Chromene type meroterpenoid compound as well as extraction method and application thereof

Abstract

The invention discloses chromene type meroterpenoids as well as an extraction method and application thereof, belongs to the field of traditional Chinese medicine extraction, and particularly relates to chromene type meroterpenoids which are separated from rhododendron dauricum and are shown in general formulas (I)-(IV) and an extraction method of the chromene type meroterpenoids, and also provides the chromene type meroterpenoids or isomers of the chromene type meroterpenoids. The compound, or a pharmaceutically acceptable salt of the compound or a pharmaceutical composition containing the compound has an inhibition effect on inflammation, and can be used for preparing anti-inflammatory drugs. The invention further enriches the structural diversity of active substances of rhododendron dauricum, lays a foundation for relevant biological activity tests of subsequent monomeric compounds, provides an active lead compound for new drug development, and also provides a theoretical basis for deep research and development of rhododendron dauricum medicinal materials. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine extraction, and particularly relates to a chromene-type diterpenoid compound isolated from Rhododendron dauricum, an extraction method thereof, and its application in the preparation of anti-inflammatory drugs. Background Art

[0002] Rhododendron dauricum L., also known as Dazixiang, is a semi-evergreen shrub belonging to the genus Rhododendron in the family Ericaceae. It is generally 1 to 2 meters tall and has the characteristic of multi-branching. Its leaves are alternate, nearly leathery, elliptical to ovate-elliptical in shape, about 1.5 to 3.5 centimeters long, and the margin is entire. The flowers of Rhododendron dauricum usually have 1 to 4 flowers at the top of the branches. The corolla is funnel-shaped, red-purple in color, and about 2.5 to 3.5 centimeters in diameter. The flowering period is generally from May to June, and the fruit ripening period is from July to August. It not only has high ornamental value and landscape application value, but also has high medicinal value. It is mainly distributed in Heilongjiang, Liaoning, Jilin, Inner Mongolia and other places. It is recorded in the Chinese Pharmacopoeia 2020 Edition that the dried leaves of Rhododendron dauricum are "Manshanhong", pungent in taste and cold in nature; it belongs to the lung and spleen meridians, and has the effects of relieving cough and reducing phlegm and anti-inflammatory. It is used as a traditional medicine to treat diseases such as asthma, acute and chronic bronchitis, diabetes, cough with excessive phlegm, cancer and hypertension (National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China: Volume I [M]. Beijing: China Medical Science and Technology Press, 2020: 387.). The chemical constituents of Rhododendron dauricum are mainly diterpenoids, triterpenoids, flavonoids and polyphenolic compounds. Chromene-type meroterpenoids with polycyclic skeletons have also attracted more attention (Xie Yangyang. Study on the Chemical Constituents of Rhododendron dauricum [D]. Huazhong University of Science and Technology, 2014.; Ye C, Jin M, Li R, Sun J, Li G. Phytochemical and chemotaxonomic study on the leaves of Rhododendron dauricum L [J]. Biochemical Systematics and Ecology, 2020, 90: 104038.). According to literature reports, chromene-type meroterpenoids isolated from plants of the genus Rhododendron have good anti-inflammatory (Shi Q, Li T T, Wu Y M, Sun X, Hou A J. Meroterpenoids with diverse structures and anti-inflammatory activities from Rhododendron anthopogonoides [J]. Phytochemistry, 2020, 180: 112524.), PTP1B inhibitory (Liang C, Kjaerulff L, Hansen P R, Kongstad K T, Staerk D.Dual high-resolution α-glucosidase and PTP1B inhibition profiling combined with HPLC-PDA-HRMS-SPE-NMR analysis for the identification of potentially antidiabetic chromene meroterpenoids from Rhododendron capitatum[J].Journal of Nature Products,2021,84(9):2454-2467.) and antiviral (Kashiwada Y, Yamazaki K, Ikeshiro Y, Yamagishi T, Fujioka T, Mihashi K, Mizuki K, Cosentino L M, Fowke K, Morris-Natschke S L, Lee K H. Isolation of rhododaurichromanic acid B and the anti-HIV principles rhododaurichromanic acid A and rhododaurichromenic acid from Rhododendron dauricum[J]. Tetrahedron,2001,57(8):1559-1563.) activities. To maximize the medicinal value of Rhododendron dauricum, a systematic study of the components of Rhododendron dauricum branches and leaves was carried out, new chromene meroterpenoids were extracted, the structures of the compounds were confirmed by means of nuclear magnetic resonance, infrared, mass spectrometry, etc., and the inhibitory effects of the extracted compounds on the production of NO in LPS-induced RAW264.7 cells were detected. Summary of the Invention

[0003] The primary object of the present invention is to provide a chromene meroterpenoid compound.

