CN109438406B

CN109438406B - Chromone derivative extracted from anshansenia glauca and preparation method and application thereof

Info

Publication number: CN109438406B
Application number: CN201811375707.5A
Authority: CN
Inventors: 李银科; 黄茹; 李雪梅; 杨光宇; 刘欣; 杨叶昆; 黄海涛; 李晶; 徐永; 张建铎
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2018-11-19
Filing date: 2018-11-19
Publication date: 2021-10-22
Anticipated expiration: 2038-11-19
Also published as: CN109438406A

Abstract

The invention discloses a chromone derivative extracted from anshansenia hansenii and a preparation method and application thereof. The chromone derivative is separated from the whole Lupinus hancei and has the molecular formula of C₁₇H₁₈O₅The compound was named: 5- (isobutyryl) -2- (2-oxopropyl) -8-methoxy-4H-4-chromone. The preparation method of the chromone derivative is characterized in that the chromone derivative is obtained by taking the whole plant of the anshansenna plant as a raw material and carrying out the steps of crushing, extracting, silica gel column chromatography and high-pressure liquid chromatography separation. The compound disclosed by the invention is proved to have a good inhibition effect on tobacco mosaic virus through a biological activity test. The compound is easy to separate and obtain, has good activity and can be used as a pilot compound for resisting tobacco mosaic virus.

Description

Chromone derivative extracted from anshansenia glauca and preparation method and application thereof

Technical Field

The invention belongs to the technical field of phytochemistry, and particularly relates to a chromone derivative extracted and separated from anshana beans as well as a preparation method and application thereof.

Background

The Lupinus columnus (Cassiaumilam.) is perennial shrubby mantle grass of Cassia of Leguminosae (Leguminosae), namely Cassia, namely mimosa, Cymbopogon formosanus, Mucuna cumingii, dreamina, sand, Japanese cypress, Japanese soapwort herb, Chinese honeylocust, lignification of the base part, 25-75 cm high, multiple branches, linear sickle shape, small convex tip at the top, branch, petiole, leaf axis, mollissima, and flowering for 8-9 months; the results are 10-12 months. The Canadenia tricuspidata is grown in shrubs or grasses which grow on mountainous regions and open lands, is mainly distributed in Guangdong, Yunnan and other provinces at home, is also distributed in India, Central and south peninsula, Malaysia and Australia at foreign countries, is used as a traditional Chinese herbal medicine, and has the effects of clearing heat and removing toxicity, strengthening spleen and promoting diuresis, and relaxing bowels. It is mainly used for treating jaundice, summer heat vomiting and diarrhea, infantile malnutrition, edema, dysuresia, habitual constipation, furuncle, carbuncle, swelling and venomous snake bite. Chromones and their derivatives are widely found in plants, and some are colored substances so that their precursors are called chromones. The chromone compound has various biological activities, is widely concerned by researchers, and the research on the structure-activity relationship shows that the physiological activity and the substitution condition of the chromone compound are very close, so that the discovery of a novel chromone derivative has important significance. The invention separates a chromone derivative with anti-tobacco mosaic virus activity from anshana glauca, and the compound has not been reported yet.

Disclosure of Invention

A first object of the present invention is to provide a chromone derivative; the second object is to provide a method for preparing the chromone derivative; the third purpose is to provide the application of the chromone derivative in the preparation of the tobacco mosaic disease resistant drug.

The first object of the invention is achieved in that the compound is isolated from Cassiaquilla arborescens (Cassiahumilalam.) and has the formula C₁₇H₁₈O₅The compound has the following structure:

this compound was a yellow gum named 5- (isobutyryl) -2- (2-oxopropyl) -8-methoxy-4H-4-chromone, english name: 5- (Isobutyryl) -2- (2-oxopyryl) -8-methoxy-4H-chromen-4-one.

The second purpose of the invention is realized by the preparation method of the chromone derivative, which takes the plants of the anshansenna as raw materials and is prepared by the steps of crushing, extract extraction, silica gel column chromatography, high pressure liquid chromatography and gel column chromatography.

