CN112794832B - Compound NBY-10 extracted from folium Arctii and having antiinflammatory activity, and its preparation method and application - Google Patents
Compound NBY-10 extracted from folium Arctii and having antiinflammatory activity, and its preparation method and application Download PDFInfo
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Abstract
The invention relates to a compound NBY-10 with anti-inflammatory activity extracted from burdock leaves, a preparation method and application thereof, which can effectively solve the problems of preparing the compound with anti-inflammatory activity from the burdock leaves and realizing the application of the compound in preparing anti-inflammatory drugs, and the method comprises the steps of extracting the burdock leaves with 30% ethanol, concentrating, extracting concentrated solution n-butanol, decompressing and concentrating a solvent, carrying out silica gel column chromatography, decompressing and recovering the solvent with dichloromethane-methanol, carrying out secondary silica gel column chromatography, carrying out gradient elution with petroleum ether-ethyl acetate, collecting 100:40 eluent, decompressing and recovering the solvent, carrying out Sephadex LH-20 column chromatography, eluting with methanol, decompressing and recovering the solvent, and recrystallizing in methanol to obtain the compound NBY-10 with anti-inflammatory activity extracted from the burdock leaves. The invention has the advantages of rich raw materials, easy operation of the preparation method, strong guidance quality, high separation speed, high efficiency and high product purity, and the compound can be effectively used for preparing anti-inflammatory drugs.
Description
Technical Field
The invention relates to a medicine, in particular to a compound NBY-10 with anti-inflammatory activity extracted from burdock leaves and a preparation method and application thereof.
Background
The burdock leaves are dry basal leaves of Arctium lappa of compositae, are widely distributed in China, have a long history of eating and medicine, and modern researches show that the burdock leaves are rich in nutrition, are rich in minerals such as inulin, cellulose, carotene, protein, calcium, phosphorus, iron and the like and multiple vitamins and have a strong health-care function. Meanwhile, the product has the effects of oxidation resistance, inflammation resistance and the like, and is a good nourishing product used as both medicine and food. Many herbal writings record the dietary therapy experience of burdock leaves. For example, niu bang ye tie is used to treat various sores and incised sores in the formula of "simple and sanitary prescription". The burdock leaves are expected to become a new resource for inhibiting inflammation, and a safe and effective compound for inhibiting inflammation is obtained from the burdock leaves and is used for preparing anti-inflammatory drugs, which is not reported publicly so far.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the compound NBY-10 with anti-inflammatory activity extracted from the burdock leaves, and the preparation method and the application thereof, which can effectively solve the problems of preparing a new compound with anti-inflammatory activity from the burdock leaves with long-term food therapy history and realizing the application of the new compound in preparing anti-inflammatory drugs.
The technical scheme of the invention is that a compound NBY-10 with anti-inflammatory activity extracted from burdock leaves has a molecular structural formula as follows:
the preparation method comprises the following steps:
(1) 20kg of burdock leaf powder, extracting 1-3 times with ethanol with the mass concentration of 30% and the weight of the burdock leaf powder being 4-6 times of the weight of the burdock leaf powder, extracting for 1-4 h each time, combining the extracting solutions, and concentrating to obtain a concentrated solution equivalent to 0.1-3 g/mL of crude drug;
(2) sequentially extracting the concentrated solution with 2-8L, 1-4L and 0.5-2L of n-butyl alcohol for 4 times, combining n-butyl alcohol extract, and concentrating the solvent under reduced pressure to obtain a component Fr.A (55-90 g); performing silica gel column chromatography on the component Fr.A, eluting by 8-25L of a solution with the volume ratio of dichloromethane to methanol being 100 to 2, wherein the inner diameter is 4-8 cm and the height is 8-25 cm, collecting eluent, and recovering a solvent under reduced pressure to obtain a component Fr.A-1 (15-30 g);
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate at a volume ratio of 100:5, 2-6L, 100:10, 2-6L, 100:20, 2-6L, 100:40 and 4-15L, with the inner diameter of the silica gel column chromatography being 3-7 cm and the height of the silica gel column chromatography being 12-30 cm, collecting eluent at a volume ratio of 100:40, and recovering the solvent under reduced pressure to obtain a component Fr.A-1-3 (2-6 g);
(4) performing Sephadex LH-20 column chromatography on the component Fr.A-1-3, eluting with methanol at an elution volume of 1-15L and an elution flow rate of 0.5-1 mL/min, and recovering the solvent under reduced pressure to obtain a component Fr.A-1-3-3 (700-2000 mg), wherein the inner diameter is 1-3 cm and the height is 80-150 cm;
(5) recrystallizing the component Fr.A-1-3-3 in methanol to obtain NBY-10 (350-1000 mg, (3aR,5R,5aS,9aR,9bR) -3a,4,5,5a,6,7,9a,9b-octahydro-5-hydroxy-8- (hydroxymethy) -5-methyl-1-methyrenephho [2,1-b ] furan-2(1H) -one) with anti-inflammatory activity extracted from burdock leaves.
