CN115286561B - Indole alkaloid compound in gene editing tobacco, and preparation method and application thereof - Google Patents
Indole alkaloid compound in gene editing tobacco, and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of phytochemistry, and particularly relates to an indole alkaloid compound, and a preparation method and application thereof. The molecular formula of the compound is as follows: c (C) 15 H 15 NO 4 It has the following structure:
the invention also discloses a preparation method and application of the compound. The invention firstly identifies a new compound from the gene editing tobacco, determines the compound as indole alkaloid compound by nuclear magnetic resonance and mass spectrometry determination methods, and characterizes the specific structure of the compound. The indole alkaloid compound has good activity of resisting tobacco mosaic virus: experiments on tobacco mosaic virus resistance show that the relative inhibition rate of the indole alkaloid compound reaches 42.8%, and the activity of the indole alkaloid compound is higher than that of a control Ningnanmycin (33.2%). The compound has good application prospect in preparing medicines for resisting tobacco mosaic virus. The compound has simple structure and good activity, and can be used as a lead compound of a tobacco mosaic virus resistant drug.
Description
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to an indole alkaloid compound in gene editing tobacco, and a preparation method and application thereof.
Background
Tobacco is a plant of the genus nicotiana of the family Solanaceae, and is one of the most widely planted commercial crops worldwide. China is the largest tobacco producing country and consuming country in the world, and the annual output can reach 4-5 hundred million tons. Besides the application of cigarette smoking, people pay attention to exerting the effects of preventing and treating diseases, and the tobacco has very good curative effects on dermatitis, diarrhea and stomatitis. Tobacco is also very effective against infectious diseases such as typhoid, malaria, influenza, pneumonia, etc. The characters record that the london pestis is rampant in 1665 years, the death of the infected person is not less, but the smoker is less, people generally consider that the tobacco can prevent and treat pestilence, and people write the kingdom to state the tobacco function, and the kingdom cancels the smoking inhibition. In addition, the tobacco can be used for biological pesticide application, and the tobacco plant pesticide can be decomposed in a plurality of days after application, has no residual toxic pollution and has no adverse effect on environment, crops, people and poultry. Pesticides applied in many countries during the mature period of fruit and vegetable are now such pesticides. Such as: the nicotine sulfate medicament is used for preventing and controlling aphids, and is most suitable for fruit tree disinfestation; the effect of preventing and controlling rice borer and mirid insects by using the nicotine preparation is not less than 'six'; the composition is used for preventing and controlling cotton red spiders, and has better effect than 1059. The pesticide 4115 prepared by using waste tobacco powder successfully in Ningbo cigarette factories in China is used for preventing and controlling pests such as rice, cotton, vegetables and the like, has high pesticide effect and is harmless to human and animals.
According to literature reports, tobacco outstanding efficacy is closely related to its abundant secondary metabolites. The compounds identified from tobacco at present are up to 4000, and the main components comprise diterpenoid compounds, sesquiterpenoid compounds, flavonoid compounds, alkaloids, coumarin and the like. In recent years, gene editing techniques have been widely used for crop variety improvement. Compared with other means, the gene editing has obvious advantages, one important characteristic is site-directed saturation mutation, can realize accurate modification of DNA level, and can change the gene expression mode and obtain new functions of the gene by deleting or inserting small fragment bases, accurate replacement of single base, allele replacement and other gene editing technologies.
Since genes are closely related to plant metabolism, changes in genes can cause changes in plant metabolic networks, and thus changes in the content and variety of metabolic components, gene editing crops become an important source for finding active metabolic components. As such, the research on the active ingredients in the gene editing tobacco is fully carried out, and the method has important significance for finding out the secondary metabolite with biological activity from the tobacco and promoting the comprehensive development and utilization of the tobacco. The invention discovers a novel indole alkaloid compound from gene editing tobacco, and the compound is remarkable in tobacco mosaic virus resistance.
