Elephantopus scaber lactone compound, preparation method and application thereof
阅读说明：本技术 一种地胆草内酯化合物、制备方法及其应用 (Elephantopus scaber lactone compound, preparation method and application thereof ) 是由 邹忠梅 张涛 付露 于猛 齐云 李凌宇 于 2020-07-16 设计创作，主要内容包括：本发明提供了一种地胆草内酯化合物,所述地胆草内酯化合物的结构通式为：<Image he="526" wi="486" file="DDA0002587804940000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>该类化合物从地胆草属植物的花、根、茎叶或全草提取得到,具有优异的抗炎活性,为该化合物未报到的活性,且强于地胆草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及其在抗炎,尤其制备神经炎症药物中的应用,适合工业生产。(The invention provides a elephantolide compound, which has a structural general formula as follows: the compound is extracted from flowers, roots, stems and leaves or whole plants of elephantopus plants, has excellent anti-inflammatory activity, is activity which is not reported by the compound, and is stronger than the elephantopus whole extract and effective parts thereof; the invention also provides a preparation method of the compound and application of the compound in anti-inflammation, in particular to preparation of neuroinflammation drugs, and is suitable for industrial production.)
1. A elephantopin lactone compound is characterized in that the elephantopin lactone compound is a compound shown as a structural formula I or a physiologically acceptable salt thereof,
C7-C11is a single bond or a double bond;
R1is one of double bond, hydroxymethyl, methyl, hydroxyl and ester group containing 1-8 carbon atoms;
R2Is one of hydrogen, methyl, ethyl and acyl containing 1-8 carbon atoms;
R3is unsubstituted, hydrogen or hydroxyl.
2. The elephantolide compound as claimed in claim 1, wherein said elephantolide compound comprises an isomer.
3. The elephantolide compound as claimed in claim 2, wherein said isomer is C2-O chemical bond and/or C7-C11The isomer of (1).
4. The elephantolide compound as claimed in claim 1, wherein R is selected from the group consisting of1Is one of double bond, hydroxymethyl, methyl, hydroxyl, acyloxymethyl containing 1-5 carbon atoms, R2Is one of hydrogen, ethyl and acyl containing 1 to 5 carbon atoms.
5. The elephantolide compound as claimed in claim 4, wherein R is1Is a double bond, hydroxymethyl, methyl, hydroxy or 2, 5-dihydroxyvaleryloxymethyl group, R2Is hydrogen, 2-methacryloyl, angeloyl or ethyl, and when R is2When it is angeloyl, R3Is hydrogen or hydroxy.
6. The elephantolide compound according to claim 5, wherein,
C7-C11when it is a double bond, R1Is hydroxy or hydroxymethyl, R2Is 2-methacryloyl, isovaleryl, or ethyl, R 3Is unsubstituted;
or C7—C11When it is a single bond, R1Is methyl, a methyl double bond, 2, 5-dihydroxyvaleryloxymethyl or hydroxymethyl, R2Is hydrogen, angeloyl or 2-methacryloyl, and R3 is unsubstituted, hydrogen or hydroxy.
7. The method for producing the elephantolide compound as claimed in claim 1, comprising the steps of:
extracting flower, root, stem and leaf or whole plant of elephantopus plant with 50-95% organic solvent, dispersing the extract with water, extracting with ethyl acetate, collecting ethyl acetate part, 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.
8. The method of claim 7, wherein the organic solvent is one of methanol, ethanol, acetone, diethyl ether and ethyl acetate.
9. The method of claim 8, wherein the column chromatography comprises: separating the extract by silica gel column chromatography, performing gradient elution with dichloromethane-acetone, collecting effective components, recovering solvent to dryness, purifying by gel column chromatography, eluting with methanol, dichloromethane-methanol or acetone as eluent, collecting eluate, and recovering solvent to dryness to obtain the compound of formula (I).
10. The use of the elephantopus scaber lactone compound of claim 1 in the preparation of an anti-inflammatory medicament.
Technical Field
The invention relates to an application of a elephantopus scaber lactone compound in preparation of an anti-inflammatory drug, and belongs to the field of medicines.
