CN109053651B - Spiro sesquiterpene dimer compound and preparation method thereof - Google Patents
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Abstract
The invention relates to a spiro sesquiterpene dimer compound and a preparation method thereof. The compound has a structure shown in a formula I, wherein R1-R18 are respectively and independently selected from hydrogen, halogen, hydroxyl and amino. When prepared, the method comprises the following steps: (1) extracting dry root of east China blue thorn by using ethanol to obtain ethanol extract; (2) subjecting the ethanol extract to silica gel column chromatography, and sequentially subjecting to gradient elution with petroleum ether-ethyl acetate and petroleum ether-acetone as eluents to obtain fraction; (3) and (3) eluting the fraction obtained in the step (2) by using hydroxypropyl sephadex with chloroform-methanol to obtain the spiro sesquiterpene dimer compound. The invention provides the compound for the first time, and finds that the compound has wide application in the fields of medicines, pesticides and the like.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a spiro sesquiterpene dimer compound and a preparation method thereof.
Background
Plant resources are an important source and a model basis for new drug development. On the one hand, the abundant chemical components and diverse biological activities in natural plants make them always an important source for people to find active medical leads; on the other hand, the plant secondary metabolite has the advantages of difficult generation of drug resistance, safety to non-target organisms, good environmental compatibility and the like, and becomes an important direction for the research and development of environment-friendly pesticides. The research and the product development of the secondary metabolite of the plant with homology of medicine and food provide a powerful weapon for resisting diseases for human beings and provide green low-toxicity substances for the development of Chinese botanical pesticides.
Echinops grijsii Hance is a plant of genus Echinops (Compositae). The dried root is also called Yuzhou uniflower swisscentaury root, has the efficacies of clearing away heat and toxic material, eliminating carbuncle, promoting lactation, relaxing muscles and tendons, promoting blood circulation and the like, and is recorded in Chinese pharmacopoeia of 2010 edition. Has great development and application prospects in treating acute mastitis swelling and pain, carbuncle, cellulitis, dorsum, scrofula and sore toxin, galactostasis, and damp arthralgia and spasm.
To date, researchers at home and abroad have separated various structural compounds from the dry root of east China blue thorn, mainly including thiophene, phenylpropanoids, terpenes (monoterpene, sesquiterpene, diterpene, etc.), steroids, alkaloids, fatty acids, and the like. The thiophene and sesquiterpene compounds are various in types and rich in content.
By spiro compound is meant a polycyclic compound in which two single rings share a common carbon atom. Because of the rigid structure and stable structure, the chiral ligand has larger specific optical rotation, and has important application in the aspects of asymmetric catalysis, luminescent materials, pesticides, hospitals, polymer adhesives and the like. Therefore, the separation and extraction of the sesquiterpene dimer structure compound containing spiro ring from the east China blue spiny root medicinal material has important practical significance.
At present, no report related to the compound of the invention exists, and no report related to the application of the compound of the invention in medical and agricultural activities is found.
Disclosure of Invention
In view of the problems in the prior art, the invention provides a spiro sesquiterpene dimer compound, a preparation method and an application thereof. The compound with the structure has application values of medicines and pesticides, and a pharmaceutical composition containing the spiro sesquiterpene dimer compound provides more drug selection ways for resisting tumors, resisting inflammation, protecting liver, killing aphids and nematodes.
The technical scheme for solving the technical problems is as follows:
a spiro sesquiterpene dimer compound comprises a structure shown in formula I:
wherein R1-R18 are independently selected from hydrogen, halogen, hydroxyl and amino.
The invention provides a compound shown in formula I, or a crystal form, a stereoisomer, a pharmaceutically acceptable salt, a solvate, a prodrug or a metabolite of the compound.
Preferably, in the formula I, R1 to R18 are all hydrogen. The inventor unexpectedly finds that when R1-R18 are all hydrogen, the spiro sesquiterpene dimer compound has four highly oxidized carbon atoms, has the advantages of good fat solubility, good permeability and the like, and is beneficial to the medicine molecules entering the organism to play a relevant role.
Preferably, the spiro sesquiterpene dimer compound comprises a structure shown in a formula II:
the inventor finds that the compound with the structure shown in the formula II has the advantages of better lipid solubility, permeability and the like.
The invention also provides a pharmaceutical preparation which comprises the spiro sesquiterpene dimer compound. The pharmaceutical preparation is a composition, and is a preparation prepared from a compound shown in the formula I or the formula II, or a crystal form, a stereoisomer, a pharmaceutically acceptable salt, a solvate, a prodrug or a metabolite thereof and pharmaceutically acceptable auxiliary materials.