Disclosure of Invention
The invention separates a new alkaloid compound with activity of resisting tobacco mosaic virus from tobacco, and the compound has no report.
The percentages used in the present invention are mass percentages unless otherwise indicated.
The first aspect of the invention provides an indole alkaloid compound, wherein the molecular formula of the compound is as follows:
C 15 H 15 NO 4 it has the following structure:
the compound was orange gum, named: tobacco indole-C, english name: nicindole-C.
The second aspect of the invention provides a preparation method of the indole alkaloid compound in the first aspect, which specifically comprises the following steps:
A. extracting extract: taking tobacco leaf samples as raw materials, ultrasonically extracting with 90-99wt% ethanol, adding the extracting solution into a mixed solution of ethyl acetate and 3wt% tartaric acid, standing the mixed solution for layering, and then using Na 2 CO 3 The pH of the aqueous layer was adjusted to 9.0, extracted again with ethyl acetate, filtered and concentrated under reduced pressure to give an extract.
B. Silica gel column chromatography: loading the extract obtained in the step A into a column by using 200-300 mesh silica gel dry method, and performing silica gel column chromatography; gradient elution is carried out on chloroform-methanol solutions with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 respectively, parts with the same polarity are combined, and eluent of each part is collected and concentrated; wherein the mass ratio of the silica gel to the extract is 2-5; collecting an eluent obtained when the chloroform-methanol solution with the volume ratio of 9:1 is used for eluting, wherein the eluent is called a first eluent; and (3) continuing to separate the first eluent by using a silica gel chromatographic column, performing gradient elution by using a series of chloroform-acetone solutions with volume ratios of 9:1, 8:2, 7:3, 6:4 and 5:5, and collecting eluent obtained when the chloroform-acetone solution with the volume ratio of 8:2 is used for eluting, wherein the eluent is called second eluent.
C. High performance liquid chromatography separation: and B, introducing the second eluent obtained in the step B into a high performance liquid chromatography for separation and purification, wherein the high performance liquid chromatography separation and purification is carried out by adopting a Zorbax PrepHT GF chromatographic column with the flow rate of 21.2mm multiplied by 250mm and 5 mu m, the flow rate is 12mL/min, the mobile phase is 55wt% of methanol aqueous solution, the detection wavelength of an ultraviolet detector is 302nm, 200 mu L of the second eluent is injected each time, and the eluent corresponding to the chromatographic peak retention time of 31.6min after each injection is collected and called a third eluent, and the third eluent is subjected to solvent removal to obtain the indole alkaloid compound.
Preferably, in the step A, the concentration of ethanol is preferably 95wt%.
Preferably, in the step B, before the extract is roughly separated by silica gel column chromatography, the extract is dissolved by methanol and then mixed with 80-120 meshes of silica gel with the weight ratio of 1.5-2.5 times.
Preferably, in the step C, after the high performance liquid chromatography separation and purification, the obtained compound is dissolved again with pure methanol, and then separated by gel column chromatography with pure methanol as a mobile phase, so as to further separate and purify.
The third aspect of the invention provides an application of the indole alkaloid compound in preparing a medicine for resisting tobacco mosaic virus.
