CN110746338A

CN110746338A - Preparation method and application of pyrrole-2-carboxylate compound in streptomyces stemonae endophytic

Info

Publication number: CN110746338A
Application number: CN201911100397.0A
Authority: CN
Inventors: 胡立宏; 王吓长; 赵慧敏; 杨爱萍; 丁宁; 李世洋; 彭任; 朱友娟
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2020-02-04

Abstract

The invention discloses a preparation method and application of pyrrole-2-carboxylate compounds in streptomyces stemonae endophytic bacteria, wherein the pyrrole-2-carboxylate compounds have a structure shown by compounds 1-10. The pyrrole-2-carboxylate compound is separated from streptomyces stemonae endophytic for the first time, and all the compounds are separated from the nature for the first time. The compounds 2,3,4,5,9,10 and 11 (stemonine), 12 (pyrrole-2-carboxylic acid), BS-1 (streptomyces crude extract), IM (imidacloprid) and BI (bifenazate) are applied to the prevention and treatment of aphids and tetranychus urticae, and data show that the compounds have high inhibitory activity to cotton aphids (LC50 range: 3.55-32.00 ppm); has moderate inhibitory activity to Tetranychus urticae (LC50 range: 197.6-685.5 ppm).

Description

Preparation method and application of pyrrole-2-carboxylate compound in streptomyces stemonae endophytic

Technical Field

The invention belongs to the technical field of endophytes of traditional Chinese medicines, and particularly relates to separation and purification of a new pyrrole-2-carboxylate compound in radix stemonae endophytes and new medical application of the new pyrrole-2-carboxylate compound in prevention and treatment of cotton aphids and tetranychus urticae.

Background

Since the last century reports that plant endophytic fungi can produce taxol, researchers at home and abroad are focusing on the plant (particularly medicinal plant) endophyte, and the fact that the plant endophyte can be suitable for the special environment in the plant and can encode and produce various bioactive substances for enhancing the resistance of the plant to the outside is discovered. In addition, the endophytes have long evolved in conjunction with the host to produce certain metabolites with the same or similar structure as the host plant and with medicinal value. In recent years, metabolites separated from fermentation products of endophytes of plants have various structural types, including alkaloids, steroids, terpenes, anthraquinones, cyclic peptides, flavonoids and the like.

The traditional Chinese medicine stemona is a traditional cough-relieving and insecticidal medicine, and the stemona collected in Chinese pharmacopoeia has 3 types: stemona sessilifolia, Stemona tuberosa and Stemona tuberosa; the radix Stemonae alkaloid contains saturated azathiopyrrole ring nucleus structure, and has antitussive, expectorant, insecticidal, and antibacterial effects. Guangxi is one of the major producing areas of radix stemonae, but the radix stemonae resource is being sharply reduced due to destructive harvesting and ecological environment destruction in recent years.

The stemona endophyte produces a compound with activity similar to that of a host plant, and the secondary metabolite of the stemona endophyte can be used as a potential source of a novel medicament or pesticide.

Disclosure of Invention

The technical problem to be solved by the invention is as follows:

provides a preparation method of pyrrole-2-carboxylate compound in streptomyces endophytic radix stemonae, is expected to solve the problem of substitute of radix stemonae medicinal material with insecticidal effect, and provides new pyrrole-2-carboxylate compound in endophytic radix stemonae for new medical application in preventing and treating aphid and tetranychus urticae.

The invention adopts the following technical scheme:

the structural formula of the pyrrole-2-carboxylate new compound is as follows:

in formula I:

r1 and R2 may be the same or different;

r1 is selected from one of hydrogen, methyl, ethyl, amino, formamido and acetamido;

r2 is selected from one of hydrogen, hydroxyl, hydroxymethyl, hydroxyethyl, amino, formamido and acetamido;

n is selected from 1 to 5.

The pyrrole-2-carboxylate compound is a compound obtained by separating streptomyces endophyticus in the traditional Chinese medicine stemona, wherein all compounds are new compounds.

The streptomyces stemonae endophytic BS-1 is obtained by the following extraction and separation methods: collecting fresh radix Stemonae, and performing surface disinfection treatment. Respectively cutting radix Stemonae, placing into a centrifuge tube, adding small amount of sterilized water, vortex, hot water bath at 80 deg.C for 1min, spreading 200uL onto ISP4 and oat culture medium (added with 50mg/L amphotericin; 25mg/L nalidixic acid), culturing at 28 deg.C for one to two months, and purifying with M2 plate.