One or more of the compounds of the present invention may be used in combination with each other, or alternatively, in combination with any other active agent. If a group of compounds is used, the compounds may be administered to the subject simultaneously, separately or sequentially.
The pharmaceutically acceptable auxiliary material of the invention refers to a substance contained in a dosage form except for an active ingredient.
The invention also provides a preparation method of the spiro sesquiterpene dimer compound, which comprises the following steps:
(1) extracting dry root of east China blue thorn by using ethanol to obtain ethanol extract;
(2) subjecting the ethanol extract to silica gel column chromatography, and sequentially subjecting to gradient elution with petroleum ether-ethyl acetate and petroleum ether-acetone as eluents to obtain fraction;
(3) and (3) eluting the fraction obtained in the step (2) by using hydroxypropyl sephadex with chloroform-methanol to obtain the spiro sesquiterpene dimer compound.
Specifically, the specific operation steps of step (2) may be:
(a) taking the ethanol extract obtained in the step (1), performing gradient elution by adopting silica gel column chromatography and taking petroleum ether-ethyl acetate volume ratios (25:1) - (0:1) as eluent, and collecting eluent as fraction;
(b) subjecting the fraction collected in the step (a) to silica gel column chromatography, performing gradient elution by using petroleum ether-acetone solvents with volume ratios of (30:1) to (1:1), and collecting the fraction;
(c) and (c) subjecting the fraction collected in the step (b) to silica gel column chromatography, performing gradient elution by petroleum ether-acetone according to the volume ratio of (8:1) to (1:1), and collecting the fraction.
In particular, the following specific steps may be used to prepare the spirocyclic sesquiterpene dimer compounds:
(1) taking dry root of east China blue thorn, crushing, carrying out reflux extraction by using 95% ethanol, combining extracting solutions, and concentrating under reduced pressure to obtain ethanol extract;
(2) the method comprises the following steps:
(a) and (2) taking the ethanol extract obtained in the step (1), performing gradient elution by using petroleum ether-ethyl acetate as an eluent in a volume ratio of 25:1, 10:1, 8:1, 5:1, 3:1, 1:1 and 0:1 in sequence by adopting silica gel column chromatography, detecting by using thin-layer chromatography, developing, combining the same elution parts, and concentrating the combined elution parts under reduced pressure until the combined elution parts are dry for later use.
(b) Subjecting the certain component in the step (a) to silica gel column chromatography, gradient eluting with petroleum ether-acetone solvent at volume ratio of (30:1) - (1:1), detecting by thin layer chromatography, developing, and mixing the same elution parts.
(c) Performing silica gel column chromatography, gradient eluting with petroleum ether-acetone at volume ratio of (8:1) - (1:1), detecting with thin layer chromatography, developing, and mixing the same eluates.
(3) And (c) passing a certain fraction obtained in the step (c) through Sephadex LH-20 (hydroxypropyl Sephadex), and eluting with 1:1 chloroform-methanol to obtain the spiro sesquiterpene compound.
Preferably, in the step (a), an eluent obtained by eluting with an eluent with a petroleum ether-ethyl acetate volume ratio of 5:1 is collected as a fraction.
Preferably, in the step (b), the eluent obtained by eluting with the eluent with the petroleum ether-acetone volume ratio of 5:1 is collected as a fraction.
Preferably, in the step (c), the eluent obtained by eluting with the eluent with the petroleum ether-acetone volume ratio of 3:1 is collected as a fraction.
During the research process, the inventor tries a plurality of experiments, and unexpectedly finds that the beneficial effects of adopting the parameters are as follows: obtain high purity and high quality echinocandin A.
The invention also provides a new application of the spiro sesquiterpene dimer compound.
The invention provides application of a compound shown in the formula I or the formula II, or a crystal form, a stereoisomer, a pharmaceutically acceptable salt, a solvate, a prodrug or a metabolite of the compound in preparation of medicines and pesticides.
The invention also provides a new application of the spiro sesquiterpene dimer compound.
The spiro sesquiterpene dimer compound can be applied to preparation of antitumor drugs, has obvious antitumor activity, and can be further prepared into antitumor drugs for cancer treatment.
The spiro sesquiterpene dimer compound can be applied to preparation of anti-inflammatory drugs. The inventor unexpectedly finds that the anti-inflammatory capability of the compound is better than that of the existing drug dexamethasone in the research process.