The compound is a new compound with anti-inflammatory activity extracted from the burdock leaves, has rich raw materials, easy operation of the preparation method, strong guidance, high separation speed, high efficiency and high product purity, can be effectively used for preparing anti-inflammatory drugs, develops new application and medicinal value of the burdock leaves, and has great economic and social benefits.
Drawings
FIG. 1 is a molecular structural formula of compound NBY-10 of the present invention;
FIG. 2 is a graph relating the major HMBC and H-H COSY of compound NBY-10 of the present invention;
FIG. 3 is a NOESY correlation plot for compound NBY-10 of the present invention;
FIG. 4 is a drawing showing the results of NBY-10 compounds of the present invention1H-NMR spectrum;
FIG. 5 is a photograph of NBY-10 according to the invention13A C-NMR spectrum;
FIG. 6 is an HSQC spectrum of compound NBY-10 of the present invention;
FIG. 7 is a HMBC spectrum of compound NBY-10 of the present invention;
FIG. 8 is a chart of the infrared spectrum of compound NBY-10 of the present invention;
FIG. 9 is a graph of the ultraviolet spectrum of compound NBY-10 of the present invention;
FIG. 10 is a mass spectrum of compound NBY-10 of the present invention;
FIG. 11 is a process flow diagram of compound NBY-10 of the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention refers to the accompanying drawings.
In particular, the invention may be embodied as set forth in the following examples.
Example 1
The invention relates to a preparation method of a compound NBY-10 with anti-inflammatory activity extracted from burdock leaves, which comprises the following steps:
(1) extracting 20kg of folium Arctii powder with 5 times of ethanol with mass concentration of 30% and weight of folium Arctii powder for 2 times (2.5 hr each time), mixing extractive solutions, and concentrating to obtain concentrated solution equivalent to 0.5g/mL of crude drug;
(2) sequentially extracting the concentrated solution with n-butanol 7L, 4L, and 2L for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A (60 g); performing silica gel column chromatography on component Fr.A, eluting with volume ratio of dichloromethane: methanol: 100:2 solution 8L with inner diameter of 4cm and height of 25cm, collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1(17 g);
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 2L, 100:10, 2L, 100:20, 3L, 100:40, 5L at volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3(3 g);
(4) subjecting the component Fr.A-1-3 to Sephadex LH-20 column chromatography with inner diameter of 1.5cm and height of 130cm, eluting with methanol at elution volume of 3.5L and flow rate of 0.5mL/min, and recovering solvent under reduced pressure to obtain component Fr.A-1-3-3(850 mg);
(5) recrystallizing component Fr.A-1-3-3 in methanol to obtain NBY-10(450mg) with antiinflammatory activity extracted from folium Arctii.
Example 2
The invention relates to a preparation method of a compound NBY-10 with anti-inflammatory activity extracted from burdock leaves, which comprises the following steps:
(1) extracting 20kg of burdock leaf powder with ethanol with mass concentration of 30% 4 times of burdock leaf powder for 3 times, each time for 3h, mixing extractive solutions, and concentrating to obtain concentrated solution equivalent to 0.6g/mL of crude drug;
(2) sequentially extracting the concentrated solution with 6L, 3L and 2L of n-butanol for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A (85 g); performing silica gel column chromatography on component Fr.A, eluting with dichloromethane/methanol (100: 2) solution 24L with inner diameter of 7cm and height of 20cm, collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1(27 g);
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 5L, 100:10, 5L, 100:20, 6L, 100:40, 15L at volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3(5.5 g);
(4) subjecting the component Fr.A-1-3 to Sephadex LH-20 column chromatography with inner diameter of 3cm and height of 90cm, eluting with methanol at elution volume of 10L and flow rate of 1mL/min, and recovering solvent under reduced pressure to obtain component Fr.A-1-3-3(1800 mg);
(5) recrystallizing component Fr.A-1-3-3 in methanol to obtain NBY-10(950mg) with antiinflammatory activity extracted from folium Arctii.
