CN114105964B - Limonin compound, preparation method and application thereof as tobacco mosaic virus resistant drug - Google Patents
Limonin compound, preparation method and application thereof as tobacco mosaic virus resistant drug Download PDFInfo
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Abstract
The invention provides a limonin compound, a preparation method and application thereof as a tobacco mosaic virus resistant medicament, and belongs to the technical field of pesticides. The invention provides limonin compounds shown in a structure of formula I; the limonin compound has good preventive effect, and the anti-TMV activity of the limonin compound is related to reducing the expression of tobacco mosaic virus coat protein (TMV-CP) and the replication of TMV-CP genes and increasing the activity of relevant defenses (phenylalanine ammonia lyase, peroxidase, superoxide dismutase and natural polyphenol oxidase). Simultaneous down-regulation of the expression of NtHsp70-1 and NtHsp70-261 further suggests that limonoids inhibit TMV infection and are also associated with reduced levels of NtHsp70-1 and NtHsp 70-261.
Description
Technical Field
The invention relates to the technical field of pesticides, in particular to limonin compounds, a preparation method and application of limonin compounds as tobacco mosaic virus resistant drugs.
Background
Rhizoma Coptidis Munronia henryi Harms, also known as rhizoma anemones Altaicae, is a plant of the genus Meliaceae (Meliaceae) genus of the genus Annula (Munronia), about 6 species, southwest, and Hainan island. The plant is used in the wet places under the forest with the altitude of 1000-1400 meters, and is distributed in Guizhou, guangxi, yunnan and other places. The whole plant of Coptis Fugu Linn can be used as medicine for treating traumatic injury, rheumarthritis, stomach ache, flatulence and abdominal pain, common cold and fever, malaria. However, no report on the extraction of limonin compounds from coptis chinensis has been found in the prior art.
Disclosure of Invention
Accordingly, the invention aims to provide limonin compounds, a preparation method and application thereof as tobacco mosaic virus resistant medicines. The limonin compound provided by the invention has good activity of resisting tobacco mosaic virus and good prevention effect on the tobacco mosaic virus.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a limonin compound, which has a structure shown in a formula I:
the R is 1 And R is 2 independently-OH or-OAc.
Preferably, said R 1 is-OH, R 2 is-OAc.
Preferably, said R 1 is-OAc, R 2 is-OH.
The invention also provides a preparation method of the limonin compound, which comprises the following steps:
reflux-extracting ethanol water solution and the whole coptis root plant of the double-leaf rehmannia, and collecting an extracting solution;
concentrating the extracting solution to obtain an extract;
re-suspending the extract in water to obtain a crude extract;
extracting the crude extract by adopting ethyl acetate to obtain an ethyl acetate extract;
subjecting the ethyl acetate extract to normal phase silica gel column chromatography by a first gradient elution procedure of: eluting with 90vol% petroleum ether-10 vol% ethyl acetate, 70vol% petroleum ether-30 vol% ethyl acetate, 50vol% petroleum ether-50 vol% ethyl acetate, 30vol% petroleum ether-70 vol% ethyl acetate, 70vol% dichloromethane-30 vol% methanol, 50vol% dichloromethane-50 vol% methanol, 30vol% dichloromethane-70 vol% methanol in sequence to obtain 7 fractions of eluents named Fr1, fr2, fr3, fr4, fr5, fr6 and Fr7 in sequence;
performing small pore resin chromatographic separation on the Fr6 through a second gradient elution, wherein the procedure of the second gradient elution is as follows: sequentially eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, collecting 6 parts of eluates, and sequentially naming Fr6A, fr6B, fr6C, fr6D, fr E and Fr6F;
subjecting the Fr6C to C18 reverse phase silica gel column chromatography by a third gradient elution procedure of: sequentially eluting with 50% methanol aqueous solution, 60% methanol aqueous solution, 70% methanol aqueous solution, 80% methanol aqueous solution and anhydrous methanol, and collecting 5 eluate (Fr 6C-1, fr6C-2, fr6C-3, fr6C-4 and Fr 6C-5);
separating and concentrating the Fr6C-3 by Sephadex LH-20 methanol gel column chromatography; collecting one chromatographic liquid every 5mL, and combining 30-35 parts of chromatographic liquids to obtain a first combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
separating the first combined chromatographic solution by first half preparative high performance liquid chromatography, eluting with 60% methanol water solution, and collecting eluent containing R in the range of 25.1-26.1 min 1 is-OAc, R 2 Limonin compounds which are-OH;
separating and concentrating the Fr6C-2 by Sephadex LH-20 methanol gel column chromatography; collecting one chromatographic liquid every 5mL, combining 28-32 parts of chromatographic liquids to obtain a second combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
separating the second combined chromatographic liquid by second semi-preparative high performance liquid chromatography, eluting with 62% methanol water solution, and collecting eluent containing R in the range of 23.0-24.3 min 1 is-OH, R 2 Limonoids which are-OAc;
the conditions for the first semi-preparative high performance liquid chromatography separation and the second semi-preparative high performance liquid chromatography separation include:
chromatographic column: waters XSelect CSH C-18column;5 μm, 10X 250mm;
the flow rate of the mobile phase is 2.5 mL-min -1 ；
The column temperature is 25 ℃;
the sample injection amount was 50. Mu.L each time.
Preferably, the volume fraction of the ethanol aqueous solution is 90% -95%.
Preferably, the dosage ratio of the ethanol aqueous solution to the whole golden thread of the golden thread is 5L to 3kg.
Preferably, the temperature of the reflux extraction is 50-70 ℃.
The invention also provides application of the limonin compound in preparation of tobacco mosaic virus resistant medicines.
The invention also provides a medicine for resisting tobacco mosaic virus, which comprises an effective active component and auxiliary materials, wherein the effective active component comprises the limonin compound, the compound with the structure shown in the formula II or pharmaceutically acceptable salt, ester or stereoisomer thereof,
preferably, the mass fraction of the effective active component in the medicament is 0.1% -99%.