The crude extract of the stemona endophytic streptomyces fermentation liquor is obtained by the following method: preparing a seed culture medium, inoculating the BS-1 strain into the seed culture medium, and culturing at 26-30 ℃ for 3-5 days to obtain a seed culture solution; and inoculating the obtained seed culture solution into a fermentation culture medium, and culturing at 26-30 ℃ and 200rpm for 6-9 days to obtain a fermentation product. Adding 2-6% XAD-16 resin, continuing to oscillate for 4-6 hours, centrifuging to remove supernatant, adding methanol for extraction, performing vortex ultrasonic treatment, centrifuging, taking supernatant methanol solution, and evaporating to dryness to obtain a crude extract of the strain.

The pyrrole-2-carboxylic ester compound in the stemona streptomyces is separated and purified by the following method: and (3) carrying out column chromatography separation on the BS-1 crude extract by sequentially adopting MCI, reverse phase silica gel and Sephadex LH-20 separation materials, and purifying by a prep-HPLC method to obtain a monomer compound.

Preferably, the method comprises the following steps: during separation and purification, the BS-1 crude extract is subjected to MCI column chromatography under reduced pressure, and is subjected to gradient elution by adopting methanol-water according to 0-100% of methanol. And then, performing column chromatographic separation by adopting reverse phase silica gel and a Sephadex LH-20 separation material, and purifying by a prep-HPLC method to obtain a monomer compound. The pyrrole-2-carboxylate compound is applied to the preparation of pesticides, in particular to the application to the preparation of drugs for killing tetranychus urticae koch and cotton aphid.

Has the advantages that: the data result shows that the

compounds

2,3,4,5,9 and 10 (the structure is shown in example 1), the tuberostemonine (11) (purchased from Nanjing Jinyibai biotechnology limited), the pyrrole-2-carboxylic acid (12) (purchased from Shanghai Honghong biotechnology limited) and the BS-1 crude extract (BS-1) have insecticidal activity on aphids and two-spotted spider mite adults; wherein, the sessilene (11), the pyrrole-2-carboxylic acid (12) and the BS-1 crude extract (BS-1) have higher insecticidal activity to aphids, and the compound 3 and the sessilene (11) have higher insecticidal activity to tetranychus urticae.

Drawings

FIG. 1 is an ESI-MS spectrum of Compound 1.

FIG. 2 is a HR-ESIMS profile of Compound 1.

FIG. 3 is a drawing of Compound 1¹H NMR spectrum.

FIG. 4 is a drawing of Compound 1¹³C NMR spectrum.

FIG. 5 is a drawing of Compound 2¹H NMR spectrum.

FIG. 6 is a drawing of Compound 3¹H NMR spectrum.

FIG. 7 is a drawing of Compound 4¹H NMR spectrum.

FIG. 8 is a drawing of Compound 5¹H NMR spectrum.

FIG. 9 is a drawing of Compound 6¹H NMR spectrum.

FIG. 10 is a drawing of Compound 7¹H NMR spectrum.

FIG. 11 is a drawing of Compound 8¹H NMR spectrum.

FIG. 12 is a drawing of Compound 9¹H NMR spectrum.

FIG. 13 is a drawing of Compound 10¹H NMR spectrum.

Detailed description of the invention

The foregoing aspects of the present invention are described in further detail by way of examples, but it should not be construed that the scope of the above-described subject matter is limited to the following examples, and that all the technologies implemented based on the above-described aspects of the present invention are within the scope of the present invention.

Example 1:

(1) separating streptomyces stemonae endophytic: collecting fresh radix Stemonae, and performing surface disinfection treatment. Cutting Stemona tuberosa stems and leaves, placing into a centrifuge tube, adding small amount of sterilized water, vortex, bathing at 80 deg.C for 1min, spreading 200uL onto ISP4 and oat culture medium, culturing at 28 deg.C for one to two months, and purifying with M2 plate.

(2) And (3) culturing the strain of BS-1: preparing ISP4 culture medium [ ISP 437 g/L; agar 15g/L](ii) a Preparing oat culture medium [ Oatmeal 65g/L]. Preparing an M2 culture medium [ glucose,4.0 g/L; yeast extract,4.0 g/L; maltextract,10.0 g/L; CaCO₃,2.0g/L；agar,18.0g/L]. And (3) quenching at 120 ℃ for 15-20 minutes.

