Application of inner ester compound of jingdao in preparing medicine for treating inflammation-caused diseases
阅读说明：本技术 金挖耳内酯化合物在制备治疗炎症引起疾病药物中的应用 (Application of inner ester compound of jingdao in preparing medicine for treating inflammation-caused diseases ) 是由 邹忠梅 张涛 李凌宇 于猛 张宏武 于 2020-11-18 设计创作，主要内容包括：本发明提供了一种金挖耳内酯化合物在制备治疗炎症引起疾病药物中的应用,所述金挖耳内酯化合物的通式为式1,该类化合物具有强烈的抗炎活性,特别是哮喘疾病,为该化合物未见报道于其抗炎活性,且强于金挖耳全提取物及其有效部位；本发明还提供了所述化合物的制备方法和应用,适合药物的开发和利用。(The invention provides an application of a chrysotile lactone compound in preparing a medicament for treating diseases caused by inflammation, wherein the general formula of the chrysotile lactone compound is shown as formula 1,)
1. An application of a golden-ear lactone compound in preparing a medicament for treating diseases caused by inflammation is characterized in that the golden-ear lactone compound is a compound shown as a general formula I or a physiologically acceptable salt thereof,
wherein R is1、R2And R3All of which are one of hydrogen radical, methyl or acyl containing 1-8 carbon atoms.
2. The use of claim 1, wherein R of said aurilactone compound is1Selected from 3-methylbutyryl, 2-methacryloyl, angeloyl or isobutyryl;
R2selected from hydrogen, 3-methylbutyryl, 2-methacryloyl, isobutyryl or angeloyl;
R3selected from hydrogen, 3-methylbutyryl or angeloyl.
3. The use of claim 2, wherein R of said aurilactone compound is1Is isobutyryl, R2Selected from hydrogen, isobutyryl, 2-methacryloyl, 3-methylbutyryl or angeloyl, and R3 is selected from hydrogen, 3-methylbutyryl or angeloyl.
4. A compound of claim 3 wherein R is1And R2Are all isobutyryl groups.
5. The use of claim 4, wherein R is3Selected from hydrogen.
6. The use of claim 1, wherein the inflammation-causing disease comprises asthma, irritable bowel disease, rhinitis, allergic asthma, allergic conjunctivitis, pharyngitis, allergic bronchitis, eczema, urticaria and allergic cough.
7. The use of claim 6, wherein the inflammation-causing disorder comprises asthma and irritable bowel disease.
8. The use of claim 1, wherein the compound is prepared by:
extracting flower, root, stem and leaf or whole plant of Carpesium plant with 50-95% organic solvent, dispersing the extract with water, extracting with ethyl acetate, collecting ethyl acetate, recovering ethyl acetate, separating the obtained extract with chromatographic column to obtain compound of formula I, and subjecting the compound of formula I to preparative liquid chromatography and semi-preparative liquid chromatography to obtain high-purity monomeric compound.
9. The use according to claim 8, wherein the organic solvent is: one of methanol, ethanol, acetone, diethyl ether and ethyl acetate.
10. The use of claim 8, wherein the column chromatography comprises: separating the extract by silica gel column chromatography, performing gradient elution by using dichloromethane-methanol 200:1, 60:1 and 30:1, collecting the part at the ratio of 30:1, recovering the solvent to be dry, purifying by using gel column chromatography, eluting by using methanol, dichloromethane-methanol 1:1 or acetone as an eluent, collecting the eluent, and recovering the solvent to be dry to obtain the compound of the general formula I.
Technical Field
The invention relates to an application of a golden-ear lactone compound in preparing a medicament for treating diseases caused by inflammation, belonging to the field of medicaments.
Background
At present, the golden larch extract is a plant extract which is generally recognized to have medicinal effect, a series of lactone compounds exist at present, but the compounds only have structural information reports at present, the physiological function of the compounds is unknown, and particularly, the related action of inflammation of the compounds is not reported.
At present, related researches of the golden-ear are few, lactone compounds are reported dispersedly, the applicant obtains a class of lactone compounds through extraction, the lactone compounds have similar structures, but different substituents and have related effects of inflammation resistance, further inflammation is obtained in an animal model, and the extracted golden-ear lactone compounds are considered to have the function of resisting inflammation, have related improvement effects on an asthma model in the animal model and have wider drug development and accurate drug development potential.
Disclosure of Invention
The invention aims to provide application of a chrysotile lactone compound in preparing a medicament for treating diseases caused by inflammation, wherein the chrysotile lactone compound is a compound shown as a general formula I or a physiologically acceptable salt thereof,
wherein R is1One selected from hydrogen group, methyl group or acyl group containing 1 to 8 carbon atoms;
R2one selected from hydrogen, methyl and acyl containing 1-8 carbon atoms;
R3one selected from hydrogen, methyl and acyl containing 1-8 carbon atoms;
further preferably, R is3Is hydrogen radical, said R1Is one of acyl groups of 3 to 5 carbon atoms, R2Is one of acyl groups having 3 to 5 carbon atoms.
Further preferably, in the above use, R of the aurilactone compound is1Selected from 3-methylbutyryl, 2-methacryloyl, angeloyl or isobutyryl;
R2selected from hydrogen, 3-methylbutyryl, 2-methacryloyl, isobutyryl or angeloyl;
R3selected from hydrogen, 3-methylbutyryl or angeloyl.
Further preferably, in the above use, R of the aurilactone compound is1Selected from 3-methylbutyryl, 2-methylbutyryl, angeloyl or isobutyryl;
R2selected from hydrogen, 3-methylbutyryl, 2-methacryloyl, isobutyryl or angeloyl;
R3selected from hydrogen, 3-methylbutyryl or angeloyl.
Further preferably, in the above use, R of the aurilactone compound is1Is isobutyryl, R2Selected from hydrogen, isobutyryl, 2-methacryloyl, 3-methylbutyryl or angeloyl, and R3 is selected from hydrogen, 3-methylbutyryl or angeloyl.
Further preferably, R is1Selected from isobutyrylRadical, R2Selected from isobutyryl.
Further preferably, in the above application, R is3Is hydrogen.
Further preferably, the compound includes isomers thereof, including stereoisomers, optical isomers and the like.
The inflammation-causing disease includes asthma, allergic enteritis, rhinitis, allergic asthma, allergic conjunctivitis, pharyngitis, allergic bronchitis, eczema, urticaria and allergic cough.
Further preferably, the inflammation-causing disease includes asthma and irritable bowel disease. Further, the inflammation-causing disease is asthma.
In the research process of plant components of carpesium, the inventor surprisingly discovers the compound with the general formula I, the compound shows good activity in the aspect of anti-inflammation, and experiments show that the compound can obviously inhibit NO generation induced by LPS, and the compound is similar to dexamethasone with the acknowledged strongest effect. The structure of the compound and the activity thereof in anti-inflammatory aspects are not reported at all.
Clinical data show that dexamethasone serving as a long-acting glucocorticoid drug has severe side effects such as nervous shock femoral head necrosis and the like, but the drug with good anti-inflammatory activity is not available at present, but the anti-inflammatory activity of the compound is not as strong as that of dexamethasone but is similar to that of dexamethasone, so that the compound is a good supplement for the deficiency of dexamethasone under the condition that no better substitute product is available, and is a good news of people who are deeply poisoned by the side effect of the dexamethasone.
