CN111440183A

CN111440183A - Compound, preparation method and application thereof, and anti-rheumatoid arthritis drug

Info

Publication number: CN111440183A
Application number: CN202010457352.5A
Authority: CN
Inventors: 何承伟; 屠言贝; 王恺
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2020-07-24
Anticipated expiration: 2039-06-27
Also published as: CN110330503A; CN110330503B; CN111440183B; CN111533755B; CN111533755A

Abstract

The invention discloses a compound, a preparation method and application thereof, and a medicament for resisting rheumatoid arthritis, and relates to the technical field of natural medicaments and medicaments.A compound is a novel compound and can be 9 extracts of glytacoumestan A-I in tobacco beans, the compound is extracted from the tobacco beans by the preparation method, and the compound is applied to the preparation of the medicament for resisting rheumatoid arthritis, and pharmacodynamic tests show that the coumestan compound can remarkably slow down cell inflammation reactions stimulated by lipopolysaccharide (L PS) and interleukin-1 β (I L-1 β), can inhibit the differentiation of osteoclasts, and prompt the application prospect of the compounds for preparing the medicament for resisting rheumatoid arthritis.

Description

Compound, preparation method and application thereof, and anti-rheumatoid arthritis drug

The application is a divisional application, the application number of a parent application is 201910569463.2, and the name is 'a compound, a preparation method and application thereof and a medicament for resisting rheumatoid arthritis'.

Technical Field

The invention relates to the technical field of natural medicines and medicines, and particularly relates to a compound, a preparation method and application thereof, and a medicine for resisting rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is a multi-system autoimmune disease with a major component of chronic inflammatory responses and joint destruction. The main pathological features of the medicine are chronic and persistent synovium inflammation, synovium cell hyperplasia, inflammatory cell infiltration, pannus formation, erosion and destruction of cartilage and bone tissues, which finally result in structural destruction, deformity and functional loss of joint tissues, cause disabilities of different degrees and seriously affect the life quality of patients. RA lesions not only attack the bone and joints, but also involve organs, with multisystemic lesions outside the joints. The global incidence of RA is between 1% and 2%, and in China, the incidence of RA is about 0.2% to 0.37%, and the RA is one of the main diseases causing disability and loss of labor force of people in China. At present, the rheumatoid arthritis is mainly treated by non-steroidal anti-inflammatory drugs, disease improvement and anti-rheumatism drugs, glucocorticoids and other drugs, but the drugs have limited curative effects and obvious toxic and side effects, so that the drugs cannot be taken for a long time, and therefore, the search and the research of novel high-efficiency low-toxicity drugs for preventing and treating the inflammatory development and the joint injury of the rheumatoid arthritis are still urgent. The natural product has the characteristics of wide sources, rich components, less adverse reactions and the like, and is more and more concerned by researchers at home and abroad. Currently, attempts to find anti-rheumatoid arthritis drugs from natural products have become a research hotspot.

Tobacco bean (L abill.) Benth is a perennial herb of soybean (L eguminosae) in the family of leguminosae, also known as Penghu soybean, Penghu monoradicis, Tilletia acuminata, longleaf yinshu, etc., and is distributed in Australia, islands in south China, southeast coast and Taiwan area, etc. tobacco bean has the functions of dispelling wind-damp, strengthening bones and muscles, benefiting spleen and kidney, and is used for treating rheumatism, bone pain and soreness of waist and knees.

Disclosure of Invention

The invention aims to provide a compound and aims to provide a novel compound with the effects of preventing and treating rheumatoid arthritis.

The invention also aims to provide a preparation method of the compound, which aims to extract an active ingredient with the effect of preventing and treating rheumatoid arthritis from tobacco beans.

The invention also provides the application of the compound and a medicament for resisting rheumatoid arthritis by using the compound as an active ingredient.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

The invention provides a compound, which has a structural formula as follows:

wherein R is₁Represents methoxy or hydroxy; r₂To represent

R₃To represent

Or H; r₄To represent

R₅Represents a methoxy group or a hydroxyl group.

The invention also provides a preparation method of the compound, which comprises the following steps: extracting the above compound from tobacco bean.

The invention also provides the application of the compound in preparing medicaments for preventing and treating inflammatory diseases; preferably, the inflammatory disease is rheumatoid arthritis.

The invention also provides a medicament for resisting rheumatoid arthritis, and the active ingredients of the medicament comprise the compound.

The compound provided by the embodiment of the invention has the beneficial effects that the inventor continuously researches and discovers a novel compound with the effect of resisting rheumatoid arthritis, can effectively inhibit the generation of nitric oxide stimulated by L PS, has strong in-vitro anti-inflammatory activity, and has good capability of relieving rheumatoid arthritis synovitis.

The invention also provides a preparation method of the compound, which extracts the active ingredients from the complex ingredients of the tobacco beans by extracting the compound from the tobacco beans and has important significance for mining and utilizing medicinal resources of the tobacco beans. The invention also provides the application of the compound and a medicament taking the compound as an active ingredient, and the compound has good market application value.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a graph of the effect of compounds 1-9 in a safe concentration range on the level of nitric oxide produced by L PS stimulation in RAW264.7 cells;

FIG. 2 is a graph of the effect of compounds 1-9 on the level of TNF- α produced by I L-1 β stimulation in SW982 cells at a safe concentration range;

figure 3 is a graph of the effect of

compounds

1, 2, 7, 8, 9 on RANK L stimulated differentiation of RAW264.7 cells into osteoclasts in a safe concentration range;

FIG. 4 is a 1H-NMR spectrum of the first compound;

FIG. 5 is a 13C-NMR spectrum of the first compound;

FIG. 6 is an HSQC spectrum of the first compound;

FIG. 7 is an HMBC spectrum of a first compound;

FIG. 8 is a 1H-NMR spectrum of the second compound;

FIG. 9 is a 13C-NMR spectrum of the second compound;

FIG. 10 is the HSQC spectrum of the second compound;

FIG. 11 is an HMBC spectrum of a second compound;

FIG. 12 is a 1H-NMR spectrum of a third compound;

FIG. 13 is a 13C-NMR spectrum of a third compound;

FIG. 14 is an HSQC spectrum of a third compound;

FIG. 15 is an HMBC spectrum of a third compound;

FIG. 16 is a 1H-NMR spectrum of a fourth compound;

FIG. 17 is a 13C-NMR spectrum of a fourth compound;

FIG. 18 is an HSQC spectrum of the fourth compound;

FIG. 19 is an HMBC spectrum of a fourth compound;

FIG. 20 is a 1H-NMR spectrum of a fifth compound;

FIG. 21 is a 13C-NMR spectrum of a fifth compound;

FIG. 22 is an HSQC spectrum of a fifth compound;

FIG. 23 is an HMBC spectrum of a fifth compound;

