阅读说明：本技术 一种具有缩醛结构的木脂素类化合物、制备方法及应用 (Lignanoid compound with acetal structure, preparation method and application ) 是由 任冬梅 张华然 王姝麒 王小宁 沈涛 于 2020-06-19 设计创作，主要内容包括：本发明具体涉及一种具有缩醛结构的木脂素类化合物、制备方法及应用。本发明针对香青兰乙醇提取物进行筛选,通过大孔树脂分离及硅胶柱色谱分离的方式获取了式(X)所示化合物,结构如下：<Image he="230" wi="700" file="DDA0002547991230000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>经验证,该结构的化合物具有良好的抗氧化应激活性,对于H<Sub>2</Sub>O<Sub>2</Sub>引发的细胞损伤具有良好的修复效果,并能够调诱导Nrf2及下游基因NQO1和GCLM基因的表达,有望作为一种抗氧化应激成分应用于抗肿瘤及心血管疾病药物的添加。(The invention particularly relates to a lignan compound with an acetal structure, and a preparation method and application thereof. The invention screens the ethanol extract of moldavica dragonhead, and obtains a compound shown in a formula (X) by means of macroporous resin separation and silica gel column chromatographic separation, wherein the structure is as follows: proved by verification, the compound of the structure hasHas good antioxidant stress activity against H 2 O 2 The induced cell damage has good repairing effect, can regulate and induce the expression of Nrf2 and downstream genes NQO1 and GCLM gene, and is expected to be used as an antioxidant stress component to be applied to the addition of anti-tumor and cardiovascular disease medicines.)

1. A lignan compound having an acetal structure, wherein the compound is selected from the group consisting of a compound represented by formula (X), a pharmaceutically acceptable salt, a solvate, a stereoisomer, a polymorph, a metabolite, and a prodrug thereof;

2. the lignan compound having an acetal structure according to claim 1, wherein the lignan compound having an acetal structure is selected from the group consisting of the following structures:

3. the process for producing the lignan compound having an acetal structure according to claim 1 or 2, wherein the process comprises separating the ethanol extract of Dracocephalum moldavica sequentially by macroporous resin column chromatography and silica gel column chromatography.

4. The method for producing the lignan compound having an acetal structure according to claim 3, wherein the ethanol extract of Dracocephalum moldavica is produced by the following steps: reflux-extracting herba Dracocephali with 20-95% ethanol for at least one time, mixing the reflux solutions, and removing solvent to obtain ethanol extract of herba Dracocephali;

preferably, the moldavica dragonhead adopts the whole grass of the overground part; furthermore, the dosage of the ethanol is 3 to 8 times of the mass of the whole herb part of the Dracocephalum moldavica ground;

preferably, in order to take efficiency and extraction rate into consideration, the extraction times are preferably 2-6 times;

preferably, the time of each reflux extraction is 0.5-1.5 hours.

5. The method for producing the lignan compound having an acetal structure according to claim 4, wherein the ethanol extract of Dracocephalum moldavica is produced by the following steps: taking the crushed moldavica dragonhead ground whole grass, carrying out reflux extraction for 3 times by using 60% ethanol, wherein the using amount of the ethanol for each time is 5 times of the mass of the moldavica dragonhead ground whole grass to be extracted, the extraction time for each time is 1 hour, merging the extracting solutions, and concentrating to be dry to obtain the moldavica dragonhead extract.

6. A process for producing a lignan compound having an acetal structure according to claim 3, wherein the separation with the macroporous resin is performed by: suspending the ethanol extract of dracocephalum moldavica with 0.5-1.5 times of water to obtain suspension, separating by macroporous resin, eluting with water for 5-10 column volumes, then sequentially eluting with 20%, 40%, 60% and 95% ethanol for 2-6 column volumes each time, and combining 40% ethanol eluate and 60% ethanol eluate;

preferably, the macroporous resin is DM130 macroporous resin.