The invention provides a limonin compound, which has a structure shown in a formula I:
the R is 1 And R is 2 independently-OH or-OAc.
The invention provides limonin compounds shown in a structure of formula I; the limonin compound has good preventive effect, and the anti-TMV activity of the limonin compound is related to reducing the expression of tobacco mosaic virus coat protein (TMV-CP) and the replication of TMV-CP genes and increasing the activity of relevant defenses (phenylalanine ammonia lyase, peroxidase, superoxide dismutase and natural polyphenol oxidase). Simultaneous down-regulation of the expression of NtHsp70-1 and NtHsp70-261 further suggests that limonoids inhibit TMV infection and are also associated with reduced levels of NtHsp70-1 and NtHsp 70-261.
In the examples, western-blot and RT-PCR experiments further prove that limonin A has good TMV resisting activity, and the TMV resisting activity is related to reducing the expression of tobacco mosaic virus coat protein (TMV-CP) and the replication of TMV-CP genes and increasing the activity of relevant defenses enzymes (phenylalanine ammonia lyase, peroxidase, superoxide dismutase and natural polyphenol oxidase). In confirming whether limonin a inhibits TMV infection with heat shock protein 70 (Hsp 70), the effect of limonin a treatment on Hsp70 total protein was not greatly different, but limonin A, B, a compound having the structure shown in formula II, was able to down-regulate expression of NtHsp70-1, indicating that limonin A, B, a compound having the structure shown in formula II, inhibited TMV infection with reduced content of NtHsp 70-1.
Drawings
FIG. 1 is a graph showing the effect of inoculating TMV 12h after spraying limonin A at 100 μg/mL on leaf surface, wherein A is the accumulation of TMV-CP in the inoculated leaf at 3 rd day of Western-blot detection, B is the accumulation of TMV-CP and the expression level of TMV-CP gene in the upper new leaf at 5 th day of Western-blot and RT-PCR detection, CK - As negative control, N is Ningnanmycin, and Actin and RbcS are used as internal references;
FIG. 2 shows the activity of PAL, POD, SOD and PPO after limonin A treatment, wherein A is PAL, B is POD, C is SOD and D is PPO, and samples are collected at 1D,3D,5D and 7D;
FIG. 3 shows that TMV is inoculated 12h after 100 mug/mL limonin A is sprayed on leaf surfaces, and the expression level of Hsp70 is detected on the fifth day;
FIG. 4 shows qRT-PCR detection of Hsp70-1 and Hsp70-261 gene expression levels.
Detailed Description
The invention provides a limonin compound, which has a structure shown in a formula I:
the R is 1 And R is 2 independently-OH or-OAc.
In the present invention, the R 1 preferably-OAc, R 2 preferably-OH, wherein the structure of the limonin compound is shown as a formula A:
in the present invention, the R 1 preferably-OH, R 2 preferably-OAc, said lemonThe structure of the limonin compound is shown as a formula B:
the invention also provides a preparation method of the limonin compound, which comprises the following steps:
reflux-extracting ethanol water solution and the whole coptis root plant of the double-leaf rehmannia, and collecting an extracting solution;
concentrating the extracting solution to obtain an extract;
re-suspending the extract in water to obtain a crude extract;
extracting the crude extract by adopting ethyl acetate to obtain an ethyl acetate extract;
subjecting the ethyl acetate extract to normal phase silica gel column chromatography by a first gradient elution procedure of: eluting with 90vol% petroleum ether-10 vol% ethyl acetate, 70vol% petroleum ether-30 vol% ethyl acetate, 50vol% petroleum ether-50 vol% ethyl acetate, 30vol% petroleum ether-70 vol% ethyl acetate, 70vol% dichloromethane-30 vol% methanol, 50vol% dichloromethane-50 vol% methanol, 30vol% dichloromethane-70 vol% methanol in sequence to obtain 7 fractions of eluents named Fr1, fr2, fr3, fr4, fr5, fr6 and Fr7 in sequence;
the Fr6 was subjected to a small pore resin chromatography by a second gradient elution, the procedure of which was: sequentially eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, collecting 6 parts of eluates, and sequentially naming Fr6A, fr6B, fr6C, fr6D, fr E and Fr6F;
subjecting the Fr6C to C18 reverse phase silica gel column chromatography by a third gradient elution procedure of: sequentially eluting with 50% methanol aqueous solution, 60% methanol aqueous solution, 70% methanol aqueous solution, 80% methanol aqueous solution and anhydrous methanol, and collecting 5 eluate (Fr 6C-1, fr6C-2, fr6C-3, fr6C-4 and Fr 6C-5);
separating and concentrating the Fr6C-3 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic liquid every 5mL, and combining 30-35 parts of chromatographic liquid to obtain combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
separating the combined chromatographic solution by using a first half of preparative high performance liquid chromatography, eluting with 60% methanol water solution, and collecting eluent containing R in the range of 25.1-26.1 min 1 is-OAc, R 2 Limonin compounds which are-OH;
separating and concentrating the Fr6C-2 by Sephadex LH-20 methanol gel column chromatography; collecting one part of chromatographic liquid every 5mL, and combining 28-32 parts of chromatographic liquid to obtain combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
separating the combined chromatographic liquid by second semi-preparative high performance liquid chromatography, eluting with 62% methanol water solution, collecting eluent of 23.0-24.3 min, wherein the eluent contains R 1 is-OH, R 2 Limonoids which are-OAc;
the conditions for the first semi-preparative high performance liquid chromatography separation and the second semi-preparative high performance liquid chromatography separation include:
chromatographic column: waters XSelect CSH C-18column;5 μm, 10X 250mm;
the flow rate of the mobile phase is 2.5 mL-min -1 ；
The column temperature is 25 ℃;
the sample injection amount was 50. Mu.L each time.