The physiologically acceptable salt mainly refers to an inorganic acid salt or an organic acid salt of the aurilactone compound, wherein the inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid or hydroiodic acid; the organic acid is tartaric acid, citric acid, formic acid, acetic acid, oxalic acid, butyric acid, oxalic acid, maleic acid, succinic acid, adipic acid, alginic acid, citric acid, aspartic acid, benzenesulphonic acid, camphoric acid, camphorsulphonic acid, digluconic acid, cyclopentanepropionic acid, dodecylsulphuric acid, ethanesulphonic acid, glucoheptonic acid, glycerophosphoric acid, hemisulphuric acid, heptanoic acid, hexanoic acid, fumaric acid, 2-hydroxyethanesulphonic acid, lactic acid, maleic acid, methanesulphonic acid, nicotinic acid, 2-naphthalenesulphonic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, propionic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulphonic acid salt, undecanoate and the like, preferably tartaric acid, citric acid, oxalic acid, maleic acid, succinic acid, citric acid, benzenesulphonic acid and the like. After salifying, the drug effect is not influenced, and the solubility of the compound can be further improved; further preferred is a hydrochloride or maleate salt.
The applicant isolated the following known compounds from the golden fungus,
auricularia auricular lactone X1: (4R,5R,6S,7R,8R,9R,10R) -4, 8-dihydroxy-5-isobutyryloxy-9-3-methylbutanoyloxy-3-one-germyl-6, 12-lactone;
auricularia auricular lactone X2: (4R,5R,6S,7R,8R,9R,10R) -4, 8-dihydroxy-5-2-methylbutanoyloxy-9-3-methylbutanoyloxy-3-one-germacrane-6, 12-lactone;
auricularia auricular lactone X3: (4R,5R,6S,7R,8R,9R,10R) -4, 8-dihydroxy-5-angeloyloxy-9-2-methylbutanoyloxy-3-one-germacrane-6, 12-lactone;
auricularia auricular lactone X4: (4R,5R,6S,7R,8R,9R,10R) -4, 8-dihydroxy-5-angeloyloxy-9-butanoyloxy-3-keto-germyl-6, 12-lactone;
the substituents of the above compounds are listed in table 1 below:
TABLE 1 substituent comparison of the above-mentioned compounds
Serial number
Compound numbering
R1
R2
R3
1
incaspitolide D
Isobutyryl radical
Isobutyryl radical
Hydrogen radicals
2
cardivarolide F
Angeloyl radical
Angeloyl radical
Hydrogen radicals
3
cardivarolide G
Angeloyl radical
3-methylbutyryl-
Hydrogen radicals
4
cardivarolide H
Isobutyryl radical
Angeloyl radical
Hydrogen radicals
5
cardivarolide I
Isobutyryl radical
Hydrogen radicals
3-methylbutyryl
6
cardivarolide J
Angeloyl radical
Hydrogen radicals
Angeloyl radical
7
cardivarolide K
Isobutyryl radical
Hydrogen radicals
Angeloyl radical
8
divarolide E
3-methylbutyryl
3-methylbutyryl
Hydrogen radicals
9
divarolide F
Isobutyryl radical
2-methacryloyl radical
Hydrogen radicals
10
divarolide G
2-methylbutyryl
2-methylbutyryl
Hydrogen radicals
11
Goldwort lactoneX1
Isobutyryl radical
3-methylbutyryl
Hydrogen radicals
12
Jatropha curcas lactone X2
2-methylbutyryl
3-methylbutyryl
Hydrogen radicals
13
Jatropha curcas lactone X3
Angeloyl radical
2-methylbutyryl
Hydrogen radicals
14
Jatropha curcas lactone X4
Angeloyl radical
Isobutyryl radical
Hydrogen radicals
At present, the golden larch is known to have the effects of clearing heat and removing toxicity, and relieving swelling and pain, but no relevant effective component with inflammation resistance is found.
However, the applicant finds that the extracted golden-ear fungus extract has the treatment-related function of diseases caused by inflammation through the extraction of corresponding plants.
The invention also relates to a process for the preparation of compounds of the general formula I: extracting flower, root, stem and leaf or whole plant of Carpesium plant with organic solvent, dispersing the extract with water, extracting with ethyl acetate, collecting ethyl acetate part, recovering solvent, and subjecting the obtained extract to column chromatography to obtain compound of formula I.
The Carpesium plants include Tremella aurantialba, Tremella macrocephala, Tremella aurantialba, Tremella minoxidil, Tremella pubescens, Tremella forbesii, Carpesium abrotanoides, Carpesium guizhouense, and plateau Carpesium abrotanum. The invention preferably uses the golden-ear (with the scientific name: Carpesium divaricatum Sieb. et Zucc) as the extraction raw material, and the medicinal part is preferably the whole herb.
The organic solvent is selected from one of methanol, ethanol, acetone, diethyl ether and ethyl acetate, preferably methanol, ethanol or acetone. In view of safety and cost, ethanol having a concentration of 50 to 95% is more preferable, and ethanol having a concentration of 90 to 95% is most preferable. The extraction method is cold soaking or hot reflux extraction.
The preparation method further comprises the following steps: extracting flower, root, stem and leaf or whole plant of Carpesium plant with 50-95% lower alcohol, dispersing the extract with water, sequentially extracting with ethyl acetate, collecting ethyl acetate fraction, recovering ethyl acetate, separating the obtained extract with chromatographic column to obtain compound of formula I, and subjecting the compound of formula I to preparative liquid chromatography and semi-preparative liquid chromatography to obtain high-purity monomeric compound, wherein the high-purity is not less than 98%.
The lower alcohol includes methanol and ethanol. Said preparative liquid chromatography (prepHPPLC), e.g. chromatographic column Daisogel-C18-100A (10 μm; 250X 30 mm; 20mL/min), 55-65% CH as mobile phase3OH, preferably 60% CH for mobile phase3OH; semi-preparative liquid chromatography (semi-prepHPPLC) such as YMC-Pack ODS-A column (5 μm; 250X 10 mm; 2mL/min) with 60% -90% (0-35min) CH3OH-H2And O is mobile phase to carry out gradient elution.
In the extraction step, 4-6 times of water is added into the extract for dispersion, and the volume ratio of the water phase to the organic phase is (1:3) - (3:1), preferably 1: 1; the extraction is finished by clarifying both layers, and the extraction times are 1-5 times. Solvent recovery methods are conventional in the art.
The obtained compound of the general formula (I) is an effective part, and the effective part contains 40-50% of the compound of the general formula (I).
The column chromatography comprises: the extract is firstly separated by silica gel column chromatography, and is subjected to gradient elution by dichloromethane-methanol (200:1, 60:1, 30:1), the 30:1 part is collected, the solvent is recovered to be dry, then the extract is purified by gel column chromatography, and is eluted by methanol, dichloromethane-methanol (1:1) or acetone as eluent, the eluent is collected, and the solvent is recovered to be dry, so that the compound with the general formula (I) is obtained, and the acetone is preferably used as the eluent.
The gradient elution with dichloromethane-methanol (200:1, 60:1, 30:1) can also be performed with petroleum ether-acetone (20:1, 6:1, 3:1), and the following steps are collected in a ratio of 3:1, the method of gel column purification was not changed. The above ratios are volume ratios.
The silica gel column chromatography is performed by selecting column chromatography silica gel with the particle size of 200-300 meshes, wherein the sample loading amount is 6.7-10%, namely the weight ratio of the sample loading amount to the silica gel is (1:10) - (1:15), and during gradient elution, 3-5 column volumes of eluent with each concentration are preferably eluted.
And (3) carrying out gel column chromatography, wherein the sample loading amount is 2-2.5%, namely the weight ratio of the sample loading amount to the gel is (1:40) - (1:50), eluting by 7-8 column volumes, and selectively collecting eluent of 3 rd-5 column volumes according to specific conditions.
