CN113796380B - Application of clausena lansium alkaloid and salt thereof in preparation of products for inhibiting phytophthora and/or ring rot activity - Google Patents

Application of clausena lansium alkaloid and salt thereof in preparation of products for inhibiting phytophthora and/or ring rot activity Download PDF

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CN113796380B
CN113796380B CN202111179904.1A CN202111179904A CN113796380B CN 113796380 B CN113796380 B CN 113796380B CN 202111179904 A CN202111179904 A CN 202111179904A CN 113796380 B CN113796380 B CN 113796380B
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alkaloid
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phytophthora
methanol
clausena lansium
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CN113796380A (en
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彭文文
章宇微
傅小香
刘欣媛
黄英金
曹端韬
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Jiangxi Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/10—Aromatic or araliphatic carboxylic acids, or thio analogues thereof; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
    • A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings

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Abstract

The invention provides an application of clausena lansium alkaloid and salt thereof in preparing products for inhibiting phytophthora and/or ring rot activity, and belongs to the technical field of natural medicines. The invention provides an application of wampee alkaloid and salt thereof in preparation of products for inhibiting phytophthora and/or ring rot fungi activity, wherein the wampee alkaloid comprises at least one of dictamnine, lantitumidae B, 6-methoxy carbazolyl-3-carboxylic acid methyl ester and carbazolyl-3-carboxylic acid methyl ester. The 4 Clausena lansium alkaloids recorded in the invention have a good inhibition effect on the activity of phytophthora capsici leonian, wherein dictamnine also has a good inhibition effect on the activity of pyricularia piricola, can be used as an inhibitor for inhibiting the growth of phytophthora and/or phyidium, and can also be used for preparing agricultural bactericides.

Description

Application of clausena lansium alkaloid and salt thereof in preparation of products for inhibiting phytophthora and/or ring rot activity
Technical Field
The invention belongs to the technical field of natural medicines, and particularly relates to application of clausena lansium alkaloids and salts thereof in preparation of products for inhibiting phytophthora and/or ring rot.
Background
In the world today, environmental protection and food safety have become two major focus of social attention. However, long-term or unscientific use of chemical pesticides has led to a series of problems including resistance to pathogenic microorganisms and pests in various crops, deterioration of the ecological environment, and reduction of the level of biodiversity. Therefore, the search for a novel environment-friendly pesticide with high efficiency, low toxicity and low residue has become a main target of pesticide research and creation at present. The botanical pesticide has become one of the first choice for developing green pesticide by virtue of high selectivity, easy degradation and no pollution in natural environment. Because the pesticide active compound searched from the natural resource treasury has better structural diversity and activity specificity, the botanical pesticide naturally becomes an important way and a research hotspot for creating new pesticides.
The Clausena lansium is plant of Clausena genus of Rutaceae family. The health-care food is mainly distributed in southern provinces of China, fruits of the health-care food have the effects of promoting digestion, guiding qi downward and the like, roots, leaves and fruit stones of the health-care food have the effects of promoting qi circulation, eliminating stagnation, relieving exterior syndrome, relieving pain, reducing phlegm and the like, chemical components of the health-care food mainly comprise compounds such as coumarins, carbazole alkaloids, amides, terpenoids and the like, and the biological activity of the health-care food mainly comprises antibiosis, tumor resistance, antimalarial, liver protection, platelet aggregation resistance, blood fat reduction and the like. Since wampee has important medicinal value, a series of chemical components such as carbazole alkaloid, amide, coumarin and the like and the activities of the compounds such as anti-tumor, neuroprotection, antibiosis and the like are also reported in the prior art. The research is mainly directed to the medicinal activity of the wampee, but the research on the agricultural activity of the wampee is not common. Although some progress has been made so far in the study of the bacteriostatic activity of wampee, most have focused on the level of crude extracts, in particular on the level of compounds, and only (E) -N-2-phenylethylcinnamamide is known. The variety and the content of alkaloids in wampee plants are large, and systematic research and evaluation on agricultural bacteriostatic activity of wampee plants are necessary, so that chemical components with higher activity are excavated, and the positive effects on better development and utilization of wampee are achieved.
