Miracil D compound, its extraction method and application

Abstract

The application discloses a kind of miraculin compounds and its extraction method and application, belong to the field of traditional Chinese medicine extraction, specifically related to a kind of miraculin compounds as shown in general formula (I) and (II) isolated from Rhododendron dauricum and the extraction method of the compound, and the compound or the isomer of the compound, or the inhibitory effect of the compound on inflammation in pharmaceutically acceptable salt or pharmaceutical composition comprising the compound can be used for preparing anti-inflammatory drugs.The application further enriches the structural diversity of active substances of Rhododendron dauricum, which lays a foundation for subsequent related biological activity tests of monomeric compounds, provides active lead compounds for new drug development, and also provides a theoretical basis for in-depth research and development of Rhododendron dauricum medicinal materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine extraction, and particularly relates to a chromene type heteroterpene compound separated from Rhododendron dauricum L. and an extraction method thereof and application of the compound in preparation of anti-inflammatory drugs. BACKGROUND

[0002] Rhododendron dauricum L. is a semi-evergreen shrub of the genus Rhododendron in the family Ericaceae. It has high ornamental and medicinal values and is mainly distributed in Heilongjiang, Liaoning, Jilin, and Inner Mongolia. The dried leaves of R. dauricum are recorded as 'Man Shan Hong' in the 2020 edition of Chinese Pharmacopoeia, with a bitter taste and cold nature, and are used to treat asthma, acute and chronic bronchitis, diabetes, cough and excessive sputum, cancer, and hypertension. The chemical constituents of R. dauricum mainly include diterpenoids, triterpenoids, flavonoids, and polyphenols. A small amount of grifolin and its derivatives have been isolated from plants in the genus Rhododendron. These compounds have good anti-leukemia and anti-cancer effects. (Nurammina Emingniazi, Palidan Eshe, Aymenisa Abudurejiman. Effects and mechanisms of grifolin on the proliferation and apoptosis of leukemia cells by regulating miR-646 / ADAM17 pathway[J]. Hebei Medicine, 2020, 42(24): 3702-3707; Pang W J, Bai W H, Li Y. Mechanism of grifolin inhibiting glycolysis and proliferation of breast cancer MDA-MB-231 cells by regulating miR-4429 expression[J]. Chinese Journal of Eugenics and Genetics, 2021, 29(9): 1289-1293; Che, X., Yan, H., Sun, H., Dongol, S., Wang, Y., Lv, Q., Jiang, J. Grifolin induces autophagic cell death by inhibiting the Akt / mTOR / S6K pathway in human ovarian cancer cells[J]. Oncology Reports 2016, 36(2): 1041-1047; Luo Y H, Zeng H T, Hu H Z, Ni G T.Proteomic study on the promotion of orcinol on apoptosis of HeLa cells[J].Chinese Pharmacological Bulletin,2013,29(3):417-420.), anti-inflammatory (Iwata,N., Wang,N., Yao,X., Structures and histamine release inhibitory effects of prenylated orcinol derivatives from Rhododendron dauricum. Journal of Natural Products[J]. 2004, 67(7), 1106-1109.), and antioxidant (Nukata,M., Hashimoto,T., Yamamoto,I., Iwasaki,N., Tanaka,M., Asakawa,Y., Neogrifolin derivatives possessing anti-oxidative activity from the mushroom Albatrellus ovinus[J].. Phytochemistry 2002, 59(7), 731-737.) biological activities. In order to maximize the medicinal value of Rhododendron dauricum, the branches and leaves of Rhododendron dauricum were systematically studied, new orcinol compounds were extracted, the structure of the compounds was confirmed by nuclear magnetic resonance, infrared, mass spectrometry and other means, and the inhibitory effect of the extracted compounds on the production of NO in RAW264.7 cells induced by lipopolysaccharide (Lipopolysaccharide, LPS) was detected. SUMMARY

[0003] The primary object of the present application is to provide an orcinol compound.

[0004] The second object of the present application is to provide an extraction method of the orcinol compound.

[0005] The third object of the present application is to provide a pharmaceutical composition containing the orcinol compound.

[0006] The fourth object of the present application is to provide an application of the orcinol compound or the isomer of the compound, or the pharmaceutically acceptable salt of the compound or the pharmaceutical composition containing the compound in the preparation of anti-inflammatory drugs.

