Flavonoids in daphne retusa and extraction method and application thereof

Abstract

The application belongs to the field of traditional Chinese medicine extraction, and particularly relates to flavonoids in Daphne retusa leaves and branches, an extraction method thereof and application of the flavonoids in preparing anti-inflammatory drugs. The flavonoids are any one of compounds shown in general formula (I), (II), (III) and (IV), or isomers of the compounds, or pharmaceutically acceptable salts of the compounds. The application further enriches the structural diversity of active substances in Daphne retusa, and lays a foundation for subsequent biological activity tests of monomer compounds, provides active lead compounds for new drug development, and provides a theoretical basis for deep research and development of Daphne retusa.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine extraction, and particularly relates to flavonoids from Daphne retusa leaves and stems, an extraction method thereof and application of the flavonoids in preparing anti-inflammatory drugs. BACKGROUND

[0002] As traditional medicinal plants in China, there are about 65 species of Daphne in Thymelaeaceae. The plants of Daphne genus have warm nature and bitter and acrid taste, and have the effects of dispelling wind and dampness, promoting blood circulation and relieving pain. Daphne giraldii Nitsche, also known as Zushima, is the root and stem bark of Daphne giraldii Nitsche, and can also be used as Daphne tangutica Maxim, Daphne retusa Hemsl., Daphne koreana Nakai and Edgeworthia chrysantha Lindl. Daphne retusa Hemsl. is mainly used for treating stomachache, headache, numbness of limbs, rheumatic arthralgia, contusion and injury, arthritis and rheumatoid arthralgia. As early as in the 1970s, scholars have studied the chemical components of the medicinal plants of Zushima, and isolated compounds such as coumarin, flavone, lignan and diterpene with novel structures and diverse biological activities. Therefore, it is of great significance to deeply explore active ingredients with medicinal value from the plants for the research and development of the drug. (Hu Xiaojia, Jin Huizi, Yan Lan, et al. Chemical constituents of Daphne retusa Hemsl. [J]. Natural Products Research and Development, 2011, 23(01): 20-24. Wang Mingwei, Li Chengyi, Li Bo. Research progress of Zushima crude drugs [C]. Chinese Medical Association Academic Conference on Identification of Chinese Medicine, Chinese Medical Association Committee on Chinese Medicine National Academic Conference on Chinese Medicine, 2007.) Based on the fact that Daphne oleoides can be used for treating rheumatoid arthritis and lumbago in folk, Erdem et al. isolated 12 compounds from Daphne oleoides, and found that the compound 5, 7, 4'-trihydroxyflavone has the effect of inhibiting the activity of matrix metalloproteinase-3 (MMP-3) (Erdem, S. S., et al. Flavonoids from Daphne oleoides and their matrix metalloproteinase-3 inhibitory activity. Phytotherapy Research, 2010, 24(10): 1476-1480.) However, the flavonoids from Daphne retusa Hemsl. have not been reported.

[66] et al. studied the anti-inflammatory activity of compounds isolated from the aerial parts of Daphne odora. The results showed that Daphne glycoside, 1,2-dehydrodaphnetoxin and Daphne glycoside inhibited the biosynthesis of interleukin-1 and tumor necrosis factor. Zheng Weifa et al.

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

[0003] The purpose of this invention is to provide a method for extracting flavonoids from the branches and leaves of Daphne odora and their application in the preparation of anti-inflammatory drugs.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0006]

[0007] A method for preparing flavonoids from the branches and leaves of Daphne odora involves extracting and purifying the dried branches and leaves of Daphne odora to obtain compounds represented by general formulas (Ⅰ), (Ⅱ), (Ⅲ), and (Ⅳ).

[0008] To elaborate further,

[0009] (1) Using dried branches and leaves of Daphne odora as raw material, add 8 to 15 times the mass of the raw material in an ethanol aqueous solution, reflux extract 2 to 4 times, each extraction for 2 to 4 hours, combine the extracts, recover the solvent under reduced pressure, and concentrate to obtain the total extract.