Since the number of factors affecting the fraction in the column chromatography is large, it is preferable to determine the desired fraction by thin layer detection (TLC) in the process. Namely, the germacrane type sesquiterpene is detected by a thin layer, dark spots are formed under 254nm, and light yellow spots are formed after the color is developed by 5% concentrated sulfuric acid (the mixture is baked for 5 minutes at 95 ℃).
The invention particularly provides application of the compound in the general formula I in preparing a medicine for treating asthma, and experiments show that the anti-inflammatory effect and the asthma treatment effect do not have corresponding necessary relationship, so that the applicant respectively performs animal model research on asthma prevention and treatment after finding that the compound has the anti-inflammatory effect, and therefore the invention provides application of other compounds in preparing the medicine for treating asthma.
The invention also aims to provide an anti-inflammatory pharmaceutical composition, which is prepared from an effective dose of the compound and pharmaceutically acceptable auxiliary materials. The effective amount is 5-50 mg/day, and the pharmaceutically acceptable auxiliary materials refer to auxiliary materials required for preparing any pharmaceutical dosage form suitable for human or animals, such as diluents, binders, wetting agents, disintegrants, lubricants and glidants when the pharmaceutical dosage form is prepared into an oral solid preparation; when the injection is prepared, the pharmaceutically acceptable auxiliary materials refer to a pH regulator, a cosolvent, an antioxidant, an isotonic agent and the like.
Restated again: the following experiments are only exemplary of the many experiments performed during the development of the present invention and do not cover and exhaust all the experiments performed by the inventors for the purpose of illustrating the antidepressant activity of the compounds of the present invention with only those data.
Experimental part
Anti-inflammatory Activity of Compounds on lipopolysaccharide
1. Experimental Material
Drugs and reagents: dimethyl sulfoxide (DMSO), available from Fisher corporation (HPLC grade); 25% trypsin, available from Sigma; DMEM medium (streptomycin inclusion) purchased from Invitrogen, usa; fetal bovine serum, purchased from Gibco; horse serum, purchased from Hyclone; PBS available from Hyclone
Cells and instruments: the RAW264.7 cell strain is offered by the pharmaceutical plant institute of Chinese academy of medicine, the cloud subject group, a cell culture bottle (Coring company), a sterile 96-well plate, a cell counting plate, a glass slide, a cover glass, a glass pipette, a nylon filter, a stainless steel filter, glass bottles of various specifications, a pipette gun, a gun head box and a centrifuge tube.
The instrument comprises the following steps: DHG-9070A electric heating constant temperature air blast drying oven (Beijing Luxi technology Co., Ltd.), MCO-15AC type CO2Isothermal cell culture chamber (SANYO, Japan), ZHJH-C1115B model clean bench (Shanghai Zhi composition analysis Instrument manufacturing Co., Ltd.), CKX41 type inverted phase contrast microscope (OLYMPUS, Japan), MQX200 type enzyme standard photometer (BioTek instruments Co., Ltd., USA), Legend Micro 17R type high-speed low-temperature centrifuge (Thermo, USA), QL-901 Micro vortex mixer (Hai-Hai)Tubel instruments, Inc. of Ministry), -80 ℃ ultra low temperature refrigerator (Thermo, USA), KQ-250B model ultrasonic cleaner (Kunshan ultrasonic instruments Inc.), SSW-420-2S model constant temperature water bath (Shanghai Boxun Co., Ltd.), CY50945 model Locator liquid nitrogen tank (Thermo, USA).
A sample to be tested: the compounds of the invention, namely icasphitolide D, cardirolide F, cardirolide G, cardirolide H, cardirolide I, cardirolide J, cardirolide K, cardirolide E, cardirolide F, cardirolide G, Auricularia aurora lactone X1, Auricularia aurora lactone X2, Auricularia aurora lactone X3 and Auricularia aurora lactone X4, are all self-made and have the purity of more than 98%;
positive drugs: dexamethasone.
2. Experimental methods
Griess reagent was used to detect NO secretion levels in cells. This method was proposed by Green et al in 1982. The principle is that NO is easily oxidized in vivo or in water to generate NO2 -Under the acidic condition, the diazo compound can be subjected to Griess reaction with diazo salt sulfanilamide to generate the diazo compound. The compound can further generate a color reaction, and has a maximum absorption peak at 540 nm. The OD value is linearly related to the concentration of NO.
In vitro experiments, RAW264.7 cells were used for the experiments. Cells were cultured in plastic culture dishes using DMEM medium plus 10% by volume of FBS (fetal bovine serum) at 37 deg.C, 5% CO2Culturing under the conditions of (1). Cells were diluted in a petri dish without using E-enzyme, and the tips were blown down, and the RAW cells were smaller and more numerous, and were counted using a counting plate. Then according to 2X 104Each well was plated on a 96-well plate. At 37 ℃ 5% CO2The cells are cultured in the incubator for 24 hours to ensure complete adherence of the cells.
Drugs were added at different concentrations, followed by 1 μ g/mL LPS to induce inflammation. After 24h of co-cultivation. And taking 50 mu L of the supernatant, adding Griess reagent with the same volume into a new 96-well plate, incubating for 10min, and detecting the NO content on a microplate reader. The wavelength used was 540 nm. The NO content was calculated using a standard curve previously determined with sodium nitrite. Each drug was tested 3 times.
The inhibition rate was calculated by the following formula:
inhibition [% ], [% ] (OD control-OD sample)/OD control × 100.
3. Results of the experiment
Table 2 results of anti-inflammatory activity of known compounds
The experimental results are shown in table 2, and the compounds of the general formula I of the present invention: incapitolide D, cariderrolide F, cariderrolide G, cariderrolide H, cariderrolide I, cariderrolide J, cariderrolide K, cariderrolide E, biderrolide F, biderrolide G, aurantiolide X1, aurantiolide X2, aurantiolide X3 and aurantiolide X4 can obviously inhibit the generation of NO induced by LPS, but the effect of dexamethasone with the strongest activity is not achieved, but is close to that of dexamethasone.
Further animal models prove that: (cell model anti-inflammatory model and animal disease model function differently, so further demonstration is required)
Experimental animals and materials:
(1) laboratory animal
According to the guidelines and protocols of the animal protection and use committee of the institute of medicinal plants of the academy of Chinese medical science, the selected male Balb/c mice of 6-8 weeks old are placed in the center of the experimental animals of the unit for SPF-level management and feeding. All mice were fed normally for 2 days to acclimate before the start of the experiment, followed by the entire experimental period.
(2) Experimental reagent
Tween 20 (national reagent company, shanghai), paraformaldehyde solution (biological company, shanghai), skimmed milk powder (mon cow company, anhui), hematoxylin-eosin dye (subsidiary hospital of Jiangsu university, Zhenjiang), ovalbumin (OVA, Sigma, Japan), xylene (national reagent company, shanghai), absolute ethanol (national reagent company, shanghai), NF- κ Bp65 primary antibody (merck company, Dammstadt, Germany), Maker (Fermentas company, Dorema), PMSF solution (Baiolabo company, Beijing), PVDF membrane (Millipore company, Massachusei, USA), protease inhibitor (Thermo Scientific company, Massachusei), TWEAK primary antibody (merck company, Dammstadt, Germany), shanghanruyi (south america), anti-mouse secondary antibody (schmitt, Germany), and goat serum blocking solution (Hongyoky) Rabbit anti-sheep secondary antibody (merck, dalschatat, germany), goat anti-rabbit secondary antibody (merck, dalschatat, germany), Trizol (isolate Invitrogen, carlsbad, usa), transmembrane fluid (self-prepared), DAB (color developer Dako, denmark), RIPA lysate (pecan, shanghai), BCA protein quantification kit (pecan, shanghai), PAGE gel kit (solibao, beijing), loading buffer (baixontai, beijing).