Wherein in the extract extraction step, the whole plant of the corynespora bunge is taken, the corynespora bunge can be crushed or cut into small segments, high-concentration methanol (w%: 80-100%) or high-concentration ethanol (w%: 80-100%) or high-concentration acetone (w%: 70-100%) is used as an extraction solvent, and the extraction solvent comprises the following steps: soaking the pedunculate gomphrena beans at a weight ratio of 2-4: 1 for 24-72 h, extracting for 3-5 times, combining the extracting solutions, filtering and concentrating into an extract;

in the step of silica gel column chromatography, dissolving the extract by using 1.5-3 times of pure methanol or pure ethanol or pure acetone, mixing the extract by using 0.8-1.5 times of 80-100 meshes of silica gel, and filling the mixture into a column by using 2-5 times of 150-200 meshes of silica gel in a dry method for silica gel column chromatography; performing gradient elution by using chloroform-methanol solution with the volume ratio of 20: 1-5: 5(20:1, 9:1, 8:2, 7:3, 6:4 and 5:5), merging parts with the same polarity, collecting eluent of each part, and concentrating;

wherein the high pressure liquid chromatography separation and purification stepWherein 9:1 part of the column chromatography eluent is further separated and purified by high pressure liquid chromatography to obtain the flavonoid compound, and the high pressure liquid chromatography separation and purification adopts C of 20mm multiplied by 250mm and 5 mu m₁₈And (3) performing chromatographic column chromatography with flow rate of 20mL/min and mobile phase of 55% methanol, detecting wavelength of 351nm by an ultraviolet detector, injecting 200 mu L of sample each time, collecting chromatographic peak of 28.6min, accumulating for multiple times, and evaporating to dryness.

Wherein in the step of gel column chromatography, the obtained compound is dissolved by pure methanol again, and then the pure methanol is taken as a mobile phase to be separated by gel column chromatography for further separation and purification.

The structure of the prepared compound was identified by the following method:

the compound is yellow jelly, and HRESI-MS shows that the peak of the quasi-molecular ion is 325.1047[ M + Na ]]⁺(calculated 325.1052), combined¹H NMR and DEPT spectra confirm that the molecular formula is C₁₇H₁₈O₅The unsaturation degree was 9.

The infrared spectrum shows hydroxyl groups (3076 cm)^-1) Carbonyl groups (1738, 1680 and 1654 cm)^-1) And aromatic rings (1612, 1547 and 1468cm^-1) The resonance absorption peak of (1). The maximum absorption of the ultraviolet spectrum at 235, 274 and 351nm also indicates that aromatic ring structures may exist in the compound.

Compound (I)¹H、¹³NMR spectra of C and DEPT (see Table 1, FIG. 1 and FIG. 2) show that they contain 16 carbons and 16 hydrogens, including 1 chromonic ring system (C-2 to C-10), 3 aromatic protons (H-3, H-6 and H-7), and 1 2-oxopropyl group (CH)₃-CO-CH₂-；C-11～C-13；H₂-11 and H₃-13), 1 isobutyryl group [ (CH)₃)₂-CH-CO-；C-14～C-16；H₆-16,17and H-15]And 1 methoxy group (. delta.)_C 56.1,δ_H3.83). H can be observed in the HMBC spectrum of the compound (see FIG. 3)₂-11(δ_H3.57) and C-2 (. delta.))_C161.8) and C-3 (. delta.))_C 112.4),H-3(δ_H6.13) and C-11 (. delta.))_C48.6) related, it can be speculated that the 2-oxopropyl moiety is substituted at the C-2 position; from H-15 (delta)_H4.26) and C-5 (. delta.))_C 128.8)，H-6(δ_H7.59) and C-14 (. delta.))_C208.1) related, it is presumed that the isobutyryl moiety is substituted at the C-5 position; from the methoxy proton (. delta.)_H3.83) and C-8 (. delta.))_C208.1) the relevant presumably hydroxyl group is substituted in the C-8 position. To this end, the structure of the compound was determined and the compound was named: 5- (isobutyryl) -2- (2-oxopropyl) -8-methoxy-4H-4-chromone.