FIG. 24 is a 1H-NMR spectrum of a sixth compound;

FIG. 25 is a 13C-NMR spectrum of a sixth compound;

fig. 26 is an HSQC spectrum of the sixth compound;

FIG. 27 is an HMBC spectrum of a sixth compound;

FIG. 28 is a 1H-NMR spectrum of a seventh compound;

FIG. 29 is a 13C-NMR spectrum of a seventh compound;

fig. 30 is an HSQC spectrum of the seventh compound;

FIG. 31 is an HMBC spectrum of a seventh compound;

FIG. 32 is a 1H-NMR spectrum of an eighth compound;

FIG. 33 is a 13C-NMR spectrum of an eighth compound;

fig. 34 is an HSQC spectrum of the eighth compound;

FIG. 35 is an HMBC spectrum of an eighth compound;

FIG. 36 is a 1H-NMR spectrum of a ninth compound;

FIG. 37 is a 13C-NMR spectrum of a ninth compound;

fig. 38 is an HSQC spectrum of the ninth compound;

fig. 39 is an HMBC spectrum of the ninth compound.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following provides a compound, a preparation method and application thereof, and a medicament for resisting rheumatoid arthritis.

The embodiment of the invention provides a compound, the structural formula of which is as follows:

wherein R is₁Represents methoxy or hydroxy; r₂To represent

R₃To represent

Or H; r4 represents

R₅Represents a methoxy group or a hydroxyl group.

The compounds are novel compounds, can obviously relieve cell inflammatory reaction stimulated by lipopolysaccharide and interleukin-1 β, have the capacity of inhibiting osteoclast differentiation, and show the application prospect of the compounds in the preparation of medicines for resisting rheumatoid arthritis.

The inventors have studied various compounds having the above structures and screened out 9 compounds having the most prominent effects, which can be summarized as the following structures:

specifically, the compound is any one selected from a first compound, a second compound, a third compound, a fourth compound, a fifth compound, a sixth compound, a seventh compound, an eighth compound (also referred to as a target compound), and a ninth compound (also referred to as an impurity compound); wherein the structural formulas of the first to ninth compounds are as follows in sequence:

the first to ninth compounds are derived from tobacco bean, and are active ingredients of tobacco bean, so that the tobacco bean has the effect of resisting rheumatoid arthritis.

The embodiment of the invention also provides a preparation method of the compound, which comprises the following steps: extracting the above compounds from tobacco beans; preferably, the extracted compounds include a first compound, a second compound, a third compound, a fourth compound, a fifth compound, a sixth compound, a seventh compound, an eighth compound, and a ninth compound. The method specifically comprises the following steps:

s1 preparation of extract

Crushing tobacco beans, extracting with alcohol, and concentrating to obtain alcohol extract. Extracting with 90-98% ethanol solvent for at least 3 times until the color of the extractive solution is light, mixing extractive solutions, filtering, and concentrating under reduced pressure.

Preferably, the ratio of material to liquid in the extraction is 1:5-10, and the ratio of material to liquid is controlled in the above range, so that the effective components in the tobacco bean can be extracted more sufficiently, and the extraction rate of the active components is improved. Specifically, the extraction method is the existing extraction method such as cold soaking extraction, heating reflux extraction or ultrasonic-assisted extraction.

S2 solvent extraction

Mixing the alcohol extract with water, and sequentially extracting with petroleum ether and ethyl acetate to obtain petroleum ether extract and ethyl acetate extract. The inventors have found that the use of this extraction sequence allows more active ingredient concentration in the ethyl acetate extraction section for subsequent separation and purification.

In some embodiments, the solvent extraction process comprises extracting with petroleum ether, ethyl acetate, and water-saturated n-butanol to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract, and water extract, respectively. The ingredients in the tobacco beans can be more effectively separated through multiple times of extraction, the purity of the finally extracted active ingredients is improved, the selection of the extracting agent and the setting of the extraction sequence are both suitable for the scheme, and the purity of the extracted product can be effectively improved under the condition.

S3 chromatography segmentation

Gradient elution is carried out on the ethyl acetate extraction part to obtain 11 elution parts, the active ingredients are concentrated in the 11 elution parts, and the elution parts can be sequentially numbered from top to bottom or from left to right.

Preferably, in the chromatographic fractionation process, a silica gel chromatographic column is used for fractionation, and a petroleum ether-ethyl acetate system is used for gradient elution, so that the inventor finds that the petroleum ether-ethyl acetate system can ensure that the obtained eluted part is more convenient for subsequent separation and purification, and the extraction rate and the purity of each compound are ideal.

Preferably, when gradient elution is performed with a petroleum ether-ethyl acetate system, the ratio of petroleum ether: the elution gradient for ethyl acetate was 10:1, 5:1, 3:1, 2:1, 1:2, and 1: 5. The inventor optimizes the proportion of an elution system in gradient elution, and the elution part can be well layered by adopting the elution gradient.

S4 purification and separation

Combining and purifying the first elution part, the second elution part and the third elution part to obtain a third compound; purifying with a fifth eluting portion to obtain a first compound; purifying with a sixth eluting portion to obtain a fourth compound; purifying by using a seventh elution part to obtain a sixth compound, a seventh compound and a second compound; and purifying by using a ninth elution part to obtain a fifth compound, an eighth compound and a ninth compound. The inventor optimizes the purification steps through a large amount of experimental researches, and the extraction rate and the purity of the compound can be improved to a greater extent by adopting the purification process, so that the waste of raw materials is reduced.

In order to further improve the extraction rate and purity of the 9 compounds, the inventors optimized the separation and purification steps of each elution part.

Wherein, the process of obtaining the third compound by combining and purifying the first elution part, the second elution part and the third elution part comprises the following steps: combining the three elution parts, performing column chromatography elution, and purifying by using a preparative thin layer chromatography. Preferably, the eluent corresponding to the three elution parts is a mixed solution of dichloromethane and methanol; more preferably, the volume ratio of dichloromethane to methanol is 1: 0.5-1.5. The yield of the product is higher by adopting the mixed solution of dichloromethane and methanol as the eluent, and the inventor further improves the yield of the third compound by regulating and controlling the mixture ratio of the eluent.

Specifically, in the chromatographic conditions of the multiple preparation thin-layer chromatography, the volume ratio of petroleum ether to ethyl acetate is 4-6:1, the volume ratio of petroleum ether to dichloromethane is 1-3:3, the column chromatography corresponding to three elution parts is Sephadex L H-20 column chromatography, the chromatographic conditions of the preparation thin-layer chromatography have certain influence on the yield and the purity of the third compound, the third compound can be separated more efficiently by controlling the chromatographic conditions within the range, and the yield is about 0.0003 thousandth.