[0004] The second object of the present invention is to provide a method for extracting a chromene meroterpenoid compound.

[0005] The third object of the present invention is to provide a pharmaceutical composition containing a chromene meroterpenoid compound.

[0006] The fourth object of the present invention is to provide the use of a chromene meroterpenoid compound or an isomer of the compound, or a pharmaceutically acceptable salt of the compound or a pharmaceutical composition containing the compound in the preparation of an anti-inflammatory drug.

[0007] The technical solution of the present invention includes the following:

[0008] A chromene-type diterpenoid compound, which is any one of the compounds represented by the general formula (Ⅰ)-(IV), or an isomer of the compound, or a pharmaceutically acceptable salt of the compound.

[0009]

[0010] Wherein: R 1 and R 2 are each independently selected from H, hydroxyl, methoxy, acetoxy, carboxyl, R 3 is α-methyl or β-methyl, R 4 and R 5 are each independently α-H or β-H, R 6 and R 7 are each independently H, α-methyl or β-methyl, α-hydroxyl or β-hydroxyl, α-methoxy or β-methoxy, R 8 and R 10 are each independently H, α-hydroxyl or β-hydroxyl, α-methoxy or β-methoxy, R 9 is H or keto carbonyl, R 11 is H, α-hydroxyl or β-hydroxyl; in the general formula (I)-(IV), the dotted line indicates that this position can be a double bond structure.

[0011] A chromene-type diterpenoid compound described in the present invention is any one of the compounds shown in the following structural formulas 1-9, or an isomer of the compound, or a pharmaceutically acceptable salt of the compound;

[0012]

[0013] The pharmaceutically acceptable salts described in the present invention include: sodium salts, potassium salts, ammonium salts, hydrochloride salts and sulfate salts.

[0014] The isomers described in the present invention include: optical isomers, cis-trans isomers, racemates and mixtures thereof.

[0015] The present invention also provides a method for extracting chromene-type diterpenoid compounds, including the following steps:

[0016] (1) Using the branches and leaves of Rhododendron dauricum as raw materials, adding an ethanol aqueous solution, refluxing and extracting 2-4 times, each extraction for 2-4 hours, combining to obtain an extraction solution, recovering the solvent under reduced pressure, and concentrating to obtain a total extract;

[0017] (2) Disperse the total extract into 5-10 times the volume of water by mass, extract successively with 5-10 times the volume of petroleum ether and ethyl acetate by mass, recover the solvent, and obtain a petroleum ether extraction concentrate, an ethyl acetate extraction concentrate and an aqueous phase respectively;

[0018] (3) The petroleum ether extraction concentrate is separated by silica gel column chromatography, and eluted with a gradient of petroleum ether - ethyl acetate with a volume ratio of 30:1 - 0:1 as the eluent, and the fraction PE5 with a volume ratio of 5:1 - 1:1 is collected; the ethyl acetate extraction concentrate is separated by silica gel column chromatography, and eluted with a gradient of dichloromethane - methanol with a volume ratio of 100:1 - 0:1 as the eluent, and the fraction E2 with a volume ratio of 90:1 - 80:1 is collected;

[0019] (4) Concentrate the fraction PE5, and the concentrate is separated by gel column chromatography to obtain the chromene - type diterpenoids 5, 6, 7, 8 and 9 through purification; concentrate the fraction E2, and the concentrate is separated by gel column chromatography to obtain the chromene - type diterpenoids 1, 2, 3 and 4 through purification.

[0020] The above extraction method, wherein:

[0021] In the said step (1), an ethanol aqueous solution with a volume concentration of 70% - 95% and 8 - 15 times the volume of the raw material is added for extraction. The 8 - 15 times the volume of the raw material specifically means that the volume - mass ratio of the added ethanol aqueous solution to the raw material is (8 - 15):1 in mL / g.