The structure of the indole alkaloid compound prepared by the method is determined by the following method:
appearance observation finds that: the compound is orange glue;
the ultraviolet-visible absorption spectrum shows that the compound has maximum absorption at 265 nm and 302nm, which proves that the compound has an aromatic ring structure;
infrared spectra (potassium bromide tablets) showed carbonyl groups (1686 and 1655 cm) in the compound -1 ) Aromatic ring (1618, 1564, 1468 cm) -1 ) A characteristic functional group; high resolution mass spectrometry (HRESIMS) gave excimer ion peak 296.0894[ M+Na ]] + The molecular formula of the compound can be determined to be C 15 H 15 NO 4 The unsaturation was 9. Bonding of 1 H and 13 c and HSQC NMR data showed that the compound included a 1,2,3,4, 5-pentasubstituted benzene ring (C-4-C-7, C-3a, C-7a and H-7), an N-2-oxopropyl group (C-4 '-C-6', H) 2 -4' and H 3 -6'), one-COCH 2 CH 2 Structural fragment (C-1 '-C-3', H) 2 -2' and H 2 -3'), a carbonyl group (C-1), an N-methylene group (C-3 and H) 2 -3) and a methoxy group (delta) C 56.2q；δ H 3.80s)。
The unsaturation 7 of the benzene ring and three carbonyl groups is removed, and there should also be two rings in the compound in order to satisfy the 9 unsaturations of the compound. The combined literature reports: it is presumed that the benzene ring, carbonyl group (C-1), methylene group (C-3) and nitrogen atom should form an isoindole ring, and this can be presumed by H 2 -3 and C-1/C-4/C-3a/C-7a, H-7 and C-1/C-3The HMBC correlation of a/C-7a was confirmed. In addition-COCH 2 CH 2 The structural fragment should also be linked to the benzene ring to form a five-membered ring, which can be deduced also from H 2 -2' and C-1'/C-3', H 2 The HMBC correlation of-3 ' and C-1'/C-2' was confirmed.
After the parent backbone of the compound is determined, the remaining substituent positions can be determined by further analysis of its HMBC correlation. -CH 2 CH 2 The CO-structural fragment is connected with C-4 and C-5 positions of benzene ring to form five-membered cyclic ketone, and C-1 'is connected with C-5, C-3' is connected with C-4, and H can be used for preparing the five-membered cyclic ketone 2 -2' and C-4/C-5,H 2 The HMBC correlation of 3' and C-4/C-5/C-3a was confirmed. Methoxy substituted at C-6 as a traversable methoxy hydrogen (. Delta.) H 3.80 s) and HMBC related validation of C-6. In addition, the N-2-oxopropyl group and the nitrogen atom may be linked by H-4' and C-1\C-3, as well as H 2 The HMBC correlation of-3 and C-4' is confirmed. The structure of the compound is confirmed, and the compound is identified as tobacco indole-C, and the English name is: nicindole-C.
The compounds of Table 1 1 H NMR 13 C NMR data (CDCl) 3 )
Compound uv, ir spectra and mass spectral data: c (C) 15 H 15 NO 4 Is orange glue; lambda of ultraviolet spectrum (methanol medium) max nm (log ε) 210 (4.02), 265 (3.47), 302 (3.03); infrared spectrum (KBr tablet) v max 2962、1686、1655、1618、1564、1468、1349、1168、1054、938、826cm -1 The method comprises the steps of carrying out a first treatment on the surface of the Positive ion mode ESIMS m/z 296[ M+Na ]] + Positive ion mode HRESIMS m/z 296.0894[ M+Na ]] + (calculated value C) 15 H 15 NNaO 4 ,296.0899)。
The beneficial effects of the invention are as follows:
1. according to the invention, a new compound is firstly separated from tobacco, and is determined to be an indole alkaloid compound by a nuclear magnetic resonance and mass spectrometry method, and the specific structure of the indole alkaloid compound is characterized. The indole alkaloid compound has good activity of resisting tobacco mosaic virus: experiments on tobacco mosaic virus resistance show that the relative inhibition rate of the indole alkaloid compound reaches 42.8%, and the activity of the indole alkaloid compound is higher than that of a control Ningnanmycin (33.2%). The results show that the compound has good application prospect in preparing the tobacco mosaic virus resistant medicines. The compound has simple structure and good activity, and can be used as a lead compound of a tobacco mosaic virus resistant drug.
2. Because the raw materials of the compound are easy to obtain; the extraction method of the compound is simple, the compound is easy to separate and obtain, and the industrialized preparation is easy to realize.
3. The compound has simple molecular structure, is easy to realize artificial synthesis, and can be realized through artificial synthesis in the subsequent industrialization.