Wherein the process of obtaining the first compound by using the fifth elution part comprises: the fifth eluate fraction is concentrated for 5-8 days and the crystals are filtered. After a long period of concentration, a large amount of crystals are formed, and the part of crystals is collected and filtered to obtain the pure first compound, wherein the yield is about 0.015 per thousand.

The process for obtaining the fourth compound by using the sixth elution part comprises the steps of performing column chromatography on the sixth elution part, identifying eluent by T L C, merging to obtain five fractions, taking the fourth fraction, purifying by using preparative thin-layer chromatography, preferably, the chromatographic conditions of the preparative thin-layer chromatography corresponding to the fourth fraction are that the volume ratio of petroleum ether to acetone is 2-4:1, preferably, the column chromatography corresponding to the sixth elution part is Sephadex L H-20 column chromatography, and the separation of the fourth compound needs to be performed by multiple steps of separation to achieve higher purity, wherein the separation steps are repeatedly performed by the inventor, so that the yield and the purity of the fourth compound are optimal by selecting the specific fraction and optimizing the chromatographic conditions, and the yield is approximately 0.0005 thousandth.

The process of obtaining the sixth compound, the seventh compound and the second compound by purifying the seventh elution part comprises the steps of carrying out Sephadex L H-20 column chromatography on the seventh elution part, merging eluent after being detected by T L C to obtain five fractions 7A-7E, carrying out Toyopeal HW-40 column chromatography on the fraction 7A, carrying out T L C detection on the eluent and merging the five fractions to obtain 7A1-7A5, carrying out preparative liquid chromatography on the fractions 7A3 and 7A4 respectively to obtain the sixth compound and the seventh compound (the yields are respectively 0.0005 thousandth and 0.0009 thousandth), carrying out Sephadex L H-20 column chromatography on the fraction 7C again to obtain the second compound (the yield is approximately 0.027 thousandth), purifying the sixth compound, the seventh compound and the second compound from the seventh elution part, and the purification steps are complicated, so that the inventor continuously carries out higher yield, and the optimized separation steps are optimized.

The inventor further optimizes the preparation conditions of the liquid chromatography to further improve the separation effect, and the preparation conditions of the liquid chromatography in the process of obtaining the sixth compound and the seventh compound are that an 18C-reverse phase chromatographic column is adopted, the flow rate is 7-9m L/min, preferably, the eluent corresponding to the sixth compound is a mixture of methanol and water, and the volume ratio of the methanol to the water is 90:8-12, preferably, the eluent corresponding to the seventh compound is a mixture of the methanol and the water, and the volume ratio of the methanol to the water is 95: 4-6.

The process for obtaining the fifth compound, the eighth compound and the ninth compound by purifying the ninth elution part comprises the steps of carrying out silica gel chromatographic column chromatography on the ninth elution part, carrying out gradient elution by using a petroleum ether-acetone system to obtain seven elution fractions 9A-9G, carrying out Sephadex L H-20 column chromatography on the fraction 9D, carrying out T L C detection on eluent, merging to obtain three fractions 9D1-9D3, carrying out liquid chromatographic separation on the fraction 9D2 to obtain the fifth compound (the yield is approximately 0.0007 per thousand), carrying out Sephadex L H-20 column chromatography on the fraction 9E, carrying out T L C detection on eluent, merging to obtain three fractions 9E1-9E3, carrying out Toyopeal-40 liquid chromatographic separation on the fraction 9E3 to obtain three fractions 9E3A-9E3C, carrying out liquid chromatographic purification on the fraction 9E3B to obtain the eighth compound and the ninth compound (the yield is 0.0004, the ninth compound and the ninth compound, and the ninth compound are obtained by repeatedly researched steps of the ninth purification process.

Preferably, when gradient elution is performed with a petroleum ether-acetone system, the ratio of petroleum ether: the elution gradient of acetone was 10:1, 6:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1: 2. The ninth elution part can be better separated by adopting the elution gradient, and the yield of the three compounds is improved.

The inventor further optimizes the preparation conditions of the liquid chromatography to further improve the separation effect in the process of obtaining the fifth compound, the eighth compound and the ninth compound, wherein the preparation conditions of the liquid chromatography are that an 18C-reverse phase chromatographic column is adopted, the flow rate is 7-9m L/min, more preferably, the eluent corresponding to the fifth compound is a mixture of methanol and water, and the volume ratio of the methanol to the water is 90:8-12, more preferably, the eluent corresponding to the eighth compound is a mixture of the methanol and the water, and the volume ratio of the methanol to the water is 70:25-35, more preferably, the eluent corresponding to the ninth compound is a mixture of the methanol and the water, and the volume ratio of the methanol to the water is 70: 25-35.

The eluent for the Sephadex L H-20 column chromatography is a mixed solution of dichloromethane and methanol, preferably, the volume ratio of the dichloromethane to the methanol is 1:0.5-1.5, the eluent for the Toyopeal HW-40 column chromatography is methanol, and the first to ninth compounds are separated and purified according to the optimized steps and conditions, so that the purity of the obtained compounds is more than 90%.

S5, characterization

Performing nuclear magnetic spectrum and mass spectrum characterization on the extracted compound after purification and separation to identify the structure of the compound; more preferably, the nuclear magnetic spectrum is selected from¹H-NMR、¹³C-NMR, HMBC, HSQC or NOESY; more preferably, the mass spectrum is selected from HR-ESI-MS or ESI-MS. Characterization can be performed after the isolation and purification is complete to determine the composition of the extracted compounds.

The embodiment of the invention also provides the application of the compound in preparing medicaments for preventing and treating inflammatory diseases; preferably, the inflammatory disease is rheumatoid arthritis.

In vitro anti-inflammatory and anti-rheumatoid arthritis activities of 9 coumestan novel compounds are tested, pharmacodynamic experiments show that in a RAW264.7 cell (mouse mononuclear macrophage leukemia cell) inflammation model stimulated by lipopolysaccharide (L PS), 9 compounds can inhibit the generation of nitric oxide stimulated by L PS to different degrees within the safe concentration range of the compounds and have an dose-effect relationship, which shows that 9 compounds have strong in vitro anti-inflammatory activity, in an SW982 cell (human synovial sarcoma cell) inflammation model stimulated by interleukin-1 β (I L-1 β), in the safe concentration range (5-20 mu M) of each compound, 9 compounds can inhibit the generation of proinflammatory factor TNF- α stimulated by I L-1-7 to different degrees, which shows that 9 compounds have the capacity of relieving the inflammation of rheumatoid arthritis, and simultaneously establishing a RAW 264.264 cell model stimulated by nuclear factor kappa B receptor activator ligand (RANB L), a RAW264 cell model stimulated by I L-1 and a TRAW 7 cell differentiation model which shows that the differentiation of the compounds to the seventh compound, the osteoclast phosphatase concentration range (TRAK L) and the ninth compound can reduce the activity of the compound and the ninth compound in the ninth compound and the ninth compound (TRAP) in the safe concentration range (5-20 mu M) of the ninth compound.

