) shown in the compound.

[0015] In the step (5), the concentrated fraction E3 is collected and separated by silica gel column chromatography using dichloromethane-methanol in a volume ratio of 30:1-1:0, and a fraction with a volume ratio of dichloromethane to methanol of 15:1 is collected, which is recorded as E32; after the fraction E32 is concentrated, it is separated by silica gel column chromatography using a mixed solution of dichloromethane and methanol in a volume ratio of 10:1 as an eluent, and a fraction is collected, which is recorded as E322; after the fraction E322 is concentrated, it is separated by ODS column chromatography, and gradient elution is performed using methanol-water in volume ratios of 1:9, 3:7, 5:5, 7:3, and 9:1 as an eluent, and a fraction with a volume ratio of 3:7 is collected, which is recorded as E3222; the fraction E3222 is concentrated and purified by preparative HPLC chromatography using methanol-water in a volume ratio of 35:65 as a mobile phase to obtain the compound represented by general formula (II).

[0016] A pharmaceutical composition comprises the flavonoid compound, or an isomer of the compound, or a pharmaceutically acceptable salt of the compound.

[0017] A pharmaceutical preparation, comprising the flavonoid compound, or the isomer of the compound, or the pharmaceutically acceptable salt of the compound; or the combination as active ingredients, and pharmaceutically acceptable adjuvants, wherein the active ingredients account for 0.1-99% of the preparation mass.

[0018] The preparation is in the form of tablets, capsules, powders, syrups or injections.

[0019] A compound, a composition or a preparation for use, and a compound, a composition or a preparation for use in preparing an anti-inflammatory drug.

[0020] Advantages of the present invention:

[0021] The flavonoid compound or its isomer, a pharmaceutically acceptable salt or a pharmaceutical composition containing the compound of the present invention has a significant effect on inhibiting NO production in mouse mononuclear macrophage RAW 264.7 after LPS modeling, and can be used to prepare anti-inflammatory drugs. The present invention further enriches the structural diversity of active substances in the branches and leaves of Daphne koreana, and on this basis, lays a foundation for the subsequent relevant biological activity tests of the obtained monomer compounds, provides active lead compounds for the development of new drugs, and also provides a theoretical basis for the in-depth research and development of the branches and leaves of Daphne koreana. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The compounds represented by the general formula (I), (II), (III) and (IV) provided in the embodiments of the present invention affect the NO inhibition rate of mouse mononuclear macrophage RAW264.7 after LPS modeling. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0024] Example 1

[0025] The method for extracting flavonoid compounds from the branches and leaves of Daphne koreana comprises the following steps:

[0026] (1) Using the dried branches and leaves of Daphne koreana (25 kg) as raw materials, adding 95% ethanol aqueous solution with a volume concentration of 8 to 15 times the mass of the raw materials, reflux extraction 3 times, each extraction for 3 hours, combining to obtain the extract, reducing the pressure to recover the solvent, and concentrating to obtain the total extract (4954 g);

[0027] (2) dispersing the total extract into 6 times the mass of water, and extracting with petroleum ether and ethyl acetate, respectively, to obtain a petroleum ether layer, an ethyl acetate layer and a water layer;

[0028] (3) The ethyl acetate extract concentrate (1499 g) was separated by silica gel column chromatography using dichloromethane-methanol in a volume ratio of 100:0 to 0:1 as the eluent for gradient elution, and fractions E1 with a volume ratio of 100:1, E2 with a volume ratio of 50:1, E3 with a volume ratio of 20:1, and E4 with a volume ratio of 10:1 were collected;

[0029] (4) The concentrated fraction E2 (242.0 g) was collected and separated by silica gel column chromatography (10*100 cm), eluted with dichloromethane-methanol in a volume ratio of 80:1-1:0, and the fraction with a volume ratio of dichloromethane to methanol of 20:1 was collected and recorded as E22;

[0030] Fraction E22 (121.5 g) was concentrated by vacuum distillation and then separated by MCI chromatography (8*80 cm) using an aqueous solution containing 80%-100% methanol as the eluent. The fraction eluted with 80% methanol aqueous solution was collected and recorded as E221.

