CN110483535B - Isoquinoline tricyclic alkaloid compound and preparation method and application thereof - Google Patents

Isoquinoline tricyclic alkaloid compound and preparation method and application thereof Download PDF

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CN110483535B
CN110483535B CN201910810821.4A CN201910810821A CN110483535B CN 110483535 B CN110483535 B CN 110483535B CN 201910810821 A CN201910810821 A CN 201910810821A CN 110483535 B CN110483535 B CN 110483535B
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isoquinoline
silica gel
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李雪梅
黄海涛
高茜
杨光宇
李晶
孔维松
王晋
许�永
胡秋芬
刘欣
宋春满
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China Tobacco Yunnan Industrial Co Ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention discloses an isoquinoline tricyclic alkaloid compound and a preparation method and application thereof. The compound is obtained by taking the whole plant of the hypericum perforatum as a raw material and carrying out ethyl acetate extraction, silica gel column chromatography and high-pressure liquid chromatography separation and purification. Molecular formula C16H15NO2Having the following structural formula:
Figure DDA0002988226310000011
the compound was named: 1- (2,2-dimethyl-2H-pyran [2, 3-g)]Isoquinolin-6-yl) ethanone. The compound has an inhibiting effect on phytophthora nicotianae which is obviously better than that of agricultural streptomycin, and the control effect on the black shank of tobacco reaches (71.5 +/-3.2)%. The invention has the advantages of wide distribution of production raw materials, large biological yield, high alkaloid content, simple compound preparation process, low production cost and easy realization of industrial application.

Description

Isoquinoline tricyclic alkaloid compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological pesticides, and particularly relates to an isoquinoline tricyclic alkaloid compound which is extracted and separated from hypericum japonicum for the first time. Meanwhile, the invention also relates to a preparation method of the compound and application of the compound in prevention and treatment of tobacco black shank.
Background
Thalictrum L, a perennial herb of the Ranunculaceae family. The genus is about 200 plants worldwide, widely distributed in asia, europe, africa, north america and south america; there are about 67 kinds of China, and most of China is distributed in the southwest of China. About 29 of the plants can be used for medicine, and many of the plants have the functions of clearing heat, treating dampness, sweating, stopping dysentery, treating conjunctival congestion and the like. The meadow bugle (Latin's name: Thalictrum glandulosimum (Fine & Gagnep.) W.T.Wang & S.H.Wang) is a herb of Thalictrum, is distributed on the mountain lawn with the elevation of 2500 m in Yunnan Dali and Binchuan of China. Herba Hyperici Japonici has effects of clearing heat, resisting bacteria, and lowering blood pressure, and can be used to replace Coptidis rhizoma. The root and stem of the Chinese medicinal composition are commonly used in Yunnan Bai nationality, and can be used for treating enteritis, dysentery, jaundice, conjunctival congestion, swelling and pain, etc. Early studies indicate that the herba clematis is rich in alkaloid active ingredients.
The tobacco black shank is one of the most devastating diseases in tobacco production, and is also called tobacco epidemic disease, and the tobacco growers are called black stalk crazy, black root and aconite disease. The main tobacco producing areas in China occur in different degrees, wherein Anhui, Shandong and Henan provinces are historically serious disease areas; the occurrence of tobacco in southern areas such as Yunnan, Guizhou, Sichuan, Hunan, Guangdong, Guangxi and Fujian is also quite common. At present, the control of the black shank is mainly realized by methods such as crop rotation cultivation, variety gene improvement, biological pesticide and the like. Where control with biopesticides is the most common and most easily achieved method.
The discovery of new active substances with plant disease control efficacy from medicinal plant resources has been a hot spot of biological pesticide research at home and abroad. The novel isoquinoline tricyclic alkaloid compound is obtained by researching chemical components of the scandent schefflera stem and leaf and performing active screening, extraction and separation, and the compound has no related report so far, and is worthy of being mentioned as having remarkable activity of resisting the tobacco black shank virus.
Disclosure of Invention
The invention aims to provide a novel isoquinoline tricyclic alkaloid compound.
Another object of the present invention is to provide a method for preparing said isoquinoline tricyclic alkaloid compounds.
The invention also aims to provide application of the isoquinoline tricyclic alkaloid compound in prevention and treatment of tobacco black shank.
The purpose of the invention is realized by the following technical scheme.