The spiro sesquiterpene dimer compound can be applied to preparation of liver-protecting medicines. The spiro sesquiterpene dimer compound can be applied to preparation of drugs for inhibiting expression of key pathogenic factors IFN-gamma. The inventor proves that the spiro sesquiterpene dimer compound can obviously inhibit the expression of a key pathogenic factor IFN-gamma through a Con A-induced acute liver injury model, can reduce the secretion of an inflammatory medium induced by Con A through the treatment of the spiro sesquiterpene dimer compound, reduces the immune liver injury of a mouse, and has the effect of protecting the liver.
The spiro sesquiterpene dimer compound can be applied to preparation of pesticides.
The spiro sesquiterpene dimer compound can be applied to the preparation of aphicide. The research of the inventor shows that the spiro sesquiterpene dimer compound shows obvious toxicity on four aphids, namely the Sinorhizus persicae, the Aphis graminicola and the Aphis fabae, has a wide insecticidal spectrum and obvious insecticidal activity, and can be used as an active ingredient to be further prepared into an aphid-killing botanical pesticide.
The spiro sesquiterpene dimer compound can be applied to preparation of nematocides. The research of the inventor shows that the spiro sesquiterpene dimer compound has insecticidal capacity on meloidogyne incognita, and the nematicidal capacity of the spiro sesquiterpene dimer compound is unexpectedly found to be stronger than that of the existing commercial pesticide fosthiazate, so that the spiro sesquiterpene dimer compound has a better application prospect in the nematicidal direction as a plant-derived pesticide.
In the application process, the spiro sesquiterpene dimer compound can be prepared into a proper dosage form or added with one or more conventional auxiliary materials in the field to prepare a pharmaceutical composition for application according to the actual use requirement.
Test results show that the compound has good anti-tumor, anti-inflammatory, aphid-killing and nematode-killing effects, provides a new choice for clinically screening and/or preparing anti-tumor and anti-inflammatory medicines, and provides a new choice for screening and/or preparing aphid-killing and nematode-killing medicines in agriculture.
Obviously, according to the above-mentioned contents of the present invention, many other modifications, substitutions or changes can be made without departing from the basic technical idea of the present invention, and the modifications, substitutions or changes are also within the protection scope of the present invention.
Drawings
FIG. 1 is a HRESIMS plot of a compound of formula II.
FIG. 2 is an Infrared (IR) spectrum of a compound of formula II.
FIG. 3 is a drawing of a compound of formula II1H NMR spectrum.
FIG. 4 shows a compound of formula II13C NMR spectrum.
FIG. 5 is a diagram of the NMR HSQC spectrum of the compound of formula II.
FIG. 6 is a nuclear magnetic resonance HMBC spectrum of the compound of formula II.
FIG. 7 is a diagram of the COSY and HMBC signals of the compound of formula II.
FIG. 8 is a single crystal X of a compound of formula II.
FIG. 9 shows the results of comparing the serum IFN-. gamma.expression levels of the experimental group and the control group in example 4.
Detailed Description
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Unless otherwise specified, the raw materials and equipment used in the embodiments of the present invention are known and commercially available products are obtained.
EXAMPLE 1 preparation of the Compounds of the invention
1. Experimental materials:
① A Chinese medicinal composition
The root of Baphicacanthus cusia is purchased from Anguo Yao of Hebei province in 2017, and identified as the dried root of Baphicacanthus cusia (Echinops grijsii handle) of Baphicacanthus of Compositae by the inventor of the present invention, Liuting Ting Baboshi.
② reagent and filler
Column chromatography silica gel of 200-300 mesh (reagent grade) purchased from Qingdao ocean silica gel desiccant factory;
thin layer chromatography silica gel GF254 (chemical purity) purchased from Qingdao ocean silica gel desiccant factory;
sephadex LH-20 sepharose, available from Amersham, Sweden;
a GF254 silica gel preparation thin layer purchased from Yangtze river friend silica gel development Co., Ltd;
analytically pure reagents such as petroleum ether, n-hexane, trichloromethane, ethyl acetate, acetone, methanol and the like are purchased from Beijing chemical plants.
③ Experimental instrument
Bruker-AVIIIHD-600 NMR (Bruker, Switzerland); nicolet 5700 Infrared Spectroscopy (Thermo corporation, USA); Perkin-Elmer 341 polarimeter (Perkin Elmer, USA); BP211D one tenth ten million electronic balance (Sartorius, switzerland); r-210 rotary evaporator (BUCHI, Switzerland); model DZG-6050 vacuum drying oven (shanghai semen).