[0031] Fraction E221 (102.2 g) was concentrated by vacuum distillation and separated by ODS column chromatography (5*70 cm) using a gradient elution with methanol-water in a volume ratio of 2:8, 4:6, 6:4, 7:3, and 1:0, respectively. The fraction with a volume ratio of 4:6 was collected and recorded as E2212.

[0032] The fraction E2212 (25.9 g) was concentrated and purified by preparative HPLC chromatography [C18 column (5 μm)] using methanol-water with a volume ratio of 30:70 as the mobile phase to obtain the general formula (Ⅰ) (8.1 mg, t R =23.8min), general formula (III) (1.1mg, t R =31.2min)、(Ⅳ)(3.3mg,tR =39.0min) shown in the compound.

[0033] (5) The concentrated fraction E3 (465.0) was subjected to silica gel column chromatography (10*100 cm) to separate CH2Cl2-MeOH with a volume ratio of 30:1 to 1:0 of ​​dichloromethane-methanol as the eluent, and the fraction with a volume ratio of dichloromethane to methanol of 15:1 was collected and recorded as E32;

[0034] Fraction E32 (87.0 g) was concentrated and separated by silica gel column chromatography (10*100 cm), and the fraction with a volume ratio of dichloromethane to methanol of 10:1 was collected and recorded as E322;

[0035] Fraction E322 (20.6 g) was concentrated by vacuum distillation, and then separated by ODS column chromatography (5*60 cm), with gradient elution using methanol-water with volume ratios of 1:9, 3:7, 5:5, 7:3, and 9:1, respectively, and the fraction with a volume ratio of 3:7 was collected and recorded as E3222;

[0036] Fraction E3222 (120.7 mg) was concentrated by distillation under reduced pressure, and purified by preparative HPLC chromatography [C18 column (5 μm)] using methanol-water with a volume ratio of 35:65 as the mobile phase to obtain the compound represented by the general formula (II) (3.5 mg, t R =54.3min).

[0037] The structures of the flavonoid compounds represented by the general formulas (I), (II), (III) and (IV) extracted and separated were identified, and the specific physicochemical data are as follows:

[0038] Rosacatechin C (compound of general formula (Ⅰ)): Yellow amorphous powder (CH3OH); -61.1(c=0.20,CH3OH); HR-ESI-MS m / z 599.1990[M+H] + (calcd for C 34 H 31 O 10 + ,599.1990);UV(MeOH)ν max :260and 280nm; CD(CH3OH):210nm(negative)and 280nm(negative); 1 H-NMR (600MHz, DMSO-d6) and 13 C-NMR (150 MHz, DMSO-d6) data are shown in Tables 1 and 2.

[0039] Daphnodorin Q (compound of general formula (II)): White amorphous powder (CH3OH); -41.1 (c=0.20, CH3OH); HR-ESI-MS m / z 603.1154[MH] - (calcd.for C 31 H 23 O 13 - ,603.1144);UV(MeOH)ν max :210,260nm; CD(CH3OH):210nm(negative)and 240nm(positive); 1 H-NMR (600MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) data are shown in Tables 1 and 2.

[0040] Rosacatechin D (compound of general formula (III)): Yellow amorphous powder (CH3OH); +222.2(c=0.20,CH3OH); HR-ESI-MS m / z 331.0818[MH] - (calcd for C 17 H 15 O7 - ,331.0823);UV(MeOH)ν max :220and 370nm; CD(CH3OH):240nm(positive)and 280nm(negative); 1 H-NMR (600MHz, DMSO-d6) and 13 C-NMR (150 MHz, DMSO-d6) data are shown in Tables 1 and 2.

[0041] Rosacatechin E (compound of general formula (IV)): Yellow amorphous powder (CH3OH); -250.0 (c=0.20, CH3OH); HR-ESI-MS m / z 387.1131[M+Na] + (calcd for C 18 H 20 8Na + ,387.1050);UV(MeOH)νmax :225,270nm; CD(CH3OH):210nm(positive),240nm(negative)and280nm(positive); 1 H-NMR (600MHz, DMSO-d6) and 13 C-NMR (150 MHz, DMSO-d6) data are shown in Tables 1 and 2.