All percentages used in the present invention are mass percentages unless otherwise indicated.
An isoquinoline tricyclic alkaloid compound with a molecular formula of C16H15NO2Having the following structural formula:
Figure GDA0002988226300000021
the compound was named: 1- (2,2-dimethyl-2H-pyran [2,3-g ] isoquinolin-6-yl) ethanone, english name: 1- (2,2-dimethyl-2H-pyrano [2,3-g ] isoquinolin-6-yl) ethanone.
A method of preparing said isoquinoline tricyclic alkaloid compounds comprising the steps of:
(1) extracting the extractum: drying the whole plant of herba Ceratopteridis Tectori in the sun, pulverizing to 30-50 mesh, placing the pulverized sample in a glass reaction kettle, reflux-extracting with 95% ethanol for 2 times, mixing the two extractive solutions, concentrating to small volume, diluting with 3% tartaric acid solution, and extracting with ethyl acetate for 2 times; saturating the water phase with sodium carbonate, extracting with ethyl acetate for 2 times, mixing the extracted ethyl acetate phases, and concentrating under reduced pressure to obtain alkaloid extract;
(2) silica gel column chromatography: dissolving the extract obtained in the step (1) by using 1.5-3 times of pure methanol or pure ethanol or pure acetone, mixing the extract with 0.8-2.5 times of 80-100 meshes of silica gel, and drying; the silica gel filled in the column is 150-200 meshes, and the using amount of the silica gel is 3-8 times of the weight of the extract; performing gradient elution with chloroform-acetone solutions at weight ratios of 20:1, 9:1, 8:2, 7:3, 6:4 and 5:5, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions;
(3) high-pressure liquid chromatography separation and purification: and (3) taking 8:2 parts of the column chromatography gradient eluent to perform high-pressure liquid chromatography separation and purification: using Zorbax PrepHT GF of Agilent company, a 21.2mm multiplied by 25cm reverse phase column, using 58% methanol water solution as a mobile phase, the flow rate is 15mL/min, the detection wavelength of an ultraviolet detector is 346nm, collecting a chromatographic peak of 35.6min, accumulating for a plurality of times, and evaporating to dryness to obtain the isoquinoline tricyclic alkaloid compound.
And (3) dissolving the compound obtained after the separation and purification by the high pressure liquid chromatography in pure methanol, and separating by a sephadex column chromatography with the pure methanol as a mobile phase to obtain a yellow jelly, namely a pure compound.
Structural identification of the compounds:
the compound is yellow jelly, and HRESI-MS shows that the peak of the quasi-molecular ion is 276.1008[ M + Na ]]+Is combined with1H and13c NMR spectrum to confirm the molecular formula as C16H15NO2Is a alkaloid compound, and the unsaturation degree of the compound is 10. Its infrared spectrum shows that there is carbonyl (1644 cm) in the compound-1) And aromatic rings (1610, 1538 and 1469 cm)-1) The signals, the absorption maxima of 225, 276, 308 and 346nm of the ultraviolet spectrum also confirm the presence of aromatic ring structures in the compounds. Process for preparing compounds1H、13C-NMR and DEPT spectra (Table-1) showed that it contained 16 carbons and 15 hydrogens. The method comprises the following steps: a 1,6, 7-substituted isoquinoline core (C-1 to C-10; H-3, H-4, H-5, and H-8), a synthosphane ring (-CH ═ CH-C (CH)3)2-O-, C-1 'to C-5', H-1 ', H-2' and H6-4 ", 5"), one acetyl group (-CO-CH)3(ii) a C-1' and C-2′；H3-2'). The isoquinoline nucleus can exist through H-3 and C-1, C-4, C-10; h-4 and C-3, C-5, C-9, C-10; h-5 and C-4, C-9, C-10; and HMBC correlation of H-8 and C-1, C-9, C-10 (FIG. 3). The harmonic dimethylchromene ring can be present by H-1 'and C-2', C-3 ', H-2' and C-1 ', C-3', C-4 ', 5', and H6HMBC correlation of-4 ", 5" with C-2 ", C-3" was confirmed. Further analysis of its HMBC correlation spectrum, according to H-1' and C-6, C-7, C-8; h-2 ' and C-7, and H-8 and C-1 ' are related to HMBC, it is speculated that the gem-dimethyl chromene ring is substituted at the C-6 and C-7 positions of the phenyl ring of the isoquinoline, and the carbon at the C-1 ' position is attached at the C-7 position of the phenyl ring of the isoquinoline. The parent structure of the compound can be determined to be isoquinoline tricyclic alkaloid with gem-dimethyl chromene ring. After the parent nucleus of the compound is determined, the remaining acetyl group can be defined as a substituent by the acetylmethyl hydride H3HMBC at-2' and C-1 correlate and the substitution of the acetyl group at C-1 can be determined. The structure of this compound was confirmed and the compound was named: 1- (2,2-dimethyl-2H-pyran [2, 3-g)]Isoquinolin-6-yl) ethanone.