The alkaloids in the wampee plant mainly comprise carbazoles and amides, the two types of alkaloids are distributed in roots, stems, leaves, fruits and seeds of the wampee plant, the polarity is mostly medium and small, and the wampee alkaloids have strong ultraviolet absorption due to the existence of a large conjugated system. In addition, the carbazole alkaloid also has the obvious characteristic of thin-layer chromatography (TLC) color development, and the compound is colored into light blue spots by heating under a 5-10% sulfuric acid-absolute ethyl alcohol color developing agent. The above characteristics can be fully utilized in the separation and purification of the wampee alkaloid. To date, the methods for extracting and separating wampee alkaloids are summarized mainly in the following three types: (1) extracting by normal hexane reflux, detecting by TLC, and repeatedly performing column chromatography with silica gel to obtain wampee alkaloid, which has the disadvantages of insufficient extraction and easy leakage of trace alkaloid; (2) extracting petroleum ether and ethyl acetate respectively, extracting chloroform and ethyl acetate respectively, and performing repeated silica gel column chromatography to obtain alkaloid, wherein the alkaloid has the defect that only a single normal phase separation material is used, and a reverse phase material is not used, so that the loss of a sample is easily caused; (3) acetone or methanol is adopted for reflux extraction, and then a thin layer plate is prepared by silica gel column chromatography and TLC for separation, which has the defect that the preparation of the thin layer plate is only suitable for the separation of trace components and is not suitable for a large amount of components. The extraction and separation methods are all established on the systematic research of the novelty and diversity of the alkaloid structure in the wampee plants, the emphasis is on the structure of the wampee alkaloid, and the pertinence of the separation of the active ingredients is not strong; in addition, the separation method mainly uses silica gel materials, and rarely uses MCI, reversed phase materials and the like, so that the loss of samples and the dispersion of active ingredients are easily caused; in addition, the detection method is single, mainly depends on TLC detection, and has low sensitivity, and trace components are easy to miss detection.
Disclosure of Invention
In view of the above, the invention aims to provide an application of a clausena lansium alkaloid and a salt thereof in preparing a product for inhibiting the activity of phytophthora and/or ring rot.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an application of clausena lansiumamide and salt thereof in preparing a product for inhibiting phytophthora and/or ring rot activity, wherein the clausena lansiumamide comprises at least one of dictamnine, lansiumamide B, 6-methoxy carbazolyl-3-methyl carboxylate and carbazolyl-3-methyl carboxylate.
Preferably, the salt comprises a salt formed by reacting the wampee alkaloid with an inorganic acid, an organic acid, an alkali metal, an alkaline earth metal or a basic amino acid.
Preferably, the product comprises a pesticide, a growth inhibitor.
Preferably, the preparation method of the clausena lansium alkaloid comprises the following steps:
mixing and reflux-extracting stem and leaf of clausena lansium with methanol, and concentrating under reduced pressure to obtain methanol extract; performing acid extraction and alkali precipitation on the methanol extract to obtain total alkaloids and non-alkaloids parts of the wampee, performing silica gel column chromatography coarse separation on the total alkaloids part of the wampee to obtain secondary parts, performing phytophthora root rot inhibition activity tests on the secondary parts respectively, performing TLC detection and activity tracking separation on the secondary part with stronger activity, separating active alkaloids by combining MCI column chromatography and RP-18 column chromatography separation methods, and finally performing HPLC separation and purification to obtain the active wampee alkaloid component.
Preferably, the methanol is a methanol solution with a volume fraction of 95-99%.
Preferably, the acid is hydrochloric acid with the volume fraction of 4-6%, and the mass volume ratio of the methanol extract to the hydrochloric acid is 1:8-1: 10.
Preferably, the alkali precipitation is carried out by adjusting the pH to 9-10 with ammonia water.
The invention also provides a pesticide for inhibiting the activity of phytophthora and/or ring rot, and the pesticide comprises at least one of the wampee alkaloids and an agriculturally and pharmaceutically acceptable salt or an acceptable carrier thereof.
Preferably, the phytophthora includes phytophthora capsici, cucumber and cucurbita.
Preferably, the ring rot germs include pear and apple ring rot germs.
The invention has the beneficial effects that:
the existing method for extracting and separating the wampee alkaloids in the field focuses on exploiting diversity and novelty of alkaloid structure, and the invention focuses on the aspects of extraction, detection, enrichment and activity tracking and separation of bacteriostatic active wampee alkaloids for the first time, so as to obtain 4 kinds of wampee alkaloid compounds with strong inhibitory activity on the growth of phytophthora and ring rot germs, and belongs to the initiative of the inventor. In addition, the 4 Clausena lansium alkaloid compounds can be used as inhibitors for inhibiting the growth of Phytophthora and Phyllospora striae, and can also be used for preparing agricultural bactericides.