Example 3
The invention relates to a preparation method of a compound NBY-10 with anti-inflammatory activity extracted from burdock leaves, which comprises the following steps:
(1) extracting 20kg of burdock leaf powder with 6 times of burdock leaf powder weight and 30% ethanol for 2 times, each time for 2h, mixing extractive solutions, and concentrating to obtain concentrated solution equivalent to 0.5g/mL of crude drug;
(2) sequentially extracting the concentrated solution with n-butanol 3L, 4L, and 1L for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A (75 g); performing silica gel column chromatography on component Fr.A, eluting with a volume ratio of dichloromethane to methanol of 100:2 of 20L, with an inner diameter of 6cm and a height of 17cm, collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1(23 g);
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 4L, 100:10, 4L, 100:20, 5L, 100:40, 13L at volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3(4.5 g);
(4) subjecting the component Fr.A-1-3 to Sephadex LH-20 column chromatography with inner diameter of 2.5cm and height of 100cm, eluting with methanol with elution volume of 7.8L and elution flow rate of 0.8mL/min, and recovering solvent under reduced pressure to obtain component Fr.A-1-3-3(1400 mg);
(5) recrystallizing component Fr.A-1-3-3 in methanol to obtain NBY-10(800mg) with antiinflammatory activity extracted from folium Arctii.
Example 4
The invention relates to a preparation method of a compound NBY-10 with anti-inflammatory activity extracted from burdock leaves, which comprises the following steps:
(1) extracting 20kg of folium Arctii powder with 6 times of ethanol with mass concentration of 30% and weight of folium Arctii powder for 2 times, each time for 1.5 hr, mixing extractive solutions, and concentrating to obtain concentrated solution equivalent to 0.4g/mL of crude drug;
(2) sequentially extracting the concentrated solution with n-butanol 5L, 3L, and 2L for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A (65 g); performing silica gel column chromatography on component Fr.A, eluting with dichloromethane/methanol (volume ratio) 100:2 solution 13L with inner diameter of 5cm and height of 15cm, collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1(20 g);
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 3L, 100:10, 3L, 100:20, 4L, 100:40, 10L in volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3(4.5 g);
(4) subjecting the component Fr.A-1-3 to Sephadex LH-20 column chromatography with inner diameter of 2cm and height of 100cm, eluting with methanol at volume of 5L and flow rate of 0.6mL/min, and recovering solvent under reduced pressure to obtain component Fr.A-1-3-3(1000 mg);
(5) recrystallizing component Fr.A-1-3-3 in methanol to obtain NBY-10(650mg) with antiinflammatory activity extracted from folium Arctii.
It is to be noted that the above examples are only for illustrating the specific embodiments of the present invention, and the detailed description of the compound extracted from burdock leaves and the extraction method thereof is illustrative, not intended to limit the scope of the present invention, and all changes and modifications that do not depart from the general concept of the present invention shall fall within the scope of the present invention.
The obtained compound is determined and identified aS a new compound extracted from burdock leaves, and the compound NBY-10((3aR,5R,5aS,9aR,9bR) -3a,4,5,5a,6,7,9a,9b-octahydro-5-hydroxy-8- (hydroxymethyl) -5-methyl-1-methyenapitho [2,1-b ] furan-2(1H) -one) has a molecular structural formula shown in figure 1, has an anti-inflammatory effect through experiments, and has the following specific determination and experimental data:
first, structural identification
Instruments and materials used:
shimadzu double-beam 210A uv spectrometer (Shimadzu, Kyoto, japan);
LTQ orbitrap high resolution mass spectrometer (Thermo Fisher Scientific, Bremen, germany);
nuclear magnetic resonance apparatus: bruker AVANCE III 500-NMR spectrometer (Bruker, Billerica, German), TMS as internal standard;
thermo Fisher Scientific U3000 high performance liquid chromatograph;
BT25S precision balance (sidoris ltd, germany).