TABLE-1 preparation of the compounds1H and13c NMR data (solvent CDCl3, 500 and 125MHz)
Figure GDA0002988226300000041
Infrared, ultraviolet and mass spectral data of compounds: UV (CH)3OH)λmax(logε)225(4.22)、276(3.62)、308(3.39)、346(348)；IR(KBr)νmax 3074、2962、1644、1610、1538、1469、1357、1266、1164、1047、895、762cm-1；1H NMR and13c NMR data (CDCl)3500 and 125MHz) are shown in Table-1; ESIMS M/z 276[ M + Na ] in positive ion mode]+，HRESIMS m/z 276.1008[M+Na]+(calculation value C)16H15NNaO2,276.1001)。
The isoquinoline tricyclic alkaloid compound is applied to prevention and treatment of tobacco black shank.
1. The tobacco black shank is caused by phytophthora nicotianae infection, and the capacity of the compound for inhibiting the activity of the phytophthora nicotianae is measured, wherein the capacity comprises the following steps:
(1) preparation of oatmeal agar medium: adding 1000mL of water into oatmeal, heating the oatmeal in a boiling water bath for 1 hour, filtering the oatmeal by using gauze, adding the water for supplementing 1000mL, then adding sugar and agar, heating the oatmeal to completely melt the agar, filtering the oatmeal in a triangular flask by using the gauze (absorbent cotton is added in the middle) while the oatmeal is hot, sterilizing the oatmeal for 20min at 121 ℃ and 15 pounds, taking out the oatmeal and cooling the oatmeal to about 45 ℃, adding ampicillin (5mg/100mL) into an aseptic operation table, uniformly mixing the ampicillin and the ampicillin, pouring the ampicillin into the flat dish, culturing the ampicillin for 48 hours at 28 ℃, and checking the.
(2) Bacteriostatic experiments: circular filter paper with diameter of 5mm is placed in a culture dish, sterilized under high pressure for 30min under 15 pounds, and then is respectively immersed in 20 mu M compound, 75% ethanol solution and sterilized distilled water after being dried. Respectively sucking 0.2mL of fresh bacterial liquid of phytophthora nicotianae on oatmeal agar medium plates by using a sterile suction pipe on a sterile operating table. Coating with triangular glass coating rod, lightly sticking the filter paper sheets onto corresponding flat plates with tweezers, culturing at 28 deg.C, observing experimental results, and measuring the size of antibacterial zone. Meanwhile, agricultural streptomycin is used as a positive control.
The test result shows that: the diameter of the inhibition zone of the isoquinoline tricyclic alkaloid compound is 15.2 +/-1.2 mm, and the diameter of the inhibition zone of the positive control agricultural streptomycin is 12.2 +/-1.0 mm. The compound has the effect of inhibiting phytophthora nicotianae remarkably superior to that of positive control agricultural streptomycin and has outstanding activity of inhibiting black shank.
2. The compound of the invention has the following effects of preventing and treating tobacco black shank:
transplanting tobacco seedlings into flowerpots with the diameter of 10cm and the height of 10cm, wherein the culture medium is as follows: sterilizing soil, peat and perlite (2:2:1), and culturing 1 strain per pot. After transplanting and seedling slowing, 10g of bacterial grains are added into roots, tobacco seedlings are placed in an artificial climate room for cultivation, the temperature is 30 ℃ in the daytime, the temperature is 28 ℃ in the night, the illumination is dark (12h:12h), and the relative humidity is 95%, so that the tobacco seedlings are attacked. Before the black shank disease occurs, the tobacco seedlings are subjected to root irrigation treatment by using 20 mu M of the compound, and 10mL of the compound is irrigated to each tobacco seedling; the number of the plants is 2, 10 plants are treated, the treatment is repeated for 3 times, and after 14 days, the disease condition is investigated and the disease index is calculated. The results show that: the compound has the effect of controlling the tobacco black shank of (71.5 +/-3.2)%, and has obvious effect of controlling the tobacco black shank.