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

[0011] (3) The ethyl acetate extract concentrate was collected and separated by silica gel column chromatography. The dichloromethane-methanol mixture with a volume ratio of 100:0 to 0:1 was used as the eluent for gradient elution. The fractions E1 (100:1), E2 (50:1), E3 (20:1), and E4 (10:1) were collected.

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

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

[0014] In step (4), the concentrated fraction E2 is separated by silica gel column chromatography using dichloromethane-methanol at a volume ratio of 80:1-1:0, and the fraction with a volume ratio of dichloromethane to methanol of 20:1 is collected and denoted as E22. After concentration, fraction E22 is separated by MCI chromatography using an aqueous solution containing 80%-100% methanol as the eluent, and the fraction eluted by the 80% methanol aqueous solution is collected and denoted as E221. After concentration, fraction E221 is separated by ODS column chromatography, using a gradient elution of methanol-water at volume ratios of 2:8, 4:6, 6:4, 7:3, and 1:0, and the fraction with a volume ratio of 4:6 is collected and denoted as E2212. Fraction E2212 is purified by preparative HPLC chromatography using methanol-water at a volume ratio of 30:70 to obtain the general formula (Ⅰ)(t). R =23.8min), general formula (III)(t R =31.2min), (Ⅳ)(t R The compound shown is 39.0 min.

[0015] In step (5), the concentrated fraction E3 was collected by silica gel column chromatography using a dichloromethane-methanol mixture with a volume ratio of 30:1 to 1:0. The fraction with a volume ratio of 15:1 of dichloromethane and methanol was collected and denoted as E32. After concentration, fraction E32 was separated by silica gel column chromatography using a mixture of dichloromethane and methanol with a volume ratio of 10:1 as the eluent. The fraction was collected and denoted as E322. After concentration, fraction E322 was separated by ODS column chromatography using a gradient elution of methanol-water with volume ratios of 1:9, 3:7, 5:5, 7:3, and 9:1. The fraction with a volume ratio of 3:7 was collected and denoted as E3222. Fraction E3222 was purified by preparative HPLC using methanol-water with a volume ratio of 35:65 as the mobile phase to obtain the compound shown in general formula (II).

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

[0017] A pharmaceutical preparation comprising, as an active ingredient, the flavonoid compound, or an isomer of the compound, or a pharmaceutically acceptable salt of the compound; or the composition thereof, and a pharmaceutically acceptable adjuvant, wherein the active ingredient comprises 0.1-99% by weight of the preparation.

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

[0019] The use of a compound, the composition, or the formulation thereof, and the use of the compound, the composition, or the formulation thereof in the preparation of an anti-inflammatory medicament.

[0020] Advantages of this invention:

[0021] The flavonoids or their isomers, pharmaceutically acceptable salts, or pharmaceutical compositions containing these compounds of the present invention significantly inhibit NO production in mouse RAW 264.7 mononuclear macrophages after LPS modeling, and can be used to prepare anti-inflammatory drugs. This invention further enriches the structural diversity of active substances in the branches and leaves of *Daphne odora*, laying the foundation for subsequent bioactivity testing of the obtained monomeric compounds, providing active lead compounds for new drug development, and also providing a theoretical basis for in-depth research and development of *Daphne odora* branches and leaves. Attached Figure Description

[0022] Figure 1 The effects of compounds of general formulas (Ⅰ), (Ⅱ), (Ⅲ), and (Ⅳ) provided for embodiments of the present invention on the NO inhibition rate of mouse monocytes and macrophages RAW264.7 after LPS modeling. Detailed Implementation

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

[0024] Example 1

[0025] The method for extracting flavonoids from the branches and leaves of Daphne odora includes the following steps:

[0026] (1) Using dried branches and leaves of Daphne odora (25kg) as raw material, add 8 to 15 times the mass of the raw material in a 95% ethanol aqueous solution, reflux extract 3 times, each extraction for 3 hours, combine the extracts, recover the solvent under reduced pressure, concentrate to obtain total extract (4954g).