[0022] In the said step (4), the specific separation and purification process is as follows:

[0023] After the fraction E2 is concentrated, it is separated by MCI column chromatography, and eluted with a gradient of methanol - water with a volume ratio of 10:90 - 100:0 as the eluent, and the fraction with a volume ratio of 50:50 - 70:30 is collected, denoted as E22; the fraction with a volume ratio of 80:20 is collected, denoted as E23;

[0024] After the fraction E22 is concentrated, it is separated by ODS column chromatography, and eluted with a gradient of methanol - water with a volume ratio of 40:60 - 100:0 as the eluent, and the fraction with a volume ratio of 70:30 - 80:20 is collected, denoted as E222;

[0025] After the fraction E222 is concentrated, it is separated by silica gel column chromatography, and eluted with a gradient of petroleum ether - acetone with a volume ratio of 60:1 - 0:1 as the eluent, and the fraction with a volume ratio of 40:1 - 20:1 is collected, denoted as E2222;

[0026] After the fraction E2222 is concentrated, it is separated by Sephadex LH - 20 column chromatography, and eluted with methanol as the eluent, and the fraction is collected, denoted as E22221;

[0027] After the fraction E22221 is concentrated, it is purified by preparative HPLC chromatography with acetonitrile - water with a volume ratio of 60:40 as the mobile phase to obtain compounds 3 and 4;

[0028] Fraction E23 was concentrated and then separated by silica gel column chromatography. Petroleum ether - acetone with a volume ratio of 60:1 - 0:1 was used as the eluent for gradient elution. The fraction with a volume ratio of 40:1 - 20:1 was collected and denoted as E232;

[0029] Fraction E232 was concentrated and then separated by ODS column chromatography. Methanol - water with a volume ratio of 70:30 - 100:0 was used as the eluent for gradient elution. The fraction with a volume ratio of 70:30 - 80:20 was collected and denoted as E2321;

[0030] Fraction E2321 was concentrated and then purified by preparative HPLC chromatography using acetonitrile - water with a volume ratio of 65:35 as the mobile phase to obtain compounds 1 and 2;

[0031] Fraction PE5 was concentrated and then separated by silica gel column chromatography. Petroleum ether - ethyl acetate with a volume ratio of 10:1 - 0:1 for elution was used as the eluent for gradient elution. The fraction with a volume ratio of 3:1 - 1:1 was collected and denoted as PE54;

[0032] Fraction PE54 was concentrated and then separated by ODS column chromatography. Methanol - water with a volume ratio of 70:30 - 100:0 was used as the eluent for gradient elution. The fraction with a volume ratio of 70:30 - 80:20 was collected and denoted as PE543;

[0033] Fraction PE543 was concentrated and then purified by preparative HPLC chromatography using methanol - water with a volume ratio of 60:40 as the mobile phase to obtain PE5432 and PE5434;

[0034] Fraction PE5432 was concentrated and then purified by preparative HPLC chromatography using acetonitrile - water with a volume ratio of 50:50 as the mobile phase to obtain compounds 6, 7, 8 and 9;

[0035] Fraction PE5434 was concentrated and then purified by preparative HPLC chromatography using acetonitrile - water with a volume ratio of 50:50 as the mobile phase to obtain compound 5.

[0036] The chromene - type meroterpenoids in the present invention are extracts from the branches and leaves of Rhododendron dauricum.

[0037] Use of the extract from the branches and leaves of Rhododendron dauricum in the present invention for preparing anti - inflammatory drugs.

[0038] A pharmaceutical composition, which comprises one or more of the chromene - type meroterpenoids described in the present invention, isomers of the compound, and pharmaceutically acceptable salts of the compound; and also comprises one or a combination of pharmaceutically acceptable carriers, excipients, and diluents. The administration route of the pharmaceutical composition is oral or injection, and the dosage forms include: tablets, capsules, powders, syrups, and injections.

[0039] The present invention also provides the use of the chromene-type diterpenoids, isomers of the compound, pharmaceutically acceptable salts of the compound, or the pharmaceutical composition in the preparation of anti-inflammatory drugs.