7. The process for producing a lignan compound having an acetal structure according to claim 3, wherein the separation by silica gel column chromatography is carried out by: concentrating the macroporous resin eluent, separating by silica gel column chromatography, sequentially eluting with dichloromethane/methanol at a volume ratio of 100:0-0:100, and eluting and separating the part eluted with dichloromethane/methanol at a volume ratio of 100:20 by gel chromatography;

preferably, the gel chromatographic column Sephadex LH-20 column is eluted by 100% methanol;

preferably, the gel chromatography eluate is traced by thin layer chromatography, and the eluates of the same substance are combined.

8. The process for producing a lignan compound having an acetal structure according to claim 3, wherein the process further comprises the step of separating the mixture of lignan compounds I to IV from the eluate obtained by silica gel column chromatography by HPLC; preferably, compounds I-IV are obtained by chiral resolution.

9. A composition or a pharmaceutical preparation, wherein the composition or the pharmaceutical preparation comprises the lignan compound with an acetal structure or a pharmaceutically acceptable salt thereof of claim 1 or 2, or an extract or a concentrated solution of moldavica dragonhead comprising the lignan compound with an acetal structure of claim 1 or 2, and at least one pharmaceutically acceptable adjuvant or carrier.

10. Use of the lignan compound having an acetal structure or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or an extract or a concentrated solution of Dracocephalum moldavica Maxim comprising the lignan compound having an acetal structure, or the composition or pharmaceutical preparation according to claim 9 for preparing a medicament for resisting oxidation;

preferably, the lignan compound with an acetal structure or a pharmaceutically acceptable salt thereof of claim 1 or 2, or an extract or a concentrated solution of dracocephalum moldavica linn comprising the lignan compound with an acetal structure, or the composition or the pharmaceutical preparation of claim 9 is used for preparing a medicament for protecting against H₂O₂Use in medicine for inducing oxidative damage to cells;

and, the use of the lignan compound having an acetal structure or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or an extract or a concentrated solution of dracocephalum moldavica linn comprising the lignan compound having an acetal structure, or the use of the composition or the pharmaceutical preparation according to claim 9 for preparing a medicament or a biochemical agent for inducing the expression of Nrf2 and/or its downstream genes; preferably, the Nrf2 downstream gene is selected from NQO1 and GCLM;

and, the lignan compound having an acetal structure or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or an extract or a concentrated solution of dracocephalum moldavica linn comprising the lignan compound having an acetal structure, or a composition or a pharmaceutical preparation according to claim 9 for preparing Nrf2 signal pathway agonist drugs or biochemical reagents.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a lignan compound with an acetal structure, a preparation method thereof and application of the lignan compound as an antioxidant active ingredient.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Oxidative Stress (OS) refers to a state of imbalance between Oxidative and antioxidant effects in the body, tending to oxidize, resulting in inflammatory infiltration of neutrophils, increased protease secretion, and the production of a number of Oxidative intermediates. Oxidative stress is a negative effect produced in vivo by free radicals and is considered to be an important factor in aging and diseases.

Modern medical studies have demonstrated that oxidative stress is associated with cell death, tissue damage, cardiovascular disease, tumors, and neuronal degeneration. Nrf2 is an important transcription factor that regulates cellular antioxidant stress, and can exert a protective effect by increasing cellular resistance to oxidative stress by regulating the expression of phase ii detoxification enzymes (SOD, MDA, NOS, CAT, GSH-PX, etc.), genes of antioxidant enzymes (HO-1, Nrf2, γ -GCS, SOD, NQO1, etc.), downstream antioxidant genes GR, GCLM, etc.