The invention carries out reflux extraction on ethanol aqueous solution and the whole golden thread of the compound leaf and collects extract.
In the present invention, the volume fraction of the aqueous ethanol solution is preferably 90% to 95%.
In the invention, the dosage ratio of the ethanol aqueous solution to the whole golden thread plant of the golden thread is preferably 5L to 3kg.
In the present invention, the temperature of the reflux extraction is preferably 50 to 70 ℃, and the number of times of the reflux extraction is preferably 3.
In the invention, the whole plant of the Coptis multocida is preferably obtained from Guizhou.
In the invention, the whole golden thread of the double leaf is preferably dried and crushed to obtain a crushed material, and then the crushed material is mixed with an ethanol water solution for reflux extraction.
In the present invention, the particle size of the pulverized product is preferably 80 to 100 mesh.
After the extracting solution is obtained, the extracting solution is concentrated to obtain extractum.
In the present invention, the concentration is preferably concentration under reduced pressure. In the present invention, in the process of the reduced pressure concentration, it is preferable to obtain an extract and recover a solvent; the condensation temperature of the reduced pressure concentration is preferably 4 ℃, and the pressure of the reduced pressure concentration is preferably 0.2-0.25 MPa; the mass of the extract is preferably 1-1.5 kg, more preferably 1.2kg, based on 9kg of the whole golden thread plant.
After the extract is obtained, the extract is resuspended in water to obtain crude extract.
In the invention, the mass ratio of the extract to the water is preferably 1: (6.3 to 11).
After the crude extract is obtained, the invention adopts ethyl acetate to extract the crude extract to obtain an ethyl acetate extract.
In the present invention, the number of extractions is preferably 3, and the volume ratio of ethyl acetate to the residue used for each extraction is preferably 1:1.
after ethyl acetate extract is obtained, normal phase silica gel column chromatographic separation is carried out on the ethyl acetate extract through first gradient elution, and the procedure of the first gradient elution is as follows: the elution was performed sequentially with 90vol% petroleum ether-10 vol% ethyl acetate, 70vol% petroleum ether-30 vol% ethyl acetate, 50vol% petroleum ether-50 vol% ethyl acetate, 30vol% petroleum ether-70 vol% ethyl acetate, 70vol% methylene chloride-30 vol% methanol, 50vol% methylene chloride-50 vol% methanol, 30vol% methylene chloride-70 vol% methanol, to obtain 7 fractions of eluents, once designated as Fr1, fr2, fr3, fr4, fr5, fr6 and Fr7.
In the invention, the mass ratio of the ethyl acetate extract to the silica gel is preferably 1:20.
in the present invention, the volume of the eluent used for each gradient in the second gradient elution is preferably 5L, and the flow rate of the eluent is preferably 12mL/min.
Obtaining Fr6, and carrying out small-pore resin chromatographic separation on the Fr6 through a second gradient elution, wherein the procedure of the second gradient elution is as follows: sequentially eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, collecting 6 parts of eluates, and sequentially named Fr6A, fr6B, fr6C, fr6D, fr E and Fr6F.
In the invention, the purpose of the small-pore resin chromatographic separation is to carry out preliminary refining on the Fr6 fraction rich in limonoid compounds and triterpene compounds, fr6E and Fr6F are rich in residual chlorophyll and other small polar compounds, and Fr6C is rich in limonoid compounds and other types of compounds such as lignin.
After Fr6C is obtained, the Fr6C is subjected to C18 reverse phase silica gel column chromatographic separation through third gradient elution, and the third gradient elution is carried out according to the following procedures: sequentially eluting with 50% methanol aqueous solution, 60% methanol aqueous solution, 70% methanol aqueous solution, 80% methanol aqueous solution and anhydrous methanol, and collecting 5 eluate, which are named Fr6C-1, fr6C-2, fr6C-3, fr6C-4 and Fr6C-5.
In the invention, the mass ratio of the Fr6C and C18 reverse phase silica gel is preferably 2:100.
in the present invention, the volume of the eluent used for each gradient in the third elution is preferably 7L, and the flow rate of the eluent is preferably 8mL/min.
In the invention, the purpose of separating the C18 reverse phase silica gel column chromatography is to distinguish limonin compounds and lignin compounds, and further achieve the purpose of enriching the target limonin compounds.
After Fr6C-3 is obtained, separating and concentrating the Fr6C-3 by Sephadex LH-20 methanol gel column chromatography; collecting one chromatographic liquid every 5mL, and combining 30-35 parts of chromatographic liquids to obtain a first combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L.
In the invention, sephadex LH-20 methanol gel chromatography is mainly used for separation according to the difference of molecular weight, plant secondary metabolites with the molecular weight of more than 600 flow out before 30 bottles, plant secondary metabolites with the molecular weight of less than 450 flow out after 35 bottles, and 25-32 parts of chromatographic liquid are mainly used for enriching the secondary metabolites with the molecular weight of 700-750.
After combining the chromatographic liquids, the invention preferably dissolves the first combined chromatographic liquid in chromatographic methanol solution to obtain methanol chromatographic dissolution liquid; the volume of the chromatographic methanol solution is preferably 1mL, based on the mass of the first combined chromatographic solution of 0.1 g.
After the first combined chromatographic liquid is obtained, the invention leads the firstSeparating by high performance liquid chromatography with methanol aqueous solution with volume fraction of 60%, collecting eluate containing R in the range of 25.1-26.1 min 1 is-OAc, R 2 Limonin compound as-OH, namely limonin A.
The invention preferably collects the eluent between 21.3 and 22.7min, wherein the eluent contains a compound with a structure shown in a formula II:
in the present invention, the apparatus used for the first half preparative high performance liquid chromatography separation is preferably Agilent 1260 high performance liquid chromatography (Agilent in the united states).