(3) And (3) culturing the fermentation liquor of the BS-1: [ corn flour,40.0 g/L; glucose,10.0 g/L; glutenpowder,5.0 g/L; k₂HPO₄·3H₂O,0.5g/L；bran,10.0g/L；CaCO₃,2.0g/L；(NH₄)₂SO₄,1.0g/L]. The fermentation liquor is evenly distributed in 150 conical flasks with 250mL, and is sterilized for 15-20 minutes at 120 ℃. Inoculating the BS-1 strain into a prepared seed culture medium, and culturing at 28 ℃ for 3 days to obtain a seed culture solution; the seeds were also inoculated into the fermentation broth at a rate of 5%.

Example 2: preparation of pyrrole-2-carboxylate compounds: separating the fermentation liquor and the thalli obtained in the example 1, extracting the fermentation liquor and the thalli for 3 times by using methanol with the same volume respectively, combining the extraction liquids, and concentrating under reduced pressure to obtain an extract; subjecting the extract to MCI column chromatography under reduced pressure, gradient eluting with methanol-water according to 0-100% methanol, and mixing into 12 fractions (A-L) by HPLC analysis. The component B is prepared by a preparation liquid phase, and the elution conditions are as follows: isocratic elution with 35% methanol-water gave compound 2(8.8 mg). Component C was prepared using a preparative liquid phase, elution conditions: isocratic elution with 35% methanol-water gave compound 7(7 mg). Fraction F was gelled and divided into 3 fractions (F1-F3), F2 fraction was prepared with the preparative liquid phase, elution conditions: isocratic elution with 30% methanol-water gave compound 6(2 mg). Component G was prepared using a preparative liquid phase, elution conditions: from 0 to 30min, 40% by volume methanol was linearly increased to 60% methanol to give compound 4(14mg) and compound 1(7.7 mg). The component I is prepared by a preparation liquid phase, and the elution conditions are as follows: isocratic elution with 60% methanol-water gave compound 3(23 mg). Fraction J was passed through a gel column and divided into three fractions (J1-J3), fraction J1 was prepared using the preparative liquid phase, elution conditions: isocratic elution with 60% methanol-water gave compound 10(34.6 mg). Part J2 was prepared using the preparative liquid phase, method: from 0 to 30min, the volume fraction of 50% methanol was linearly increased to 80% methanol to give Compound 5(19.0 mg). Part J3 was prepared using the preparative liquid phase, elution conditions: from 0 to 30min, 40% by volume methanol was linearly increased to 70% methanol to give compound 8(11.7 mg). The component K is prepared by a preparation liquid phase, and the elution conditions are as follows: isocratic elution with 75% methanol-water gave compound 9(61 mg).

The structure of the compound is as follows:

example 3:

taking the compound 1 prepared in the above example 2, the identification process thereof is as follows: from high resolution Mass Spectrometry data 212.0901[ M + H]⁺The molecular formula of the compound 1 is calculated to be C₁₀H₁₃NO₄The unsaturation degree is 5. The hydrogen spectrum data shows that there are two methyl groups (. delta.)_H2.02,1.27), one methylene group (. delta.) is used_H2.57), three methine groups (. delta.)_H6.77,6.53,5.24). The carbon spectrum data shows that there are two aromatic carbons (delta)_C121.6,119.0), two carbonyl groups (. delta.))_C159.7,171.7). COSY spectrum data suggests two structural units: NH-1/H-5/4-CH₃H-3 and 7-CH₃/H-7/H₂-8. Then the HMBC spectrogram information, H-3 (delta)_H6.77) are related to C-2/C-4/C-5; h-5 (delta)_H6.53) C-3/C-4 related conclusions about the key backbone 4-methyl l-1H-pyrrole-2-carboxylic acid. The two above building blocks are connected by HMBC correlation of H-3/C-6 and H-7/C-6, see FIGS. 1,2,3, 4.

Fig. 5,6,7,8,9,10,11,12,13 correspond to the hydrogen spectra of

compounds

2,3,4,5,6,7,8,9,10, respectively. The identification process is consistent with that of the compound 1, and only the fat chain length is increased or the C-4/C-9 substituent group is replaced on the basis of the compound 1.

1H NMR data for Compounds 1-10 are shown in Table 1, for Compounds 1-10¹³The C NMR data are shown in Table 2.

TABLE 1 preparation of compounds 1 to 10¹H NMR data

^#measured in CD₃OD.