(3) Laboratory apparatus
An atomizing suction pump (Omron corporation, japan), a cryogenic centrifuge (bayoto corporation, beijing), a clean bench (sujing cleansing corporation, zhejiang), an electronic balance (Sartorius corporation, japan), a tissue microtome (Leica corporation, germany), a cytocentrifugal smear machine (Thermo Shandon corporation, ma, usa), an ultra pure water separator (Millipore corporation, ma, usa), an ice machine (Scotsman corporation, italy), -20 ℃, -80 ℃ refrigerator (hel corporation, Qingdao), a water bath (jump medical optical instrument factory, shanghai), a large, medium, and small gun heads (Axygen corporation, california, usa), a centrifuge tube (Axygen corporation, california, usa), a micro-adjustable liquid transfer gun (Eppendorf corporation, hamburg, germany), an EP tube (Eppendorf corporation, hamburg, germany), an eight-tube centrifuge, south-beam mill, a vortex mixer (health care corporation, tay), electrophoresis apparatus (BIO-RAD, california, usa), shaker (new health medical, tay), optical microscope (Olympus, japan), forceps, scissors, hemostats, etc. (surgical instrument factory, shanghai), drop-off preventing slide and coverslip (santai instrument, south ton).
1. Experimental methods
(1) Grouping of experimental animals and model making
70 male Balb/c mice were randomly and evenly divided into a normal control group, an asthma model group and a dexamethasone intervention group.
Asthma model group: injecting the antigen mixed solution (containing ovalbumin, OVA and 10% aluminum hydroxide, 50 mu g of each) into the abdominal cavity of the mouse on days 1, 8 and 15; mice were given asthma challenge daily from day 22 to day 28 (1/day, 30 min/each given 2% OVA suspension nebulization).
Dexamethasone intervention group: sensitization and challenge were performed in the asthma group, but prior to challenge mice were challenged by intraperitoneal injection of 0.2mg dexamethasone.
Sample group: sensitization and challenge were performed in the asthma group, but prior to challenge, mice were intervened by intraperitoneal injection of 5mg samples.
Control group: on days 1, 8 and 15, 0.2m LPBS was injected into the abdominal cavity of the mice, and the mice were inhaled by nebulizing PBS solution (1 time/day, 30 minutes/time) every day from day 22 to day 28.
2. Data analysis
The detection data is processed by SPSS17.0 statistical software package, and the data is processed byShowing that the data has uniform variance, performing statistical analysis in groups by using F test, performing pairwise comparison between multiple groups by using LSD-t test, and obtaining P value<A value of 0.05 indicates that the difference is statistically significant.
3. Conclusion
Table 3 immunohistochemical monitoring of TWEAK and NF- κ B protein expression n ═ 10 in each group,
as a result, it was found that: (1) compared with a dexamethasone vaccine group and a control group, the expression quantity of TWEAK in an incapiolide group D, a carirolide group F, a carirolide group G, a carirolide group H, a carirolide group I, a carirolide group E, a carirolide group F, a carirolide group G, a carirolide group X1, a carirolide group X2, a carirolide group X3 and a carirolide group X4 and an asthma model group has statistical significance in difference; (2) NF-kB has obvious balance on the expression of mouse lung tissues in an incasPitolide D group, a cardiorolide F group, a cardiorolide G group, a cardiorolide H group, a cardiorolide I group and a cardiorolide F group and an asthma model group, which are close to a dexamethasone intervention group and have obvious difference compared with the parameters of the asthma group.
The above data show that: in addition to cardiorolide J and cardiorolide K, icasapient D, cardiorolide F, cardiorolide G, cardiorolide H, cardiorolide I, divarolide E, divarolide F, divarolide G, aurantiolide X1, aurantiolide X2, aurantiolide X3 and aurantiolide X4 all had the function of elevating lung tissue-related asthma parameters in mice and had anti-asthmatic activity.
Therefore, according to the invention, four compounds of icasplatide D, cardiorolide F, cardiorolide G, cardiorolide H, cardiorolide I, cardiorolide J, cardiorolide K, divarolide E, divarolide F, divarolide G, aurilactone X1, aurilactone X2, aurilactone X3 and aurilactone X4 are extracted from the golden larch, all have anti-inflammatory functions, and in an animal model, 12 compounds all play a role in improving asthma, while the icasplatide D has the best function, and 12 compounds are applied to the preparation of asthma medicaments.
Detailed Description
Example 1:
preparation of the Compound of general formula (I) (Total lactones):
extracting 9.0kg of dried herba Carpesii Divaricati (Carpesium divaricatum) with 8 times of 95% ethanol under reflux for 3 times (3 hr each time), mixing extractive solutions, recovering ethanol to obtain 720g dry extract, dispersing the extract (dry extract) with 3L water, and sequentially dispersingExtraction was performed 3 times with equal volumes of petroleum ether and ethyl acetate, and the ethyl acetate fraction was collected and the solvent was recovered to obtain 270g of an ethyl acetate extract. Purifying the ethyl acetate extract with silica gel column (200-300 mesh column chromatography silica gel, sample-silica gel mass ratio of 1:10), and purifying with CH2Cl2MeOH (200:1, 60:1, 30:1) was eluted in gradients of 3 column volumes each, and CH was collected2Cl2MeOH (30:1) eluent, recovery of the solvent, to give the crude extract of the compound of general formula (I). Separating the crude extract by Sephadex LH-20 column chromatography (mass ratio of sample to gel is 1:40), eluting with methanol for 8 column volumes, collecting the eluate of 5-6 column volumes, and recovering solvent to obtain 65g of total lactone of general formula (I).
Example 2:
preparation of the Compound of general formula (I) (Total lactones):
soaking dried herba Carpesii Cernui (C.divaricatum) 9.0kg in 8 times of 95% ethanol for 3 times (each for 2 days), mixing extractive solutions, recovering ethanol to obtain dry extract 270g, dispersing the extract (dry extract) in water with 2.5L, adding twice volume of ethyl acetate, extracting for 5 times, collecting ethyl acetate part, recovering solvent, and obtaining 35g of ethyl acetate extract. Purifying the ethyl acetate extract with silica gel column (200-300 mesh column chromatography silica gel, sample-to-silica gel mass ratio of 1:15), and purifying with CH2Cl2MeOH (200:1, 60:1, 30:1) was eluted in gradients of 3 column volumes each, and CH was collected2Cl2MeOH (30:1) eluent, recovery of the solvent, to give the crude extract of the compound of general formula (I). Separating the crude extract by Sephadex LH-20 column chromatography (mass ratio of sample to gel is 1:40), eluting with methanol for 8 column volumes, collecting the eluate of 5-6 column volumes, and recovering solvent to obtain 6.8g of total lactone of general formula (I).
Example 3
The difference from example 1 is that the extract is Carpesium macrocephalum (C. macrocephalum), the medicinal part is whole herb, and the solvent is methanol. 30g of total lactones of general formula (I) are obtained.
Example 4
The difference from example 2 is that the extract is black flower golden Tremella (C.triste), the medicinal part is stem and leaf, and the solvent is acetone. 7.5g of total lactones of general formula (I) are obtained.