In the present invention, the conditions for the first half preparative high performance liquid chromatography separation include:
chromatographic column: waters XSelect CSH C-18column;5 μm, 10X 250mm;
the flow rate of the mobile phase is 2.5 mL-min -1 ；
The column temperature is 25 ℃;
the sample injection amount was 50. Mu.L each time.
The invention adopts Sephadex LH-20 methanol gel column chromatography to separate and concentrate the Fr 6C-2; collecting one chromatographic liquid every 5mL, combining 28-32 parts of chromatographic liquids to obtain a second combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L.
After combining the chromatographic liquids, the second combined chromatographic liquid is preferably dissolved in chromatographic methanol solution to obtain methanol chromatographic dissolution liquid; the volume of the chromatographic methanol solution is preferably 1mL, based on the mass of the second combined chromatographic solution of 0.1 g.
After the second combined chromatographic liquid is obtained, the second combined chromatographic liquid is subjected to second semi-preparative high performance liquid chromatography separation, and is eluted by adopting a methanol water solution with the volume fraction of 62 percent, and eluent with the volume fraction of 23.0-24.3 min is collected, wherein the eluent contains the R 1 is-OH, R 2 Limonin compound as-OAc, namely limonin B;
the conditions for the second semi-preparative high performance liquid chromatography separation include:
chromatographic column: waters XSelect CSH C-18column;5 μm, 10X 250mm;
the flow rate of the mobile phase is 2.5 mL-min -1 ；
The column temperature is 25 ℃;
the sample injection amount was 50. Mu.L each time.
In the present invention, the apparatus used for the second semi-preparative high performance liquid chromatography separation is preferably Agilent 1260 high performance liquid chromatography (Agilent in the united states).
The invention also provides application of the limonin compound in preparation of tobacco mosaic virus resistant medicines.
The invention also provides a medicine for resisting tobacco mosaic virus, which comprises an effective active component and auxiliary materials, wherein the effective active component comprises the limonin compound, the compound with the structure shown in the formula II or pharmaceutically acceptable salt, ester or stereoisomer thereof,
the source of the compound having the structure shown in formula II is not particularly limited, and may be commercially available products known to those skilled in the art or may be prepared by the method disclosed in the present invention.
In the invention, the mass fraction of the effective active component in the medicine is preferably 0.1% -99%.
For further explanation of the present invention, the limonoids, the preparation method and application thereof, and the tobacco mosaic virus resistant drugs provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method of limonin A, B and the compound with the structure shown in the formula II comprises the following steps:
1) Reflux-extracting the whole coptis chinensis with 95% ethanol water solution, and collecting the extractive solution; concentrating the extracting solution to obtain an extract; re-suspending the extract in water to obtain a crude extract; the whole plant of Coptis chinensis Franch is collected from Guizhou; drying and crushing the whole coptis root of the double leaf to obtain crushed materials, mixing the crushed materials with an ethanol water solution, and carrying out reflux extraction; the particle size of the crushed material is 80-100 meshes, and the reflux extraction temperature is 50 ℃; the number of times of reflux extraction is 3; the mass of the crushed materials is 9kg, and the volume of ethanol water solution adopted by each reflux extraction is 15L; concentrating under reduced pressure to obtain extract and recovering solvent; condensing temperature of reduced pressure concentration is 4 ℃, and pressure of reduced pressure concentration is 0.2MPa; the mass of the extract is 1.2kg calculated by the mass of the whole plant of the Coptis chinensis Roxb.f.; the mass ratio of the extract to the water is 1:6.3.
2) Extracting the crude extract by adopting ethyl acetate, and collecting an ethyl acetate part to obtain an ethyl acetate extract; the number of extractions was 3, and the volume ratio of ethyl acetate to the remainder used for each extraction was 1:1, a step of;
3) Subjecting the ethyl acetate extract to normal phase silica gel column chromatography, and performing first gradient elution, wherein the procedure of the first gradient elution is as follows: eluting with 90vol% petroleum ether-10 vol% ethyl acetate, 70vol% petroleum ether-30 vol% ethyl acetate, 50vol% petroleum ether-50 vol% ethyl acetate, 30vol% petroleum ether-70 vol% ethyl acetate, 70vol% dichloromethane-30 vol% methanol, 50vol% dichloromethane-50 vol% methanol, 30vol% dichloromethane-70 vol% methanol in sequence to obtain 7 fractions of eluates, named Fr1, fr2, fr3, fr4, fr5, fr6 and Fr7; the mass ratio of the ethyl acetate extract to the silica gel is 1:20, a step of; the volume of eluent adopted by each gradient in the second gradient elution is 5L, and the flow rate of the eluent is 12mL/min;
3) Performing small-pore resin chromatographic separation on the Fr6, and performing second gradient elution, wherein the second gradient elution comprises the following steps: sequentially eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, collecting 6 parts of eluates, and sequentially naming Fr6A, fr6B, fr6C, fr6D, fr E and Fr6F;
5) C18 reverse phase silica gel column chromatography separation is carried out on the Fr6C, and the third gradient elution is carried out according to the following procedure: sequentially eluting with 50% methanol aqueous solution, 60% methanol aqueous solution, 70% methanol aqueous solution, 80% methanol aqueous solution and anhydrous methanol, and collecting 5 eluate (Fr 6C-1, fr6C-2, fr6C-3, fr6C-4 and Fr 6C-5); the mass ratio of Fr6C to the C18 reverse phase silica gel is 2:100; the volume of eluent used for each gradient in the third elution is preferably 7L and the flow rate of eluent is preferably 8mL/min.