2. Separation and purification of components:
1) taking 5kg of dry root of east China blue thorn, crushing, extracting with 95% ethanol in volume fraction for 3 times under reflux, combining the extracting solutions, and concentrating under reduced pressure to obtain 400g of ethanol extract;
2) taking the ethanol extract obtained in the step 1), performing gradient elution by adopting silica gel column chromatography sequentially with petroleum ether-ethyl acetate volume ratios of 25:1, 10:1, 8:1, 5:1, 3:1, 1:1 and 0:1 as eluent, detecting by thin-layer chromatography, developing, and combining the same elution parts to obtain 7 fractions Fr.1-Fr.7. (Fr.1 is a fraction of petroleum ether-ethyl acetate 25:1, Fr.2 is a fraction of petroleum ether-ethyl acetate 10:1, Fr.3 is a fraction of petroleum ether-ethyl acetate 8:1, Fr.4 is a fraction of petroleum ether-ethyl acetate 5:1, and so on for a total of 7 fractions)
3) Subjecting Fr.4 obtained in step 2) to silica gel column chromatography, gradient eluting with petroleum ether-acetone at volume ratio of (30:1) to (1:1), detecting by thin layer chromatography, developing color with 10% sulphuric acid ethanol developer, and combining the same eluate according to Rf value and color development to obtain 7 fractions Fr.4.1-Fr.4.7(Fr.4.1 is petroleum ether-acetone 30:1 fraction, Fr.4.2 is petroleum ether-acetone 15:1 fraction, Fr.4.3 is petroleum ether-acetone 8:1 fraction, Fr.4.4 is petroleum ether-acetone 5:1 fraction, Fr.4.5 is petroleum ether-acetone 3:1 fraction, Fr.4.6 is petroleum ether-acetone 2:1 fraction, and Fr.4.7 is petroleum ether-acetone 1:1 fraction). Fr.4.4 is further subjected to silica gel column chromatography, gradient elution with petroleum ether-acetone at a volume ratio of (8:1) to (1:1), detection by thin layer chromatography, color development, and combination of the same elution fractions to obtain Fr.4.4.1-Fr.4.4.4(Fr.4.4.1 is a fraction of petroleum ether-acetone 8:1, Fr.4.4.2 is a fraction of petroleum ether-acetone 5:1, Fr.4.4.3 is a fraction of petroleum ether-acetone 3:1, and Fr.4.4.4 is a fraction of petroleum ether-acetone 1: 1).
4) Subjecting Fr.4.4.3 obtained in step 3) to Sephadex LH-20 (hydroxypropyl dextran gel), and eluting with 1:1 chloroform-methanol to obtain echinocandin A.
5) The thin-layer chromatography color development condition of the invention is as follows: observing dark spots under an ultraviolet lamp (254nm), spraying 10% ethanol sulfate, and baking at 105 deg.C until color development.
3. Identification of the compound of interest:
echinocandin A, white crystal, infrared data showed the presence of hydroxyl groups (3433 cm)-1) And a carbonyl group (1731 cm)-1) The signal (as shown in fig. 2). As shown in fig. 1, high scoreThe structural formula of the compound is shown in the resolution mass spectrum as ([ M + Na ]]+m/z489.2975, calcd 489.2981), unsaturation of 10. The carbon spectral data (fig. 4) for this compound shows that it has 30 carbon atoms, including 4 methyl groups, 12 methylene groups, 5 methine groups, and 9 quaternary carbons, including a significant 2 carbonyl signal, 6 exocyclic double bond signal, and two vicinal oxygen carbon signals. The compound can be divided into part a and part B by combining its one-dimensional nuclear magnetic data (fig. 3 and 4) and two-dimensional nuclear magnetic data (fig. 5, 6 and 7). Part a includes 2 methyl groups, 6 methylene groups (2 are alkene carbon signals), 2 vicinal oxymethylene groups and 5 quaternary carbons (including 1 carbonyl signal and 2 alkene carbon signals), which signals suggest that our part a contains 2 terminal double bonds.1H-1The presence of H (5) -H can be clearly seen in the H COSY signal2(6)-H(7)-H2(8)-H2(9) (fig. 7). Further HMBC remote correlation gives H2-1 has remote association with C-2, C-3 and C-14; h215 has remote correlations with C-3, C-4 and C-5, and these data suggest that we have a carbonyl group attached at the 2-position and an oxygen atom attached at the 3-position (FIG. 7). Parts B and A are very similar, except that at C-4 'and C-15' are attached a methylene group and a methine group, respectively. The further HMBC signal H-4' is related to the remote location of C-3; h2Remote association of-15' with C-3, C-4 and C-2 gives that the attachment of part A to part B is via a novel carbon-carbon direct attachment. H-4 and H2-15' of1H-1The H COSY correlation further confirms the connection mode of the H COSY and the H COSY. Since the chemical shift of carbon to the oxygen atom is relatively low field, it was determined that the hydroxyl group is attached at the C-3' position. The structural formula of the compound was further confirmed by X-ray single crystal diffraction (FIG. 8). The compound is a new skeleton eudesmane dimer compound, which has a unique C-3 and C-15' connection mode and has a special 1-oxaspiro [4.5 ]]Can-6-onering, named as echinocandin A. Therefore, the chemical structure of the novel compound is determined as shown in the formula I, and the chemical structure including the absolute configuration is also determined as shown in the formula II:
4. nuclear magnetic resonance hydrogen spectrum (1H-NMR): Bruker-AVIII HD-600spectrometer assay, data are shown in Table 1.