[0042] Table 1 Compounds 13 C-NMR(150MHz,CD3OD(2),DMSO-d6(1,3-4),δin ppm)

[0043]

[0044] Table 2 Compounds 1 H-NMR(600MHz,CD3OD(2),DMSO-d6(1,3-4),δin ppm,J in Hz)

[0045]

[0046]

[0047] Through physical and chemical data and modern spectroscopic methods (HRESIMS and NMR), combined with relevant data from public literature, the structures of the above compounds were identified and it was determined that the compounds were all new compounds that had not been reported in the literature, as shown below:

[0048]

[0049] The effects of the compounds extracted above on the inhibition of NO production in mouse mononuclear macrophage RAW 264.7 cells after LPS modeling were studied as follows:

[0050] 1. Principle: As an important inducing factor of systemic inflammatory response, lipopolysaccharide (LPS) is widely used in the construction of in vivo and in vitro inflammatory models. Under the stimulation of excessive LPS, macrophage surface antigens recognize receptors and bind to them, thereby activating nitric oxide synthase (NOS) and producing excessive NO. NO is rapidly oxidized to form NO2 - , NO2 -It can undergo diazotization reaction with sulfanilamide, and then undergo coupling reaction with N-1-naphthylethylene-diaminedihydrochloride to generate purple-red azo compounds. The absorbance value measured at 540nm by an enzyme marker can be used to measure the NO content in the sample, and compared with the standard to test whether the compound has anti-inflammatory effect.

[0051] 2. Methods: Mouse mononuclear macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin sodium, and incubated and grown in a constant temperature incubator at 37°C and 5% CO2.

[0052] Mouse mononuclear macrophage RAW264.7 cells in logarithmic phase were taken and the cell concentration was adjusted to 3.5×10 4 After 100 μL of cell suspension was added to each well of the plate, the cells were inoculated into 96-well plates. A control group (RAW264.7 cells, DMSO, NO inhibition rate 100%), a model group (RAW264.7 cells, DMSO, 0.2 μg / mL LPS, NO inhibition rate 0%), a positive drug group (RAW264.7 cells, dexamethasone (Dex), 0.2 μg / mL LPS) and a test drug group (RAW264.7 cells, the compounds obtained above, 0.2 μg / mL LPS) were set up at the same time. The cells were incubated for 24 hours in a 5% CO2, 37°C constant temperature incubator, and 40 μL of cell supernatant was taken and placed in an ELISA plate. 40 μL of Griess reagent of equal volume was added to each well to mix with the cell supernatant and react completely. After reacting at room temperature for 10 min, use an ELISA reader to measure the absorbance of the solution in the well at 540 nm (see Figure 1 ), the inhibition rate formula is as follows:

[0053]

[0054] 3. Results: Figure 1 It can be seen that the addition of 20 μM of the compound has the effect of inhibiting NO production. At the same time, compound (II) can significantly inhibit NO production in mouse mononuclear macrophage RAW 264.7 after LPS modeling.

[0055] The above embodiments are only used to help understand the method and the central idea of ​​the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection of the claims of the present invention.

Claims

1. A flavonoid compound in the branches and leaves of Daphne koreana, characterized in that: The flavonoid compound is any one of the compounds represented by the general formula (I), (II), (III), (IV) or an isomer of the compound, or a pharmaceutically acceptable salt of the compound; 2. The method for preparing flavonoid compounds from branches and leaves of Daphne koreana according to claim 1, characterized in that: The dried branches and leaves of Daphne koreana are used as raw materials and extracted and purified to obtain the compounds represented by the general formulas (I), (II), (III) and (IV) in claim 1.