Background
Inflammation is a complex immune defense reaction involving multiple cells and multiple factors, can occur in tissues and organs of any part of an organism, and is a common pathological process in clinic. Research shows that inflammation is related to many diseases, such AS inflammatory cytokine CRP can promote the generation and development of Atherosclerosis (AS), tumor necrosis factor-alpha (TNF-alpha) plays an important role in the generation of osteoporosis, and the inflammation of diabetes is widely accepted. Many infectious diseases and chronic non-infectious major diseases such as autoimmune diseases, malignant tumors, cardiovascular diseases, diabetes and the like belong to the inflammatory disease category, and the diseases are spreading worldwide and have evolved into important public health problems seriously jeopardizing human health and socioeconomic sustainable development.
The steroid anti-inflammatory drugs and the non-steroid anti-inflammatory drugs are commonly used for treating inflammatory diseases clinically, the steroid anti-inflammatory drugs generally refer to steroid hormones including adrenocortical hormone, glucocorticoid and the like, and the drugs have strong anti-inflammatory effect, but have serious side effects such as water and sodium retention, infection risk and the like, and are limited in clinical use. The non-steroidal anti-inflammatory drugs are drugs which do not contain steroid structures and have anti-inflammatory effects, have good tolerance, but are limited by serious gastrointestinal adverse reactions in long-term use, and even have gastrointestinal bleeding and perforation in severe cases. Because the existing anti-inflammatory drugs have the problems of single effect, great toxic and side effects and the like, the clinical requirements cannot be met, and the development of a safer and more effective anti-inflammatory drug is urgently needed.
At present, the elephantopus scaber extract is a plant extract which is generally recognized to have medicinal effects, related research is few, research is mainly focused on the crude extract and the effective parts of the elephantopus scaber extract, the crude extract and the effective parts which can be obtained by different extraction methods are different and are limited to technical reasons, at present, several elephantopus scaber lactones are mainly discovered and cannot meet the requirement of clinic on ideal anti-inflammatory drugs, and a series of new elephantopus scaber lactone compounds are discovered by extracting and combing the elephantopus scaber extract again by adopting an extraction process and a preparation technology developed by an applicant, and the elephantopus scaber has the same mother ring structure and has stronger anti-inflammatory activity, so that the blank of the field is filled.
Disclosure of Invention
The invention aims to provide an amide compound containing polyunsaturated fatty chains, which is a compound shown in a structural formula I or a physiologically acceptable salt thereof,
C7-C11is a single bond or a double bond; r1Is one of double bond, hydroxymethyl, methyl, hydroxyl and ester group containing 1-8 carbon atoms;
R2is one of hydrogen, methyl, ethyl and acyl containing 1-8 carbon atoms;
R3is unsubstituted, hydrogen or hydroxyl.
The ester group means that the substituent is bonded to C through an ester group11Are connected.
In the process of researching the elephantopus plant components, the inventor surprisingly discovers that the compound with the structural general formula I shows good activity in the aspect of anti-inflammation, and experiments show that the compound can obviously inhibit NO generation induced by LPS, and 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 inorganic acid salt or organic acid salt of the germacrane sesquiterpenoids, 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.
Further, the elephantopus scaber lactone compound comprises isomers thereof.
Preferably, the isomer is C2-O chemical bond and/or C7-C11The isomer of (1).
Further, said R1Is one of double bond, hydroxymethyl, methyl, hydroxyl, acyloxymethyl containing 1-5 carbon atoms, R2Is one of hydrogen, ethyl and acyl containing 1 to 5 carbon atoms.
Further, said R1Is a double bond, hydroxymethyl, methyl, hydroxy or 2, 5-dihydroxyvaleryloxymethyl; r2Is hydrogen, 2-methacryloyl, angeloyl or ethyl, and when R is2When it is angeloyl, R3Is hydrogen or hydroxy.
Further, C7-C11When it is a double bond, R1Is hydroxy or hydroxymethyl, R2Is 2-methacryloyl, isovaleryl, or ethyl, R3Is unsubstituted or substitutedA group;
or C7—C11When it is a single bond, R1Is methyl, a methyl double bond, 2, 5-dihydroxyvaleryloxymethyl or hydroxymethyl, R2Is hydrogen, angeloyl or 2-methacryloyl, and R3 is unsubstituted, hydrogen or hydroxy.
Further, the general formula I of the invention is the sum of the compounds comprising the following elephantolide B-K.
According to the extraction method of the invention, the inventor separates new elephantolide (Elephantopinolide D and B-K) from elephantopus scaber, wherein the elephantopus scaber lactone B-K is a new compound named by the applicant according to the literature naming rules, the structure and the efficacy of the elephantopus scaber are not reported, but the efficacy of the elephantopus scaber is quite different from that of the compound disclosed by the invention.