4. The preparation method combining the conventional column chromatography and the high performance liquid chromatography is adopted, the preparation operation flow of the compound is simple, the purity of the obtained compound is high, and the quality and purity of the compound in the subsequent industrial production are ensured.
5. The compound disclosed by the invention is safe and nontoxic, shows good activity of resisting the tobacco mosaic virus, and can provide ideal new skeleton type medicine source molecules for preventing and treating the tobacco mosaic disease.
Drawings
FIG. 1 is a nuclear magnetic resonance carbon spectrum of the indole alkaloid compound.
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the indole alkaloid compound.
Fig. 3 is the main HMBC-related for the indole alkaloids.
Detailed Description
The present invention will be further illustrated by the following examples, but is not limited to the examples. Experimental methods, in which specific conditions are not specified in examples, are generally available commercially according to conventional conditions as well as those described in handbooks, or according to general-purpose equipment, materials, reagents, etc. used under conditions suggested by manufacturers, unless otherwise specified. The starting materials required in the examples below are all commercially available.
Indole alkaloid compound C 15 H 15 NO 4 The preparation method comprises the steps of extract extraction, silica gel column chromatography and high-pressure liquid chromatography separation, and specifically comprises the following steps:
A. extracting extract: ultrasonic extracting tobacco leaf sample with 90-99% alcohol, adding the extractive solution into mixed solution of ethyl acetate and 3% tartaric acid, standing for layering, and Na 2 CO 3 The pH of the aqueous layer was adjusted to 9.0, extracted again with ethyl acetate, filtered and concentrated under reduced pressure to give an extract.
B. Silica gel column chromatography: loading the extract into a column by using 200-300 mesh silica gel dry method, and performing silica gel column chromatography; gradient elution is carried out on chloroform-methanol solutions with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 respectively, parts with the same polarity are combined, and eluent of each part is collected and concentrated; wherein the mass ratio of the silica gel to the extract is 2-5. Eluting the concentrated part of chloroform-methanol 9:1 eluent by using chloroform-acetone solutions with volume ratios of 9:1, 8:2, 7:3, 6:4 and 5:5 respectively, collecting the 8:2 part of the eluent, concentrating, and separating and purifying by high performance liquid chromatography.
C. The high performance liquid chromatography separation and purification method adopts a Zorbax PrepHT GF chromatographic column with the flow rate of 21.2mm multiplied by 250mm and 5 mu m, the flow rate of 12mL/min, the mobile phase of 55% methanol aqueous solution, the detection wavelength of an ultraviolet detector of 302nm, 200 mu L of sample injection each time, and collection of chromatographic peaks with the length of 31.6min, and the crude product of the compound can be obtained after accumulation for a plurality of times and evaporation.
D. And C, after the separation and purification by a high performance liquid chromatography, dissolving the crude product of the compound by using pure methanol again, and separating by using a gel column chromatography by using the pure methanol as a mobile phase to obtain the indole alkaloid compound by further separation and purification.
The raw materials used in the invention are not affected by fields, planting conditions and the like, and the invention is further described by new indole alkaloids which are derived from tobacco gene editing in Yunnan and are separated and identified in tobacco:
example 1
The tobacco used is BBL gene knockoutLines with significantly reduced nicotine content. The tobacco sample is cultivated in a pot in a water culture room, and the planting place is Kunming. Sampling 2.5kg sample, extracting with 95% ethanol under ultrasound for 3 times each for 30min, mixing the extractive solutions, adding into mixed solution of ethyl acetate and 3% tartaric acid (ethyl acetate: tartaric acid=97:3, mass ratio), standing for layering, and adding Na 2 CO 3 The pH value of the aqueous layer is adjusted to 9.0, and the aqueous layer is extracted again by ethyl acetate, filtered and concentrated into extract under reduced pressure to obtain 160g of extract. Subjecting the extract to silica gel column chromatography with 200 meshes of silica gel of 1.0kg, performing gradient elution with chloroform-methanol of volume ratio of 9:1, 8:2, 7:3, 6:4 and 5:5, performing TLC monitoring, combining the same parts to obtain 5 parts, concentrating the chloroform-methanol elution part of volume ratio of 9:1, eluting with chloroform-acetone solution of volume ratio of 9:1, 8:2, 7:3, 6:4 and 5:5 respectively, collecting eluent obtained when the chloroform-acetone solution of volume ratio of 8:2 is used for eluting, preparing high performance liquid chromatography with An Jielun, preparing a column with 55% methanol as a mobile phase, preparing the column with Zorbax PrepHT GF column (21.2x250mm, 5 μm) as a stationary phase, performing detection wavelength of an ultraviolet detector of 302nm, performing sample injection 200 μl each time, collecting chromatographic peaks of 31.6min, and performing accumulation for multiple times to obtain a crude product; dissolving the crude product with pure methanol again, and separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain the new compound pure product.