Example 5
The difference from example 1 is that the extract is a black flower golden Tremella (C. triste), the medicinal part is a root, and the solvent used is 50% ethanol. 7.5g of total lactones of general formula (I) are obtained.
Example 6
Preparation of compound icasipitolide D:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain the icasipellide D.
Physicochemical data for incaspetolide D are as follows: white needle crystal (methanol), [ alpha ]]2D 0-83.8 (c 0.167, MeOH), readily soluble in methanol, soluble in chloroform, dichloromethane, ethanol. Positive ion HR-ESI-MS shows: m/z 477.2111[ M + Na ]]+(calcd for C23H34O9Na,477.2101)，1H NMR(CD3OD, 600MHz) and13C NMR(CD3OD, 150MHz) data are shown in table 1.
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,600MHz)δ1.87(1H,m,H-1a)，1.75(1H,m,H-1b)，3.85(1H,br d,J＝10.2Hz,H-2a)，2.20(1H,o,H-2b)，5.43(1H,dd,J＝9.6,2.4Hz,H-5)，4.70(1H,dd,J＝9.6,6.6Hz,H-6)，3.05(1H,m,H-7)，4.44(1H,d,J＝10.2Hz,H-8)，5.17(1H,d,J＝10.2Hz,H-9)，2.22(1H,o,H-10)，6.35(1H,d,J＝3.0Hz,H-13a)，5.70(1H,d,J＝2.4Hz,H-13b)，0.99(3H,d,J＝6.6Hz,H-14)，1.26(3H,s,H-15)，2.71(1H,o,H-2′)，1.28(3H,d,J＝6.6Hz,H-3′)，1.27(3H,d,J＝6.6Hz,H-4′)，2.71(1H,o,H-2″)，1.26(3H,d,J＝7.2Hz,H-3″)，1.26(3H,d,J＝7.2Hz,H-4″)。13C NMR(CD3OD,150MHz)δ25.3(C-1)，32.9(C-2)，217.6(C-3)，80.4(C-4)，78.1(C-5)，80.0(C-6)，41.5(C-7)，70.3(C-8)，78.5(C-9)，30.0(C-10)，132.7(C-11)，169.6(C-12)，123.8(C-13)，19.9(C-14)，23.3(C-15)，176.4(C-1′)，34.0(C-2′)，18.0(C-3′)，18.0(C-4′)，177.2(C-1″)，34.1(C-2″)，18.5(C-3″)，17.9(C-4″)。
example 7
Preparation of cardiorolide F:
the total lactones obtained in example 2 were then individually prepared using prepHPPLC (60% CH)3OH) to yield cardiothiolade F.
The physicochemical data for cardiorolide F are as follows: white needle crystal (methanol), [ alpha ]]2D 0-84.7 (c 0.085, MeOH), readily soluble in methanol, soluble in chloroform, dichloromethane, ethanol. UV (MeOH) lambdamax(logε):199(4.88)nm，IR(neat)nmax:3443,1757,1718,1690cm-1. Positive ion HR-ESI-MS shows: m/z 501.2101[ M + Na ]]+(calcd for C25H34O9Na,501.2101)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.87(1H,m,H-1a)，1.74(1H,m,H-1b)，3.87(1H,m,H-2a)，2.24(1H,o,H-2b)，5.51(1H,dd,J＝10.0,2.0Hz,H-5)，4.73(1H,dd,J＝9.0,6.5Hz,H-6)，3.08(1H,m,H-7)，4.48(1H,d,J＝10.5Hz,H-8)，5.27(1H,d,J＝10.5Hz,H-9)，2.24(1H,o,H-10)，6.34(1H,d,J＝3.0Hz,H-13a)，5.71(1H,d,J＝1.0Hz,H-13b)，0.99(3H,d,J＝6.5Hz,H-14)，1.25(3H,s,H-15)，6.18(1H,o,H-3′)，2.00(3H,s,H-4′)，1.96(3H,br d,J＝10.5Hz,H-5′)，6.18(1H,o,H-3″)，2.00(3H,s,H-4″)，1.96(3H,d,J＝10.5Hz,H-5″)。13C NMR(CD3OD,125MHz)δ25.7(C-1)，33.0(C-2)，217.9(C-3)，80.4(C-4)，78.1(C-5)，80.0(C-6)，41.7(C-7)，70.5(C-8)，78.5(C-9)，30.1(C-10)，132.8(C-11)，169.7(C-12)，124.0(C-13)，20.0(C-14)，23.4(C-15)，167.1(C-1′)，127.9(C-2′)，137.7(C-3′)，19.3(C-4′)，14.6(C-5′)，167.7(C-1″)，127.5(C-2″)，138.4(C-3″)，19.5(C-4″)，14.7(C-5″)。
example 8
Preparation of cardiorolide G:
the total lactones obtained in example 2 were then individually prepared using prepHPPLC (60% CH)3OH) to yield cardivasrolide G.
The physicochemical data for cardiorolide G are as follows: white needle crystal (methanol) easily dissolved inMethanol, which can be dissolved in chloroform, dichloromethane and ethanol. Positive ion HR-ESI-MS shows: m/z 503.2257[ M + Na ]]+(calcd for C23H32O9Na,503.2263)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.87(1H,m,H-1a)，1.65(1H,m,H-1b)，3.88(1H,m,H-2a)，2.23(1H,o,H-2b)，5.52(1H,dd,J＝9.5,2.0Hz,H-5)，4.73(1H,dd,J＝9.5,1.5Hz,H-6)，3.06(1H,m,H-7)，4.44(1H,d,J＝10.5Hz,H-8)，5.18(1H,d,J＝10.0Hz,H-9)，2.23(1H,m,H-10)，6.34(1H,d,J＝3.0Hz,H-13a)，5.69(1H,d,J＝3.0Hz,H-13b)，0.99(3H,d,J＝6.5Hz,H-14)，1.26(3H,s,H-15)，6.20(1H,qq,J＝7.0,1.5Hz,H-3′)，1.99(3H,q,J＝1.5Hz,H-4′)，2.02(3H,dq,J＝7.0,1.5Hz,H-5′)，2.32(2H,o,H-2″)，2.13(3H,o,H-3″)，1.01(3H,d,J＝6.6Hz,H-4″)，1.01(3H,d,J＝6.6Hz,H-5″)。13C NMR(CD3OD,125MHz)δ25.4(C-1)，33.2(C-2)，217.8(C-3)，80.4(C-4)，78.1(C-5)，80.0(C-6)，41.6(C-7)，70.3(C-8)，78.7(C-9)，29.9(C-10)，132.8(C-11)，169.7(C-12)，123.9(C-13)，20.0(C-14)，23.4(C-15)，167.1(C-1′)，127.5(C-2′)，138.4(C-3′)，19.3(C-4′)，14.8(C-5′)，173.2(C-1″)，43.2(C-2″)，25.4(C-3″)，21.4(C-4″)，21.5(C-5″)。
example 9
Preparation of cardiothiolade H:
the total lactones obtained in example 3 were then individually prepared using prepHPPLC (60% CH)3OH) to yield cardivasrolide H.