6) Separating and concentrating the Fr6C-3 by Sephadex LH-20 methanol gel column chromatography; collecting one chromatographic liquid every 5mL, and combining 30-35 parts of chromatographic liquids to obtain a first combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm x 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
the eluent is used in an amount of 0.5g based on the mass of Fr 6C-3: 1L;
after combining the chromatographic liquids, the invention dissolves the first combined chromatographic liquid in chromatographic methanol solution to obtain methanol chromatographic dissolution liquid; the volume of the chromatographic methanol solution is preferably 1mL, based on 0.1g of the mass of the first combined chromatographic solution;
7) Semi-preparative high performance liquid chromatography separation is carried out on the first combined chromatographic liquid, methanol aqueous solution with the volume fraction of 60% is adopted for eluting, and eluent with the volume fraction of 25.1-26.1 min is collected, wherein the eluent contains limonin A; collecting eluent with the concentration of 21.3-22.7 min, wherein the eluent contains a compound with a structure shown in a formula II; the conditions for semi-preparative high performance liquid chromatography separation include:
chromatographic column: waters XSelect CSH C-18column (5 μm, 10X 250 mm);
the flow rate of the mobile phase is 2.5 mL-min -1 ；
The column temperature is 25 ℃;
the sample injection amount is 50 mu L each time;
the equipment adopted by semi-preparative high performance liquid chromatography separation is Agilent 1260 high performance liquid chromatography (Agilent in U.S);
8) Separating and concentrating the Fr6C-2 by Sephadex LH-20 methanol gel column chromatography; collecting one chromatographic liquid every 5mL, combining 28-32 parts of chromatographic liquids to obtain a second combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
the eluent is used in an amount of 0.5g based on the mass of the Fr 6C-2: 1L;
after combining the chromatographic liquids, dissolving the second combined chromatographic liquid in chromatographic methanol solution to obtain methanol chromatographic dissolution liquid; the volume of the chromatographic methanol solution is preferably 1mL based on the mass of the second combined chromatographic solution of 0.1 g;
9) Semi-preparative high performance liquid chromatography separation is carried out on the second combined chromatographic liquid, methanol aqueous solution with the volume fraction of 62% is adopted for eluting, eluent of 23.0-24.3 min is collected, and limonin B is contained in the eluent; the conditions for semi-preparative high performance liquid chromatography separation include:
chromatographic column: waters XSelect CSH C-18column (5 μm, 10X 250 mm);
the flow rate of the mobile phase is 2.5 mL-min -1 ；
The column temperature is 25 ℃;
the sample injection amount is 50 mu L each time;
the equipment used for semi-preparative high performance liquid chromatography separation is Agilent 1260 high performance liquid chromatography (Agilent, america).
The structural data of limonin A, B obtained by semi-preparative high performance liquid chromatography separation are as follows:
limonin a:(c 0.21,MeOH)，
IR(KBr)ν max 3447,3324,2899,1704,1682,1403,1345,1262,1212,1177,1118,1005,852cm –1 ；ESI-MS(+)m/z 769[M+Na] + ；HR-ESI-MS(+)m/z 769.3050[M+Na] + (calcd for C 38 H 50 O 15 Na，769.3042)； 1 H NMR(CDCl 3 ,600MHz)δ：5.58(1H,dd,J＝10.8,3.6Hz,H-1),2.76(1H,d,J＝14.2Hz,H-2a),2.55(1H,dd,J＝14.2,11.3Hz,H-2b),2.79(1H,dd,J＝12.0,2.4Hz,H-5),2.80(1H,m,H-6a),2.62(1H,m,H-6b),3.38(1H,d,J＝9.0,H-9),5.45(1H,m,H-11),5.87(1H,d,J＝9.0Hz,H-12),3.89(1H,s,H-15),2.29(1H,dd,J＝16.8,9.0Hz,H-16a),1.85(1H,dd,J＝16.8,7.8Hz,H-16b),3.02(1H,dd,J＝10.8,7.2Hz,H-17),0.94(3H,s,H-18),1.51(3H,s,H-19),7.11(1H,s,H-21),6.15(1H,d,J＝1.0Hz,H-22),7.34(1H,t,J＝1.2Hz,H-23),1.65(3H,s,H-28),4.27(1H,d,J＝12.0Hz,H-29a),4.13(1H,d,J＝12.0Hz,H-29b),5.48(1H,s,H-30a),5.40(1H,s,H-30b),3.43(1H,d,J＝3.6Hz,H-2'),1.56(1H,m,H-3'),1.18(1H,m,H-4a'),1.06(1H,m,H-4b'),0.82(1H,t,J＝7.2Hz,H-5'),0.89(1H,d,J＝6.6Hz,H-6'),3.63(1H,s,OMe-3),2.05(1H,s,OAc-1),2.09(1H,s,OAc-2'),8.03(1H,s,HCOO-11)； 13 C NMR(CDCl 3 ,150MHz)δ：74.1(C-1),36.5(C-2),170.7(C-3),87.7(C-4),45.7(C-5),34.5(C-6),174.7(C-7),137.2(C-8),53.9(C-9),48.4(C-10),70.5(C-11),75.6(C-12),45.7(C-13),70.5(C-14),59(C-15),33.6(C-16),37.9(C-17),13.7(C-18),17.9(C-19),121.8(C-20),140.4(C-21),111.1(C-22),143.0(C-23),20.0(C-28),68.5(C-29),122.9(C-30),51.7(OCH 3 -3),170.2(OAc-1),169.8(OAc-2'),174.8(C-1'),74.9(C-2'),38.0(C-3'),22.8(C-4'),11.5(C-5'),15.2(C-6'),161.0(HCOO-11)。
limonin B: [ alpha ]21D+289.1 (c 0.3, meOH):