5. Nuclear magnetic resonance carbon spectrum (13C-NMR): Bruker-AVIII HD-600spectrometer assay, data are shown in Table 1.
TABLE 1 of Echinocamphetamine A1H-NMR(600MHz)、13C-NMR (150MHz) nuclear magnetic data (determination solvent: CDCl)3；δ：ppm；J：Hz)
6. X-single crystal: the Agilent Gemini E double-light source X-ray single crystal diffractometer is used for measuring, and the data are as follows:
the molecular formula is as follows: c30H42O4M is 466.31, triclinic,
α＝108.971(5)°,β＝90.502(4)°,γ＝107.466(4)°,
106.6K for T, space group P1, 2 for Z, 0.562mm for μ (CuK α)-1,19415Reflections collected,10018 Independent reflections(Rint＝0.0496).Thefinal wR(F2)values were 0.1482(all data).The goodness of fit on F2was1.023.Flack parameter＝-0.17(16).
The absolute configuration of the glaucopterin A is determined by the crystal data, and is shown as a formula II.
To illustrate the advantageous effects of the present invention, the present invention further provides the following examples.
Example 2 in vitro antitumor experiments
1. Test cell
Human hepatoma cells (HepG2), human cervical carcinoma cells (Hela) and human laryngeal carcinoma cells (Hep2) purchased from the laboratory of the central hospital of the Beijing coordination of Chinese academy of medical sciences.
2. Experimental drugs
The spirocyclic sesquiterpene dimer (glaucontin a) prepared in example 1, and camptothecin (purchased from the national pharmaceutical group chemical agents ltd) were used as positive control drugs. The culture medium is used for preparing a drug solution with the concentration of 5-80 mu M.
3. Determination of the Activity of pharmaceutical anticancer cells (MTT method)
Respectively culturing human liver cancer cells (HepG2), human cervical cancer cells (Hela) and human laryngeal cancer cells (Hep2) by using a high-glucose DMEM culture medium containing 10% by mass of fetal bovine serum. Then, three kinds of cells in logarithmic growth phase were cultured at 2X 104The cells were plated at a density of 100. mu.L/well in 96-well plates. After 24 hours, the test compounds (1, 3, 6, 12, 24, 48. mu.M) were added to the three tumor cells at different concentrations, and the culture was continued for 72 hours. The cell culture medium was decanted, 20. mu.L of MTT solution (in PBS, 5mg/ml) was added to each well, incubation was continued for 4 hours, 50. mu.L of 20% SDS solution was added to each well, and the cells were left overnight in a CO2 incubator. Measuring absorbance at 570nm wavelength with microplate reader, calculating growth inhibition rate of the compound to be detected with different concentrations on tumor cells, and calculating drug concentration (IC) of the compound to be detected on half growth inhibition of tumor cells50) Each experiment was repeated three times.
4. Results of the experiment
The result of the screening test of the in vitro anti-tumor activity proves that the glaucocalyxin A has obvious inhibition effect on three tumor cell lines of human liver cancer cells (HepG2), human cervical cancer cells (Hela) and human laryngeal cancer cells (Hep2) (shown in table 2), and particularly has anti-tumor activity (IC) on the tumor cell lines of human cervical cancer cells (Hela)5026.17 mu M) is superior to the existing antitumor drug camptothecin (IC)50＝31.66μM)。
TABLE 2 inhibitory Effect of Echinocamphetamine A on three tumor cell lines (IC)50，μM)
The test results show that the high-temperature-resistant steel,
as can be seen from the data in Table 2, the echinocandin A has inhibition effect on three tumor cells, namely HepG2, Hela and Hep2, and the inventors unexpectedly found that the echinocandin A has better inhibition effect on Hela cells than camptothecin.