The physicochemical data for cardiothiolade H are as follows: white needle crystal (methanol), which is easily soluble in methanol, and soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 489.2101[ M + Na ]]+(calcd for C24H34O9Na,489.2101)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,600MHz)δ1.88(1H,m,H-1a)，1.73(1H,m,H-1b)，3.84(1H,br d,J＝11.4Hz,H-2a)，2.24(1H,o,H-2b)，5.41(1H,dd,J＝9.6,1.8Hz,H-5)，4.70(1H,dd,J＝9.6,6.6Hz,H-6)，3.07(1H,m,H-7)，4.47(1H,d,J＝10.2Hz,H-8)，5.28(1H,d,J＝10.2Hz,H-9)，2.24(1H,m,H-10)，6.35(1H,d,J＝3.0Hz,H-13a)，5.72(1H,d,J＝3.0Hz,H-13b)，1.00(3H,d,J＝6.6Hz,H-14)，1.25(3H,s,H-15)，2.72(1H,m,H-2′)，1.26(3H,d,J＝6.6Hz,H-3′)，1.25(3H,d,J＝6.6Hz,H-4′)，6.17(2H,qq,J＝7.2,1.8Hz,H-3″)，1.97(3H,dq,J＝1.8,1.2Hz,H-4″)，2.01(3H,dq,J＝7.2,1.8Hz,H-5″)。13C NMR(CD3OD,150MHz)δ25.5(C-1)，32.8(C-2)，217.6(C-3)，80.4(C-4)，78.1(C-5)，80.0(C-6)，41.6(C-7)，70.5(C-8)，78.5(C-9)，30.0(C-10)，132.7(C-11)，169.6(C-12)，123.9(C-13)，20.0(C-14)，23.3(C-15)，176.4(C-1′)，34.0(C-2′)，18.0(C-3′)，17.9(C-4′)，167.7(C-1″)，127.8(C-2″)，137.7(C-3″)，19.4(C-4″)，14.6(C-5″)。
example 10
Preparation of cardiorolide I:
the total lactones obtained in example 4 were then individually prepared using prepHPPLC (60% CH)3OH) to yield cardivasrolide I.
The physicochemical data for cardiorolide I are as follows: white needle crystal (methanol), which is easily soluble in methanol, and soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 491.2249[ M + Na ]]+(calcd for C24H36O9Na,491.2257)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.89(1H,m,H-1a)，1.71(1H,m,H-1b)，3.76(1H,m,H-2a)，2.30(1H,m,H-2b)，5.20(1H,dd,J＝8.0,2.0Hz,H-5)，4.88(1H,m,H-6)，3.11(1H,m,H-7)，5.56(1H,d,J＝8.5Hz,H-8)，3.70(1H,d,J＝8.5Hz,H-9)，2.06(1H,m,H-10)，6.33(1H,d,J＝3.0Hz,H-13a)，5.66(1H,d,J＝3.0Hz,H-13b)，1.11(3H,d,J＝6.0Hz,H-14)，1.22(3H,s,H-15)，2.69(1H,m,H-2′)，1.26(3H,d,J＝6.0Hz,H-3′)，1.23(3H,d,J＝6.0Hz,H-4′)，2.16(2H,m,H-2″)，1.98(1H,m,H-3″)，0.89(3H,d,J＝5.0Hz,H-4″)，0.90(3H,d,J＝5.0Hz,H-5″)。13C NMR(CD3OD,125MHz)δ24.5(C-1)，35.2(C-2)，217.9(C-3)，80.6(C-4)，78.5(C-5)，80.2(C-6)，40.5(C-7)，76.4(C-8)，75.5(C-9)，31.1(C-10)，133.0(C-11)，169.6(C-12)，125.0(C-13)，20.2(C-14)，23.5(C-15)，176.8(C-1′)，34.3(C-2′)，18.3(C-3′)，18.4(C-4′)，173.3(C-1″)，43.3(C-2″)，25.8(C-3″)，21.6(C-4″)，21.7(C-5″)。
example 11
Preparation of cardiorolide J:
the total lactones obtained in example 5 were then individually prepared using prepHPPLC (60% CH)3OH) to yield cardivasrolide J.
The physicochemical data for cardiorolide J are as follows: white needle crystal (methanol), which is easily soluble in methanol, and soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 501.2101[ M + Na ]]+(calcd for C25H34O9Na,501.2103)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.94(1H,m,H-1a)，1.76(1H,m,H-1b)，3.83(1H,m,H-2a)，2.35(1H,m,H-2b)，5.30(1H,d,J＝10.0Hz,H-5)，4.95(1H,m,H-6)，3.17(1H,m,H-7)，5.68(1H,d,J＝10.0Hz,H-8)，3.77(1H,d,J＝10.0Hz,H-9)，2.11(1H,m,H-10)，6.34(1H,d,J＝3.0Hz,H-13a)，5.70(1H,d,J＝3.0Hz,H-13b)，1.12(3H,d,J＝6.5Hz,H-14)，1.25(3H,s,H-15)，6.08(1H,m,H-3′)，1.97(3H,s,H-4′)，1.96(3H,d,J＝10.0Hz,H-5′)，6.17(1H,m,H-3″)，1.80(1H,s,H-4″)，1.97(3H,d,J＝10.0Hz,H-5″)。13C NMR(CD3OD,125MHz)δ24.1(C-1)，34.9(C-2)，217.8(C-3)，80.3(C-4)，78.0(C-5)，79.9(C-6)，40.2(C-7)，76.2(C-8)，75.3(C-9)，30.9(C-10)，133.1(C-11)，169.3(C-12)，124.6(C-13)，20.2(C-14)，23.5(C-15)，167.1(C-1′)，127.4(C-2′)，138.2(C-3′)，19.2(C-4′)，14.6(C-5′)，167.5(C-1″)，127.4(C-2″)，138.4(C-3″)，19.3(C-4″)，14.6(C-5″)。
example 12
Preparation of cardiothiolade K:
the total lactones obtained in example 5 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain cardiothiolade K.
The physicochemical data for cardiothiolade K are as follows: white needle crystal (Jia)Alcohol), is easily soluble in methanol, and is soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 489.2101[ M + Na ]]+(calcd for C24H34O9Na,489.2101)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.90(1H,m,H-1a)，1.72(1H,m,H-1b)，3.77(1H,m,H-2a)，2.31(1H,m,H-2b)，5.18(1H,d,J＝10.0Hz,H-5)，4.90(1H,m,H-6)，3.14(1H,m,H-7)，5.65(1H,d,J＝10.0Hz,H-8)，3.76(1H,d,J＝10.5Hz,H-9)，2.09(1H,m,H-10)，6.32(1H,d,J＝3.0Hz,H-13a)，5.70(1H,d,J＝3.0Hz,H-13b)，1.11(3H,d,J＝6.5Hz,H-14)，1.23(3H,s,H-15)，2.68(1H,m,H-2′)，1.22(3H,d,J＝6.0Hz,H-3′)，1.23(3H,d,J＝6.0Hz,H-4′)，6.09(1H,m,H-3″)，1.79(1H,s,H-4″)，1.90(3H,d,J＝10.0Hz,H-5″)。13C NMR(CD3OD,125MHz)δ24.5(C-1)，35.2(C-2)，217.5(C-3)，80.2(C-4)，78.1(C-5)，79.9(C-6)，40.2(C-7)，76.2(C-8)，75.3(C-9)，30.9(C-10)，133.0(C-11)，169.2(C-12)，124.6(C-13)，19.8(C-14)，23.1(C-15)，176.4(C-1′)，34.0(C-2′)，17.9(C-3′)，18.0(C-4′)，167.5(C-1″)，127.3(C-2″)，138.2(C-3″)，19.2(C-4″)，14.6(C-5″)。
example 13
Preparation of divarolide E:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain divarolide E.