The invention firstly uses methanol for crude extraction, then uses an acid-extraction alkali-precipitation method for extracting and enriching total alkaloids, then uses a method of combining TCL characteristic detection, enrichment and activity screening for tracing and separating bacteriostatic active alkaloids, successfully uses modern chromatographic technology for removing pigment and other impurities in the yellow skin stem and leaf, and comprehensively uses the modern chromatographic technology for separation to obtain the active alkaloids. The extraction and separation method has the advantages of strong pertinence, good controllability and reproducibility, low cost and simple and convenient operation, and active ingredients are not easy to disperse and lose, and the method is suitable for industrial production.
Drawings
FIG. 1 is a flow chart of a preparation method of bacteriostatic alkaloid in clausena lansium stems and leaves.
Detailed Description
The invention provides an application of clausena lansiumamide and salt thereof in preparing a product for inhibiting phytophthora and/or ring rot activity, wherein the clausena lansiumamide comprises at least one of dictamnine, lansiumamide B, 6-methoxy carbazolyl-3-methyl carboxylate and carbazolyl-3-methyl carboxylate.
In the present invention, when the pathogen is phytophthora, the clausena lanuginosa alkaloids include at least one of dictamnine, lansiumamide B, methyl 6-methoxycarbazolyl-3-carboxylate, and methyl carbazolyl-3-carboxylate, and when the pathogen is verticillium, the clausena lanuginosa alkaloids include at least dictamnine.
In the invention, the structural formulas of the dictamnine, the lantumimide B, the 6-methoxy carbazolyl-3-carboxylic acid methyl ester and the carbazolyl-3-carboxylic acid methyl ester compound are respectively as follows:
Figure RE-GDA0003346408320000041
in the present invention, the salt preferably includes a salt formed by reacting the wampee alkaloid with an inorganic acid, an organic acid, an alkali metal, an alkaline earth metal or a basic amino acid, and the present invention is not particularly limited to the specific types of the inorganic acid, the organic acid, the alkali metal, the alkaline earth metal or the basic amino acid, for example, the inorganic acid includes sulfuric acid, hydrochloric acid, phosphoric acid and nitric acid, the organic acid includes acetic acid, methanesulfonic acid, citric acid, tartaric acid, lactic acid and palmitic acid, the alkali metal includes potassium and sodium, the alkaline earth metal includes calcium and magnesium, and the basic amino acid includes lysine. The present invention is not particularly limited in kind of the product, and preferably includes a pesticide and a growth inhibitor. The specific types of phytophthora and ring rot fungi are not particularly limited, the phytophthora preferably comprises phytophthora capsici, cucumber and cucurbita moschata, and the ring rot fungi preferably comprises ring rot fungi of pears and apples.
In the present invention, the preparation method of the clausena lansium alkaloid preferably comprises the following steps: mixing and reflux-extracting stem and leaf of clausena lansium with methanol, and concentrating under reduced pressure to obtain methanol extract; performing acid extraction and alkali precipitation on the methanol extract to obtain total alkaloids and non-alkaloids parts of the wampee, performing silica gel column chromatography coarse separation on the total alkaloids part of the wampee to obtain secondary parts, performing phytophthora root rot inhibition activity tests on the secondary parts respectively, performing TLC detection and activity tracking separation on the secondary part with stronger activity, separating active alkaloids by combining MCI column chromatography and RP-18 column chromatography separation methods, and finally performing HPLC separation and purification to obtain the active wampee alkaloid component.
The sources of the yellow skin stems and leaves are not particularly limited, and the yellow skin stems and leaves can be purchased from the market or can be obtained by self-collection. In the invention, the stem and leaf of the clausena lansium preferably need to be air-dried and crushed, the methanol is preferably a methanol solution with the volume fraction of 95-99%, the reflux extraction temperature is preferably 55-65 ℃, the reflux extraction frequency is preferably 2-3, and the time of each reflux extraction is preferably 2-3 h.