[0040] Advantages of the present invention:

[0041] The present invention relates to a chromene-type diterpenoid or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound. Based on the inhibitory effect of these compounds on the production of NO in LPS-induced RAW264.7 cells, it can be applied to the preparation of anti-inflammatory drugs. This discovery not only further enriches the structural diversity of the active substances in Rhododendron dauricum, but also lays a foundation for subsequent biological activity tests of monomeric compounds, provides active lead compounds for new drug development, and also provides a theoretical basis for the in-depth research and development of Rhododendron dauricum medicinal materials. Description of the Drawings

[0042] Figure 1 It is the inhibitory activity of Compounds 1-9 in the present invention on the production of NO in LPS-induced RAW264.7. Detailed Embodiments

[0043] The technical solutions of the present invention will be further described below in conjunction with specific embodiments. The specific embodiments and examples described herein are only used to illustrate the present invention in detail, and do not limit the present invention. For those steps or conditions not specified in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be used. For the reagents and equipment not specified by the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0044] Example 1

[0045] The extraction method of chromene-type diterpenoids from Rhododendron dauricum includes the following steps:

[0046] (1) Using the branches and leaves of Rhododendron dauricum with a total dry weight of 12.9 kg as raw materials, adding an 8-fold volume of 95% ethanol aqueous solution (110 L) based on the volume of the raw materials, refluxing and extracting 2 times, each extraction for 2 hours, combining the obtained extraction solutions, recovering the solvent under reduced pressure, and concentrating to obtain a total extract of the 95% ethanol layer (2.3 kg);

[0047] (2) Disperse the obtained total extract into 5-fold volume of water (12 L), and extract successively with 5-fold volume of petroleum ether and ethyl acetate. The extraction solutions are concentrated to recover the solvent, and a petroleum ether extraction concentrate (400 g), an ethyl acetate extraction concentrate (560 g), and an aqueous phase are obtained respectively;

[0048] (3) The petroleum ether extraction concentrate was separated by silica gel column chromatography, and gradient elution was carried out with petroleum ether - ethyl acetate with a volume ratio of 30:1 - 0:1 as the eluent, and the fraction PE5 with a volume ratio of 5:1 - 1:1 was collected; the ethyl acetate extraction concentrate was separated by silica gel column chromatography, and gradient elution was carried out with dichloromethane - methanol with a volume ratio of 100:0 - 0:1 as the eluent, and the fraction E2 with a volume ratio of 90:1 - 80:1 was collected;

[0049] (4) The fraction PE5 was concentrated, and the concentrate was separated by gel column chromatography to further purify chromene - type meroterpenoids 5, 6, 7, 8 and 9; the fraction E2 was concentrated, and the concentrate was separated by gel column chromatography to further purify chromene - type meroterpenoids 1, 2, 3 and 4. The specific separation and purification process is as follows:

[0050] After the fraction E2 was concentrated, it was separated by MCI column chromatography, and gradient elution was carried out with methanol - water with a volume ratio of 10:90 - 100:0 as the eluent, and the fraction with a volume ratio of 50:50 - 70:30 was collected, denoted as E22; the fraction with a volume ratio of 80:20 was collected, denoted as E23;

[0051] After the fraction E22 was concentrated, it was separated by ODS column chromatography, and gradient elution was carried out with methanol - water with a volume ratio of 40:60 - 100:0 as the eluent, and the fraction with a volume ratio of 70:30 - 80:20 was collected, denoted as E222;

[0052] After the fraction E222 was concentrated, it was separated by silica gel column chromatography, and gradient elution was carried out with petroleum ether - acetone with a volume ratio of 60:1 - 0:1 as the eluent, and the fraction with a volume ratio of 40:1 - 20:1 was collected, denoted as E2222;

[0053] After the fraction E2222 was concentrated, it was separated by Sephadex LH - 20 column chromatography, and methanol was used as the eluent to collect the fraction, denoted as E22221;

[0054] After the fraction E22221 was concentrated, it was purified by preparative HPLC chromatography with acetonitrile - water with a volume ratio of 60:40 as the mobile phase to obtain 3.2 mg of compound 3 and 2.5 mg of compound 4;

[0055] After the fraction E23 was concentrated, it was separated by silica gel column chromatography, and gradient elution was carried out with petroleum ether - acetone with a volume ratio of 60:1 - 0:1 as the eluent, and the fraction with a volume ratio of 40:1 - 20:1 was collected, denoted as E232;

[0056] After the fraction E232 was concentrated, it was separated by ODS column chromatography, and gradient elution was carried out with methanol - water with a volume ratio of 70:30 - 100:0 as the eluent, and the fraction with a volume ratio of 70:30 - 80:20 was collected, denoted as E2321;

[0057] After concentration, fraction E2321 was purified by preparative HPLC chromatography using acetonitrile - water with a volume ratio of 65:35 as the mobile phase, to obtain 4.3 mg of compound 1 and 2.9 mg of compound 2;