Compared with the prior art, the invention has the following advantages:
(1) the isoquinoline tricyclic alkaloid compound is extracted and separated from the herba nasutae firstly, the inhibition effect of the isoquinoline tricyclic alkaloid compound on phytophthora nicotianae is obviously superior to that of agricultural streptomycin, and the control effect on the black shank of the tobacco reaches (71.5 +/-3.2)%. The application of the compound provides a novel efficient and safe biological pesticide molecular structure for preventing and treating the black-stem disease of tobacco.
(2) The production raw material (golden string horsetail) of the invention has wide distribution, large biological yield, high alkaloid content, simple compound preparation process, low production cost and easy realization of industrialized application.
Drawings
FIG. 1 is a nuclear magnetic resonance carbon spectrum of an isoquinoline tricyclic alkaloid compound;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of an isoquinoline tricyclic alkaloid compound;
FIG. 3 is a graph relating the major HMBC derivatives of the isoquinoline tricyclic alkaloids of the present invention.
Detailed Description
The present invention is further described in detail with reference to the drawings and examples, which are not intended to limit the technical scope of the present invention, and all changes and equivalents which are made based on the teachings of the present invention should fall within the protective scope of the present invention.
Example 1
Preparation of isoquinoline tricyclic alkaloid Compound C16H15NO2The method comprises the steps of extract extraction, silica gel column chromatography and high-pressure liquid chromatography separation, and specifically comprises the following steps:
(1) extracting the extractum: taking the whole plant of the herba nasutae, drying in the sun, and crushing to 30-50 meshes. Weighing 3.0-4.5 kg of crushed sample, placing the crushed sample in a 20L glass reaction kettle, adding 10-15L of 95% ethanol, performing reflux extraction for 30-50 min, and filtering out an extracting solution; and adding 10-15L of 95% ethanol into the filter residue again, performing reflux extraction for 30-50 min, and filtering an extracting solution. And combining the two extracting solutions, concentrating to a small volume, diluting with 5-8L of 3% tartaric acid solution, and extracting with 5-8L of ethyl acetate for 2 times. And after extraction, saturating the water phase with sodium carbonate, extracting for 2 times with 5-8L of ethyl acetate, combining the ethyl acetate phases obtained by the second extraction, and concentrating under reduced pressure to obtain 45.6-54.7 g of an alkaloid part extract.
(2) Silica gel column chromatography: dissolving the extract with 1.5-3 times of pure methanol, pure ethanol or pure acetone, mixing the extract with 40-60 g (80-100 meshes) of crude silica gel, drying, performing column chromatography with 150-250 g of silica gel (150-200 meshes), and purifying with chloroform: acetone (20:1, 9:1, 8:2, 7:3, 6:4, 5:5) was gradient eluted and divided into 6 fractions.
(3) And (3) separating and purifying by high pressure liquid chromatography: and (3) selecting 8:2 elution parts for further separation by HPLC: using Zorbax PrepHT GF (21.2mm multiplied by 25cm) reverse phase column of Agilent company, using 58% methanol water solution as mobile phase, flow rate is 15mL/min, ultraviolet detector detection wavelength is 346nm, collecting chromatographic peak of 35.6min, accumulating for many times, evaporating to dryness to obtain crude compound. Dissolving the crude product with methanol, purifying with Sephadex column to obtain pure compound.