The physicochemical data for divarolide E are as follows: white needle crystal (methanol), [ alpha ]]2D 0-95.2 (c 0.125, MeOH), readily soluble in methanol, soluble in chloroform, dichloromethane, ethanol. UV (MeOH) lambdamax(logε):210(3.38)nm，IR(KBr)nmax:3458,1744,1718,1661cm-1. Positive ion HR-ESI-MS shows: m/z 505.2427[ M + Na ]]+(calcd for C25H38O9Na,505.2414)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.79(1H,m,H-1a)，1.65(1H,m,H-1b)，3.76(1H,br d,J＝7.5Hz,H-2a)，2.22(1H,m,H-2b)，5.36(1H,dd,J＝8.5,2.0Hz,H-5)，4.60(1H,dd,J＝8.0,5.0Hz,H-6)，2.97(1H,m,H-7)，4.35(1H,d,J＝10.5Hz,H-8)，5.11(1H,d,J＝10.5Hz,H-9)，2.15(1H,m,H-10)，6.27(1H,d,J＝3.0Hz,H-13a)，5.62(1H,d,J＝2.5Hz,H-13b)，0.92(3H,d,J＝7.0Hz,H-14)，1.18(3H,s,H-15)，2.31(1H,d,J＝7.0Hz,H-2′a)，2.26(1H,o,H-2′b)，2.09(1H,o,H-3′)，0.96(3H,d,J＝7.0Hz,H-4′)，0.95(3H,d,J＝7.0Hz,H-5′)，2.06(2H,o,H-2″)，2.09(1H,o,H-3″)，0.96(3H,d,J＝7.8Hz,H-4″)，0.95(3H,d,J＝7.8Hz,H-5″)。13C NMR(CD3OD,125MHz)δ25.3(C-1)，32.8(C-2)，217.8(C-3)，80.3(C-4)，78.2(C-5)，79.8(C-6)，41.5(C-7)，70.3(C-8)，78.7(C-9)，29.8(C-10)，132.6(C-11)，169.6(C-12)，123.9(C-13)，20.0(C-14)，23.4(C-15)，172.5(C-1′)，42.7(C-2′)，25.3(C-3′)，21.3(C-4′)，21.4(C-5′)，173.3(C-1″)，43.0(C-2″)，25.4(C-3″)，21.4(C-4″)，21.4(C-5″)。
example 14
Preparation of divarolide F:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain divarolide F.
The physicochemical data for divarolide F are as follows: white needle crystal (methanol), which is easily soluble in methanol, and soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 475.1939[ M + Na ]]+(calcd for C23H32O9Na,475.1944)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,600MHz)δ1.83(1H,m,H-1a)，1.71(1H,m,H-1b)，3.81(1H,br d,J＝12.0Hz,H-2a)，2.16(1H,m,H-2b)，5.37(1H,dd,J＝9.6,1.2Hz,H-5)，4.65(1H,dd,J＝9.6,6.0Hz,H-6)，3.01(1H,m,H-7)，4.43(1H,d,J＝10.2Hz,H-8)，5.18(1H,d,J＝10.2Hz,H-9)，2.21(1H,m,H-10)，6.30(1H,d,J＝3.0Hz,H-13a)，5.67(1H,d,J＝3.0Hz,H-13b)，0.94(3H,d,J＝6.6Hz,H-14)，1.21(3H,s,H-15)，2.67(1H,m,H-2′)，1.21(1H,d,J＝6.6Hz H-3′)，1.20(3H,d,J＝6.6Hz,H-4′)，5.63(1H,dq,J＝3.6,1.8Hz,H-2″)，6.13(3H,dq,J＝3.0,1.8Hz,H-3″)，1.96(3H,br s,H-4″)。13C NMR(CD3OD,150MHz)δ25.3(C-1)，32.9(C-2)，217.6(C-3)，80.4(C-4)，78.1(C-5)，79.9(C-6)，41.6(C-7)，70.3(C-8)，79.3(C-9)，30.1(C-10)，132.7(C-11)，169.6(C-12)，123.8(C-13)，19.9(C-14)，23.3(C-15)，176.3(C-1′)，33.9(C-2′)，18.0(C-3′)，17.9(C-4′)，167.2(C-1″)，136.4(C-2″)，124.7(C-3″)，17.1(C-4″)。
example 15
Preparation of divarolide G:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to yield divarolide G.
The physicochemical data for divarolide G are as follows: white needle crystal (methanol), which is easily soluble in methanol, and soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 505.2414[ M + Na ]]+(calcd for C25H38O9Na,505.2414)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.88(1H,m,H-1a)，1.75(1H,m,H-1b)，3.87(1H,m,H-2a)，2.29(1H,m,H-2b)，5.39(1H,d,J＝10.0Hz,H-5)，4.69(1H,d,J＝10.0Hz,H-6)，3.02(1H,m,H-7)，4.40(1H,d,J＝10.0Hz,H-8)，5.15(1H,d,J＝10.0Hz,H-9)，2.23(1H,m,H-10)，6.32(1H,d,J＝3.0Hz,H-13a)，5.67(1H,d,J＝3.0Hz,H-13b)，0.98(3H,d,J＝6.5Hz,H-14)，1.24(3H,s,H-15)，2.52(1H,m,H-2′)，1.76(1H,m,H-3′a)，1.52(1H,m,H-3′b)，1.24(3H,d,J＝7.0Hz,H-4′)，0.98(3H,d,J＝7.0Hz,H-5′)，2.52(1H,m,H-2″)，1.76(1H,m,H-3″a)，1.52(1H,m,H-3″b)，1.26(3H,d,J＝7.0Hz,H-4″)，0.96(3H,d,J＝7.0Hz,H-5″)。13C NMR(CD3OD,125MHz)δ25.3(C-1)，33.2(C-2)，217.6(C-3)，80.3(C-4)，78.1(C-5)，79.9(C-6)，41.7(C-7)，70.5(C-8)，78.4(C-9)，30.0(C-10)，132.7(C-11)，169.5(C-12)，123.8(C-13)，20.0(C-14)，23.5(C-15)，175.9(C-1′)，41.3(C-2′)，26.3(C-3′)，16.1(C-4′)，10.7(C-5′)，176.7(C-1″)，41.5(C-2″)，26.2(C-3″)，16.2(C-4″)，10.6(C-5″)。
example 16
Preparation of aurilactone X1:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain the aurantiamarin X1.
The physicochemical data of the eupolyphaga lactone X1 are as follows: white needle crystal (methanol), white powder, easily soluble in methanol, soluble in chloroform, dichloromethane, ethanol. Positive ion HR-ESI-MS shows: m/z 491.2269[ M + Na ]]+(calcd for C24H36O9Na,491.2257)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,600MHz)δ1.85(1H,m,H-1a)，1.73(1H,m,H-1b)，3.83(1H,br d,J＝9.0Hz,H-2a)，2.20(1H,o,H-2b)，5.42(1H,dd,J＝9.6,2.4Hz,H-5)，4.69(1H,dd,J＝9.6,2.4Hz,H-6)，3.04(1H,m,H-7)，4.42(1H,d,J＝10.0Hz,H-8)，5.18(1H,d,J＝10.2Hz,H-9)，2.20(1H,o,H-10)，6.34(1H,d,J＝3.0Hz,H-13a)，5.69(1H,d,J＝3.0Hz,H-13b)，1.02(3H,d,J＝7.2Hz,H-14)，1.25(3H,s,H-15)，2.65(1H,m,H-2′)，1.26(1H,d,J＝6.6Hz,H-3′)，1.25(3H,d,J＝6.6Hz,H-4′)，2.33(1H,d,J＝7.8Hz,H-2″a)，2.20(1H,o,H-2″b)，2.12(1H,m,H-3″)，1.02(3H,d,J＝7.2Hz,H-4″)，1.00(3H,d,J＝6.6Hz,H-5″)。13C NMR(CD3OD,150MHz)δ25.3(C-1)，32.8(C-2)，217.7(C-3)，80.3(C-4)，78.1(C-5)，79.9(C-6)，41.3(C-7)，70.2(C-8)，78.6(C-9)，29.9(C-10)，132.7(C-11)，169.7(C-12)，123.8(C-13)，19.9(C-14)，23.3(C-15)，176.3(C-1′)，33.9(C-2′)，17.39(C-3′)，17.9(C-4′)，173.2(C-1″)，43.0(C-2″)，25.3(C-3″)，21.4(C-4″)，21.4(C-5″)。
example 17
Preparation of aurilactone X2:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain the aurantiamarin X2.