Example 2
The tobacco sample used was a strain with BBL gene knockout and significantly reduced nicotine content. Tobacco samples were grown in a greenhouse with a Kunming planting site. Sampling 2.2kg, extracting with 95% ethanol under ultrasound for 3 times, each for 30min, mixing the extractive solutions, adding into mixed solution of ethyl acetate and 3% tartaric acid (ethyl acetate: tartaric acid=97:3, mass ratio), standing for layering, and adding Na 2 CO 3 The pH value of the aqueous layer is adjusted to 9.0, and the aqueous layer is extracted again by ethyl acetate, filtered and concentrated into extract under reduced pressure to obtain 160g of extract. 88.6g of extract is obtained by extracting the extract with EtOAc, the extract is subjected to silica gel column chromatography by using a 200 mesh silica gel column of 1.0kg,performing gradient elution with chloroform-methanol with volume ratio of 9:1, 8:2, 7:3, 6:4 and 5:5, performing TLC monitoring, combining the same parts to obtain 5 parts, concentrating chloroform-methanol elution part with volume ratio of 9:1, eluting with chloroform-acetone solution with volume ratio of 9:1, 8:2, 7:3, 6:4 and 5:5 respectively, collecting eluate obtained when eluting with chloroform-acetone solution with volume ratio of 8:2, performing high performance liquid chromatography separation with An Jielun, preparing column with 55% methanol as mobile phase and Zorbax PrepHT GF column (21.2X250 mm,5 μm) as stationary phase, measuring wavelength with ultraviolet detector of 302nm, sampling 200 μl each time, collecting chromatographic peak with volume ratio of 31.6min, and evaporating to obtain crude compound; dissolving the crude product with pure methanol again, and separating with Sephadex LH-20 gel column chromatography with pure methanol as mobile phase to obtain the new compound pure product.
Example 3
The compound prepared in example 1 was taken as orange gum.
The measuring method comprises the following steps: nuclear magnetic resonance, in combination with other spectroscopic techniques, is used to identify structures.
The ultraviolet-visible absorption spectrum shows that the compound has maximum absorption at 265 nm and 302nm, which proves that the compound has an aromatic ring structure;
infrared spectra (potassium bromide tablets) showed carbonyl groups (1686 and 1655 cm) in the compound -1 ) Aromatic ring (1618, 1564, 1468 cm) -1 ) A characteristic functional group; high resolution mass spectrometry (HRESIMS) gave excimer ion peak 296.0894[ M+Na ]] + The molecular formula of the compound can be determined to be C 15 H 15 NO 4 The unsaturation was 9. Bonding of 1 H and 13 c and HSQC NMR data showed that the compound included a 1,2,3,4, 5-pentasubstituted benzene ring (C-4-C-7, C-3a, C-7a and H-7), an N-2-oxopropyl group (C-4 '-C-6', H) 2 -4' and H 3 -6'), one-COCH 2 CH 2 Structural fragment (C-1 '-C-3', H) 2 -2' and H 2 -3'), a carbonyl group (C-1), an N-methylene group (C-3 and H) 2 -3) and a methoxy group (delta) C 56.2q；δ H 3.80s)。
The unsaturation 7 of the benzene ring and three carbonyl groups is removed, and there should also be two rings in the compound in order to satisfy the 9 unsaturations of the compound. The combined literature reports: it is presumed that the benzene ring, carbonyl group (C-1), methylene group (C-3) and nitrogen atom should form an isoindole ring, and this can be presumed by H 2 The HMBC correlation of-3 with C-1/C-4/C-3a/C-7a, H-7 with C-1, C-3a, C-7a was confirmed. In addition-COCH 2 CH 2 The structural fragment should also be linked to the benzene ring to form a five-membered ring, which can be deduced also from H 2 -2' and C-1'/C-3', H 2 The HMBC correlation of-3 ' and C-1'/C-2' was confirmed.