Column chromatography silica gel (100-; sephadex LH-20 (research institute of optometry and Fine chemistry, Tianjin).
A deuterated reagent: DMSO-d6、CD3OD and CDCl3(Cambridge Isotope Laboratories, USA) chromatographic grade methanol (MeOH) and acetonitrile (MeCN) were purchased from TEDIA reagents Tiandi USA, and reagents such as petroleum ether, methanol, dichloromethane, ethyl acetate were analytically pure (Tianjin Fuyu Fine chemical Co., Ltd.).
DMEM high-glucose medium (Israel BI, lot # 0024419);
fetal bovine serum (Israel BI, lot number: 02-411-9);
lipopolysaccharide (LPS, Sigma-Aldrich, USA, batch number: L2994, adding physiological saline to prepare a stock solution);
OX-LDL (Shanghai-derived leaf Biotechnology Co., Ltd., batch No. S24897);
BCA protein quantification kit (Wuhan Boston biology, lot: AR 1110);
RIPA lysate (Roche Bio Inc., batch: AR 0102);
a biosafety cabinet (Saimer aircraft, USA, model 1384-A2);
carbon dioxide incubator (american type 3111, siemer fly);
enzyme-labeling instrument (Saimer Fei USA, model MULTISKAN FC).
The compound prepared by the method of example 1-4 is identified to be the same compound, named as new compound NBY-10 with anti-inflammatory activity extracted from burdock leaf, the compound NBY-10 is light yellow oil,
(c＝0.10g/100ml,CH3OH); by HR-ESI-MS [ M + Na ]]+The calculated value m/z is 287.12595, and the molecular formula is presumed to be C15H20O4The unsaturation degree was 6. Ultraviolet spectrum display (CH)3OH)λmax(log ε) 202(1.020),220(0.738) nm; infrared spectroscopy showed carbonyl groups with hydroxyl groups and esters (3375,1752 cm)-1) The characteristic absorbs the signal.1H-NMR(CD3OD,500MHz) and13C-NMR(CD3OD,125MHz) shows a methyl signal [ delta ]H 1.24(3H,s,H3-11)；δC 22.2(C-11)]Four methylene signals [ delta ]H 1.47(1H,m,H-6a)，1.67(1H,m,H-6b)，2.15(2H,m,H-7)，1.25(1H,m,H-4a)，2.04(1H,dd,J＝5.5,13.5Hz,H-4b)，4.07(1H,d,J＝13.4Hz,H-12a)，4.03(1H,d,J＝13.4Hz,H-12b)；δC 20.1(C-6),20.6(C-7)，39.4(C-4)，66.7(C-12)]4 times of nailsBase signal [ delta ]H3.13(1H,m,H-9a)，3.35(1H,m,H-9b)，4.89(1H,m,H-3a)，1.65(1H,m,H-5a)；δC 34.2(C-9a)，41.0(C-9b)，78.5(C-3a)，46.4(C-5a)]One trisubstituted double bond [ delta ]H 5.93(1H,d,J＝5.0Hz,H-9)；δC 140.8(C-8),124.0(C-9)]One 1, 1-disubstituted double bond [ delta ]H 5.98(1H,d,J＝3.3Hz,H-10)，6.17(1H,d,J＝3.3Hz,H-10a)；δC 140.2(C-1)，125.1(C-10)]One sp3Quaternary carbon deltaC73.2(C-5), one ester carbonyl carbon δC173.4 (C-2). Process for preparation of compound NBY-101H and13C-NMR data are similar to Cadinanolide, but NMR spectra show the presence of an oxymethylene signal [ delta ]H 4.07(1H,d,J＝13.4Hz,H-12a)，4.03(1H,d,J＝13.4Hz,H-12b)；δC 66.7(C-12)]Instead of methyl. H-12 shows HMBC correlation with C-7 and C-9. HMBC Spectroscopy H2-7 and C-5 a/C-6/C-9; h-9a and C-5 a/C-6/C-8/C-9; h-9b and C-9a/C-3a/C-4/C-5 a; h2-4 and C-3 a/C-5/C-11; h-2 and C-9 b/C-2/C-1; h-10 and C-5 a; h3Correlation of-11 with C-4/C-5/C-5 a. The H-H COSY spectra show the correlation between H-6 and H-7/H-5a, H-9b and H-9a/H-3a, H-3a and H-4. Thus, the planar structure of compound NBY-10 was determined. The relative configuration of compound NBY-10 was deduced on the basis of NOESY experiments. NOE associations from H-9a to H-9b, H-3a indicate that they are on the same side, while those from H3NOE associations of-11 to H-5a are displayed on the other face. The absolute configuration of compound NBY-10 was determined by theoretical ECD calculations. The ECD spectrum of compound NBY-10 showed a positive Cotton effect at 220nm, consistent with the experimental results. Thus, the absolute configuration of compound NBY-10 is 3aR,5R,5aS,9aR,9 bR.