Dracocephalum moldavica L is a plant of Dracocephalum of Labiatae, and has effects of dispelling pathogenic wind, clearing heat, relieving sore throat, relieving cough, cooling liver and stopping bleeding. In previous researches of research teams of the inventor, a heptenedimethylene diacid dimethyl ester compound is obtained by screening from an ethanol extract of cymbidium sinense, and the heptenedimethylene diacid dimethyl ester compound has good antioxidant effect. Screening of active entity compounds in natural medicines is an important research idea in the field, active substances in the natural medicines are further developed, and the method has good medicine development significance.

Disclosure of Invention

Based on the above background technology, the invention further screens the active ingredients in the ethanol extract of dracocephalum moldavica, and obtains a compound with a novel structure through continuous trial and search, and the compound also has good antioxidant activity.

Based on the technical effects, the invention provides the following technical scheme:

in a first aspect of the present invention, there is provided a lignan compound having an acetal structure, wherein the compound is selected from the group consisting of a compound represented by formula (X), a pharmaceutically acceptable salt, a solvate, a stereoisomer, a polymorph, a metabolite, and a prodrug thereof;

according to the invention, the compound is further screened, and the lignan compound with the acetal structure is a stereoisomer and is selected from the following structures:

after the preparation method is adjusted, the compound with the structure is obtained from the ethanol extract of the moldavica dragonhead, the compound with the structure has obvious structural difference with the compound obtained in the previous research, but still shows good antioxidant activity, and is used as an antioxidant active ingredient to be added into the development of corresponding medicines.

In a second aspect of the present invention, there is provided a method for preparing the lignan compound having an acetal structure according to the first aspect, wherein the method comprises sequentially separating an ethanol extract of moldavica dragonhead with macroporous resin and silica gel column chromatography.

In a third aspect of the present invention, the present invention provides a composition or a pharmaceutical preparation, the composition or the pharmaceutical preparation comprises the lignan compound with an acetal structure or a pharmaceutically acceptable salt thereof as described in the above first aspect of the present invention, or an extract or a concentrated solution of dracocephalum moldavica linn as described in the above first aspect of the present invention, and may comprise at least one pharmaceutically acceptable adjuvant or carrier.

In a fourth aspect of the present invention, the present invention further provides a lignan compound having an acetal structure described in the first aspect, or a pharmaceutically acceptable salt thereof, or an extract or a concentrated solution of moldavica dragonhead comprising the lignan compound having an acetal structure described in the first aspect, or an application of the composition or the pharmaceutical preparation described in the third aspect in preparing a medicament for resisting oxidation.

According to the conventional research thought in the field, the compound provided by the invention has antioxidant activity, and can be added into therapeutic drugs for resisting oxidative stress related diseases, such as antitumor drugs, anti-cardiovascular disease drugs, anti-neurodegenerative disease drugs and the like.

The beneficial effects of one or more technical schemes are as follows:

the invention provides a compound with a novel structure, which has catalase-like activity and can repair H₂O₂The induced cell damage also has the effect of inducing Nrf2 and downstream channel genes. The compound is expected to be added to the development of related medicines as an antioxidant active substance; in addition, the research result of the invention has reference significance for screening and developing natural medicines.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Figure 1 is a chiral resolution and CD profile of the compound described in example 1.

FIG. 2 is a histogram of induction of Nrf2 and downstream genes NQO1 and GCLM by compounds I-IV described in example 2.

FIG. 3 is a graph of the compounds I-IV vs. H described in example 3₂O₂Protective effect of induced PC12 cell damage.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the invention. As used herein, the singular is intended to include the plural unless the context clearly dictates otherwise, and it should be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of features, steps, operations, devices, components, and/or combinations thereof.

As described in the background art, in order to solve the above technical problems, the present invention proposes the use of a lignan compound having an acetal structure as an antioxidant active ingredient.

In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples, wherein the reagents described in the following examples are all commercially available products.