The embodiment of the invention also provides a medicament for resisting rheumatoid arthritis, and the active ingredients of the medicament comprise the compound; the 9 compounds can be used as active ingredients of medicaments to prepare medicaments for resisting rheumatoid arthritis, and the medicaments are prepared into pills, granules, tablets, sachets or injections.

The features and properties of the present invention are described in further detail below with reference to examples.

The instrument and the material are that the tobacco bean medicinal material is purchased from Fuqing city of Fujian province, the plant species is identified by DNA bar code (completed in doctor Korea laboratory of medicinal plant institute of Chinese medical academy of sciences), silica gel (200 meshes and 300 meshes) is used for column chromatography, silica gel GF254(10-40 mu m) is used for thin-layer chromatography (Qingdao ocean chemical Co., Ltd.), Sephadex L H-20 filler is used for column chromatography, Toyopeal HW-40 filler is used for column chromatography, COSMOSI L5C 18-MS-II 20ID × 250mm is used for preparing chromatographic column, liquid chromatogram IMADZU Preparative System L C-20AP, nuclear magnetic resonance instrument Bruker, 600MHz and high resolution mass spectrum Thermo L TQ bitorap X L hybrid FTMS.

Example 1

This example provides a method of preparing a compound, comprising the steps of:

(1) extracting an ethanol extract: drying 5kg tobacco bean herb, pulverizing, placing into an extraction barrel, cold-soaking with 95% ethanol at a material-liquid ratio of 1:5 for 3 times, each time for 3 days until the color of the extractive solution is lighter, mixing filtrates, filtering, and concentrating under reduced pressure to obtain 184g ethanol extract.

(2) And (3) solvent extraction, namely preparing water suspension from the ethanol extract of the tobacco beans by using 800m L distilled water, and extracting by using petroleum ether, ethyl acetate and water saturated n-butanol respectively to obtain 29.0g of a petroleum ether extraction part, 22.8g of an ethyl acetate extraction part, 30.1g of an n-butanol extraction part and 74.3g of a water extraction part.

(3) Silica gel column chromatography fractionation the ethyl acetate fraction of the tobacco beans was fractionated on a 200-mesh 300 mesh silica gel column (70 × 245mm) and gradient eluted with a petroleum ether-ethyl acetate system (10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, ethyl acetate) to give 11 eluted fractions Fr.1(0.25g), Fr.2(0.14g), Fr.3(0.15g), Fr.4(0.51g), Fr.5(0.30g), Fr.6(0.51g), Fr.7(1.06g), Fr.8(4.09g), Fr.9(4.19g), Fr.10(3.73g), Fr.11(5.73 g).

(4) Concentrating and standing an elution part Fr.5 for 7 days, filtering and crystallizing to obtain a first compound (72mg) after a large amount of crystals are generated, combining elution parts Fr.1, Fr.2 and Fr.3, then carrying out Sephadex L H-20 column chromatography, eluting dichloromethane: methanol: 1, carrying out T L C detection on eluent, combining eluent, carrying out multiple preparative thin-layer chromatography (petroleum ether: ethyl acetate: 5: 1; petroleum ether: dichloromethane: 2:3), purifying to obtain a third compound (1.4mg), carrying out Sephadex L H-20 column chromatography, eluting dichloromethane: methanol: 1, carrying out T L C detection on eluent, combining eluent to obtain a flow 6A-6E, carrying out multiple preparative thin-layer chromatography (petroleum ether: acetone: 3:1) on a flow 6D, purifying to obtain a fourth compound (2.3mg), carrying out column chromatography on Sephadex 367: 4H-6E, carrying out T367: 90H-5 column chromatography, carrying out T367: 5H-5 chromatography, carrying out T367 methanol: 7: 1: 35H-20 column chromatography, carrying out T367H-20 column chromatography, combining eluent, carrying out multiple preparative thin-5 chromatography, carrying out T357 chromatography, carrying out T7H-5 chromatography, carrying out chromatography, combining eluent, carrying out multiple preparative thin-5 chromatography, carrying out chromatography (methanol-35A chromatography) chromatography, combining methanol-35H-35 chromatography, carrying out chromatography.

(5) Subjecting the fraction Fr.9 to silica gel chromatography (36 × mm) for continuous segmentation, performing gradient elution with a petroleum ether-acetone system (10:1, 6:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1:2) to obtain 7 eluted fractions 9A-9G, subjecting the 7 eluted fractions to Sephadex L H-20 column chromatography, eluting with dichloromethane: methanol 1:1, subjecting the eluates to T L C detection, combining to obtain fractions 9D1-9D3, subjecting the fractions 9D2 to preparative liquid chromatography to obtain a fifth compound (3.2mg, methanol: water: 86590: 10), subjecting the fractions 9E to Sephadex L H-20, eluting with dichloromethane: methanol 1:1 to obtain fractions 9E1-9E3, subjecting the fractions 9E3 to Topeyhal chromatography, subjecting the fractions to Topeyhal chromatography, eluting with methanol 3: 9 HW-40, subjecting the fractions 9E to preparative liquid chromatography to obtain fractions 9E 3: 9 HW, subjecting the fractions to preparative liquid chromatography, purifying with methanol 3: 364, 3mg, 3H-3670H-364 and 3670 parts of the ninth compound (9).

Example 2

(1) extracting an ethanol extract: drying 5kg tobacco bean herb, pulverizing, placing into an extraction barrel, cold-soaking and extracting with 90% ethanol at a material-liquid ratio of 1:10 for 3 times, each time for 3 days until the color of the extractive solution is lighter, mixing filtrates, filtering, and concentrating under reduced pressure to obtain ethanol extract.

(2) And (3) solvent extraction, namely preparing the ethanol extract of the tobacco beans into water suspension by using distilled water of 800m L, and extracting by using petroleum ether, ethyl acetate and water saturated n-butanol respectively to obtain a petroleum ether extraction part, an ethyl acetate extraction part, an n-butanol extraction part and a water extraction part.

(3) Silica gel column chromatography segmentation the ethyl acetate fraction of the tobacco beans was fractionated on a 200-mesh 300-mesh silica gel column (70 × 245mm) and gradient eluted with a petroleum ether-ethyl acetate system (10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, ethyl acetate) to give 11 eluted fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, Fr.7, Fr.8, Fr.9, Fr.10, Fr.11.