[0058] After concentration, fraction PE5 was separated by silica gel column chromatography and eluted with a gradient of petroleum ether - ethyl acetate with a volume ratio of 10:1 - 0:1 as the eluent, and the fractions with a volume ratio of 3:1 - 1:1 were collected and denoted as PE54;

[0059] After concentration, fraction PE54 was separated by ODS column chromatography and eluted with a gradient of methanol - water with a volume ratio of 70:30 - 100:0 as the eluent, and the fractions with a volume ratio of 70:30 - 80:20 were collected and denoted as PE543;

[0060] After concentration, fraction PE543 was purified by preparative HPLC chromatography using methanol - water with a volume ratio of 60:40 as the mobile phase, to obtain PE5432 and PE5434;

[0061] After concentration, fraction PE5432 was purified by preparative HPLC chromatography using acetonitrile - water with a volume ratio of 50:50 as the mobile phase, to obtain 6.2 mg of compound 6, 5.1 mg of compound 7, 5.6 mg of compound 8 and 8.8 mg of compound 9;

[0062] After concentration, fraction PE5434 was purified by preparative HPLC chromatography using acetonitrile - water with a volume ratio of 50:50 as the mobile phase, to obtain 3.2 mg of compound 5.

[0063] The chromene - type diterpenoids 1 - 9 extracted were subjected to structure identification, and the specific physicochemical data are as follows:

[0064] Compound 1: Yellow oil, (c = 0.46, MeOH), HRESIMS(negative) m / z 339.1969 [M - H] - (calcd for C 22 H 27 O 3 , 339.1960), and the molecular formula of compound 1 was determined to be C 22 H 28 O 3 , 1 H - NMR(600 MHz, CDCl 3 ) and 13 C - NMR(150 MHz, CDCl 3 ) data are shown in Table 1.

[0065] Compound 2: Yellow oil, (c = 0.30, MeOH), HRESIMS (negative) m / z 339.1969 [M-H] - (calcd for C 22 H 27 O 3 , 339.1960), determining the molecular formula of Compound 2 as C 22 H 28 O 3 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 )) data are shown in Table 1.

[0066] Compound 3: Yellow oil, (c = 0.20, MeOH), HRESIMS (negative) m / z 341.2110 [M-H] - (calcd for C 22 H 27 O 3 , 341.2117), determining the molecular formula of Compound 3 as C 22 H 30 O 3 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) data are shown in Table 2.

[0067] Compound 4: Yellow oil, (c = 0.60, MeOH), HRESIMS (negative) m / z 341.2110 [M-H] - (calcd for C 22 H 29 O 3 , 341.2117), determining the molecular formula of Compound 4 as C 22 H 30 O 3 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 )) data are shown in Table 2.

[0068] Compound 5: Yellow oil, (c = 0.29, MeOH), HRESIMS (negative) m / z 359.2222 [M-H] - (calcd for C 22 H 31 O 4 , 359.2220), determining the molecular formula of compound 5 as C 22 H 32 O 4 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) data are shown in Table 2.

[0069] Compound 6: Yellow oil, (c = 0.30, MeOH), HRESIMS (negative) m / z 357.2070 [M-H] - (calcd for C 22 H 29 O 4 , 357.2066), determining the molecular formula of compound 6 as C 22 H 30 O 4 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) data are shown in Table 3.

[0070] Compound 7: Yellow oil, (c = 0.21, MeOH), HRESIMS (negative) m / z m / z 357.2070 [M-H] - (calcd for C 22 H 29 O 4 , 357.2066), determining the molecular formula of compound 7 as C 22 H 30 O 4 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) data are shown in Table 3.

[0071] Compound 8: Yellow oil, (c = 0.25, MeOH), HRESIMS (negative) m / z 357.2070 [M-H] - (calcd for C 22 H 29 O 4 , 357.2066), determining the molecular formula of compound 8 as C 22 H 30 O 4 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 )) data are shown in Table 4.

[0072] Compound 9: Yellow oil, (c = 0.55, MeOH), HRESIMS (negative) m / z 357.2070 [M-H] - (calcd for C 22 H 29 O 4 , 357.2066), determining the molecular formula of compound 9 as C 22 H 30 O 4 , 1 H-NMR (600 MHz, CDCl 3 ) and 13 C-NMR (150 MHz, CDCl 3 ) data are shown in Table 4.