1H-NMR and 13C-NMR date for compound 10in MeOD(J in Hz)
Second, Activity test
1.1 cell culture
Culturing RAW264.7 cells in DMEM complete medium (containing 10% newborn calf serum and 90% DMEM incomplete culture solution), standing at 37 deg.C and containing 5% CO2Culturing in an incubator. Cells in the logarithmic growth phase were selected for the experiments.
Mouse mononuclear macrophages (RAW264.7) were cultured by cell imaging laboratory of university of medicine in south river.
1.2 MTT method for detecting RAW264.7 cell viability
Taking RAW264.7 cells in logarithmic growth phase, preparing into 1 × 10 cells with complete culture medium5The cell suspension was inoculated into a 96-well plate, 100. mu.l of the cell suspension was added to each well, and the plate was left at 37 ℃ with 5% CO2Culturing in an incubator. After 24h, the supernatant from the wells was discarded and 100. mu.l of complete medium containing compounds NBY-10 at concentrations of 160, 80, 40, 20, 10, 0mM, respectively; the drug administration group and blank group are provided with 3 multiple wells at 37 deg.C and 5% CO2Culturing in an incubator. After 24h, adding 10 mu l of MTT into each well, continuing to culture for 3h, discarding supernatant, adding 100 mu l of DMSO into each well, shaking on a bed for 10min, fully dissolving purple crystals, and measuring the OD value of each well at 490nm by using an enzyme-labeling instrument. And cell viability was calculated as (a test/a space) × 100%.
TABLE 1 Effect of Compounds NBY-10 on RAW264.7 cell Activity
Note: p <0.01, p < 0.001 compared to blank.
And (4) analyzing results:
effect of different concentrations of compound NBY-10 on RAW264.7 cell viability as shown in table 1 at compound NBY-10 concentration of 40 μ M, cell viability was less than 100% and at other concentrations cell viability was greater than 100%. We chose compound NBY-10 to be administered to cells at a concentration of 10, 20. mu.M, and used it for further experimental studies on cells.
1.3 Griess method for detecting NO level generated by RAW264.7 cell induced by LPS
Cells in logarithmic growth phase were seeded in 24-well plates (2X 10)5Individual cells/well), 37 ℃ 5% CO2After culturing for 12h in an incubator in the environment, adding samples to be detected with different mass concentrations, and adding 1 mu g/mL LPS after incubation for 1 h. Meanwhile, a blank control group (culture medium), a model (LPS + culture medium) group, a positive drug group (dexamethasone + culture medium) and a drug action group are arranged. And continuously carrying out incubation culture for 24 h. Each group was replicated 3 independent experiments. The supernatant (100mL) was mixed with an equal volume of Grignard reagent, and the OD at 540nm of the mixture was measured with an ELISA detector to calculate the NO inhibition rate, the results of which are shown in Table 2.
TABLE 2 Effect of Compounds NBY-10 on LPS-induced NO levels in RAW264.7 cells
Note: positive drug (dexamethasone)
And (4) analyzing results:
as can be seen from Table 2, the NO release of the model group was significantly higher than that of the blank control group (P <0.01), indicating successful molding. Compared with the model group, the concentration treatment can remarkably reduce the release amount of NO (P <0.01) and is concentration-dependent. As shown in Table 2, compound NBY-10 was found to inhibit the production of inflammatory factor NO.