(4) Concentrating and standing an elution part Fr.5 for 5 days, filtering and crystallizing to obtain a first compound after a large amount of crystals are generated, combining elution parts Fr.1, Fr.2 and Fr.3, performing Sephadex L H-20 column chromatography, eluting dichloromethane with methanol at ratio of 1:0.5, detecting eluent with T L C, combining the eluates, performing thin layer preparative chromatography (petroleum ether: ethyl acetate: 4: 1; petroleum ether: dichloromethane: 1:3) for multiple times, purifying to obtain a third compound, purifying a component Fr.6 with Sephadex L H-20 column chromatography, eluting dichloromethane: methanol at ratio of 1:0.5, detecting eluent with T L C, combining the eluates to obtain fractions 6A-6E, purifying a fraction 6D with thin layer preparative chromatography (petroleum ether: acetone: 2:1) for multiple times, purifying a component Fr.7 with Sephadex L H-20 column chromatography, detecting eluent with T L C, combining the eluates to obtain a fourth compound, purifying a component Fr.7 with water at ratio of 5:5, purifying a component Twent 7: 5: 7, purifying a component Fr.7 with methanol at ratio of 5, combining the eluates with water at ratio of 5, purifying a wash, obtaining a component T7, after detecting methanol at ratio of T7: 5, combining the eluent with methanol at ratio of 5, obtaining a wash after the eluent with water, obtaining a wash, and wash.

(5) Subjecting the fraction Fr.9 to silica gel chromatography (36 × mm) for continuous segmentation, performing gradient elution with a petroleum ether-acetone system (10:1, 6:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1:2) to obtain 7 eluted fractions 9A-9G, subjecting the 7 eluted fractions to Sephadex L H-20 column chromatography, eluting with dichloromethane: methanol ═ 1:0.5, subjecting the eluates to T L C detection, combining to obtain fractions 9D1-9D3, subjecting the fraction 9D2 to preparative liquid chromatography to obtain a fifth compound (methanol: water ═ 90:8), subjecting the fraction 9E to Sephadex L H-20 column chromatography, eluting with dichloromethane: methanol ═ 1:0.5 to obtain fractions 9E1-9E3, subjecting the fraction 9E3 to Topeyopeal HW-40 column chromatography, eluting with methanol ═ 9 HW ═ 3:3, purifying with dichloromethane: 0.3: 9H-20 to obtain a ninth compound (methanol: 3670).

Example 3

(1) extracting an ethanol extract: drying 5kg tobacco bean herb, pulverizing, placing into extraction barrel, extracting with 98% ethanol at a material-liquid ratio of 1:8 for 3 times, each time for 3 days until the color of the extractive solution is lighter, mixing filtrates, filtering, and concentrating under reduced pressure to obtain ethanol extract.

(4) Concentrating and standing an elution part Fr.5 for 8 days, filtering and crystallizing to obtain a first compound after a large amount of crystals are generated, combining elution parts Fr.1, Fr.2 and Fr.3, performing Sephadex L H-20 column chromatography, eluting dichloromethane with methanol 1:1.5, performing T L C detection on eluent, combining the eluent, performing thin layer preparative chromatography (petroleum ether: ethyl acetate: 6: 1; petroleum ether: dichloromethane: 3) for multiple times, purifying to obtain a third compound, performing Sephadex L H-20 column chromatography on the component Fr.6, performing T L C detection on the eluent, combining the eluent to obtain fractions 6A-6E, performing thin layer preparative chromatography (petroleum ether: acetone: 4:1) for multiple times on the fraction 6D, purifying to obtain a fourth compound, performing Sephadex L H-20 column chromatography on the component Fr.7, performing T L C detection on the eluent, combining the eluent with methanol 1:5, performing T7-5 column chromatography, performing T-Sephadex chromatographic column chromatography, performing T-5 column chromatography, performing T-Sephadex chromatography on the eluent, combining the eluent with methanol 3: 1:3, purifying to obtain a third compound, performing TLC-20 column chromatography, performing HW 357 column chromatography, performing TLC chromatography on the component Fr.7, performing TLC chromatography, performing chromatographic separation on the component Fr.7, performing chromatographic separation on the component 6A-7, performing chromatographic separation on the component 35H-7, performing the eluent, performing chromatographic separation on the component 7, performing the eluent, performing the chromatographic column chromatography, performing the component 35H-7, performing the eluent, performing chromatographic column chromatography, performing the chromatographic column chromatography, the chromatographic column chromatography.

(5) Subjecting the fraction Fr.9 to silica gel chromatography (36 × mm) for continuous segmentation, performing gradient elution with a petroleum ether-acetone system (10:1, 6:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1:2) to obtain 7 eluted fractions 9A-9G, subjecting the 7 eluted fractions to Sephadex L H-20 column chromatography, eluting with dichloromethane: methanol ═ 1:1.5, subjecting the eluates to T L C detection, combining the eluates to obtain fractions 9D1-9D3, subjecting the fraction 9D2 to preparative liquid chromatography to obtain a fifth compound (methanol: water ═ 90:12), subjecting the fraction 9E to Sephadex L H-20 column chromatography, eluting with dichloromethane: methanol ═ 1:1.5 to obtain fractions 9E1-9E3, subjecting the fraction 9E3 to TopeyalHW-40 column chromatography, eluting with methanol ═ 9E ═ 1:1.5 to obtain a fraction 9E3, subjecting the fraction 9E ═ 9 to preparative liquid chromatography, purifying with methanol: (3635: 3: 9E) to obtain a ninth compound (3670).

Test example 1

Effect of coumestan-like novel compounds on L PS-stimulated nitric oxide production in RAW264.7 cells.

The main experimental materials: 9 novel compounds of the coumestan class obtained by extraction and separation in the above example 1; RAW264.7 cells were purchased from the chinese academy of sciences cell bank; the nitric oxide determination kit is purchased from Biyuntian biotechnology company; fetal Bovine Serum (FBS) and DMEM media were purchased from Gibco.

The experimental method comprises setting control group, L PS group and each drug experimental group, taking 70% -80% RAW264.7 cells, scraping cells, blowing into single cell suspension, adding 10% FBS-containing DMEM medium to adjust cell concentration to 1 × 105/m L, inoculating into 96-well plate with each well being 100 μ L, placing at 37 deg.C and 5% CO₂After the cells are attached to the wall, the supernatant is aspirated, the control group is added with 100 mu L DMEM medium, the L PS model group is added with 100 mu L DMEM medium containing 1 mu g/m L L PS, each drug experiment group is added with 100 mu L DMEM medium containing compounds with various concentrations and 1 mu g/m L L PS, the temperature is 37 ℃, and 5% CO is added₂And (3) incubating for 24 hours, taking 100 mu L of supernatant after the culture is finished, transferring the supernatant into a clean 96-well plate, respectively adding 50 mu L of Griess reagent A liquid and B liquid, developing for 5min in a dark place, measuring the absorbance (OD) value of each well by using a microplate reader at 540nm, and repeating the experiment for 3 times.