[0073] Table 1 Carbon and hydrogen spectrum data of compounds 1 - 2

[0074]

[0075]

[0076] Table 2 Carbon and hydrogen spectrum data of compounds 3 - 5

[0077]

[0078]

[0079] Table 3 Carbon and hydrogen spectrum data of compounds 6 - 7

[0080]

[0081] Table 4 Carbon and hydrogen spectrum data of compounds 8 - 9

[0082]

[0083]

[0084] By means of physicochemical data and modern spectroscopy (HRESIMS and NMR), combined with the relevant data in the public literature, the structures of the above compounds were identified, and it was determined that Compounds 1-9 are all new compounds not reported in the literature.

[0085]

[0086] Example 2

[0087] The extraction method of chromene-type diterpenoids from Rhododendron dauricum L. includes the following steps:

[0088] (1) Using the branches and leaves of Rhododendron dauricum L. with a total dry weight of 15 kg as raw materials, adding an ethanol aqueous solution with a volume concentration of 95% (120 L) at 8 times the volume of the raw materials, refluxing and extracting 2 times, each extraction for 2 hours, combining the obtained extraction solutions, recovering the solvent under reduced pressure, and concentrating to obtain a total extract of the 95% ethanol layer (2.5 kg);

[0089] (2) Dispersing the obtained total extract into water (13 L) at 5 times the volume, successively extracting with petroleum ether and ethyl acetate at 5 times the volume, concentrating the extraction solutions to recover the solvents, and obtaining a petroleum ether extraction concentrate (464 g), an ethyl acetate extraction concentrate (600 g) and an aqueous phase respectively;

[0090] (3) The petroleum ether extraction concentrate was separated by silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate with a volume ratio of 30:1 - 0:1, and fractions PE5 with a volume ratio of 5:1 - 1:1 were collected; the ethyl acetate extraction concentrate was separated by silica gel column chromatography, eluted with a gradient of dichloromethane-methanol with a volume ratio of 100:0 - 0:1, and fractions E2 with a volume ratio of 90:1 - 80:1 were collected;

[0091] (4) Concentrating fraction PE5, the concentrated solution was separated by gel column chromatography to further purify to obtain 4.0 mg of Compound 5, 7.4 mg of Compound 6, 5.9 mg of Compound 7, 6.4 mg of Compound 8 and 9.5 mg of Compound 9; concentrating fraction E2, the concentrated solution was separated by gel column chromatography to further purify to obtain 6.0 mg of Compound 1, 4.6 mg of Compound 2, 3.9 mg of Compound 3 and 3.0 mg of Compound 4. The specific separation and purification process is the same as that in Example 1.

[0092] Example 3

[0093] The extraction method of chromene-type diterpenoids from Rhododendron dauricum L. includes the following steps:

[0094] (1) Using the branches and leaves of Rhododendron dauricum with a total dry weight of 20 kg as raw materials, add 8 times the volume of 95% ethanol aqueous solution (160 L) by volume of the raw materials, reflux and extract 2 times, each extraction for 2 hours, combine to obtain the extract, recover the solvent under reduced pressure, and obtain the total extract of the 95% ethanol layer (3.125 kg) after concentration;

[0095] (2) Disperse the obtained total extract into 5 times the volume of water (16 L), extract successively with 5 times the volume of petroleum ether and ethyl acetate, concentrate the extract to recover the solvent, and obtain the concentrated petroleum ether extract (620 g), the concentrated ethyl acetate extract (750 g) and the aqueous phase respectively;

[0096] (3) The concentrated petroleum ether extract is separated by silica gel column chromatography, eluted with petroleum ether-ethyl acetate with a volume ratio of 30:1 - 0:1 as the eluent gradient, and collect the fractions PE5 with a volume ratio of 5:1 - 1:1; the concentrated ethyl acetate extract is separated by silica gel column chromatography, eluted with dichloromethane-methanol with a volume ratio of 100:0 - 0:1 as the eluent gradient, and collect the fractions E2 with a volume ratio of 90:1 - 80:1;

[0097] (4) Concentrate the fraction PE5, and the concentrated solution is separated by gel column chromatography to further purify to obtain 5.4 mg of compound 5, 9.9 mg of compound 6, 7.9 mg of compound 7, 8.5 mg of compound 8 and 12.7 mg of compound 9; concentrate the fraction E2, and the concentrated solution is separated by gel column chromatography to further purify to obtain 8.1 mg of compound 1, 6.2 mg of compound 2, 5.3 mg of compound 3 and 4.0 mg of compound 4. The specific separation and purification process is the same as in Example 1.