After the parent backbone of the compound is determined, the remaining substituent positions can be determined by further analysis of its HMBC correlation. -CH 2 CH 2 The CO-structural fragment is connected with C-4 and C-5 positions of benzene ring to form five-membered cyclic ketone, and C-1 'is connected with C-5, C-3' is connected with C-4, and H can be used for preparing the five-membered cyclic ketone 2 -2' and C-4/C-5,H 2 The HMBC correlation of 3' and C-4/C-5/C-3a was confirmed. Methoxy substituted at C-6 as a traversable methoxy hydrogen (. Delta.) H 3.80 s) and HMBC related validation of C-6. In addition, the N-2-oxopropyl group and the nitrogen atom may be linked by H-4' and C-1\C-3, as well as H 2 The HMBC correlation of-3 and C-4' is confirmed. The structure of the compound is confirmed, and the compound is identified as tobacco indole-C, and the English name is: nicindole-C.
Example 4
The compound prepared in example 2 was taken as 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 indole alkaloid compound, tobacco indole-C.
Example 5
Taking any indole alkaloid compound prepared in the examples 1-2 to perform an activity test for resisting tobacco mosaic virus, wherein the test conditions are as follows:
the tobacco mosaic virus resistance activity of the compound is measured by adopting a half leaf method when the mass concentration of the medicament is 20 mu M. Selecting leaves (leaf shape is normal, disease and insect free) suitable for testing on 5-6 tobacco plants, uniformly scattering fine silicon carbide on the leaves, and preparing with a writing brushTobacco mosaic virus source (3.0X10) -3 ) Uniformly smearing on the leaves scattered with silicon carbide, immediately placing the leaves in a culture dish containing liquid medicine for treatment for 20min after the poison receiving of all the selected leaves is finished, taking out, sprinkling water drops on the leaves and the liquid, recovering and discharging two half leaves, covering glass in enamel covered with toilet paper for moisture preservation, controlling the temperature (23+/-2 ℃), placing the enamel in a greenhouse for natural light irradiation, and obtaining the cumic spots after 2-3 days, wherein each treatment is provided with the other half leaves as a contrast, and 1 group of treatments of commodity Ningnanmycin as a contrast, and calculating the relative inhibition rate according to a formula.
XI％＝(CK-T)/CK×100％
X: relative inhibition (%), CK: the number of the dead spots of the half-leaf virus-collecting leaves soaked in clear water is T, and the number of the dead spots of the half-leaf virus-collecting leaves soaked in the liquid medicine is T.
The result shows that the relative inhibition rate of the compound is 42.8 percent, which is higher than that of the control Ningnan mycin by 33.2 percent, thus indicating that the compound has good tobacco mosaic virus resistance.