[0007] The technical scheme of the present application includes the following:

[0008] The orcinol compound is any one of the compounds as shown in general formula (I)-(II) or the isomer of the compound, or the pharmaceutically acceptable salt of the compound.

[0009]

[0010] wherein: R1 and R3 are each independently selected from hydroxyl, methoxyl, acetyloxy, R2 is selected from H, carboxyl, R4 is selected from H, alpha-hydroxyl or beta-hydroxyl, alpha-methoxyl or beta-methoxyl, R5 is selected from H, ketone carbonyl, R6 is H, alpha-hydroxyl or beta-hydroxyl, alpha-methoxyl or beta-methoxyl, ketone carbonyl, R7 is selected from H, methyl, hydroxyl, methoxyl, acetyloxy; in general formula (I)-(II), the dotted line indicates that the position can be a double bond structure.

[0011] The one kind of aspergillus nomame compound in the application is any one of the following structural formula 1-4 or the isomer of the compound, any one of the pharmaceutically acceptable salt of the compound;

[0012]

[0013] The pharmaceutically acceptable salt in the application includes: sodium salt, potassium salt, ammonia salt, hydrochloride and sulfate.

[0014] The isomer in the application includes: optical isomer, cis-trans isomer, racemate and their mixture.

[0015] The application further provides an extraction method of the aspergillus nomame compound, comprising the following steps:

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

[0017] (2) dispersing the total extract into 5-10 mass times of water, sequentially extracting with 5-10 mass times of petroleum ether and ethyl acetate, recovering the solvent, and respectively obtaining petroleum ether extraction concentrated liquid, ethyl acetate extraction concentrated liquid and water phase;

[0018] (3) subjecting the ethyl acetate extraction concentrated liquid to silica gel column chromatography separation, using dichloromethane-methanol with a volume ratio of 100:1-0:1 as eluent gradient elution, and collecting fraction E2 with a volume ratio of 90:1-80:1;

[0019] (4) concentrating fraction E2, and subjecting the concentrated liquid to MCI column chromatography, ODS column chromatography and gel column chromatography separation, and purifying to obtain aspergillus nomame compounds 1, 2, 3 and 4.

[0020] The above extraction method, wherein:

[0021] In the step (1), the raw material is extracted by adding 8-15 times of volume of ethanol aqueous solution with a volume concentration of 70%-95%.

[0022] In the 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, eluted by gradient elution 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 is collected and recorded as E22.

[0024] After the fraction E22 is concentrated, it is separated by ODS column chromatography, eluted by gradient elution 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 is collected and recorded as E222.

[0025] After the fraction E222 is concentrated, it is separated by silica gel column chromatography, eluted by gradient elution 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 is collected and recorded as E2222.

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

[0027] After the fraction E22221 is concentrated, it is purified by preparative HPLC chromatography with methanol-water with a volume ratio of 70:30 as the mobile phase, to obtain compounds 1, 2, 3 and a fraction E222214.

[0028] After the fraction E222214 is concentrated, it is purified by preparative HPLC chromatography with methanol-water with a volume ratio of 65:35 as the mobile phase, to obtain compound 4.

[0029] The miracillicin compound in the present application is extracted from the branch and leaf part of Rhododendron dauricum.

[0030] The application of the branch and leaf part extract of Rhododendron dauricum in the present application in the preparation of anti-inflammatory drugs.

[0031] A pharmaceutical composition comprising one or more of the miracillicin compound, isomers of the compound, and pharmaceutically acceptable salts of the compound; and one or more of a pharmaceutically acceptable carrier, excipient, and diluent, or a combination thereof.

[0032] The application further provides application of the aurofusarin compound, the isomer of the compound, the pharmaceutically acceptable salt of the compound or the pharmaceutical composition in preparation of an anti-inflammatory drug.

[0033] The application has the following beneficial effects:

[0034] The aurofusarin compound or the isomer thereof, or the pharmaceutically acceptable salt thereof or the pharmaceutical composition containing the compound can be applied to preparation of an anti-inflammatory drug by inhibiting the production of NO in LPS-induced RAW264.7 cells. The method of the application further enriches the structural diversity of active substances of Rhododendron dauricum, and lays a foundation for subsequent biological activity test of monomer compounds, provides an active lead compound for new drug development, and provides a theoretical basis for deep research and development of Rhododendron dauricum. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The application has the following beneficial effects: DETAILED DESCRIPTION

[0036] Further, the application is described in detail below by means of specific embodiments and examples with reference to the accompanying drawings. The specific embodiments and examples described herein are only used to illustrate the application in detail, and are not used to limit the application.