In a first aspect of the present invention, there is provided a lignan compound having an acetal structure, wherein the compound is selected from the group consisting of a compound represented by formula (X), a pharmaceutically acceptable salt, a solvate, a stereoisomer, a polymorph, a metabolite, and a prodrug thereof;

preferably, the lignan compound having an acetal structure is selected from the following structures:

in a second aspect of the present invention, there is provided a method for preparing the lignan compound having an acetal structure according to the first aspect, wherein the method comprises sequentially separating an ethanol extract of moldavica dragonhead with macroporous resin and silica gel column chromatography.

Preferably, the preparation method of the ethanol extract of moldavica dragonhead comprises the following steps: reflux-extracting herba Dracocephali with 20-95% ethanol for at least one time, mixing the reflux solutions, and removing solvent to obtain the ethanol extract.

More preferably, the moldavica dragonhead adopts the whole grass of the overground part; furthermore, the amount of the ethanol is 3-8 times of the mass of the whole herb part of the Dracocephalum moldavica.

More preferably, the number of times of extraction is preferably 2 to 6 times in order to achieve both efficiency and extraction yield.

Further preferably, each reflux extraction time is 0.5 to 1.5 hours.

In some embodiments of the above preferred embodiments, the ethanol extract of Dracocephalum moldavica is prepared by the following steps: taking the crushed moldavica dragonhead ground whole grass, carrying out reflux extraction for 3 times by using 60% ethanol, wherein the using amount of the ethanol for each time is 5 times of the mass of the moldavica dragonhead ground whole grass to be extracted, the extraction time for each time is 1 hour, combining the extracting solutions, and concentrating to be dry to obtain the moldavica dragonhead extract.

Preferably, the operation of separating by using macroporous resin is as follows: suspending the ethanol extract of dracocephalum moldavica with 0.5-1.5 times of water to obtain suspension, separating by macroporous resin, eluting with water for 5-10 column volumes, then sequentially eluting with 20%, 40%, 60% and 95% ethanol for 2-6 column volumes each time, and combining 40% ethanol eluate and 60% ethanol eluate.

Further preferably, the macroporous resin is DM130 macroporous resin.

Preferably, the silica gel column chromatography separation method is as follows: concentrating the macroporous resin eluent, separating by silica gel column chromatography, eluting with dichloromethane/methanol at a volume ratio of 100:0-0:100, and eluting the part eluted with dichloromethane/methanol at a volume ratio of 100:20 by gel chromatography.

Further preferably, the gel chromatographic column Sephadex LH-20 column is eluted by using 100% methanol.

Further preferably, the gel chromatography eluate is traced by thin layer chromatography, and the eluates of the same substance are combined.

Preferably, the preparation method further comprises the step of separating the mixture of lignan compounds I to IV from the eluate obtained from the silica gel column chromatography by HPLC; further, compounds I-IV are obtained by chiral resolution.

In a third aspect of the present invention, the present invention provides a composition or a pharmaceutical preparation, the composition or the pharmaceutical preparation comprises the lignan compound with an acetal structure or a pharmaceutically acceptable salt thereof as described in the above first aspect of the present invention, or an extract or a concentrated solution of dracocephalum moldavica linn as described in the above first aspect of the present invention, and may comprise at least one pharmaceutically acceptable adjuvant or carrier.

In an embodiment of the present invention, the extract or the concentrated solution of Dracocephalum moldavica containing the lignan compound having an acetal structure according to the above first aspect of the present invention may be obtained by the preparation method according to the above second aspect of the present invention, and the extract or the concentrated solution may be one obtained by separating specific compounds or one obtained by simultaneously containing all the compounds according to the above first aspect of the present invention, i.e., the extract or the concentrated solution of Dracocephalum moldavica containing the lignan compound having an acetal structure according to the above first aspect of the present invention may simultaneously contain one or more (e.g., two, three or four) of the compounds I-IV according to the present invention.