3. The method for preparing flavonoid compounds from branches and leaves of Daphne koreana according to claim 2, characterized in that: (1) Using the dried branches and leaves of Daphne koreana as raw materials, adding ethanol aqueous solution of 8 to 15 times the mass of the raw materials, reflux extraction 2 to 4 times, each extraction time 2 to 4 hours, combining to obtain extracts, recovering the solvent under reduced pressure, and concentrating to obtain a total extract; (2) dispersing the total extract into water of 5 to 10 times its mass, and extracting with petroleum ether and ethyl acetate respectively to obtain a petroleum ether layer, an ethyl acetate layer and a water layer; (3) collecting the ethyl acetate extract concentrate and subjecting it to silica gel column chromatography, using dichloromethane-methanol in a volume ratio of 100:0 to 0:1 as an eluent for gradient elution, collecting fractions E1 with a volume ratio of 100:1, E2 with a volume ratio of 50:1, E3 with a volume ratio of 20:1, and E4 with a volume ratio of 10:1; (4) The concentrated fraction E2 collected above is separated by silica gel column chromatography using dichloromethane-methanol with a volume ratio of 80:1-1:0 to purify the obtained compound to obtain the compound represented by the general formula (I), the general formula (III) and the general formula (IV); (5) The concentrated fraction E3 collected above was further purified by silica gel column chromatography using dichloromethane-methanol in a volume ratio of 30:1-1:0 to obtain the compound represented by general formula (II).

4. The method for preparing flavonoid compounds from branches and leaves of Daphne koreana according to claim 3, characterized in that: In the step (4), the concentrated fraction E2 is subjected to silica gel column chromatography using dichloromethane-methanol with a volume ratio of 80:1-1:0, and the fraction with a volume ratio of dichloromethane to methanol of 20:1 is collected, which is recorded as E22; after the fraction E22 is concentrated, it is separated by MCI chromatography using an aqueous solution containing 80%-100% methanol as an eluent, and the fraction eluted with 80% methanol aqueous solution is collected, which is recorded as E221; Fraction E221 was concentrated and separated by ODS column chromatography, and gradient elution was performed using methanol-water with a volume ratio of 2:8, 4:6, 6:4, 7:3, and 1:0 as the eluent, and the fraction with a volume ratio of 4:6 was collected and recorded as E2212; Fraction E2212 was concentrated and purified by preparative HPLC chromatography using methanol-water with a volume ratio of 30:70 as the mobile phase to obtain the general formula (I) (t R =23.8min), general formula (III) (t R =31.2min)、(Ⅳ)(t R =39.0min) shown in the compound.

5. The method for preparing flavonoid compounds from branches and leaves of Daphne koreana according to claim 3, characterized in that: In the step (5), the concentrated fraction E3 is collected and separated by silica gel column chromatography using dichloromethane-methanol in a volume ratio of 30:1-1:0, and a fraction with a volume ratio of dichloromethane to methanol of 15:1 is collected, which is recorded as E32; after the fraction E32 is concentrated, it is separated by silica gel column chromatography using a mixed solution of dichloromethane and methanol in a volume ratio of 10:1 as an eluent, and a fraction is collected, which is recorded as E322; after the fraction E322 is concentrated, it is separated by ODS column chromatography, and gradient elution is performed using methanol-water in a volume ratio of 1:9, 3:7, 5:5, 7:3, and 9:1 as an eluent, and a fraction with a volume ratio of 3:7 is collected, which is recorded as E3222; the fraction E3222 is concentrated and purified by preparative HPLC chromatography using methanol-water in a volume ratio of 35:65 as a mobile phase to obtain the compound represented by general formula (II).

6. A pharmaceutical composition, characterized in that It includes the flavonoid compound according to claim 1, or an isomer of the compound, or a pharmaceutically acceptable salt of the compound.

7. A pharmaceutical preparation, characterized in that: The flavonoid compound according to claim 1, or the isomer of the compound, or the pharmaceutically acceptable salt of the compound; or the composition according to claim 6 is used as an active ingredient, and a pharmaceutically acceptable auxiliary agent, wherein the active ingredient accounts for 0.1-99% of the mass of the preparation.

8. The pharmaceutical preparation according to claim 7, characterized in that: The preparation is in the form of tablets, capsules, powders, syrups or injections.

9. Use of the compound of claim 1, the composition of claim 6 or the preparation of claim 7, characterized in that: Use of the compound of claim 1, the composition of claim 6 or the preparation of claim 7 in the preparation of anti-inflammatory drugs.

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