The experimental result is shown in figure 1, each coumestan new compound has no cytotoxicity on RAW264.7 cells within the concentration shown in figure 1, compared with a control group, after the RAW264.7 cells are stimulated by L PS, the level of nitric oxide in cell supernatant is obviously increased, and the modeling is proved to be very successful.

Test example 2

Experiment on the effect of coumestan-like novel compounds on the production of TNF- α by SW982 cells stimulated with I L-1 β.

Main experimental materials 9 coumestan-type novel compounds extracted and separated in the above example 1, SW982 cells were purchased from cell banks of Chinese academy of sciences, TNF- α E L ISA kit was purchased from Biolegend, and Fetal Bovine Serum (FBS) and DMEM medium were purchased from Gibco.

The experimental method comprises setting control group, model group I L-1 β, and each drug experimental group, collecting SW982 cells with length of 70% -80%, trypsinizing, blowing into single cell suspension, adding 10% FBS-containing DMEM medium to adjust cell concentration to 2 × 105/m L, inoculating into 96-well plate with concentration of 100 μ L, 37 deg.C, 5% CO in each well₂After the cells are attached to the wall, the supernatant is removed, a blank group is added with 100 mu L DMEM medium, an I L-1L 0 model group is added with 100 mu L DMEM medium containing 10ng/m L I L-1 β, each drug experiment group is added with 100 mu L DMEM medium containing compounds with the concentrations of 10ng/m L I L-1 β, the temperature is 37 ℃, and 5% CO is added₂After incubation for 18 hours, 100. mu. L of the supernatant was transferred to a clean 96-well plate and the TNF- α level in the cell supernatant was determined by the procedure of the E L ISA assay kit as set forth below, and the experiment was repeated 3 times.

a. And (3) preparing the coating antibody freshly, adding 100 mu L of the coating antibody into each detection hole of the E L ISA kit respectively, and carrying out closed incubation at 4 ℃ for 16-18 h.

b. The coated antibody was aspirated and washed by adding 300. mu. L wash buffer to each well and repeated 4 times 200. mu. L1 × AssayDiluent incubation at room temperature for 1 hour.

c. The Assay Diluent was aspirated away and each well was washed with 300. mu. L wash buffer, repeated 4 times, and 100. mu. L cell supernatants or standards were pipetted into each well and incubated for 2 hours at room temperature with blocking.

d. Cell supernatants were aspirated and washed 4 times with 300. mu. L wash buffer per well, 100. mu. L TNF- α Detection Antibody solution per well and incubated for 1 hour at room temperature with blocking.

e. TNF- α Detection Antibody was aspirated and washed 4 times with 300. mu. L wash buffer per well, 100. mu. L Avidin-HRP solution per well and incubated for 30 min at room temperature with blocking.

f. TNF- α Avidin-HRP Solution was aspirated and washed 5 times with 300. mu. L wash buffer per well, 100. mu. L freshly prepared TMB Substrate Solution per well and incubated 15 minutes at room temperature in the dark.

g. Adding 100 mu L reaction stop solution into each well, slightly shaking the detection plate, mixing uniformly, rapidly detecting the absorbance value (OD value) at the wavelength of 450nm in an enzyme-labeled photometer, and drawing a standard curve according to the obtained reading for calculation.

Experimental results the experimental results are shown in figure 2. each of the coumestan-like novel compounds is non-cytotoxic to SW982 cells within the concentrations shown in figure 2. compared to the control group, the levels of TNF- α in the cell supernatants were significantly increased after SW982 cells were stimulated with I L-1 β, demonstrating that the modeling was very successful, but in each of the drug experimental groups, the levels of TNF- α in the cell supernatants of each group were reduced to different degrees after being treated with the coumestan-like novel compounds within the safe concentration range of the compounds, wherein

compounds

1, 2, 9 (i.e., the first compound, the second compound, and the ninth compound) significantly inhibited the production of TNF- α, the second compound of

compounds

3, 5, 6, and 8, and the activity of

compounds

4 and 7 was weak.

Test example 3

Effect of coumestan-like novel compounds on differentiation of RANK L-stimulated RAW264.7 cells into osteoclasts.

The main experimental materials: nine 9 novel compounds of the coumestan class obtained by extraction and separation in the above example 1; RAW264.7 cells were purchased from the chinese academy of sciences cell bank; the tartrate-resistant acid phosphatase (TRAP) detection kit is purchased from pelagin biotechnology; fetal Bovine Serum (FBS) and DMEM media were purchased from Gibco.

The experimental method comprises setting control group, RANK L model group and drug experimental group, taking 70% -80% RAW264.7 cells, scraping cells, blowing into single cell suspension, adding 10% FBS-containing DMEM medium to adjust cell concentration to 2 × 104/m L, inoculating in 96-well plate with each well being 100 μ L, 37 deg.C, 5% CO₂After the cells are attached to the wall, the supernatant is aspirated, the blank group is added with 100 mu L DMEM medium, the RANK L model group is added with 100 mu L DMEM medium containing 50ng/m L RANK L, and the drug experiment group is added with 100 mu L DMEM medium containing eachDMEM medium with compound concentration of 50ng/m L RANK L was replaced every 2 days, cultured for 4-5 days, after induction was completed, the supernatant was aspirated and discarded, PBS was rinsed twice, cell lysis solution (without inhibitor) was added to lyse the cells, the intracellular TRAP activity was measured according to the procedures of the tartrate-resistant acid phosphatase (TRAP) assay kit as set forth below, and the experiment was repeated 3 times.

a. Sample preparation and dilution: the cells are lysed with a cell lysate (without inhibitor), and if the sample contains TRAP with higher activity, the original lysate or PBS can be used for dilution for later use.

b. Reference is made to the following table using a 96 well plate to set up the blank, standard and sample wells, the standards being used in amounts of 4, 8, 16, 24, 32 and 40 microliters respectively, and the samples typically being added directly to 40 μ L.

TABLE 1 Standard for the arrangement of blank control well, Standard well and sample well

	Blank control	Standard article	Sample (I)
				Detection buffer solution	40μL	(80-x)μL	(40-y)μL
Chromogenic substrates	40μL	-	40μL
				Tartaric acid solution	5μL	5μL	5μL
Sample (I)	-	-	yμL
				Working solution for standard substance	-	xμL	-

c. The mixture is lightly blown and beaten by a gun head and is mixed evenly, and can also be mixed evenly by a shaking table.

d.37C for 20 min.

e. The reaction was stopped by adding 160. mu. L of stop solution to each well, at which time the standard or the wells with tartrate acid phosphatase activity showed a different shade of yellow.

f. Absorbance was measured at 405 nm.

g. Definition of acid phosphatase activity units: the amount of acid phosphatase required for hydrolyzing the para-nitrophenyl phosphate chromogenic substrate to produce 1. mu. mol of p-nitrophenol per minute at pH 4.8, 37 ℃ was defined as one unit of enzyme activity.

h. The tartrate-resistant acid phosphatase activity in the sample was calculated according to the enzyme activity definition.