[0037] If the specific experimental steps or conditions are not indicated in the examples, the operations or conditions can be carried out according to the conventional experimental steps described in the literature in the art. If the reagents and instruments are not indicated by the manufacturer, they are all conventional reagent products that can be obtained by purchase.

[0038] Example 1

[0039] The extraction method of the aurofusarin compound in Rhododendron dauricum includes the following steps:

[0040] (1) 12.9 kg of Rhododendron dauricum branches and leaves with a total dry weight of 12.9 kg are used as raw materials, 8 times the volume of 95% ethanol aqueous solution (110 L) by mass is added, and reflux extraction is carried out twice, each for 2 hours. The obtained extraction solutions are combined, the solvent is recovered under reduced pressure, and after concentration, 95% ethanol layer total extract (2.3 kg) is obtained;

[0041] (2) The obtained total extract is dispersed into 5 times the volume of water (12 L) by mass, and 5 times the volume of petroleum ether and ethyl acetate by mass are sequentially used for extraction. The extraction solutions are concentrated to recover the solvent, and petroleum ether extraction concentrated solution, ethyl acetate extraction concentrated solution (560 g) and water phase are obtained, respectively.

[0042] (3) The concentrated extract of ethyl acetate was separated by silica gel column chromatography, eluted with dichloromethane-methanol (100:0 to 0:1, by volume) as eluent, and the fraction E2 with a volume ratio of 90:1 to 80:1 was collected;

[0043] (4) The fraction E2 was concentrated, and the concentrated extract was further purified by MCI column chromatography, ODS column chromatography and gel column chromatography to obtain atropisomers 1, 2, 3 and 4. The specific purification process was as follows:

[0044] After the fraction E2 was concentrated, it was separated by MCI column chromatography, eluted with methanol-water (10:90 to 100:0, by volume) as eluent, and the fraction with a volume ratio of 50:50 to 70:30 was collected and recorded as E22;

[0045] After the fraction E22 was concentrated, it was separated by ODS column chromatography, eluted with methanol-water (40:60 to 100:0, by volume) as eluent, and the fraction with a volume ratio of 70:30 to 80:20 was collected and recorded as E222;

[0046] After the fraction E222 was concentrated, it was separated by silica gel column chromatography, eluted with petroleum ether-acetone (60:1 to 0:1, by volume) as eluent, and the fraction with a volume ratio of 40:1 to 20:1 was collected and recorded as E2222;

[0047] After the fraction E2222 was concentrated, it was separated by Sephadex LH-20 column chromatography, eluted with methanol as eluent, and the fraction was collected and recorded as E22221;

[0048] After the fraction E22221 was concentrated, it was purified by preparative HPLC chromatography, using methanol-water (70:30, by volume) as mobile phase, to obtain compound 1, 2, 3 and fraction E222214;

[0049] After the fraction E222214 was concentrated, it was purified by preparative HPLC chromatography, using methanol-water (65:35, by volume) as mobile phase, to obtain compound 4. In this embodiment 1, 5.8 mg of compound 1, 7.8 mg of compound 2, 5.1 mg of compound 3 and 4.6 mg of compound 4 were obtained.

[0050] The extracted atropisomers 1-4 were structurally identified, and the specific physical and chemical data were as follows:

[0051] Compound 1: yellow oil, (c = 0.35, MeOH), HRESIMS (positive) m / z 367.2246 [M+Na] + (calcd for C 22 H 32 O3Na +,367.2249), the molecular formula of compound 1 was determined to be C 22 H 32 O3, 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13) data are shown in Table 1.

[0052] Compound 2: yellow oil, HRESIMS (positive) m / z 365.2094 [M+Na] + (calcd for C 22 H 30 O3Na + ,365.2093), the molecular formula of compound 2 was determined to be C 22 H 30 O3, 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13)) data are shown in Table 1.