The pharmaceutical compositions of the present invention are generally safe, non-toxic and biologically desirable, and therefore the pharmaceutically acceptable carriers or excipients of the present invention are non-toxic and safe, as well as combinations thereof with the compounds of the present invention. The pharmaceutically acceptable carriers and excipients according to the invention are generally known to the person skilled in the art or can be determined by the person skilled in the art on the basis of the actual circumstances.

The pharmaceutical composition or pharmaceutical preparation of the compound of the present invention can be administered by those skilled in the art in any of the following ways, as the case may be: oral, aerosol inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous administration being preferred.

The compounds of the present invention or pharmaceutical compositions or formulations containing these compounds may be administered in unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.

The pharmaceutical combination or formulation of the present invention may also contain conventional carriers, such as, but not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.

Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.

Oral liquids may be prepared as suspensions of water and oil, solutions, emulsions, syrups, and the like, and may also be prepared as a dry product to be supplemented with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.

Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.

For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.

It will be appreciated that the optimum dosage and interval for administration of a compound of the invention will be determined by the nature of the compound and external conditions, such as the form, route and condition of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.

In a fourth aspect of the present invention, the present invention further provides a lignan compound having an acetal structure described in the first aspect, or a pharmaceutically acceptable salt thereof, or an extract or a concentrated solution of moldavica dragonhead comprising the lignan compound having an acetal structure described in the first aspect, or an application of the composition or the pharmaceutical preparation described in the third aspect in preparing a medicament for resisting oxidation.

The invention also provides the lignanoid compound with an acetal structure or the pharmaceutically acceptable salt thereof in the first aspect, or an extract or a concentrated solution of the dracocephalum moldavica linn which contains the lignanoid compound with the acetal structure, or a composition or a pharmaceutical preparation in the third aspect for preparing a medicine for preventing H₂O₂Use in medicine for inducing oxidative damage to cells; preferably, the cell is a PC12 cell.

The invention also provides the application of the lignan compound with an acetal structure or a pharmaceutically acceptable salt thereof in the first aspect, or an extract or a concentrated solution of the dracocephalum moldavica linn which contains the lignan compound with the acetal structure, or the application of the composition or the pharmaceutical preparation in the third aspect in preparing a medicine or a biochemical reagent for inducing the expression of the Nrf2 and/or the downstream gene thereof; preferably, the Nrf2 downstream gene is selected from NQO1 and GCLM.

The invention also provides the lignan compound with an acetal structure or a pharmaceutically acceptable salt thereof in the first aspect, or an extract or a concentrated solution of moldavica dragonhead comprising the lignan compound with the acetal structure, or an application of the composition or the pharmaceutical preparation in the third aspect in preparing Nrf2 signal pathway agonist drugs or biochemical reagents.

In an embodiment of the invention, the compounds I, II, III, IV according to the invention are capable of inducing expression of Nrf2 and downstream genes NQO1 and GCLM. In some embodiments of the invention, the effect of the compound I-IV of the present invention on inducing expression of Nrf2 and downstream genes NQO1 and GCLM is concentration-dependent at a concentration of 0-50 μ M, the concentration-dependent effect of Nrf2 and downstream genes NQO1 and GCLM is gradually increased with the increase of the compound concentration, especially at a concentration of 12.5-50 μ M, the increase of the expression level of Nrf2 and downstream genes NQO1 and GCLM is most obvious, the expression level of Nrf2 and downstream genes NQO1 and GCLM is still increased at a concentration of 25-50 μ M, but the concentration-dependent effect of the expression level is reduced and the increase rate of the expression level is reduced with the increase of the concentration in the concentration range.

In an embodiment of the invention, the compounds I, II, III, IV according to the invention are shown to be H₂O₂Protection against the cellular damage caused. In some embodiments, compounds I-IV of the invention are administered to 200 μ MH at a concentration of not less than 25 μ M₂O₂The induced cell damage shows protective effect, the protective effect of the compounds I-IV is not very different, which shows that the configuration of chiral carbon has little influence on the protective effect of the compound on the oxidative damage.