[0098] Test the inhibitory effects of the chromene-type diterpenoids 1 - 9 obtained by extraction on the production of NO in LPS-induced RAW264.7 cells, as follows:

[0099] (1) Cell culture

[0100] Culture mouse mononuclear macrophage RAW264.7 in DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin sodium, and incubate and grow in a constant temperature incubator at 37 °C and 5% CO 2 2.

[0101] (2) Use the Griess method to detect the effects of compounds 1 - 9 on inhibiting the release of nitric oxide (NO) from lipopolysaccharide-induced mouse RAW264.7 macrophages.

[0102] 1. Principle: Excessive LPS will induce the activation and expression of nitric oxide synthase (NOS) in macrophages to produce NO. And NO reacts rapidly with oxygen free radicals to form NO 2- , the formed NO 2 - can be quantitatively detected by Griess Reagent under acidic conditions. First, NO 2 - undergoes a diazotization reaction with sulfanilamide, then a coupling reaction with N-1-naphthylethylene-diamine dihydrochloride to form a purple-red azo compound. Finally, using an enzyme-linked immunosorbent assay (ELISA) reader, the absorbance value is measured at 540 nm, and combined with the standard curve, the NO content in the sample can be obtained.

[0103] 2. Method: Mouse mononuclear macrophage RAW264.7 cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 3.5×10 4 cells / well and seeded in a 96-well plate, and 100 μL of cell suspension was added to each well. In the experiment, a control group (RAW264.7 cells, DMSO), a model group (RAW264.7 cells, DMSO, 0.5 μg / mL LPS), a positive drug group (RAW264.7 cells, dexamethasone (20 μM), 0.5 μg / mL LPS), and a test drug group (RAW264.7 cells, each test compound (20 μM), 0.5 μg / mL LPS) were established simultaneously. Incubate in a 5% CO 2 2, 37 °C constant temperature incubator for 24 h, then aspirate 40 μL of cell supernatant and place it in an ELISA plate, add an equal volume of Griess reagent, and measure the nitrite accumulation in the culture medium at 540 nm using an ELISA reader. The results are as Figure 1 shown. Compounds 1, 2, and 7 showed good inhibitory activity on NO release.

[0104] The description of the above results is only used to help understand the method and its central idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

Claims

1. A chromene-type heteroterpenoid compound, characterized in that: It is a compound represented by general formula (I)-(IV) or any one of its pharmaceutically acceptable salts; Wherein: R1 and R2 are each independently selected from H, hydroxyl, methoxy, acetoxy, carboxyl, R3 is α-methyl or β-methyl, R4 and R5 are each independently α-H or β-H, R6 and R7 are each independently H, α-methyl or β-methyl, α-hydroxyl or β-hydroxyl, α-methoxy or β-methoxy, R8 and R 10 Each of them is independently H, α-hydroxy or β-hydroxy, α-methoxy or β-methoxy, R9 is H or ketocarbonyl, R 11 is H, α-hydroxyl or β-hydroxyl; in the general formulas (I)-(IV), the dotted line indicates that the position may be a double bond structure.

2. A chromene-type heteroterpenoid compound according to claim 1, characterized in that: It is any one of the compounds represented by the following structural formulas 1 to 9 or any one of the pharmaceutically acceptable salts of the compound; 3. The method for extracting chromene-type heteroterpenoid compounds according to claim 2, characterized in that: The steps include: (1) Using Rhododendron dahurica branches and leaves as raw materials, adding ethanol aqueous solution, reflux extraction 2 to 4 times, each extraction time 2 to 4 hours, combining to obtain extracts, reducing pressure to recover the solvent, and concentrating to obtain a total extract; (2) dispersing the total extract into 5 to 10 times the mass volume of water, extracting with petroleum ether and ethyl acetate in sequence, concentrating the extract to recover the solvent, and obtaining a petroleum ether extract concentrate, an ethyl acetate extract concentrate and an aqueous phase, respectively; (3) The petroleum ether extract concentrate was separated by silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate with a volume ratio of 30:1-0:1 as the eluent, and fraction PE5 with a volume ratio of 5:1-1:1 was collected; the ethyl acetate extract concentrate was separated by silica gel column chromatography, and gradient elution was performed using dichloromethane-methanol with a volume ratio of 100:1-0:1 as the eluent, and fraction E2 with a volume ratio of 90:1-80:1 was collected; (4) Concentrated fraction PE5, the concentrated solution was separated by gel column chromatography to purify chromene-type heteroterpenoid compounds 5, 6, 7, 8 and 9; concentrated fraction E2, the concentrated solution was separated by gel column chromatography to purify chromene-type heteroterpenoid compounds 1, 2, 3 and 4.