The novel spiro sesquiterpene dimer compound (glaucocalyxin A) has obvious antitumor activity and can be further prepared into antitumor drugs for treating cancers.
Example 3 anti-inflammatory Activity-Effect on LPS-induced secretion of inflammatory factors by mouse macrophages
1. Test cell
Mouse macrophage strain RAW264.7 was purchased from the department of chinese academy coordination cell resource center.
2. Experimental drugs and reagents
Pancreatin, dimethyl sulfoxide (DMSO) (Gibco); RP-MI1640 cell culture medium (Hyclone Co.); nitric Oxide (NO) test kit (bi yun tian); MTS reagent (Promega corporation); LPS (Sigma Co.), other reagents were all commercially available analytical grade.
3. Cell culture
RAW264.7 mouse mononuclear macrophages were cultured in RP-MI1640 medium containing 10% fetal calf serum, penicillin (1X 105U/L) and streptomycin (100mg/L) at 37 deg.C and 5% CO2Culturing in the incubator, and carrying out subculture every other day.
4. Cytotoxicity test
Taking prepared mouse RAW264.7 cells, and mixing at 5 × 104cells/well in 96 well plates at 37 ℃ with 5% CO2After 24h of culture, drugs with different concentrations were added, and a blank control group was set. The culture was continued for 24h by adding 20. mu. LMTS reagent per well at 37 ℃ with 5% CO2And culturing for 1 h. And measuring absorbance at 490nm of an enzyme-labeling instrument, and observing whether the drug has cytotoxic effect.
5. Effect of Echinocamphetin A on NO secretion from RAW264.7 cells induced by LPS
Taking prepared mouse RAW264.7 cells, and mixing at 2 × 105cells/well device 9Placing in 6-well culture plate at 37 deg.C and 5% CO2After 24 hours of incubation, the medium was discarded and the drug and LPS were administered separately. The group was divided into a normal control group (complete medium), an LPS model group (medium containing 1. mu.g/mL LPS), and a treatment group (medium containing 1. mu.g/mL LPS and different concentrations of glaucopiate A). Setting a positive drug group (containing 1 mu g/mL of dexamethasone with 100 mu M of LPS), culturing in an incubator for 24h, absorbing 50 mu L of culture solution supernatant, operating according to the instruction of the NO kit, reading the absorbance value at 540nm, and calculating the content of NO in each hole.
6. Results of the experiment
After the echinocandin A acts on RAW264.7 cells for 24h, no obvious cytotoxicity is generated at 100 mu M compared with the blank (Table 3). After 1 mu g/mL of LPS stimulation, macrophages can release a large amount of NO, the positive drug dexamethasone can reduce the NO level, and the experimental result shows that 25 mu M of glaucotin A can inhibit the NO release of RAW264.7 cells induced by LPS by more than 50% (NO inhibition rate 55.09%), which indicates that the inhibition rate of the lower-concentration glaucotin A (25 mu M) can inhibit the NO release of RAW264.7 cells induced by LPS by more than 50%. The 50 μ M echinocandin a had significantly better ability to inhibit LPS-induced release of NO from RAW264.7 cells than 100 μ M dexamethasone (see table 4). The echinocandin A is proved to have the anti-inflammatory capability superior to that of the existing drug dexamethasone.
TABLE 3 Echinacea inhibition of RAW264.7 cell proliferation
TABLE 4 Effect of Echinocamphetamine A on LPS stimulation of NO release from RAW264.7 cells
Example 4 anti-inflammatory Activity-ConA induced liver injury model in mice
1. Laboratory animal
C57BL/6 Male mice, 20-22 weeks old, 28-32 grams in body weight, purchased from laboratory animals of Wintolite, Beijing.
2. Experimental drugs and reagents
Canavalin a (ConA, Sigma); IFN-gamma ELISA detection kit was purchased from R & D.
3. Grouping and modeling
Grouping and modeling mice were randomly divided into experimental and control groups of 24 mice each, with free access during the experiment. Dissolving Echinocamphrena A with DMSO to storage concentration, and dissolving with sterile Phosphate Buffer Solution (PBS) to use concentration before use; ConA was dissolved in sterile PBS. The experimental group mice are injected with 50mg/kg of glaucocephalin A through the abdominal cavity, repeated for 1 time after 2 hours, and then induced by 15mg/kg of ConA through tail vein to establish an acute liver injury mouse model. Mice in the control group were first injected intraperitoneally with the same dose of control solvent (DMSO) as the experimental group, and repeated 1 injection after 2h, followed by modeling with 15mg/kg ConA via tail vein injection. 8h after modeling, the mouse is anesthetized by chloral hydrate, blood is taken from the heart, serum is separated, and the mouse is stored at the temperature of minus 80 ℃ for standby.