TABLE 2 preparation of compounds 1 to 10¹³C NMR data

^#measured in CD₃OD.

The absolute configuration of the compound 1 is determined by a synthesis method, the hydroxy methyl butyrate with R/S configuration reacts with pyrrole carboxylic acid to generate pyrrole carboxylic ester with R/S configuration, and a product 1a/1b with corresponding configuration is obtained, and the specific optical rotation (1a, [ α ]]²⁰ _D-12.0；1b,[α]²⁰ _D+9.0) and a specific optical rotation of compound 1 of [ α]²⁰ _D-25.7, thus determining the absolute configuration of compound 1 as 7R. Named endostemonine A.

The other 9 pyrrole-2-carboxylate compounds and the compound 1 are the same skeleton compounds, and only have slight change on the structural basis, and the structural analysis process of the compound refers to the compound 1 and is named as endostemonine B-J.

Example 4:

the invention relates to a method for measuring the insect-resistant activity of pyrrole-2-carboxylate compounds.

The test method comprises the following steps: taking

compounds

2,3,4,5,9 and 10 (structure shown in example 1), stemonine (11) (from Nanjing gold leopard Biotech Co., Ltd.), pyrrole-2-carboxylic acid (12) (from Shanghai hong Biotech Co., Ltd.), BS-1 crude extract (BS-1), and control drugs (imidacloprid IM, bifenazate BI) (from Suke agriculture and chemical Co., Ltd. of Jiangsu province) to carry out activity test on each pair of aphids and tetranychus urticae.

Preparing a medicament: the sample to be tested was dissolved in a small amount of anhydrous methanol and then diluted to the desired concentration with 0.1% tween-80 water.

Aphid activity test: and selecting adult aphids, inoculating the adult aphids to cucumber leaves, breeding for two days, and removing parent aphids and smaller aphids to keep about 25 aphids on the leaves. Cutting off leaves, soaking in the medicinal liquid together with aphid for 10s, sucking the rest medicinal liquid with filter paper, and air drying water stain. Wrapping petiole with absorbent cotton, and adding water to moisturize; put into a culture dish of 90mm, covered by a preservative film to prevent aphids from climbing out. After the treatment, the number of live insects and the number of dead insects were investigated at 1d, 2d and 3d for 3 times. The body of the insect was touched with a writing brush under the stereoscope, and the insect was dead.

Testing the activity of tetranychus urticae: selecting kidney bean seedling leaves with two-spotted spider mite adult mites, shearing the leaves, removing the adult mites on the leaves, keeping about 30 heads of the adult mites on the leaves, and connecting the adult mites to the insect-free leaves; cutting off leaves (25 left and right leaves/leaf) with adult mites on the next day, soaking the leaves and adult mites into the liquid medicine for 10s, sucking redundant liquid medicine by using filter paper, and airing water stains; the petiole is wrapped by absorbent paper and put into a disposable water cup, 20ml of water is added into the cup, and the absorbent paper can be padded under the cup in order to prevent the leaf from being immersed in the water. After the treatment, the number of live insects and the number of dead insects were investigated at 1d, 2d and 3d for 3 times. The body of the insect was touched with a writing brush under the stereoscope, and the insect was dead.

The calculation method comprises the following steps:

P_ttreatment group mortality in percent (%); p₀Control mortality in percent (%); the test results are as follows:

TABLE 3 corrected mortality assay for aphids

TABLE 4 corrected mortality assay for Tetranychus urticae

Experiments are carried out on aphids and two-spotted spider mites by adopting a leaf dipping method, the toxicity of the pyrrole-2-carboxylic ester compound on the aphids and the two-spotted spider mites is determined as shown in tables 3 and 4, and results and analysis: the

compounds

2,3,4,5,9 and 10, tuberostemonine (11), pyrrole-2-carboxylic acid (12) and a BS-1 crude extract (BS-1) have insecticidal activity on aphids and two-spotted spider mite adults; wherein, the sessilene (11), the pyrrole-2-carboxylic acid (12) and the BS-1 crude extract (BS-1) have higher insecticidal activity to aphids, and the compound 3 and the sessilene (11) have higher insecticidal activity to tetranychus urticae.

The pyrrole-2-carboxylic acid esters with different concentrations have certain toxicity to aphids and tetranychus urticae, and the death rate of the pyrrole-2-carboxylic acid esters gradually increases along with the increase of the concentration of the insecticide in the same time.