Infrared, ultraviolet and mass spectral data of compounds: ultraviolet spectrum (methanol), lambda_max(log ε)210(4.02),235(3.68),274(3.82),351(3.72) nm; infrared spectroscopy (potassium bromide pellet): v is_max 3076,2935,2857,1738,1680,1654,1612,1547,1468,1340,1143,1052,935,824cm^-1；¹H and¹³c NMR data (500 and 125MHz in CDCl as solvent)₃) See Table-1; positive ion mode ESIMS M/z 325[ M + Na ]]⁺(ii) a Positive ion mode HRESIMS M/z 325.1047[ M + Na ]]⁺(calculated 325.1052, C₁₇H₁₈NaO₅)。

Process for preparation of the Compounds of Table-1¹H NMR and¹³c NMR data (CDCl)₃)

The third purpose of the invention is realized by applying the chromone derivative to the preparation of the tobacco mosaic disease drugs.

The compound of the invention is separated for the first time, is determined to be a chromone derivative by a nuclear magnetic resonance and mass spectrometry method, and represents the specific structure of the chromone derivative. Through the experiment of resisting tobacco mosaic virus, the relative inhibition rate reaches 34.8 percent, and the tobacco mosaic virus resisting activity is good and exceeds the relative inhibition rate (30.8 percent) of the positive control nandinmycin. The results show that the chromone derivative has good application prospect in preparing the anti-tobacco mosaic virus medicine. The compound has simple structure and good activity, and can be used as a guiding compound of a tobacco mosaic virus resistant medicament.

Drawings

FIG. 1 is a nuclear magnetic resonance carbon spectrum of a compound.

FIG. 2 shows the NMR spectrum of a compound.

Figure 3 is the major HMBC correlation of compounds.

Detailed Description

The present invention is further described with reference to the following examples and accompanying drawings, but the present invention is not limited in any way, and any changes or modifications based on the teaching of the present invention will fall within the scope of the present invention.

All percentages used in the present invention are mass percentages unless otherwise indicated.

The preparation method of the chromone derivative 5- (isobutyryl) -2- (2-oxopropyl) -8-methoxy-4H-4-chromone comprises the steps of crushing, extract extraction, silica gel column chromatography, high pressure liquid chromatography and gel column chromatography, and specifically comprises the following steps:

A. extracting the extractum: taking the whole stigmata arborescens plant, crushing or cutting the stigmata arborescens into small sections, using high-concentration methanol (w%: 80-100%) or high-concentration ethanol (w%: 80-100%) or high-concentration acetone (w%: 70-100%) as an extraction solvent, and extracting the solvent: soaking the three hair leaves in a weight ratio of 2-4: 1 for 24-72 h, extracting for 3-5 times, combining the extracting solutions, filtering and concentrating into an extract;

B. silica gel column chromatography: dissolving the extract with 1.5-3 times of pure methanol or pure ethanol or pure acetone, mixing with 0.8-1.5 times of 80-100 mesh silica gel, and performing silica gel column chromatography with 2-5 times of 150-200 mesh silica gel by dry method; performing gradient elution by using chloroform-methanol solution with the volume ratio of 20: 1-5: 5(20:1, 9:1, 8:2, 7:3, 6:4 and 5:5), merging same polar parts, collecting eluent of each part and concentrating;

C. high-pressure liquid chromatography separation: separating and purifying 9:1 part of the column chromatography eluent by high pressure liquid chromatography to obtain the chromone derivative, wherein the high pressure liquid chromatography separation and purification adopts C of 20mm × 250mm and 5 μm₁₈Chromatographic column with flow rate of 20mL/min, mobile phase of 55% methanol, ultraviolet detector with detection wavelength of 351nm, sampling 200 μ L each time, collecting 28.6min chromatographic peak,and (5) accumulating for multiple times and then evaporating to dryness.

D. Gel column chromatography: and C, separating and purifying the substance separated and purified by the high-pressure liquid chromatography in the step C by the high-performance liquid chromatography, dissolving the obtained compound by using pure methanol again, and performing chromatographic separation by using a gel column by using the pure methanol as a mobile phase to further separate and purify the substance.

The chromone derivative is applied to the preparation of the tobacco mosaic virus resistant medicament.