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

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

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

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

[0031] The fraction E221 (102.2g) was concentrated by vacuum distillation and then separated by ODS column chromatography (5*70cm). The fraction was eluted with methanol-water gradients at volume ratios of 2:8, 4:6, 6:4, 7:3, and 1:0, and the fraction with a volume ratio of 4:6 was collected and designated as E2212.

[0032] The concentrated fraction E2212 (25.9 g) was purified by preparative HPLC [C18 column (5 μm)] using a methanol-water mixture (30:70 v / v) 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 The compound shown is 39.0 min.

[0033] (5) Concentrated fraction E3 (465.0), the concentrate was separated by silica gel column chromatography (10*100cm) to separate dichloromethane-methanol with a volume ratio of CH2Cl2-MeOH of 30:1-1:0 as the eluent, and the fraction with a volume ratio of dichloromethane and methanol of 15:1 was collected and denoted as E32.

[0034] The fraction E32 (87.0 g) was concentrated and then separated by silica gel column chromatography (10*100 cm). The fraction with a volume ratio of dichloromethane to methanol of 10:1 was collected and denoted as E322.

[0035] The fraction E322 (20.6g) was concentrated by vacuum distillation and then separated by ODS column chromatography (5*60cm). The fraction was eluted with methanol-water gradients at volume ratios of 1:9, 3:7, 5:5, 7:3, and 9:1. The fraction with a volume ratio of 3:7 was collected and designated as E3222.

[0036] The fraction E3222 (120.7 mg) was concentrated by vacuum distillation, and the concentrated product was purified by preparative HPLC [C18 column (5 μm)] with methanol-water (v / v) as the mobile phase of 35:65 to obtain the compound (3.5 mg, t) of general formula (II). R =54.3min).

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

[0038] Rosacatechin C (compound of general formula (Ⅰ): Yellow amorphous powder (CH3OH); HR-ESI-MSm / 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 (150MHz, DMSO-d6) data are shown in Tables 1 and 2.

[0039] Daphnodorin Q (compound of general formula (II)): White amorphous powder (CH3OH); HR-ESI-MSm / 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 (150MHz, CD3OD) data are shown in Tables 1 and 2.

[0040] Rosacatechin D (compound of general formula (III): Yellow amorphous powder (CH3OH); HR-ESI-MSm / 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 (150MHz, DMSO-d6) data are shown in Tables 1 and 2.

[0041] Rosacatechin E (compound of general formula (IV)): Yellow amorphous powder (CH3OH); HR-ESI-MSm / z 387.1131[M+Na] + (calcd forC 18 H 20 O8Na + ,387.1050); UV(MeOH)ν max :225,270nm; CD(CH3OH):210nm(positive),240nm(negative)and 280nm(positive);1 H-NMR (600MHz, DMSO-d6) and 13 C-NMR (150MHz, 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] Using physicochemical data and modern spectroscopic techniques (HRESIMS and NMR), combined with relevant data from published literature, the structures of the above compounds were identified, confirming that all compounds are novel compounds not previously reported in the literature, as shown below:

[0048]

[0049] The effects of the extracted compounds on the inhibition of NO production in RAW 264.7 mouse monocytes / macrophages after LPS modeling are studied in detail below:

[0050] 1. Principle: As an important inducing factor of systemic inflammatory response, lipopolysaccharide (LPS) is widely used in the construction of in vitro and in vivo inflammation models. Under excessive lipopolysaccharide stimulation, macrophage surface antigens recognize and bind to receptors, thereby activating nitric oxide synthase (NOS) and producing excessive NO. NO is rapidly oxidized to form NO2. - NO2 - It undergoes a diazotization reaction with sulfanilamide, and then a coupling reaction with N-1-naphthylethylene-diamine dihydrochloride produces a purple-red azo compound. The NO content in the sample can be determined by measuring the absorbance at 540 nm using a microplate reader, and the compound can be compared with a standard to test whether it has anti-inflammatory effects.