The toxicity test results of pyrrole-2-carboxylate on aphid and tetranychus urticae are obtained by fitting the data in tables 3 and 4 with a linear equation of two-dimentional system, and are shown in tables 5 and 6.

TABLE 5 determination of aphid virulence

TABLE 6 toxicity assay for Tetranychus urticae

"-" has no insecticidal activity

The data in tables 5 and 6 show that the compounds have high inhibitory activity (LC) on cotton aphid₅₀The range is as follows: 3.55-32.00 ppm); has moderate inhibitory activity (LC) on Tetranychus urticae Koch₅₀The range is as follows: 197.6-685.5ppm) and has potential effect of preventing and treating aphids and mites. The stemonine (11) is a main component with insecticidal activity in stemona plants, and from the data in the table, the control effect of a stemona endophyte crude extract BS-1 on aphids is stronger than that of stemonine, and the control effect of the stemonine crude extract BS-1 on tetranychus urticae is equivalent to that of stemonine.

Experiments show that: the pyrrole-2-carboxylate compound can kill tetranychus urticae koch and aphid. The pyrrole-2-carboxylate compound is expected to replace stemona medicinal material to exert the original insecticidal effect of the medicinal material.

Claims

1. A pyrrole-2-carboxylic ester compound is characterized in that the pyrrole-2-carboxylic ester compound has a structure shown in a figure I:

in formula I:

R₁and R₂May be the same or different;

R₁one selected from hydrogen, methyl, ethyl, amino, formamido and acetamido;

R₂one selected from hydrogen, hydroxyl, hydroxymethyl, hydroxyethyl, amino, formamido and acetamido;

n is selected from 1 to 5.

2. The pyrrole-2-carboxylate compound according to claim 1, wherein the preparation method comprises the steps of:

(1) preparing a seed culture medium: inoculating radix Stemonae endophytic streptomyces strain BS-1 into seed culture medium, and culturing at 26-30 deg.C for 3-5 days to obtain seed culture solution;

(2) inoculating the seed culture solution obtained in the step (1) into a fermentation culture medium, and standing and culturing at 26-30 ℃ for 6-9 days to obtain a fermented product;

(3) separating the fermentation broth and the mycelium in the fermentation product prepared in the step (2), ultrasonically extracting the mycelium for 3-4 times by adopting methanol, and recovering the methanol to obtain a crude extract A; adding 2-6% XAD-16 resin into the supernatant, stirring for 4-6 hr, filtering, discarding the filtrate, eluting the remaining resin with methanol to colorless, recovering methanol to obtain crude extract B, mixing A, B extractive solutions, and concentrating under reduced pressure to obtain extract;

(4) and (3) sequentially adopting MCI, reversed phase silica gel and Sephadex LH-20 separation materials to carry out column chromatography separation on the BS-1 extract crude extract in the step (3), and purifying by a prep-HPLC method to obtain a monomer compound.

3. The pyrrole-2-carboxylate compound according to claim 2, wherein streptomyces stemonae endophytic BS-1 is obtained by the following extraction and separation method:

(1) cleaning radix Stemonae, soaking in pure water, ultrasonic treating, and sterilizing the surface;

(2) cutting the root, stem and leaf of the stemona root medicinal material in the step (1), putting into a centrifuge tube, adding 0.5-1mL of sterilized water, vortex, bathing for 1min at 70-80 ℃, and coating 100-200uL of the mixture on ISP4 and an oat culture medium.

4. The pyrrole-2-carboxylate compound according to claim 2, wherein the preparation method comprises the step of adding: corn flour 40g/L, glucose 10.0g/L, gluten powder 5.0g/L, bran 10.0g/L, K₂HPO₄·3H₂O 0.5g/L,CaCO₃2.0g/L,(NH₄)₂SO₄1.0g/L。

5. The pyrrole-2-carboxylate compound according to claim 2, wherein the fermentation medium comprises: corn flour 70g/L, gluten powder 5.0g/L, bran 10.0g/L, K₂HPO₄·3H₂O 0.5g/L,CaCO₃2.0g/L,(NH₄)₂SO₄1.0g/L。

6. The pyrrole-2-carboxylate compound according to claim 2, wherein the preparation method comprises sterilizing the seed medium and the fermentation medium at 120 ℃ for 15-20 minutes.

7. Use of pyrrole-2-carboxylate compounds according to claim 1 for the preparation of pesticidal agents.

8. Use of pyrrole-2-carboxylate compounds according to claim 1 for the preparation of aphid and acaricidal medicaments.