The raw material of the herba clematidis chinensis used in the invention is not limited by regions and varieties, and the invention can be realized, and the raw material of the herba clematidis chinensis from the Yunan theory is further explained as follows:
example 2
The canary creeper sample is from the Ministry of Longmingjiqing in Yunan. Sun drying herba seu radix Nothopanacis Delavayi, and pulverizing to 35 mesh. Weighing 3.0kg of the crushed sample, placing the crushed sample in a 20L glass reaction kettle, adding 12L of 95% ethanol, performing reflux extraction for 40min, and filtering out an extracting solution; adding 95% ethanol 12L into the residue, reflux extracting for 40min, and filtering to remove the extractive solution. The combined extracts were concentrated to a small volume, then diluted with 6L of 3% tartaric acid solution and extracted 2 times with 6L of ethyl acetate. After extraction, the water phase is saturated with sodium carbonate, extracted for 2 times with 6L ethyl acetate again, the extracted ethyl acetate phases are combined, and the mixture is concentrated under reduced pressure to obtain 48.2g of alkaloid part extract. Mixing the extract with 60g (80-100 mesh) crude silica gel, oven drying, performing column chromatography with 180g silica gel (150-200 mesh), chloroform: acetone (20:1, 9:1, 8:2, 7:3, 6:4, 5:5) was gradient eluted and divided into 6 fractions. And (3) selecting 8:2 elution parts for further separation by HPLC: using Zorbax PrepHT GF (21.2mm multiplied by 25cm) reverse phase column of Agilent company, using 58% methanol water solution as mobile phase, flow rate is 15mL/min, ultraviolet detector detection wavelength is 346nm, collecting chromatographic peak of 35.6min, accumulating for many times, evaporating to dryness to obtain crude compound. Dissolving the crude product with methanol, purifying with Sephadex column to obtain pure compound.
Example 3
The canary creeper sample is from yunan Dali Binchuan county. Sun drying herba seu radix Nothopanacis Delavayi, and pulverizing to 40 mesh. Weighing 3.8kg of the crushed sample, placing the crushed sample in a 20L glass reaction kettle, adding 14L of 95% ethanol, performing reflux extraction for 30min, and filtering out an extracting solution; adding 14L of 95% ethanol into the residue, reflux extracting for 30min, and filtering to remove the extractive solution. The combined extracts were concentrated to a small volume, then diluted with 8L of 3% tartaric acid solution and extracted 2 times with 8L of ethyl acetate. After extraction, the water phase is saturated with sodium carbonate, and is extracted with 8L ethyl acetate for 2 times, and the extracted ethyl acetate phases are combined and concentrated under reduced pressure to obtain 46.7g of alkaloid part extract. Mixing the extract with 60g (80-100 mesh) of crude silica gel, oven drying, performing column chromatography with 220g of silica gel (150-200 mesh), chloroform: acetone (20:1, 9:1, 8:2, 7:3, 6:4, 5:5) was gradient eluted and divided into 6 fractions. And (3) selecting 8:2 elution parts for further separation by HPLC: using Zorbax PrepHT GF (21.2mm multiplied by 25cm) reverse phase column of Agilent company, using 58% methanol water solution as mobile phase, flow rate is 15mL/min, ultraviolet detector detection wavelength is 346nm, collecting chromatographic peak of 35.6min, accumulating for many times, evaporating to dryness to obtain crude compound. Dissolving the crude product with methanol, purifying with Sephadex column to obtain pure compound.
Example 4
The canary creeper sample is from Jianchuan county of Yunnan. Sun drying herba seu radix Nothopanacis Delavayi, and pulverizing to 45 mesh. Weighing 4.2kg of the crushed sample, placing the crushed sample in a 20L glass reaction kettle, adding 14L of 95% ethanol, performing reflux extraction for 50min, and filtering out an extracting solution; adding 14L of 95% ethanol into the residue, reflux extracting for 50min, and filtering to obtain extractive solution. The combined extracts were concentrated to a small volume, then diluted with 8L of 3% tartaric acid solution and extracted 2 times with 8L of ethyl acetate. After extraction, the water phase is saturated with sodium carbonate, and is extracted with 8L ethyl acetate for 2 times, and the extracted ethyl acetate phases are combined and concentrated under reduced pressure to obtain 50.6g of alkaloid part extract. Mixing the extract with 45g (80-100 mesh) crude silica gel, oven drying, performing column chromatography with 220g silica gel (150-200 mesh), chloroform: acetone (20:1, 9:1, 8:2, 7:3, 6:4, 5:5) was gradient eluted and divided into 6 fractions. And (3) selecting 8:2 elution parts for further separation by HPLC: using Zorbax PrepHT GF (21.2mm multiplied by 25cm) reverse phase column of Agilent company, using 58% methanol water solution as mobile phase, flow rate is 15mL/min, ultraviolet detector detection wavelength is 346nm, collecting chromatographic peak of 35.6min, accumulating for many times, evaporating to dryness to obtain crude compound. Dissolving the crude product with methanol, purifying with Sephadex column to obtain pure compound.