4. The method for extracting chromene-type heteroterpenoid compounds according to claim 3, characterized in that: In the step (1), 8 to 15 times the mass volume of the raw material and an ethanol aqueous solution with a volume concentration of 70% to 95% are added for extraction.

5. The method for extracting chromene-type heteroterpenoid compounds according to claim 3, characterized in that: The specific separation and purification process of fractions PE5 and E2 in step (4) is as follows: Fraction E2 was concentrated and separated by MCI column chromatography, with methanol-water in a volume ratio of 10:90-100:0 as the eluent for gradient elution, and fractions in a volume ratio of 50:50-70:30 were collected and recorded as E22; fractions in a volume ratio of 80:20 were collected and recorded as E23; Fraction E22 was concentrated and separated by ODS column chromatography, using methanol-water with a volume ratio of 40:60-100:0 as the eluent for gradient elution, and the fraction with a volume ratio of 70:30-80:20 was collected and recorded as E222; Fraction E222 was concentrated and separated by silica gel column chromatography, using petroleum ether-acetone with a volume ratio of 60:1-0:1 as the eluent for gradient elution, and fractions with a volume ratio of 40:1-20:1 were collected and recorded as E2222; Fraction E2222 was concentrated and separated by Sephadex LH-20 column chromatography using methanol as the eluent, and the fractions were collected and recorded as E22221; Fraction E22221 was concentrated and purified by preparative HPLC chromatography using acetonitrile-water in a volume ratio of 60:40 as the mobile phase to obtain compounds 3 and 4; Fraction E23 was concentrated and separated by silica gel column chromatography, using petroleum ether-acetone with a volume ratio of 60:1-0:1 as the eluent for gradient elution, and the fraction with a volume ratio of 40:1-20:1 was collected and recorded as E232; Fraction E232 was concentrated and separated by ODS column chromatography, using methanol-water with a volume ratio of 70:30 to 100:0 as the eluent for gradient elution, and the fraction with a volume ratio of 70:30 to 80:20 was collected and recorded as E2321; Fraction E2321 was concentrated and purified by preparative HPLC chromatography using acetonitrile-water in a volume ratio of 65:35 as the mobile phase to obtain compounds 1 and 2; Fraction PE5 was concentrated and separated by silica gel column chromatography, using petroleum ether-ethyl acetate in a volume ratio of 10:1-0:1 as the eluent for gradient elution, and fractions in a volume ratio of 3:1-1:1 were collected and recorded as PE54; Fraction PE54 was concentrated and separated by ODS column chromatography, using methanol-water with a volume ratio of 70:30-100:0 as the eluent for gradient elution, and the fraction with a volume ratio of 70:30-80:20 was collected and recorded as PE543; Fraction PE543 was concentrated and purified by preparative HPLC chromatography using methanol-water in a volume ratio of 60:40 as the mobile phase to obtain PE5432 and PE5434; Fraction PE5432 was concentrated and purified by preparative HPLC chromatography using acetonitrile-water with a volume ratio of 50:50 as the mobile phase to obtain compounds 6, 7, 8 and 9; Fraction PE5434 was concentrated and purified by preparative HPLC chromatography using acetonitrile-water in a volume ratio of 50:50 as the mobile phase to obtain compound 5.

6. Use of a chromene heteroterpenoid compound according to claim 1 or 2, or a pharmaceutically acceptable salt of the compound, in the preparation of an anti-inflammatory drug.

7. A pharmaceutical composition, characterized in that The invention comprises a chromene-type heteroterpenoid compound as described in claim 1 or 2, or one or more of the pharmaceutically acceptable salts of the compound; and further comprises one or a combination of pharmaceutically acceptable carriers, excipients and diluents.

8. A pharmaceutical composition according to claim 7, characterized in that: The administration route of the pharmaceutical composition is oral administration or injection administration, and the dosage forms include: tablets, capsules, powders, syrups and injections.

9. Use of the pharmaceutical composition according to claim 7 or 8 in the preparation of anti-inflammatory drugs.

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