The raw materials used in the invention are not limited by regions and varieties, and the invention can be realized, and the invention is further explained by the raw materials of the anshanshan hyacinth beans from various parts of Yunnan province:

example 1

The Lupinus hancei sample is from Xinping county of Yunnan, 2.0kg of the Lupinus hancei sample is crushed and extracted with 95% methanol for 5 times, each time is extracted for 24 hours, extracting solutions are combined, filtered and concentrated under reduced pressure to obtain extract 106 g. Dissolving the extract with 2.0 times of pure methanol, mixing with 120g of 80 mesh crude silica gel, loading 0.7kg of 160 mesh silica gel into column, performing silica gel column chromatography, gradient eluting with chloroform-methanol at volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, 5:5, monitoring by TLC, mixing the same parts to obtain 6 parts, wherein the chloroform-methanol eluting part at volume ratio of 9:1 is separated by LC-8A semi-preparative high performance liquid chromatography of Shimadzu, with 55% methanol as mobile phase and Venusil MP C₁₈Preparing a column (20 × 250mm,5 μm) as stationary phase at flow rate of 20ml/min, detecting wavelength of 351nm with ultraviolet detector, introducing sample 200 μ L each time, collecting chromatographic peak of 28.6min, accumulating for multiple times, and evaporating to dryness; dissolving the obtained product with pure methanol again, taking the pure methanol as a mobile phase, and carrying out Sephadex LH-20 gel column chromatography separation to obtain the new compound.

Example 2

The Lupinus hancei sample is from Yunnan Shuangjiang county, 4.0kg of Lupinus hancei is sampled and cut up, extracted for 4 times by 95% ethanol for 48 hours each time, the extracting solutions are combined, filtered and concentrated under reduced pressure to obtain an extract 196 g. Dissolving the extract with 2.0 times of pure methanol, mixing with 200g of 100 mesh crude silica gel, loading 1kg of 200 mesh silica gel into column, and performing silica gel treatmentPerforming column chromatography, performing gradient elution with chloroform-methanol at volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, and monitoring by TLC to combine the same parts to obtain 6 parts, wherein chloroform-acetone at volume ratio of 9:1 is separated by LC-8A semi-preparative high performance liquid chromatography of Shimadzu, 58% methanol is used as mobile phase, and Venusil MP C is used as carrier₁₈Preparing a column (20 × 250mm,5 μm) as stationary phase at flow rate of 20ml/min, detecting wavelength of 351nm with ultraviolet detector, introducing sample 200 μ L each time, collecting chromatographic peak of 28.6min, accumulating for multiple times, and evaporating to dryness; dissolving the obtained product with pure methanol again, taking the pure methanol as a mobile phase, and carrying out Sephadex LH-20 gel column chromatography separation to obtain the new compound.

Example 3

The Lupinus hancei sample is from Yunnan Jinghong city, 6kg of the Lupinus hancei sample is crushed, extracted by 80% acetone for 3 times with ultrasonic waves, each time for 72 hours, the extracting solutions are combined, filtered and concentrated under reduced pressure to obtain 319g of extract. Dissolving the extract with 1.6 times of pure methanol, mixing with 400g of 80 mesh crude silica gel, loading 2kg of 180 mesh silica gel into a column, performing silica gel column chromatography, gradient eluting with chloroform-methanol at volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, monitoring by TLC, mixing the same parts to obtain 6 parts, wherein the chloroform-acetone eluting part at volume ratio of 9:1 is separated by LC-8A semi-preparative high performance liquid chromatography of Shimadzu, 55% methanol is used as mobile phase, and Venusil MP C₁₈Preparing a column (20 × 250mm,5 μm) as stationary phase at flow rate of 20ml/min, detecting wavelength of 351nm with ultraviolet detector, introducing sample 200 μ L each time, collecting chromatographic peak of 28.6min, accumulating for multiple times, and evaporating to dryness; dissolving the obtained product with pure methanol again, taking the pure methanol as a mobile phase, and carrying out Sephadex LH-20 gel column chromatography separation to obtain the new compound.

Example 4

The compound prepared in example 1 was taken as a yellow gum.

Example 5

The compound prepared in example 2 was taken as an orange gum. The measurement method was the same as in example 4, and it was confirmed that the compound prepared in example 2 was the flavonoid compound: 5- (isobutyryl) -2- (2-oxopropyl) -8-methoxy-4H-4-chromone.

Example 6

The compound prepared in example 3 was taken as an orange gum. The measurement method was the same as in example 4, and it was confirmed that the compound prepared in example 3 was the flavonoid compound: 5- (isobutyryl) -2- (2-oxopropyl) -8-methoxy-4H-4-chromone.