Example 5
Identification of the structure of the compounds
The isoquinoline tricyclic alkaloid compounds prepared in example 2 were collected and measured by the following method. The compound is yellow jelly, and HRESI-MS shows that the peak of the quasi-molecular ion is 276.1008[ M + Na ]]+Is combined with1H and13c NMR spectrum to confirm the molecular formula as C16H15NO2Is a alkaloid compound, and the unsaturation degree of the compound is 10. Its infrared spectrum shows that there is carbonyl (1644 cm) in the compound-1) And aromatic rings (1610, 1538 and 1469 cm)-1) The signals, the absorption maxima of 225, 276, 308 and 346nm of the ultraviolet spectrum also confirm the presence of aromatic ring structures in the compounds. Process for preparing compounds1H、13C-NMR and DEPT spectra (Table-1) showed that it contained 16 carbons and 15 hydrogens. The method comprises the following steps: a 1,6, 7-substituted isoquinoline core (C-1 to C-10; H-3, H-4, H-5, and H-8), a synthosphane ring (-CH ═ CH-C (CH)3)2-O-, C-1 'to C-5', H-1 ', H-2' and H6-4 ", 5"), one acetyl group (-CO-CH)3；C-1'and C-2'; h3-2'). The isoquinoline nucleus can exist through H-3 and C-1, C-4, C-10; h-4 and C-3, C-5, C-9, C-10; h-5 and C-4, C-9, C-10; and HMBC correlation of H-8 and C-1, C-9, C-10 (FIG. 3). The harmonic dimethylchromene ring can be present by H-1 'and C-2', C-3 ', H-2' and C-1 ', C-3', C-4 ', 5', and H6HMBC correlation of-4 ", 5" with C-2 ", C-3" was confirmed. Further analysis of its HMBC correlation spectrum, according to H-1' and C-6, C-7, C-8; h-2 ' and C-7, and H-8 and C-1 ' are related to HMBC, it is speculated that the gem-dimethyl chromene ring is substituted at the C-6 and C-7 positions of the phenyl ring of the isoquinoline, and the carbon at the C-1 ' position is attached at the C-7 position of the phenyl ring of the isoquinoline. The parent structure of the compound can be determined to be isoquinoline tricyclic alkaloid with gem-dimethyl chromene ring. After the parent nucleus of the compound is determined, the remaining acetyl group can be defined as a substituent by the acetylmethyl hydride H3HMBC at-2' and C-1 correlate and the substitution of the acetyl group at C-1 can be determined. The structure of this compound was confirmed and the compound was named: 1- (2,2-dimethyl-2H-pyran [2, 3-g)]Isoquinolin-6-yl) ethanone.
Example 6
The compound prepared in example 3 was taken as a yellow gum. The determination method was the same as in example 5, and it was confirmed that the compound prepared in example 3 was 1- (2,2-dimethyl-2H-pyran [2,3-g ] isoquinolin-6-yl) ethanone, which is the isoquinoline tricyclic alkaloid compound.
Example 7
The compound prepared in example 4 was taken as a yellow gum. The procedure was carried out in the same manner as in example 5, and it was confirmed that the compound prepared in example 4 was the above-mentioned 1- (2,2-dimethyl-2H-pyran [2,3-g ] isoquinolin-6-yl) ethanone.
Example 8
Any of the isoquinoline tricyclic alkaloid compounds prepared in examples 1-4 are tested for activity against tobacco black shank as follows:
(1) adding 1000mL of water into oatmeal, heating the oatmeal in a boiling water bath for 1 hour, filtering the oatmeal by using gauze, adding the water for supplementing 1000mL, then adding sugar and agar, heating the oatmeal to completely melt the agar, filtering the oatmeal in a triangular flask by using the gauze (absorbent cotton is added in the middle) while the oatmeal is hot, sterilizing the oatmeal for 20min at 121 ℃ and 15 pounds, taking out the oatmeal and cooling the oatmeal to about 45 ℃, adding ampicillin (5mg/100mL) into an aseptic operation table, uniformly mixing the ampicillin and the ampicillin, pouring the ampicillin into the flat dish, culturing the ampicillin for 48 hours at 28 ℃, and checking the.