IR(KBr)ν max 3455,3323,2896,1705,1466,1404,1345,1206,1180,1119,1005,970,854cm –1 ；ESI-MS(+)m/z 769[M+Na] + ；HR-ESI-MS(+)m/z 769.3050[M+Na] + (calcd for769.3042)； 1 H NMR(CDCl 3 ,600MHz)δ：5.56(1H,br d,J＝11.0Hz,H-1),2.94(1H,d,J＝14.5Hz,H-2a),2.60(1H,dd,J＝14.5,11.2Hz,H-2b),2.81(1H,m,H-5),2.76(1H,dd,J＝18.0,8.0Hz,H-6a),2.52(1H,dd,J＝18.0,12.3Hz,,H-6b),3.68(1H,d,J＝9.0Hz,H-9),5.36(1H,m,H-11),5.77(1H,d,J＝9.0Hz,H-12),3.90(1H,s,H-15),2.30(1H,dd,J＝16.8,8.2Hz,H-16a),1.91(1H,dd,J＝16.8,9.0Hz,H-16b),3.03(1H,dd,J＝11.0,7.5Hz,H-17),0.96(3H,s,H-18),1.49(3H,s,H-19),7.27(1H,s,H-21),6.25(1H,s,H-22),7.35(1H,s,H-23),1.61(3H,s,H-28),3.80(1H,m,H-29a),3.65(1H,m,H-29b),5.48(1H,s,H-30a),5.32(1H,s,H-30b),4.78(1H,d,J＝4.0Hz,H-2'),1.67(1H,m,H-3'),1.21(1H,m,H-4a'),1.11(1H,m,H-4b'),0.81(1H,t,J＝7.0Hz,H-5'),0.87(1H,d,J＝6.5Hz,H-6'),3.68(1H,s,OMe-3),2.06(1H,s,OAc-1),2.12(1H,s,OAc-29),8.04(1H,s,HCOO-11)； 13 C NMR(CDCl 3 ,150MHz)δ：73.2(C-1),35.1(C-2),171.2(C-3),91.4(C-4),42.8(C-5),34.3(C-6),175.5(C-7),137.3(C-8),52.2(C-9),47.9(C-10),70.5(C-11),75.6(C-12),45.5(C-13),70.5(C-14),59.1(C-15),33.7(C-16),38.2(C-17),13.5(C-18),15.9(C-19),121.8(C-20),140.9(C-21),111.2(C-22),142.8(C-23),19.0(C-28),66.1(C-29),122.5(C-30),168.7(C-1'),75.9(C-2'),36.1(C-3'),23.9(C-4'),11.5(C-5'),15.4(C-6'),52.2(OCH 3 -3),170.3(OAc-1),170.3(OAc-29),161.8(HCOO-11)。
the structural characterization of the compound with the structure shown in the formula II proves that the compound with the structure shown in the formula II is obtained.
Test example 1
Results of the test for the action of limonin A, B, a compound having a structure shown in formula II, prepared in example 1 against tobacco mosaic virus:
experimental method
Passivation experiments of compounds on TMV
And selecting the heart leaf cigarettes with 5-6 leaf periods and consistent healthy growth vigor, and placing the heart leaf cigarettes in a darkroom for one night. The test compound was mixed with 32. Mu.g/mL TMV1:1 (v/v) to the desired concentration and left to stand for 0.5h. Selecting 4-6 leaves with similar sizes from each cigarette, and performing treatment by using 100 mu L of mixed solution of a friction inoculation compound and TMV on half of each leaf; the other half was rubbed with 32. Mu.g/mL TMV 100. Mu.L as positive control; leaves inoculated with DMSO solutions of the corresponding concentrations were rubbed as blanks. After 2 hours, the leaf surface was washed with corundum using sterile water. Placing the plant material into an insect-free greenhouse, and calculating the TMV inhibition rate according to a formula after 3-4 days: [1- (average number of spots treated/average number of spots of positive control) ]. Times.100. Each compound was repeated three times.
Experiment of the protective Effect of Compounds on TMV
And selecting the heart leaf cigarettes with 5-6 leaf periods and consistent healthy growth vigor, and placing the heart leaf cigarettes in a darkroom for one night. 4-6 leaves with similar sizes are selected for each cigarette, the tested compound is diluted to the required concentration by sterile water, 100 mu L of each leaf is uniformly applied as a treatment, 50 mu g/mL TMV 200 mu L is rubbed and inoculated on each leaf after 24 hours, half of the non-applied leaves are used as positive control, and the leaves which are not applied and rubbed and inoculated with DMSO solutions with corresponding concentrations are used as blank. After 2 hours, the leaf surface was washed with corundum using sterile water. Placing the plant material into an insect-free greenhouse, and calculating the TMV inhibition rate according to a formula after 3-4 days: 1- (average number of dead spots treated/average number of dead spots of positive control). Times.100. Each compound was repeated three times.
Therapeutic Effect of Compounds on TMV
And selecting the heart leaf cigarettes with 5-6 leaf periods and consistent healthy growth vigor, and placing the heart leaf cigarettes in a darkroom for one night. Selecting 4-6 leaves with similar sizes from each cigarette, performing friction inoculation on the leaves with 32 mug/mL TMV with 200 mug/mL TMV, and uniformly applying 100 mug of half of each leaf after 2 hours for treatment; the other half is used as positive control; leaves that were not dosed and rubbed with DMSO solutions of corresponding concentrations served as blanks. After 2 hours, the leaf surface was washed with corundum using sterile water. Placing the plant material into an insect-free greenhouse, and calculating the TMV inhibition rate according to a formula after 3-4 days: [1- (average number of spots treated/average number of spots of positive control) ]. Times.100. Each compound was repeated three times.