Example 7

Any chromone derivative prepared in example 1-3 is used for activity test of tobacco mosaic virus, and the test conditions are as follows:

the activity of the compound of the invention against tobacco mosaic virus is measured by a half-leaf method when the mass concentration of the medicament is 50 mg/L. Selecting leaves suitable for testing (normal leaves, no disease and no insect) on plants of 5-6-year-old flue-cured tobacco, uniformly spraying fine carborundum on the leaves, and using a writing brush to apply a standby tobacco mosaic virus source (3.0 multiplied by 10)^-3) Uniformly smearing on the leaves scattered with carborundum, immediately placing the selected leaves in a culture dish containing liquid medicine for processing for 20min after all the selected leaves are disinfected, taking out the leaves, wiping off water drops and the liquid medicine on the leaves, restoring and arranging two half leaves in a glass jar paved with toilet paper for moisture preservation, covering the glass jar, controlling the temperature to be (23 +/-2) DEG C, placing the glass jar in a greenhouse for natural light irradiation, and enabling the dead spots to be visible for 2-3 d.

XI％＝(CK-T)/CK×100％

X: relative inhibition ratio (%), CK: the number of dead spots of half leaf after being soaked in clear water is one, and the number of dead spots of half leaf after being soaked in liquid medicine is one.

The result shows that the relative inhibition rate of the compound is 34.8 percent and is 30.8 percent higher than that of the control ningnanmycin, which indicates that the compound has better activity against tobacco mosaic virus.

Claims

1. A preparation method of chromone derivative is characterized in that the molecular formula of the chromone derivative is C₁₇H₁₈O₅Has the following structure:

the preparation method comprises the following steps: dolichos tonkinensis plant → crushing → extract extraction → silica gel column chromatography → high pressure liquid chromatography;

wherein whole plant of Cassia simsii (Cassia pulima Lam.) belonging to Cassia of Leguminosae is used as raw material, and is pulverized or cut into segments;

in the extract extraction step, a methanol water solution with the mass concentration of 80-100%, an ethanol water solution with the mass concentration of 80-100% or an acetone water solution with the mass concentration of 70-100% is used as an extraction solvent, and the extraction solvent is prepared by the following steps: soaking the anshan senna for 24-72 h in an amount of 2-4: 1, extracting for 3-5 times, combining the extracting solutions, filtering and concentrating into an extract;

in the step of silica gel column chromatography, mixing the extract silica gel, and then filling the mixture into a column by using a 150-200-mesh silica gel dry method in an amount which is 2-5 times the weight of the mixture for silica gel column chromatography; performing gradient elution by using a chloroform-methanol solution system with the volume ratio of 20: 1-5: 5, merging the same polar parts, collecting eluent of each part and concentrating;

wherein before the extractum is subjected to silica gel column chromatography and coarse separation, 1.5-3 times of pure methanol or pure ethanol or pure acetone is used for dissolving the extractum, and then 0.8-1.5 times of 80-100 meshes of silica gel is used for mixing the extractum;

wherein in the step of silica gel column chromatography, the volume ratio of chloroform-methanol solution is 20:1, 9:1, 8:2, 7:3, 6:4 and 5: 5;

wherein in the step of column chromatography, the 9:1 part of the eluent is further separated and purified by high pressure liquid chromatography, wherein in the step of high pressure liquid chromatography, the high pressure liquid chromatography is used for separating and purifying C with the specification of 20mm multiplied by 250mm and 5 mu m₁₈A chromatographic column with the flow rate of 20mL/min and the mobile phase of 55% methanol water, the detection wavelength of an ultraviolet detector of 351nm, the sample injection of 200 mu L each time, the chromatographic peak of 28.6min is collected, and the chromatographic peak is evaporated to dryness after multiple accumulation to obtain the chromone derivative;

and after the high pressure liquid chromatography separation and purification step, dissolving the obtained compound by using pure methanol again, and separating by using gel column chromatography by using the pure methanol as a mobile phase to further separate and purify the compound.

2. The use of the chromone derivative obtained by the preparation method of claim 1 in the preparation of a medicament for resisting tobacco mosaic virus.