[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 in a constant temperature incubator at 37℃ and 5% CO2.

[0052] RAW264.7 mouse mononuclear macrophages in the logarithmic growth phase were harvested and their concentration adjusted to 3.5 × 10⁻⁶ cells. 4 After cell / well inoculation, cells were seeded into 96-well plates, with 100 μL of cell suspension added to each well. The experiment included a control group (RAW264.7 cells, DMSO, 100% NO inhibition), a model group (RAW264.7 cells, DMSO, 0.2 μg / mL LPS, 0% NO inhibition), a positive control group (RAW264.7 cells, dexamethasone (Dex), 0.2 μg / mL LPS), and a test drug group (RAW264.7 cells, the compounds obtained above, and 0.2 μg / mL LPS). Cells were incubated for 24 hours in a 5% CO2, 37°C incubator. Then, 40 μL of cell supernatant was transferred to each well of an ELISA plate, and 40 μL of Griess reagent was added to each well, allowing it to mix thoroughly with the cell supernatant for complete reaction. After reacting at room temperature for 10 min, the absorbance of the solution in the wells was measured at 540 nm using a microplate reader (see [link]). Figure 1 The inhibition rate formula is as follows:

[0053]

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

[0055] The above description of the embodiments is only for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims

1. A flavonoid compound found in the branches and leaves of Daphne odora, characterized in that, Flavonoids are compounds of formula (II) or pharmaceutically acceptable salts thereof; 。 2. The method for preparing flavonoids from the branches and leaves of *Daphne odora* according to claim 1, characterized in that, (1) Using dried branches and leaves of Daphne odora as raw material, add 8 to 15 times the mass of the raw material in an ethanol aqueous solution, reflux extract 2 to 4 times, each extraction for 2 to 4 hours, combine the extracts, recover the solvent under reduced pressure, concentrate and obtain the total extract. (2) Disperse the total extract into 5 to 10 times its mass of water, and extract with petroleum ether and ethyl acetate respectively to obtain a petroleum ether layer, an ethyl acetate layer and an aqueous layer; (3) The ethyl acetate extract concentrate was collected and separated by silica gel column chromatography. The dichloromethane-methanol mixture with a volume ratio of 100:0~0:1 was used as the eluent for gradient elution. The fractions E1 (100:1), E2 (50:1), E3 (20:1), and E4 (10:1) were collected. (4) After concentrating the collected fraction E3, the fraction was separated by silica gel column chromatography using a dichloromethane-methanol mixture with a volume ratio of 30:1–1:

0. The fraction with a volume ratio of 15:1 of dichloromethane and methanol was collected and recorded as E32. After concentrating fraction E32, the fraction was separated by silica gel column chromatography using a mixture of dichloromethane and methanol with a volume ratio of 10:1 as the eluent. The fraction was collected and recorded as E322. After concentrating fraction E322, the fraction was separated by ODS column chromatography using a gradient elution of methanol-water with volume ratios of 1:9, 3:7, 5:5, 7:3, and 9:

1. The fraction with a volume ratio of 3:7 was collected and recorded as E3222. After concentrating fraction E3222, the fraction was purified by preparative HPLC using a methanol-water mixture with a volume ratio of 35:65 as the mobile phase to obtain the compound shown in formula (II).

3. A pharmaceutical composition, characterized in that, Includes the flavonoids of claim 1 or pharmaceutically acceptable salts thereof.

4. A pharmaceutical preparation, characterized in that: The active ingredient is a flavonoid compound as described in claim 1 or a pharmaceutically acceptable salt thereof, wherein the active ingredient accounts for 0.1-99% of the mass of the preparation.

5. The pharmaceutical preparation according to claim 4, characterized in that: The dosage form of the preparation is tablets, capsules, powders, syrups, or injections.

6. The use of the flavonoid compound of claim 1 or the pharmaceutical composition of claim 3 in the preparation of an anti-inflammatory medicament.

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