Experiments show that the compound NBY-10 has obvious anti-inflammatory activity (action), obviously reduces the release amount of NO, inhibits the generation of inflammatory factors NO, and is effectively used for preparing the medicine for treating acute pneumonia.
In conclusion, the novel compound NBY-10 with anti-inflammatory activity extracted from the burdock leaves has the advantages of abundant raw materials, easy operation of the preparation method, strong guidance quality and over 96 percent of product purity, can be effectively used for preparing anti-inflammatory drugs, develops the novel application and medicinal value of the burdock leaves, and has great economic and social benefits.
Claims (8)
1. A compound NBY-10 with anti-inflammatory activity extracted from folium Arctii, wherein the molecular structural formula of the compound NBY-10 is:
2. the method for preparing compound NBY-10 with anti-inflammatory activity extracted from burdock leaves as claimed in claim 1, characterized by comprising the following steps:
(1) 20kg of burdock leaf powder, extracting 1-3 times with ethanol with the mass concentration of 30% and the weight of the burdock leaf powder being 4-6 times of the weight of the burdock leaf powder, extracting for 1-4 h each time, combining the extracting solutions, and concentrating to obtain a concentrated solution equivalent to 0.1-3 g/mL of crude drug;
(2) sequentially extracting the concentrated solution with 2-8L, 1-4L and 0.5-2L of n-butyl alcohol for 4 times, combining n-butyl alcohol extraction solutions, and concentrating the solvent under reduced pressure to obtain a component Fr.A; performing silica gel column chromatography on the component Fr.A, eluting by using a dichloromethane/methanol (100: 2) solution with a volume ratio of 8-25L, wherein the inner diameter is 4-8 cm, the height is 8-25 cm, collecting eluent, and recovering a solvent under reduced pressure to obtain a component Fr.A-1;
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate at a volume ratio of 100:5, 2-6L, 100:10, 2-6L, 100:20, 2-6L, 100:40 and 4-15L, with an inner diameter of 3-7 cm and a height of 12-30 cm, collecting eluent at a volume ratio of 100:40, and recovering the solvent under reduced pressure to obtain a component Fr.A-1-3;
(4) performing Sephadex LH-20 column chromatography on the component Fr.A-1-3, eluting with methanol at an elution volume of 1-15L and an elution flow rate of 0.5-1 mL/min, and recovering the solvent under reduced pressure to obtain a component Fr.A-1-3-3, wherein the inner diameter of the component Fr.A-1-3 cm and the height of the component Fr.A-1-3 cm are 80-150 cm;
(5) recrystallizing the component Fr.A-1-3-3 in methanol to obtain compound NBY-10 with antiinflammatory activity extracted from folium Arctii.
3. The method for preparing compound NBY-10 with anti-inflammatory activity extracted from folium Arctii according to claim 2, comprising the following steps:
(1) extracting 20kg of folium Arctii powder with 5 times of ethanol with mass concentration of 30% and weight of folium Arctii powder for 2 times (2.5 hr each time), mixing extractive solutions, and concentrating to obtain concentrated solution equivalent to 0.5g/mL of crude drug;
(2) sequentially extracting the concentrated solution with n-butanol 7L, 4L, and 2L for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A; performing silica gel column chromatography on component Fr.A, eluting with a volume ratio of dichloromethane to methanol of 100 to 2 of 8L, with an inner diameter of 4cm and a height of 25cm, collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1;
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 2L, 100:10, 2L, 100:20, 3L, 100:40, 5L at volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3;
(4) subjecting the component Fr.A-1-3 to Sephadex LH-20 column chromatography with inner diameter of 1.5cm and height of 130cm, eluting with methanol at elution volume of 3.5L and flow rate of 0.5mL/min, and recovering solvent under reduced pressure to obtain component Fr.A-1-3-3;
(5) recrystallizing the component Fr.A-1-3-3 in methanol to obtain compound NBY-10 with antiinflammatory activity extracted from folium Arctii.