[0053] Compound 3: yellow oil, HRESIMS (positive) m / z 365.2059 [M+Na] + ,(calcd for C 22 H 30 O3Na + ,365.4688), the molecular formula of compound 3 was determined to be C 22 H 30 O3, 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13) data are shown in Table 2.

[0054] Compound 4: yellow oil, HRESIMS (positive) m / z 381.2271 [M+Na] + (calcd for C 23 H 32 O3Na + ,381.2406), the molecular formula of compound 4 was determined to be C 23 H 34 O3, 1 H-NMR (600 MHz, CDC13) and 13 C-NMR (150 MHz, CDC13)) data are shown in Table 2.

[0055] Table 1 Carbon and hydrogen spectral data of compounds 1-2

[0056]

[0057]

[0058] Table 2 Carbon and hydrogen spectrum data of compound 3-4

[0059]

[0060] The structures of the above compounds are identified by physicochemical data and modern spectroscopy methods (HRESIMS and NMR) in combination with relevant data in the public literature, and it is determined that compounds 1-4 are all new compounds not reported in the literature.

[0061]

[0062] Example 2

[0063] The extraction method of chrysenyl heteroterpene compounds in Rhododendron dauricum includes the following steps:

[0064] (1) 15 kg of Rhododendron dauricum branches and leaves with a total dry weight are used as raw materials, 8 times the volume of 95% ethanol aqueous solution (120 L) by mass of the raw materials is added, and reflux extraction is performed twice, each for 2 hours. The obtained extraction solutions are combined, the solvent is recovered under reduced pressure, and after concentration, 95% ethanol layer total extract (2.5 kg) is obtained;

[0065] (2) The obtained total extract is dispersed into 5 times the volume of water (13 L) by mass, and is sequentially extracted with 5 times the volume of petroleum ether and ethyl acetate by mass. The extraction solutions are concentrated to recover the solvents, and petroleum ether extraction concentrated solution, ethyl acetate extraction concentrated solution (600 g) and water phase are obtained, respectively;

[0066] (3) The ethyl acetate extraction concentrated solution is separated by silica gel column chromatography, and eluted with dichloromethane-methanol (100:0-0:1 by volume ratio) as eluent in gradient elution, and the fraction E2 with a volume ratio of 90:1-80:1 is collected;

[0067] (4) The fraction E2 is concentrated, and the concentrated solution is further purified by MCI column chromatography, ODS column chromatography and gel column chromatography to obtain 6.1 mg of compound 1, 8.8 mg of compound 2, 5.8 mg of compound 3 and 5.3 mg of compound 4. The specific separation and purification process is the same as that in Example 1.

[0068] Example 3

[0069] The extraction method of chrysenyl heteroterpene compounds in Rhododendron dauricum includes the following steps:

[0070] (1) Using Rhododendron dauricum branches and leaves with a total dry weight of 20 kg as raw material, add 8 times the volume of the raw material in 95% ethanol aqueous solution (160 L), reflux extract twice, each extraction for 2 hours, combine the extracts, recover the solvent under reduced pressure, concentrate to obtain 95% ethanol layer total extract (3.12 kg).

[0071] (2) The total extract obtained was dispersed in 5 times the mass volume of water (16L), and extracted successively with 5 times the mass volume of petroleum ether and ethyl acetate. The extract was concentrated to recover the solvent, and petroleum ether extract concentrate, ethyl acetate extract concentrate (750g) and aqueous phase were obtained respectively.

[0072] (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, and the dichloromethane-methanol mixture with a volume ratio of 100:0 to 0:1 was used as the eluent for gradient elution. The fraction E2 with a volume ratio of 90:1 to 80:1 was collected.

[0073] (4) The concentrated fraction E2 was further purified by MCI column chromatography, ODS column chromatography, and gel column chromatography to obtain 8.2 mg of compound 1, 11.8 mg of compound 2, 7.8 mg of compound 3, and 7.1 mg of compound 4. The specific separation and purification process was the same as in Example 1.

[0074] The inhibitory effects of the extracted kiwifruitin compounds 1–4 on LPS-induced NO production in RAW264.7 cells were tested, as follows:

[0075] (1) Cell Culture

[0076] Mouse mononuclear macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 U / mL penicillin sodium, and incubated in a constant temperature incubator at 37°C and 5% CO2.