(B-chemical formula of K)
The substituents are as follows:
TABLE 1 list of substituents for elephantolone A-K
Compared with the existing similar compounds, the compound of the invention has improved solubility, which not only reduces the pharmaceutical difficulty, but also is beneficial to improving the bioavailability, thereby reducing the administration/use amount and reducing the toxic and side effects on the premise of ensuring the curative effect.
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 elephantopus 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 elephantopus plant comprises elephantopus scaber, elephantopus scaber and the like. In the invention, Elephantopus scaber L (scientific name) is preferably used as an extraction raw material, and the medicinal part is preferably whole grass.
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 elephantopus plant with 50-95% lower alcohol, dispersing the extract with water, sequentially extracting with ethyl acetate, collecting ethyl acetate part, 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 the upper layer and the lower layer, and the extraction frequency is 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-acetone (100:1,40:1,20:1), the 20:1 part is collected, the solvent is recovered to be dry, then the extract is purified by gel column chromatography, methanol, dichloromethane-methanol (3:1) or acetone is used as eluent for elution, the eluent is collected, the solvent is recovered to be dry, and the compound with the general formula (I) is obtained, and acetone is preferably used as the eluent.
The gradient elution with dichloromethane-acetone (100:1,40:1,20:1) can also be performed with petroleum ether-acetone (20:1,6:1,3:1), and collecting the following components (3:1, the method of gel column purification was not changed.
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 silica 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 anti-inflammatory drugs, in particular application in preparing anti-inflammatory drugs related to NO. The inflammation is especially related to nerve cells, and corresponds to senile dementia, especially Alzheimer disease.
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.
Detailed Description
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 was purchased from Hyclone.
Cells and instruments: RAW264.7 cell line (mouse monocyte macrophage leukemia cells) was offered by the cloud group of institute of medicinal and plant, academy of Chinese medical sciences, cell culture bottle (Coring corporation), sterile 96-well plate, cell counting plate, slide glass, cover glass, glass pipette, nylon filter, stainless steel filter, various specifications of glass bottle, pipette gun, gun head box and 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 CO 2Isothermal cell culture chamber (SANYO, Japan), ZHJH-C1115B model superclean bench (Shanghai Intelligent analysis Instrument manufacturing Co., Ltd.), CKX41 type inverted phase contrast microscope (OLYMPUS, Japan), MQX200 model photometer (BioTek instruments Co., Ltd.), Legend Micro 17R model high-speed low-temperature centrifuge (Thermo, USA), QL-901 Micro vortex mixer (Lenbell instruments manufacturing Co., Ltd., Hippon), 80 ℃ super-low temperature refrigerator (Thermo, USA), KQ-250B model ultrasonic cleaner (Kunshan ultrasonic Instrument manufacturing Co., Ltd.)Plant), SSW-420-2S type thermostat water bath (shanghai bosun co., ltd), CY50945 type Locator liquid nitrogen tank (Thermo corporation, usa).
A sample to be tested: the compound elephantopin A-K (purity: more than 98%) is prepared by self;
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 RAW264.7 cell line model anti-inflammatory Properties
The results of the experiments are shown in table 2, and the compounds of the general formula i of the present invention: B-K can obviously inhibit the generation of NO induced by LPS, although the effect of dexamethasone with the strongest activity is not achieved, the effect is close to that of dexamethasone, and the anti-inflammatory activity of most of lactones (B-C, F-I, K) is better than that of DET. Therefore, the A-K compounds disclosed by the general formula I all have excellent anti-NO-pathway inflammatory response.
Further validation of neuroinflammatory cell models:
the RAW264.7 cell line in the experiment is replaced by a BV-2 cell line (mouse microglia), and the BV-2 cell line is purchased from a national experimental cell resource sharing service platform.
The relevant measurements of NO secretion levels were performed again as shown in table 3:
TABLE 3 anti-inflammatory model of BV-2 nerve cells
It is shown from table 3 that BV-2 cell line belongs to mouse microglia, the inflammatory response thereof is often related to neuronal cell diseases such as alzheimer's disease and the like, and during the course of LPS-stimulated inflammatory response, compound B-K also shows superior anti-inflammatory activity to that of DET and eletripinolide D compounds, and superior anti-inflammatory activity to that of DET and eletripinolide D compounds, so that the new compound anti-inflammatory activity is superior to that of DET and eletripinolide D from inflammation detection of RAW and BV-2 cell models, and thus has potential for further development of new effective drugs.