4. ELISA method for determining serum level IFN-gamma
The ELISA microporous reaction plate with strong adsorption to antibody is selected, washed once with coated buffering liquid and patted dry. The antibody was diluted to a specific concentration with coating buffer according to the kit instructions, and each well was blocked with 100. mu.L of blocking solution and 250. mu.L of blocking solution. Adding 100 μ L of diluted serum sample per well; diluting according to the specification, and adding 100 mu L of the solution into each well; blank wells were loaded with sample diluent only. Add one antibody 100. mu.L per well, after 1 hour at room temperature, wash 4 times. After adding 100. mu.L of the enzyme-labeled secondary antibody and 1 hour at room temperature, the mixture was washed 5 times. Adding 100 mu L of substrate into each hole, standing at room temperature for 10-20 minutes, adding 100 mu L of stop solution when the color of the standard hole reaches the proper depth to stop the reaction, and measuring the absorbance at 450 nm.
5. Results of the experiment
Con A can rapidly activate immune cells in mice, and then release a large amount of cytokines to induce hepatocyte necrosis. FIG. 9 shows the effect of Echinocapratin A on the IFN-gamma content in peripheral blood of ConA-induced liver injury mice detected by an ELISA kit. The results show that the mass concentration of IFN-gamma in the serum of the control group and the experimental group is respectively (2498.45 +/-63.67) pg/mL and (450.7 +/-37.28) pg/mL, and the difference has statistical significance P < 0.01. The experimental result shows that the echinocandin A can obviously inhibit the expression of a key pathogenic factor IFN-gamma. The Con A-induced acute liver injury model proves that the glaucotin A treatment can reduce the secretion of ConA-induced inflammatory mediators, reduce the immune liver injury of mice and has the effect of protecting the liver.
Example 5 aphid killing Activity assay
1. Source of test insects
The aphids of the Sitobion aphids, Sitobion avenae, Sitobion aphids, Rhopalosiphum padi and Aphis faba (Aphis craccivora) are bred in the chemical group of pesticide toxicology and natural products of the plant protection research institute of the Chinese academy of agricultural sciences.
2. Test medicine
Spirocyclic sesquiterpene dimer (glaucoxatin A) prepared in example 1, and a 25% pymetrozine suspension (Bao Luo agriculture science group, Inc. in Beijing) were used as positive control drugs. The preparation concentrations are 1, 3, 6, 12, 24 and 48 mg/L.
3. Indoor toxicity test (immersion method)
A9 cm diameter petri dish was taken and the bottom of the dish was covered with a layer of 9cm diameter wet filter paper to maintain humidity after labeling. Selecting leaves with aphids, keeping 30 healthy aphids, respectively soaking in medicament solutions with various concentrations for 5s, placing in a culture dish after soaking, sucking redundant liquid medicine with absorbent paper, airing at room temperature, sealing with a preservative film, pricking a plurality of air holes with insects, and labeling. 3 replicates per concentration were set and the control was a 0.1% tween-80 solution containing acetone. And (3) putting the aphids into a climatic chamber with a photoperiod L: D of 14 h: 10h, a temperature of (25 +/-2) DEG C and a relative humidity of 48-52 percent for feeding, and investigating the death number and survival number of the aphids after 24 h.
Test results show that the echinocandin A shows obvious toxicity (LC) on four aphids, namely the vegetable aphid of Sinonovacula constricta, the wheat aphid of Neurovacula constricta and the broad bean aphid503.49, 16.63, 7.51, 9.36mg/L, respectively) (as shown in Table 5). LC of Echinocopterid A for Aphis citricola, Aphis graminicola and Aphis fabae50The values are all higher than those of the common pesticide pymetrozine sold in the market, and the result shows that the echinocandin A has better insecticidal activity.
TABLE 5 indoor virulence determination (LC) of Echinocapratin A against various aphids50，mg/L)
The novel spiro sesquiterpene dimer compound (glaucocalyxin A) has a wide insecticidal spectrum and obvious insecticidal activity, and can be used as an active ingredient to be further prepared into aphid-killing botanical pesticide.
Example 6 nematicidal Activity assay
1. Source of test insects
Meloidogyne incognita (melodogyne incognita), a chemical group of pesticide toxicology and natural products of the institute of plant protection, academy of agricultural sciences, china, picks up egg masses from cucumber roots, and hatches the larvae in a laboratory for later use.