4. The method for preparing compound NBY-10 with anti-inflammatory activity extracted from folium Arctii according to claim 2, comprising the following steps:
(1) extracting 20kg of burdock leaf powder with ethanol with mass concentration of 30% 4 times of burdock leaf powder for 3 times, each time for 3h, mixing extractive solutions, and concentrating to obtain concentrated solution equivalent to 0.6g/mL of crude drug;
(2) sequentially extracting the concentrated solution with 6L, 3L and 2L of n-butanol for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A; performing silica gel column chromatography on component Fr.A, eluting with dichloromethane/methanol (volume ratio of dichloromethane/methanol: 100: 2) solution 24L, collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1, wherein the inner diameter is 7cm and the height is 20 cm;
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 5L, 100:10, 5L, 100:20, 6L, 100:40, 15L in volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3;
(4) performing Sephadex LH-20 column chromatography on the component Fr.A-1-3 with inner diameter of 3cm and height of 90cm, eluting with methanol at elution volume of 10L and flow rate of 1mL/min, and recovering solvent under reduced pressure to obtain component Fr.A-1-3-3;
(5) recrystallizing the component Fr.A-1-3-3 in methanol to obtain compound NBY-10 with antiinflammatory activity extracted from folium Arctii.
5. The method for preparing the compound NBY-10 with anti-inflammatory activity extracted from burdock leaves according to claim 2, characterized by comprising the following steps:
(1) 20kg of burdock leaf powder, extracting for 2 times with ethanol with the mass concentration of 30% 6 times of the burdock leaf powder weight, extracting for 2h each time, mixing the extracting solutions, and concentrating to obtain a concentrated solution equivalent to 0.5g/mL of crude drug;
(2) sequentially extracting the concentrated solution with n-butanol 3L, 4L, and 1L for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A; performing silica gel column chromatography on component Fr.A, eluting with a volume ratio of dichloromethane to methanol of 20L (inner diameter 6cm and height 17 cm), collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1;
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 4L, 100:10, 4L, 100:20, 5L, 100:40, 13L in volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3;
(4) subjecting the component Fr.A-1-3 to Sephadex LH-20 column chromatography with inner diameter of 2.5cm and height of 100cm, eluting with methanol with elution volume of 7.8L and elution flow rate of 0.8mL/min, and recovering solvent under reduced pressure to obtain component Fr.A-1-3-3;
(5) recrystallizing the component Fr.A-1-3-3 in methanol to obtain compound NBY-10 with antiinflammatory activity extracted from folium Arctii.
6. The method for preparing the compound NBY-10 with anti-inflammatory activity extracted from burdock leaves according to claim 2, characterized by comprising the following steps:
(1) 20kg of burdock leaf powder, extracting for 2 times by using ethanol with the mass concentration of 30% and the weight 6 times of the burdock leaf powder, extracting for 1.5h each time, combining extracting solutions, and concentrating to obtain a concentrated solution equivalent to 0.4g/mL of crude drug;
(2) sequentially extracting the concentrated solution with n-butanol 5L, 3L, and 2L for 4 times, mixing n-butanol extractive solutions, and concentrating the solvent under reduced pressure to obtain component Fr.A; performing silica gel column chromatography on component Fr.A, eluting with a volume ratio of dichloromethane to methanol of 100 to 2 of 13L, with an inner diameter of 5cm and a height of 15cm, collecting eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1;
(3) performing secondary silica gel column chromatography on the component Fr.A-1, eluting with petroleum ether-ethyl acetate 100:5, 3L, 100:10, 3L, 100:20, 4L, 100:40, 10L in volume ratio, collecting 100:40 eluate, and recovering solvent under reduced pressure to obtain component Fr.A-1-3;
(4) performing Sephadex LH-20 column chromatography on the component Fr.A-1-3 with an inner diameter of 2cm and a height of 100cm, eluting with methanol at an elution volume of 5L and an elution flow rate of 0.6mL/min, and recovering solvent under reduced pressure to obtain a component Fr.A-1-3-3;
(5) recrystallizing the component Fr.A-1-3-3 in methanol to obtain compound NBY-10 with antiinflammatory activity extracted from folium Arctii.
7. The use of compound NBY-10 with anti-inflammatory activity extracted from folium Arctii as claimed in claim 1 in the preparation of anti-inflammatory drugs.
8. The use of the compound NBY-10 with anti-inflammatory activity extracted from burdock leaf of claim 1 in the preparation of a medicament for treating acute pneumonia.
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