[0077] (2) The effect of compounds 1-4 on inhibiting the release of nitric oxide (NO) from mouse RAW264.7 macrophages induced by lipopolysaccharide was detected by the Griess method.

[0078] 1. Principle: Excessive LPS induces the activation and expression of nitric oxide synthase (NOS) in macrophages, leading to the production of NO. NO then reacts with oxygen free radicals to rapidly form NO2. - NO2 formed - It can be quantitatively detected by Griess Reagent under acidic conditions. First, NO2 -The diazotization reaction with sulfanilamide, and then coupling reaction with N-1-naphthylethylene-diamine dihydrochloride, to generate a purple red azo compound, and finally using a microplate reader to measure the absorbance value at 540 nm, combined with the standard curve, the NO content in the sample can be obtained.

[0079] 2. Method: Take the logarithmic phase growth of mouse monocyte macrophage RAW264.7, adjust the cell concentration to 3.5 x 10 4 cells / well in a 96-well plate, and add 100 μL of cell suspension to each well. In the experiment, control group (RAW264.7 cells, DMSO), model group (RAW264.7 cells, DMSO, 0.5 μg / mL of LPS), positive drug group (RAW264.7 cells, dexamethasone (20 μM), 0.5 μg / mL of LPS), and the test drug group (RAW264.7 cells, each compound to be tested (20 μM), 0.5 μg / mL of LPS) were set up at the same time. Incubate in a 5% CO2, 37°C constant temperature incubator for 24 hours, then aspirate 40 μL of cell supernatant and place it in an enzyme-labeled plate, add an equal volume of Griess reagent, and measure the nitrite accumulation in the culture medium at 540 nm using a microplate reader. The results are shown in Figure 1 Table 1, compounds 2 and 4 showed better inhibitory activity on NO release.

[0080] The above results are only used to help understand the method of the present application and its central idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified, and these improvements and modifications also fall within the protection of the present application.

Claims

1. A method for extracting compounds of the genistein class, characterized in that, Includes the following steps: (1) Using Rhododendron dauricum branches and leaves as raw materials, add ethanol aqueous solution, reflux extract 2 to 4 times, each extraction for 2 to 4 hours, combine the extracts, recover the solvent under reduced pressure, concentrate and obtain total extract; (2) Disperse the total extract into 5 to 10 times its mass volume of water, and extract with petroleum ether and ethyl acetate in sequence. Concentrate the extract to recover the solvent, and obtain petroleum ether extract concentrate, ethyl acetate extract concentrate and aqueous phase respectively. (3) The ethyl acetate extract concentrate was separated by silica gel column chromatography, using dichloromethane-methanol gradient elution with a volume ratio of 100:1 to 0:1, and the fraction E2 with a volume ratio of 90:1 to 80:1 was collected. (4) The concentrated fraction E2 was separated by MCI column chromatography, ODS column chromatography and gel column chromatography to obtain 2 compounds of the kiwifruit serotonin class. After concentration, fraction E2 was separated by MCI column chromatography, using a gradient elution of methanol-water with a volume ratio of 10:90 to 100:

0. The fractions with a volume ratio of 50:50 to 70:30 were collected and designated as E22. After concentration, fraction E22 was separated by ODS column chromatography with a gradient elution of methanol-water with a volume ratio of 40:60 to 100:

0. The fraction with a volume ratio of 70:30 to 80:20 was collected and denoted as E222. After concentration, fraction E222 was separated by silica gel column chromatography, using petroleum ether-acetone with a volume ratio of 60:1 to 0:1 as the eluent gradient. Fractions with a volume ratio of 40:1 to 20:1 were collected and designated as E2222. After concentration, fraction E2222 was separated by Sephadex LH-20 column chromatography with methanol as eluent, and the fraction was collected and denoted as E22221. Fraction E22221 was concentrated and purified by preparative HPLC using a methanol-water mixture (70:30 v / v) to obtain compound 2. The structural formula of compound 2 is: 。 2. The method for extracting a compound of the genus *Symplocos* according to claim 1, characterized in that, In step (1), an ethanol aqueous solution with a volume concentration of 70% to 95% and a mass ratio of 8 to 15 times that of the raw material is added for extraction.

3. The use of compound 2 obtained by the extraction method of a kiwifruit compound according to claim 1 or 2 in the preparation of anti-inflammatory drugs.

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