2. Test medicine
The spiro sesquiterpene dimer (lanocerin a) prepared in example 1, and fosthiazate were used as positive control drugs. The preparation concentrations were 6, 12, 24, 48, 96 mg/L.
3. Preparation of Meloidogyne incognita suspension
Picking up egg masses on root knots from plants with serious root knot nematode morbidity, separating the larvae by a tray method after the root knot nematode larvae are incubated at normal temperature, diluting the nematode suspension after microscopic examination, and storing the nematode suspension at normal temperature for later use, wherein each 1ml of the suspension contains about 300 second-instar larvae.
4. Biological activity assay
The impregnation method is adopted. The drugs were each set to 5 concentration treatments, the blank set to 1, and each treatment was set to 4 replicates. Taking 24-hole biochemical test plate, placing 1ml of test solution into small holes, adding isovolumetric nematode suspension to obtain 3, 6, 12, 24, 48mg/L drug treatment solution, adding equivalent clear water into blank treatment, checking survival number and death number of second-instar larvae of Meloidogyne incognita after 48 hours treatment, and calculating LD50。
TABLE 6 determination of the virulence of meloidogyne incognita by Echinocamphetamine A
Indoor virulence determination is shown in Table 6, LD of M.meloidogyne incognita by fosthiazate5029.63mg/L, LD of Echinocamphetamine A for Meloidogyne incognita50It was 13.25 mg/L. The experimental result shows that the nematicidal ability of the echinocandin A to the meloidogyne incognita is stronger than that of the existing commercial pesticide fosthiazate, and the echinocandin A has better application prospect in the nematicidal direction as a botanical pesticide.
In conclusion, the invention provides the spiro sesquiterpene dimer compound and the extraction and separation method thereof, the compound has the unique chemical structure and is extracted from the common traditional Chinese medicine east China blue thorn, and the experimental result shows that the compound has the activities of resisting tumor, protecting liver, resisting inflammation, killing aphids and killing nematodes, so the compound is used as a plant source natural product to develop a new medicine of the traditional Chinese medicine, and has wide application prospect in the fields of medicine and pesticide.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A spiro sesquiterpene dimer compound, which is characterized by comprising a structure shown in formula I:
wherein R1-R18 are independently selected from hydrogen, halogen, hydroxyl and amino.
2. The spirocyclic sesquiterpene dimer compound of claim 1, wherein R1-R18 are each hydrogen.
3. The spirocyclic sesquiterpene dimer compound of claim 1 comprising the structure of formula ii:
4. a pharmaceutical formulation comprising a spirocyclic sesquiterpene dimer compound according to any one of claims 1-3.
5. A process for the preparation of a spirocyclic sesquiterpene dimer compound according to any of claims 1-3 comprising the steps of:
(1) extracting dry root of east China blue thorn by using ethanol to obtain ethanol extract;
(2) subjecting the ethanol extract to silica gel column chromatography, and sequentially subjecting to gradient elution with petroleum ether-ethyl acetate and petroleum ether-acetone as eluents to obtain fraction;
(3) and (3) eluting the fraction obtained in the step (2) by using hydroxypropyl sephadex with chloroform-methanol to obtain the spiro sesquiterpene dimer compound.
6. The method for preparing spiro sesquiterpene dimer compound of claim 5, wherein the specific steps in step (2) are as follows:
(a) taking the ethanol extract obtained in the step (1), performing gradient elution by adopting silica gel column chromatography and taking petroleum ether-ethyl acetate volume ratios (25:1) - (0:1) as eluent, and collecting eluent as fraction;
(b) subjecting the fraction collected in the step (a) to silica gel column chromatography, performing gradient elution by using petroleum ether-acetone solvents with volume ratios of (30:1) to (1:1), and collecting the fraction;
(c) and (c) subjecting the fraction collected in the step (b) to silica gel column chromatography, performing gradient elution by petroleum ether-acetone according to the volume ratio of (8:1) to (1:1), and collecting the fraction.
7. The method for preparing spiro sesquiterpene dimer compound of claim 6, wherein in step (a), the eluate obtained by eluting with an eluent comprising petroleum ether and ethyl acetate at a volume ratio of 5:1 is collected as a fraction.
8. The method for preparing spiro sesquiterpene dimer compound of claim 6 or 7, wherein in step (b), the eluate obtained by eluting with an eluent having a petroleum ether-acetone volume ratio of 5:1 is collected as a fraction.
9. The process for preparing spiro sesquiterpene dimer compound of claim 6 or 7, wherein in step (c), the fraction is collected as an eluate obtained by eluting with an eluent comprising petroleum ether and acetone at a volume ratio of 3: 1.
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