The experimental results are shown in figure 3. each of the novel compounds of coumestan class is not cytotoxic to RAW264.7 cells in the concentrations shown in figure 3. compared with the control group, the activity of TRAP in cells is remarkably increased after the cells of RAW264.7 are stimulated by RANK L, thus proving that the differentiation model of RAW264.7 cells into osteoclasts is successfully established, but in each administration group, in the safe concentration range of the compounds, after the cells of the novel compounds of coumestan class are incubated by 1-2 and 7-9, the activity of TRAEST in each administration group is remarkably reduced, and the effect of TRAEST in each administration group is remarkably reduced, thus showing that the compounds of coumestan class have the most remarkable effect in inhibiting the differentiation of the cells of the coumestan class.

The beneficial effects of the invention are proved by the pharmacodynamic experiments below. Experimental examples 1 to 3 the results are expressed as Mean. + -. SD. # p <0.05, # p <0.01, compared to control; p <0.05, p <0.01, p <0.001 compared to the model group. p <0.05 is statistically significant for the differences.

Test example 4

The 9 compounds prepared in example 1 were characterized, and the physicochemical properties of the first to ninth compounds were tested, and the structures were analyzed by various nuclear magnetic spectra (1H-NMR,13C-NMR, HMBC, HSQC, NOESY) and HR-ESI-MS. The physicochemical properties and the spectral data of the compounds are as follows:

a first compound: white needle crystal; HR-ESI-MS M/z 4417.16876[ M-H ] - (calcd. forC26H25O5, 417.16965); NMR (600MHz, DMSO-d6) data are shown in Table 2; 1H-NMR spectrum (600MHz, DMSO-d6), 13C-NMR spectrum (150MHz, DMSO-d 6); HSQC spectrum (600MHz, DMSO-d6) and HMBC spectrum (600MHz, DMSO-d6) are shown in FIGS. 4-7, which are not reported in literature and are named as new compounds, i.e., glytacoumestan A.

A second compound: a light colored solid; HR-ESI-MS M/z 403.15323[ M-H ] - (calcd for C25H23O5,403.15400); NMR (600MHz, DMSO-d6) data are shown in Table 2; 1H-NMR spectrum (600MHz, CD3OD), 13C-NMR spectrum (150MHz, DMSO-d 6); HSQC spectrum (600MHz, DMSO-d6) and HMBC spectrum (600MHz, DMSO-d6) are shown in FIG. 8-11, and are not reported in literature, and are new compounds named glytacoumestan B.

A third compound: a light yellow solid; HR-ESI-MS M/z 417.16968[ M + H ] + (calcd for C26H25O5, 417.169965); NMR (600MHz, CDCl3) data are shown in Table 2; 1H-NMR spectrum (600MHz, CDCl3), 13C-NMR spectrum (150MHz, CDCl 3); HSQC spectrum (600MHz, CDCl3) and HMBC spectrum (600MHz, CDCl3) are shown in FIGS. 12-15, and are not reported in literature, and are new compounds named glytacoumestan C.

A fourth compound: a white solid; HR-ESI-MS M/z 401.13763[ M-H ] - (calcd for C25H21O5,401.13835); NMR (600MHz, DMSO-d6) data are shown in Table 3; 1H-NMR spectrum (600MHz, DMSO-d6), 13C-NMR spectrum (150MHz, DMSO-d 6); HSQC spectra (600MHz, DMSO-D6) and HMBC spectra (600MHz, DMSO-D6) are shown in FIGS. 16-19, and are not reported in the literature as new compounds, named glytacoumestan D.

A fifth compound: a light colored solid; HR-ESI-MS M/z 433.16696[ M + H ] + (calcd for C26H25O6,433.16456); NMR (600MHz, CDCl3) data are shown in Table 3; 1H-NMR spectrum (600MHz, CDCl3), 13C-NMR spectrum (150MHz, CDCl 3); HSQC spectrum (600MHz, CDCl3) and HMBC spectrum (600MHz, CDCl3) are shown in FIGS. 20-23, and are not reported in literature, and are new compounds named glytacoumestan E. Fifth compound resolution: the molecular formula of the compound is deduced to be C26H26O7 according to HR-ESI-MS data, and the unsaturation degree is 17. The 1H NMR spectrum (table 3) shows two pairs of ortho-coupled aromatic protons, respectively assigned to a-ring [ H7.86 (1H, d, J ═ 9.0Hz, H-1), 7.00(1H, d, J ═ 9.0Hz), H-2) ] and B-ring [ H7.75 (1H, d, J ═ 8.4Hz, H-7), 6.91(1H, d, J ═ 8.4Hz, H-8) ]. In addition, the characteristic carbon signals of the Coumestan mother nucleus at C-6 (C158.3), C-6a (C104.1) and C-11a (C160.3) were observed in the 13C NMR spectrum, indicating that the fifth compound is a Coumestan derivative compound. Two olefinic bimodal signals [ H5.80 (1H, d, J ═ 9.6Hz), 6.87(1H, d, J ═ 9.6Hz) ], two methyl signals [ H1.51 (6H, s) ] and an oxidized quaternary carbon signal (C77.1) were observed by 1HNMR and 13C NMR, revealing the presence of a dimethylpyran ring adjacent to the B ring. Also, a 2, 3-dihydroxy-3-methylbutyl group [ H3.08 (1H, dd, J ═ 13.8 and 10.2Hz, Ha-1'),3.20(1H, dd, J ═ 13.8 and 2.4Hz, Hb-1'),3.71(1H, dd, J ═ 10.2 and 2.4Hz, H-2'),1.37(6H, s, H-4' and H-5 '); c26.3 (C-1'),78.6(C-2'),73.2(C-3'),26.3(C-4'),24.1(C-5') ], the presence of one methoxy group [ H3.98 (3H, s), C56.5 ]. In HMBC spectra, the methylene proton H-1' has a correlation with C-3(C160.2), C-4 (C116.6) and C-4a (C152.8), and the methoxy proton (H3.98) has a correlation with C-3(C160.2), revealing that the two groups are substituted at the C-4 and C-3 positions of the A ring, respectively.

A sixth compound: a white solid; HR-ESI-MS M/z 433.16541[ M + H ] + (calcd for C26H25O6,433.16456); NMR (600MHz, CD3OD) data are shown in Table 3; 1H-NMR spectrum (600MHz, CD3OD), 13C-NMR spectrum (150MHz, CD3 OD); the HSQC spectrum (600MHz, CD3OD) and the HMBC spectrum (600MHz, CD3OD) are shown in FIGS. 24-27, and are not reported in the literature and are named as novel compounds, namely glytacoumestan F.