(2) Circular filter paper with diameter of 5mm is placed in a culture dish, sterilized under high pressure for 30min under 15 pounds, and then is respectively immersed in 20 mu M compound, 75% ethanol solution and sterilized distilled water after being dried. Respectively sucking 0.2mL of fresh bacterial liquid of phytophthora nicotianae on oatmeal agar medium plates by using a sterile suction pipe on a sterile operating table. Coating with triangular glass coating rod, lightly sticking the filter paper sheets onto corresponding flat plates with tweezers, culturing at 28 deg.C, observing experimental results, and measuring the size of antibacterial zone. Meanwhile, agricultural streptomycin is used as a positive control.
(3) The test result shows that: the diameter of the inhibition zone of the isoquinoline tricyclic alkaloid compound is 15.2 +/-1.2 mm, and the diameter of the inhibition zone of the positive control agricultural streptomycin is 12.2 +/-1.0 mm. The compound has the effect of inhibiting phytophthora nicotianae remarkably superior to that of positive control agricultural streptomycin and has outstanding activity of inhibiting black shank.
(4) Transplanting tobacco seedlings into flowerpots with the diameter of 10cm and the height of 10cm, wherein the culture medium is as follows: sterilizing soil, peat and perlite (2:2:1), and culturing 1 strain per pot. After transplanting and seedling slowing, 10g of bacterial grains are added into roots, tobacco seedlings are placed in an artificial climate room for cultivation, the temperature is 30 ℃ in the daytime, the temperature is 28 ℃ in the night, the illumination is dark (12h:12h), and the relative humidity is 95%, so that the tobacco seedlings are attacked. Before the black shank disease occurs, the tobacco seedlings are subjected to root irrigation treatment by using 20 mu M of the compound, and 10mL of the compound is irrigated to each tobacco seedling; the number of the plants is 2, 10 plants are treated, the treatment is repeated for 3 times, and after 14 days, the disease condition is investigated and the disease index is calculated. The results show that: the compound has the effect of controlling the tobacco black shank of (71.5 +/-3.2)%, and has obvious effect of controlling the tobacco black shank.

Claims (4)

1. An isoquinoline tricyclic alkaloid compound with a molecular formula of C16H15NO2Having the following structural formula:
Figure FDA0002988226290000011
the compound was named: 1- (2,2-dimethyl-2H-pyran [2,3-g ] isoquinolin-6-yl) ethanone, english name: 1- (2,2-dimethyl-2H-pyrano [2,3-g ] isoquinolin-6-yl) ethanone.
2. A process for the preparation of isoquinoline tricyclic alkaloid compounds as claimed in claim 1 comprising the steps of:
(1) extracting the extractum: drying the whole plant of herba Ceratopteridis Tectori in the sun, pulverizing to 30-50 mesh, placing the pulverized sample in a glass reaction kettle, reflux-extracting with 95% ethanol for 2 times, mixing the two extractive solutions, concentrating to small volume, diluting with 3% tartaric acid solution, and extracting with ethyl acetate for 2 times; saturating the water phase with sodium carbonate, extracting with ethyl acetate for 2 times, mixing the extracted ethyl acetate phases, and concentrating under reduced pressure to obtain alkaloid extract;
(2) silica gel column chromatography: dissolving the extract obtained in the step (1) by using 1.5-3 times of pure methanol or pure ethanol or pure acetone, mixing the extract with 0.8-2.5 times of 80-100 meshes of silica gel, and drying; the silica gel filled in the column is 150-200 meshes, and the using amount of the silica gel is 3-8 times of the weight of the extract; performing gradient elution with chloroform-acetone solutions at weight ratios of 20:1, 9:1, 8:2, 7:3, 6:4 and 5:5, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions;
(3) high-pressure liquid chromatography separation and purification: and (3) taking 8:2 parts of the column chromatography gradient eluent to perform high-pressure liquid chromatography separation and purification: using Zorbax PrepHT GF of Agilent company, a 21.2mm multiplied by 25cm reverse phase column, using 58% methanol water solution as a mobile phase, the flow rate is 15mL/min, the detection wavelength of an ultraviolet detector is 346nm, collecting a chromatographic peak of 35.6min, accumulating for a plurality of times, and evaporating to dryness to obtain the isoquinoline tricyclic alkaloid compound.
3. The method of claim 2, wherein: and (3) dissolving the compound obtained after the separation and purification by the high pressure liquid chromatography in pure methanol, and separating by a sephadex column chromatography with the pure methanol as a mobile phase to obtain a yellow jelly, namely a pure compound.
4. The use of the isoquinoline tricyclic alkaloid compounds as claimed in claim 1 in the control of tobacco black shank.
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