FIG. 1 is a graph showing the effect of inoculating TMV 12h after spraying limonin A at 100 μg/mL on leaf surface, wherein A is the accumulation of TMV-CP in the inoculated leaf at 3 rd day of Western-blot detection, B is the accumulation of TMV-CP and the expression level of TMV-CP gene in the upper new leaf at 5 th day of Western-blot and RT-PCR detection, CK - As a negative control, N is ningnanmycin, action and RbcS are used as internal references, 1 is limonin A,2 is limonin B, and 3 is a compound with a structure shown in a formula II; FIG. 2 shows the activity of PAL, POD, SOD and PPO after limonin A treatment, wherein A is PAL, B is POD, C is SOD and D is PPO, and samples are collected at 1D,3D,5D and 7D; FIG. 3 shows that after foliage spraying of limonin A at 100 μg/mL, TMV was inoculated 12h, and the expression level of Hsp70 was detected on the fifth day, and Mock was a healthy plant, CK - As a negative control, actin was used as an internal reference, 1 was limonin a,2 was limonin B, and 3 was a compound having a structure shown in formula II. As shown in figures 1-3, limonin A, B and the compound with the structure shown in the formula II have good preventive effect, and the anti-TMV activity of the limonin A, B is related to reducing the expression of tobacco mosaic virus coat protein (TMV-CP) and the replication of TMV-CP genes and increasing the activity of relevant defenses (phenylalanine ammonia lyase, peroxidase, superoxide dismutase and natural polyphenol oxidase).
Western blot analysis
The method comprises the following specific steps:
1) Extracting protein: protein extraction was performed on different treatments of common smoke K326 using a total plant protein extraction kit.
2) Protein concentration was determined: the protein extract is measured by using a BCA protein quantitative kit, and the extracted protein is used for subsequent detection.
3) Sample adding pretreatment: each sample was loaded at 20 μg depending on protein concentration. Firstly, adding anhydrous mercaptoethanol serving as a protein denaturant, denaturing for 6min at 95 ℃, and standing for 5min on ice after the denaturation is finished.
4) Loading and carrying out electrophoresis: denatured protein samples were added to the corresponding wells and electrophoresis was started.
5) Film transfer treatment: the amount of the transfer solution was prepared in a ratio of the reagents required for the transfer solution of 1L (10X transfer solution: absolute methanol: water=1:2:7), and the sponge and filter paper were immersed in the transfer solution, and the PVDF membrane was immersed in absolute methanol for 1min in advance to activate it. And (3) manufacturing a membrane transfer plate (the protein is negatively charged, and is transferred to the positive electrode to be transferred to the PVDF membrane) according to the sequence of the sponge, the filter paper, the glue, the membrane, the filter paper and the sponge, putting the membrane transfer plate into a membrane transfer device, and adding a membrane transfer liquid to start membrane transfer. The transfer process needs to be performed under ice bath conditions.
6) And (3) sealing: the protein-transferred PVDF membrane was placed in TBST containing 5wt% skim milk and placed on a shaking table for slow shaking for 2 hours.
7) Incubation resistance: after the end of the blocking, the PVDF membrane was gently rinsed with TBST in order to wash off the residual skim milk on the PVDF membrane. PVDF was cut according to the target protein molecular weight and the corresponding antibody was added and incubated overnight at 4℃on a shaker.
8) Secondary antibody incubation: the next day, the primary antibody was recovered, and the PVDF membrane was rinsed with TBST for 5 min/time and 3 times, ensuring that the residual primary antibody on the PVDF membrane was washed away. The corresponding secondary antibodies (formulated with 5wt% skim milk) were then labeled according to the species source of the primary antibody. Incubate on a shaker at room temperature for 1h.
9) And (3) cleaning a secondary antibody: discarding the secondary antibody, rinsing the PVDF membrane with TBST for 5 min/time and 3 times to ensure that the residual secondary antibody on the PVDF membrane is completely washed away.
10 Developing: the target bands of the desired protein were obtained by development using a Bio-Rad fluorescence image analyzer.
RNA extraction
Total RNA was extracted using RNAeasy kit. The A260/A280 ratio of total RNA was measured on a NanoDrop biophotometer, and subsequent experiments used only samples with ratios between 1.8 and 2.0. RNA samples were also assessed for quality and integrity based on the 28S and 18S rRNA bands on 1wt% agarose gels. About 1.5. Mu.g total RNA per sample was used to prepare a library of RNA sequences.
Total RNA extraction, reverse transcription and Real-time fluorescence quantitative PCR (Real-time quantitative PCR, RT-qPCR) to detect the expression of the corresponding genes.
Extracting RNA according to a total RNA extraction kit and an experimental method. The concentration of the total RNA extracted was measured using a Nanodrop 2000 ultra-micro spectrophotometer, and then about 1.5. Mu.g of total RNA was taken and cDNA was synthesized using M-MLV reverse transcriptase, with the specific system shown in tables 1-2:
table 1A solution
Table 2B solution
The prepared solution A was placed on a PCR apparatus at 70℃for 5min, and then rapidly transferred to ice for 5min. Adding the solution B into the solution A, and placing the solution A on a PCR instrument at 37 ℃ for 1h to obtain cDNA.
3) The expression of the corresponding genes (including NtHsp70-1, ntHsp70-061 and NtHsp70-261) was detected by using a SYBR Green real-time fluorescence quantitative PCR method, as shown in FIG. 4, FIG. 4 shows that qRT-PCR detection of the expression levels of Hsp70-1 and Hsp70-261 shows that the down regulation of the expression of NtHsp70-1 and NtHsp70-261 further shows that limonin A, B and the inhibition of TMV infection by the compound with the structure shown in formula II is also related to the reduction of the contents of NtHsp70-1 and NtHsp 70-261.
4) Specific primer information is shown in Table 3.
TABLE 3 primer information
The cDNA prepared according to 2) is used as a template, and is combined with corresponding gene specific quantitative primers (table below) to carry out quantitative PCR reaction, and the specific reaction system is shown in table 4:
TABLE 4 reaction system
The reaction procedure is: 95 ℃,5min, 95 ℃,10 s,58 ℃, 30s,40 cycles. After the reaction is completed, the PCR product is drawn into a standard curve, and the expression quantity of the related genes is further calculated through the standard curve.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be comprehended within the scope of the present invention.