A seventh compound: a light yellow solid; HR-ESI-MS M/z 419.15118[ M + H ] + (calcd for C25H23O6, 419.14891); NMR (600MHz, CDCl3) data are shown in Table 4; 1H-NMR spectrum (600MHz, CDCl3), 13C-NMR spectrum (150MHz, CDCl 3); HSQC spectrum (600MHz, CDCl3) and HMBC spectrum (600MHz, CDCl3) are shown in FIGS. 28-31, and are not reported in literature, and are new compounds named glytacoumestan G.

An eighth compound: a light yellow solid; HR-ESI-MS M/z 367.08096[ M-H ] - (calcd for C2H 10H 15O7, 367.08123); NMR (600MHz, CD3OD) data are shown in Table 4; 1H-NMR spectrum (600MHz, CD3OD), 13C-NMR spectrum (150MHz, CD3 OD); the HSQC spectrum (600MHz, CD3OD) and the HMBC spectrum (600MHz, CD3OD) are shown in FIGS. 32-35, and are not reported in the literature and are named as novel compounds, namely glytacoumestan H.

A ninth compound: a white powder; HR-ESI-MS M/z 353.10162[ M-H ] - (calcd for C20H17O6,353.10196); NMR (600MHz, CD3OD) data are shown in Table 4; 1H-NMR spectrum (600MHz, CD3OD), 13C-NMR spectrum (150MHz, CD3 OD); HSQC spectrum (600MHz, CD3OD) and HMBC spectrum (600MHz, CD3OD) are shown in FIGS. 36-39, and are not reported in literature and are named as new compounds, i.e., glytacoumestan I.

TABLE 2 NMR data for novel compounds 1 to 3 proposed by the present invention

TABLE 3 NMR data on novel compounds 4 to 6 proposed by the present invention

TABLE 4 NMR data for novel compounds 7 to 9 proposed by the invention

In conclusion, the inventor continuously researches and discovers a novel compound with the effect of resisting rheumatoid arthritis, can effectively inhibit the generation of nitric oxide stimulated by L PS, has strong in-vitro anti-inflammatory activity, and has good capability of relieving the synovitis of the rheumatoid arthritis.

The invention provides a preparation method of a compound, which extracts an active ingredient from a complex ingredient composition of tobacco beans by extracting the compound from the tobacco beans. The invention also provides the application of the compound and a medicament taking the compound as an active ingredient, and the compound has good market application value.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. A compound having the formula:

2. the compound of claim 1, wherein the compound is derived from tobacco beans.

3. A process for the preparation of a compound according to claim 1 or 2, comprising the steps of:

extracting a compound as claimed in claim 1 or 2 from tobacco beans.

4. The method of claim 3, comprising the steps of:

preparing an extract: crushing tobacco beans, then carrying out alcohol extraction and concentrating to obtain alcohol extract;

solvent extraction: mixing the alcohol extract with water, and sequentially extracting with petroleum ether and ethyl acetate to obtain a petroleum ether extraction part and an ethyl acetate extraction part;

chromatography segmentation: carrying out gradient elution on the ethyl acetate extraction part to obtain 11 elution parts;

and (3) purification and separation: purifying with a ninth eluate fraction to obtain the compound;

preferably, the extracted compound is characterized by nuclear magnetic spectrum and mass spectrum after purification and separation to identify the structure of the compound; more preferably, the nuclear magnetic spectrum is selected from¹H-NMR、¹³C-NMR, HMBC, HSQC or NOESY; more preferably, the mass spectrum is selected from HR-ESI-MS or ESI-MS.

5. The method according to claim 4, wherein the step of purifying the compound using the ninth elution portion comprises: performing silica gel column chromatography on the ninth elution part, and performing gradient elution by using a petroleum ether-acetone system to obtain seven elution fractions 9A-9G;

subjecting the fraction 9E to Sephadex L H-20 column chromatography, detecting eluate with T L C, mixing to obtain three fractions 9E1-9E3, subjecting the fraction 9E3 to Toyopeal HW-40 column chromatography to obtain three fractions 9E3A-9E3C, subjecting the fraction 9E3B to preparative liquid chromatography to obtain target compound and impurity compound;

the structural formula of the impurity compound is:

preferably, when gradient elution is performed with a petroleum ether-acetone system, the ratio of petroleum ether: the elution gradient of acetone is 10:1, 6:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1: 2;

preferably, the liquid chromatography is performed under conditions of using an 18C-reverse phase chromatography column at a flow rate of 7-9m L/min, more preferably, the eluent corresponding to the target compound is a mixture of methanol and water at a methanol to water volume ratio of 70:25-35, and more preferably, the eluent corresponding to the impurity compound is a mixture of methanol and water at a methanol to water volume ratio of 70: 25-35.

6. The preparation method according to claim 4, wherein the extract is prepared by extracting for at least 3 times with 90-98% ethanol solvent, mixing extractive solutions, filtering, and concentrating under reduced pressure; preferably, the material-liquid ratio during extraction is 1: 5-10; more preferably, the extraction method is cold soaking extraction, heating reflux extraction or ultrasonic assisted extraction.

7. The method according to claim 4, wherein the solvent extraction is carried out by using petroleum ether, ethyl acetate and water saturated n-butanol to obtain petroleum ether extraction part, ethyl acetate extraction part, n-butanol extraction part and water extraction part; .

8. The preparation method according to claim 4, wherein the chromatographic fractionation process is a fractionation process using a silica gel chromatographic column, and gradient elution is performed using a petroleum ether-ethyl acetate system;

preferably, when gradient elution is performed with a petroleum ether-ethyl acetate system, the ratio of petroleum ether: elution gradients of ethyl acetate were 10:1, 5:1, 3:1, 2:1, 1:2, and 1: 5;

preferably, the eluent for the Sephadex L H-20 column chromatography is a mixed solution of dichloromethane and methanol, more preferably, the volume ratio of dichloromethane to methanol is 1: 0.5-1.5;

preferably, the eluent for Toyopeal HW-40 column chromatography is methanol.

9. Use of a compound according to claim 1 or 2 or a compound prepared by a process according to any one of claims 3 to 8 for the preparation of a medicament for the prophylaxis or treatment of inflammatory diseases;

preferably, the inflammatory disease is rheumatoid arthritis.

10. A medicament against rheumatoid arthritis, characterized in that the active ingredient comprises a compound according to claim 1 or 2 or a compound prepared by the preparation method according to any one of claims 3 to 8;

preferably, the medicament is prepared into pills, granules, tablets, sachets or injections.