The physicochemical data of the eupolyphaga lactone X2 are as follows: white needle crystal (methanol), [ alpha ]]2D 0-70.6 (c 0.085, MeOH), readily soluble in methanol, soluble in chloroform, dichloromethane, ethanol. Positive ion HR-ESI-MS shows: m/z 505.2473[ M + Na ]]+(calcd for C25H38O9Na,505.2414)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.83(1H,m,H-1a)，1.71(1H,m,H-1b)，3.81(1H,br d,J＝9.0Hz,H-2a)，2.20(1H,o,H-2b)，5.40(1H,d,J＝8.0Hz,H-5)，4.66(1H,dd,J＝8.0,5.5Hz,H-6)，3.01(1H,m,H-7)，4.40(1H,d,J＝8.5Hz,H-8)，5.15(1H,d,J＝8.5Hz,H-9)，2.20(1H,o,H-10)，6.31(1H,d,J＝3.0Hz,H-13a)，5.66(1H,d,J＝3.0Hz,H-13b)，0.97(3H,d,J＝5.5Hz,H-14)，1.22(3H,s,H-15)，2.52(1H,m,H-2′)，1.70(1H,m,H-3′a)，1.54(1H,m,H-3′b)，1.21(3H,d,J＝5.5Hz,H-4′)，0.99(3H,t,J＝7.0Hz,H-5′)，2.20(2H,d,J＝5.5Hz,H-2″)，2.15(1H,m,H-3″)，1.00(3H,d,J＝5.5Hz,H-4″)，1.00(3H,d,J＝5.5Hz,H-5″)。13C NMR(CD3OD,150MHz)δ25.4(C-1)，32.9(C-2)，217.6(C-3)，80.4(C-4)，78.1(C-5)，79.8(C-6)，41.5(C-7)，70.4(C-8)，78.7(C-9)，29.9(C-10)，132.7(C-11)，169.7(C-12)，123.7(C-13)，20.0(C-14)，23.3(C-15)，176.0(C-1′)，41.1(C-2′)，26.5(C-3′)，15.7(C-4′)，10.5(C-4′)，173.2(C-1″)，43.0(C-2″)，25.3(C-3″)，21.4(C-4″)，21.4(C-5″)。
example 18
Preparation of aurilactone X3:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain the aurantiamarin X3.
The physicochemical data of the eupolyphaga lactone X3 are as follows: white needle crystal (methanol), which is easily soluble in methanol, and soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 481.2449[ M + H ]]+(calcd for C25H37O9,481.2438)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,500MHz)δ1.88(1H,m,H-1a)，1.73(1H,o,H-1b)，3.85(1H,m,H-2a)，2.24(1H,o,H-2b)，5.51(1H,dd,J＝9.5,2.0Hz,H-5)，4.73(1H,dd,J＝9.0,6.5Hz,H-6)，3.06(1H,m,H-7)，4.44(1H,d,J＝10.5Hz,H-8)，5.17(1H,d,J＝10.5Hz,H-9)，2.24(1H,o,H-10)，6.33(1H,d,J＝3.0Hz,H-13a)，5.68(1H,d,J＝3.0Hz,H-13b)，0.98(3H,d,J＝7.5Hz,H-14)，1.25(3H,s,H-15)，6.18(1H,qq,J＝7.0,1.5Hz,H-3′)，1.98(3H,dq,J＝1.5,1.5Hz,H-4′)，1.96(3H,dq,J＝7.0,1.5Hz,H-5′)，2.48(1H,m,H-2″)，1.73(1H,o,H-3″a)，1.50(1H,m,H-3″b)，1.21(3H,d,J＝7.0Hz,H-4″)，0.97(3H,t,J＝7.0Hz,H-5″)。13C NMR(CD3OD,150MHz)δ25.4(C-1)，33.1(C-2)，217.9(C-3)，80.4(C-4)，78.1(C-5)，80.0(C-6)，41.5(C-7)，70.4(C-8)，78.4(C-9)，30.0(C-10)，132.8(C-11)，169.7(C-12)，124.0(C-13)，20.0(C-14)，23.4(C-15)，167.1(C-1′)，127.8(C-2′)，138.4(C-3′)，19.3(C-4′)，14.6(C-5′)，176.8(C-1″)，41.3(C-2″)，26.3(C-3″)，16.2(C-4″)，10.7(C-5″)。
example 19
Preparation of aurilactone X4:
the total lactones obtained in example 1 were then individually prepared using prepHPPLC (60% CH)3OH) to obtain the aurantiamarin X4.
The physicochemical data of the eupolyphaga lactone X4 are as follows: white needle crystal (methanol), which is easily soluble in methanol, and soluble in chloroform, dichloromethane, and ethanol. Positive ion HR-ESI-MS shows: m/z 489.2108[ M + Na ]]+(calcd for C24H34O9Na,489.2101)。
The structure of the compound is confirmed by a nuclear magnetic resonance method:1H NMR(CD3OD,600MHz)δ1.87(1H,m,H-1a)，1.76(1H,o,H-1b)，3.89(1H,m,H-2a)，2.24(1H,o,H-2b)，5.52(1H,d,J＝9.6Hz,H-5)，4.74(1H,dd,J＝9.6,6.6Hz,H-6)，3.07(1H,m,H-7)，4.45(1H,d,J＝10.2Hz,H-8)，5.16(1H,d,J＝10.8Hz,H-9)，2.24(1H,o,H-10)，6.34(1H,d,J＝3.6Hz,H-13a)，5.69(1H,d,J＝3.0Hz,H-13b)，0.98(3H,d,J＝7.2Hz,H-14)，1.26(3H,s,H-15)，6.20(1H,qq,J＝7.2,1.8Hz,H-3′)，1.99(3H,dq,J＝1.8,1.8Hz,H-4′)，2.01(3H,dq,J＝7.2,1.8Hz,H-5′)，2.68(1H,m,H-2″)，1.20(3H,d,J＝7.2Hz,H-3″)，1.25(3H,d,J＝7.2Hz,H-4″)。13C NMR(CD3OD,150MHz)δ25.3(C-1)，33.1(C-2)，217.9(C-3)，80.4(C-4)，78.1(C-5)，80.0(C-6)，41.6(C-7)，70.3(C-8)，78.5(C-9)，30.0(C-10)，132.7(C-11)，169.7(C-12)，124.0(C-13)，19.9(C-14)，23.4(C-15)，167.1(C-1′)，127.4(C-2′)，138.4(C-3′)，19.2(C-4′)，14.6(C-5′)，177.2(C-1″)，34.0(C-2″)，18.5(C-3″)，17.8(C-4″)。
the above compounds were all extracted to give 98% of the samples used in the experiments of the present invention.
The present invention is not limited to the above embodiments, and any other products similar or identical to the present invention, which can be obtained by anyone in the light of the present invention, are not excluded from the scope of the present invention.