Claims (6)
1. An indole alkaloid compound, characterized in that the compound has the formula: c (C) 15 H 15 NO 4 It has the following structure:
2. a process for the preparation of an indole alkaloid compound of claim 1 comprising the steps of:
A. extracting extract: ultrasonic extracting tobacco leaf sample with 90-99 wt% alcohol, adding the extracting solution into mixed solution of ethyl acetate and 3wt% tartaric acid, standing the mixed solution for layering, and using Na 2 CO 3 The pH value of the water layer is adjusted to 9.0, and the water layer is extracted again by ethyl acetate, filtered and concentrated into extractum under reduced pressure;
B. silica gel column chromatography: loading the extract obtained in the step A into a column by using 200-300 mesh silica gel dry method, and performing silica gel column chromatography; gradient elution is carried out on chloroform-methanol solutions with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 respectively, parts with the same polarity are combined, and eluent of each part is collected and concentrated; wherein the mass ratio of the silica gel to the extract is 2-5; collecting an eluent obtained when the chloroform-methanol solution with the volume ratio of 9:1 is used for eluting, wherein the eluent is called a first eluent; continuing to separate the eluent by using a silica gel chromatographic column, performing gradient elution by using a series of chloroform-acetone solutions with volume ratios of 9:1, 8:2, 7:3, 6:4 and 5:5 in sequence, and collecting eluent obtained when the eluent is eluted by using the chloroform-acetone solution with the volume ratio of 8:2, wherein the eluent is called second eluent;
C. high performance liquid chromatography separation: and B, introducing the second eluent obtained in the step B into a high performance liquid chromatography for separation and purification, wherein the high performance liquid chromatography separation and purification is carried out by adopting a Zorbax PrepHT GF chromatographic column with the flow rate of 21.2mm multiplied by 250mm and 5 mu m, the flow rate is 12mL/min, the mobile phase is 55wt% of methanol aqueous solution, the detection wavelength of an ultraviolet detector is 302nm, 200 mu L of the second eluent is injected each time, and the eluent corresponding to the chromatographic peak retention time of 31.6min after each injection is collected and called a third eluent, and the third eluent is subjected to solvent removal to obtain the indole alkaloid compound.
3. The method for preparing indole alkaloid compound of claim 2 wherein in step a, the concentration of ethanol is preferably 95wt%.
4. The method for preparing indole alkaloid compound according to claim 2, wherein in step B, the extract is mixed with 80-120 mesh silica gel in an amount of 1.5-2.5 times by weight after being dissolved in methanol before being roughly separated by silica gel column chromatography.
5. The method for preparing indole alkaloid compound according to claim 2, wherein in step C, after separation and purification by high performance liquid chromatography, the obtained compound is dissolved again with pure methanol, and then separated by gel column chromatography with pure methanol as mobile phase, so as to further separate and purify.
6. Use of an indole alkaloid compound of claim 1 in the preparation of a medicament against tobacco mosaic virus.
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| US5594009A (en) * | 1994-10-14 | 1997-01-14 | Cephalon, Inc. | Fused pyrrolocarbazoles |
| CN105399656A (en) * | 2015-12-21 | 2016-03-16 | 云南民族大学 | Isobenzazole alkaloid compound, and preparation method and applications thereof |
| CN114409661A (en) * | 2022-01-27 | 2022-04-29 | 云南中烟工业有限责任公司 | Indole alkaloid compound and preparation method and application thereof |
| CN114456102A (en) * | 2022-01-27 | 2022-05-10 | 云南中烟工业有限责任公司 | Indole alkaloid compound and preparation method and application thereof |
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| US5594009A (en) * | 1994-10-14 | 1997-01-14 | Cephalon, Inc. | Fused pyrrolocarbazoles |
| CN105399656A (en) * | 2015-12-21 | 2016-03-16 | 云南民族大学 | Isobenzazole alkaloid compound, and preparation method and applications thereof |
| CN114409661A (en) * | 2022-01-27 | 2022-04-29 | 云南中烟工业有限责任公司 | Indole alkaloid compound and preparation method and application thereof |
| CN114456102A (en) * | 2022-01-27 | 2022-05-10 | 云南中烟工业有限责任公司 | Indole alkaloid compound and preparation method and application thereof |
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