Sequence listing
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<120> limonin compound, preparation method and application thereof as tobacco mosaic virus resistant drug
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Claims (8)
1. A limonin compound having a structure represented by formula a or formula B:
2. the method for preparing limonin compounds as claimed in claim 1, comprising the steps of:
reflux-extracting ethanol water solution and the whole coptis root plant of the double-leaf rehmannia, and collecting an extracting solution;
concentrating the extracting solution to obtain an extract;
re-suspending the extract in water to obtain a crude extract;
extracting the crude extract by adopting ethyl acetate to obtain an ethyl acetate extract;
subjecting the ethyl acetate extract to normal phase silica gel column chromatography by a first gradient elution procedure of: eluting with 90vol% petroleum ether-10 vol% ethyl acetate, 70vol% petroleum ether-30 vol% ethyl acetate, 50vol% petroleum ether-50 vol% ethyl acetate, 30vol% petroleum ether-70 vol% ethyl acetate, 70vol% dichloromethane-30 vol% methanol, 50vol% dichloromethane-50 vol% methanol, 30vol% dichloromethane-70 vol% methanol in sequence to obtain 7 fractions of eluents named Fr1, fr2, fr3, fr4, fr5, fr6 and Fr7 in sequence;
performing small pore resin chromatographic separation on the Fr6 through a second gradient elution, wherein the procedure of the second gradient elution is as follows: sequentially eluting with 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, 90% methanol aqueous solution and anhydrous methanol, collecting 6 parts of eluates, and sequentially naming Fr6A, fr6B, fr6C, fr6D, fr E and Fr6F;
subjecting the Fr6C to C18 reverse phase silica gel column chromatography by a third gradient elution procedure of: sequentially eluting with 50% methanol aqueous solution, 60% methanol aqueous solution, 70% methanol aqueous solution, 80% methanol aqueous solution and anhydrous methanol, and collecting 5 eluate (Fr 6C-1, fr6C-2, fr6C-3, fr6C-4 and Fr 6C-5);
separating and concentrating the Fr6C-3 by Sephadex LH-20 methanol gel column chromatography; collecting one chromatographic liquid every 5mL, and combining 30-35 parts of chromatographic liquids to obtain a first combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
performing first half preparative high performance liquid chromatography separation on the first combined chromatographic liquid, eluting with 60% methanol water solution, and collecting eluent of 25.1-26.1 min, wherein the eluent contains limonoid compound of formula A;
separating and concentrating the Fr6C-2 by Sephadex LH-20 methanol gel column chromatography; collecting one chromatographic liquid every 5mL, combining 28-32 parts of chromatographic liquids to obtain a second combined chromatographic liquid; the conditions of Sephadex LH-20 methanol gel column chromatography include:
gel column size: 18mm by 180mm;
the flow rate of the eluent is 0.5mL/min;
the eluent is anhydrous methanol;
the eluent dosage is as follows: 1L;
performing second semi-preparative high performance liquid chromatography separation on the second combined chromatographic liquid, eluting with 62% methanol water solution, and collecting eluent of 23.0-24.3 min, wherein the eluent contains limonoid compound of formula B;
the conditions for the first semi-preparative high performance liquid chromatography separation and the second semi-preparative high performance liquid chromatography separation include:
chromatographic column: waters XSelect CSH C-18column;5 μm, 10X 250mm;
the flow rate of the mobile phase is 2.5 mL-min -1 ；
The column temperature is 25 ℃;
the sample injection amount was 50. Mu.L each time.
3. The method according to claim 2, wherein the volume fraction of the aqueous ethanol solution is 90% to 95%.
4. The preparation method according to claim 2 or 3, wherein the dosage ratio of the ethanol aqueous solution to the whole plant of Coptis mulatta is 5L/3 kg.
5. The method according to claim 2, wherein the temperature of the reflux extraction is 50 to 70 ℃.
6. The use of a limonin compound according to claim 1 for the preparation of a tobacco mosaic virus resistant medicament.
7. A tobacco mosaic virus resistant medicament comprising an effective active ingredient and an adjuvant, wherein the effective active ingredient comprises the limonoid compound of claim 1 or a pharmaceutically acceptable salt thereof.
8. The tobacco mosaic virus resistant drug according to claim 7, wherein the mass fraction of the effective active component in the drug is 0.1% -99%.
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| WO2001030776A1 (en) * | 1999-10-26 | 2001-05-03 | Centre National De La Recherche Scientifique (Cnrs) | Novel limonoids for use particularly in the treatment of cancer and method for producing the same |
| JP2009179590A (en) * | 2008-01-30 | 2009-08-13 | Takasago Yakugyo Kk | New degraded limonoid compound |
| CN105111196A (en) * | 2015-09-06 | 2015-12-02 | 叶澄 | Highly-esterified limonin compound and medical application thereof |
| CN107793403A (en) * | 2016-08-28 | 2018-03-13 | 成都宝科生物科技有限公司 | A kind of noval chemical compound with epoxy construction and preparation method thereof and medical usage |
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| WO2001030776A1 (en) * | 1999-10-26 | 2001-05-03 | Centre National De La Recherche Scientifique (Cnrs) | Novel limonoids for use particularly in the treatment of cancer and method for producing the same |
| JP2009179590A (en) * | 2008-01-30 | 2009-08-13 | Takasago Yakugyo Kk | New degraded limonoid compound |
| CN105111196A (en) * | 2015-09-06 | 2015-12-02 | 叶澄 | Highly-esterified limonin compound and medical application thereof |
| CN107793403A (en) * | 2016-08-28 | 2018-03-13 | 成都宝科生物科技有限公司 | A kind of noval chemical compound with epoxy construction and preparation method thereof and medical usage |
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