In the invention, when the methanol extract is subjected to acid-extraction and alkali-precipitation, the acid used is preferably hydrochloric acid, the volume fraction of the hydrochloric acid is preferably 4-6%, and the mass-volume ratio of the methanol extract to the hydrochloric acid is preferably 1:8-1: 10. And (3) fully suspending the methanol extract and hydrochloric acid, preferably adding ethyl acetate, uniformly mixing, standing and layering, wherein the adding amount of the ethyl acetate is 1/3 of the amount of the suspension. After separating the ethyl acetate layer, preferably, the remaining liquid is further extracted with ethyl acetate for 1-2 times, the ethyl acetate layers are combined and concentrated under reduced pressure to obtain non-alkaloid fraction. Subjecting the rest water phase to alkali precipitation treatment, wherein the alkali is preferably ammonia water, the pH value of the alkali precipitation is preferably 9-10, extracting with ethyl acetate for 3-5 times, combining ethyl acetate layers, and concentrating under reduced pressure to obtain total alkaloids fraction of Clausena lansium. The invention adopts acid-alkali extraction to concentrate the total alkaloids part of wampee, so that the target product is not easy to disperse and lose.
In the invention, the silica gel column chromatography coarse separation of the total alkaloids fraction of wampee is preferably performed by 200-300 meshes, and the eluent is preferably petroleum ether and acetone in a volume ratio of 10:0-0:10, so as to obtain 33 fractions. The fractions were checked by TLC using 5-8% sulfuric acid-absolute ethanol as developer, and the fractions developed into light blue spots under heating were combined into 9 secondary fractions Fr.1.1-Fr.2.9 according to their polarity. And respectively taking 9 secondary parts to prepare test liquid medicine, and further performing activity screening for inhibiting phytophthora original bacteria. And selecting Fr.2.3 and Fr.2.5 with the strongest bacteriostatic activity according to the screening result for further activity tracking and separation.
In the present invention, the procedure for obtaining dictamnine and lantumimide B is preferably performed by mixing fr.2.3 with 60 mesh polyamide, performing medium pressure MCI column chromatography, eluting with methanol to water of 20:80, 40:60, 60:40, 80:20, 90:10, 100:0 gradient elution, and combining the fractions with the same blue spot into 4 subfractions fr.2.3.1-fr.2.3.4 according to TLC detection. The fraction Fr.2.3.2 containing the main spot is subjected to repeated silica gel column chromatography, preferably with petroleum ether and acetone at a ratio of 5:1 to 3:1, followed by TLC detection, and the main spot fractions are combined to give a mixture Fr.2.3.2.3. Further separation and purification of fr.2.3.2.3 by HPLC, the mobile phase of which is preferably methanol to water 70:30-80:20, to give the compound dictamnine and lantisinamide B.
In the present invention, the step of obtaining 6-methoxy carbazolyl-3-carboxylic acid methyl ester and carbazolyl-3-carboxylic acid methyl ester is preferably performed by medium pressure RP-18 column chromatography of fr.2.5, and the RP-18 column chromatography eluent is preferably methanol and water (20: 80, 40:60, 60:40, 80:20, 100: 0), and the mixture is subjected to TLC detection to obtain 5 subfractions fr.2.5.1-fr.2.5.5. Further, the mixture containing two main spots is obtained by repeating silica gel column chromatography on Fr.2.5.3 containing the main spots, preferably with a eluent of 5:1 to 3:1 petroleum ether. The compound 6-methoxy carbazol-3-carboxylic acid methyl ester and carbazol-3-carboxylic acid methyl ester are obtained after multiple separation and purification by HPLC, and the mobile phase of the HPLC is preferably methanol, water, 60:40-70: 30.
By passing1H-NMR、13C-NMR and MS, and comparison of spectral data in the literature identifies the above compounds as: dictamnus dasycarpus (1) (see: Tangjitjaroenkun J, et al chemical Constants of Brand of Zanthoxylum limonella Alston [ J ]]Phytochemistry Letters,2012, 5(3): 443-: Jer-Huei Lin. Cinnanamide derivitives from Clausena lansium [ J]Phytochemistry,1989,28(2): 621-: Tian-Shung Wu, et al, Carbazole alkyloids from Clausena excataa and the biological activity, phytochemistry, 1996,43(1): 133-: li W S, et al, Carbazole alkaloids from Clausena lansium [ J].Phytochemistry,1991,30(1):343-346)。
The invention also provides a pesticide for inhibiting the activity of phytophthora and/or ring rot, and the pesticide comprises at least one of the wampee alkaloids and an agriculturally and pharmaceutically acceptable salt or an acceptable carrier thereof.
The invention has no special requirements on the dosage form of the pesticide, and the preparation cost can be any conventional dosage form of the pesticide in the field, such as missible oil, wettable powder, granules, aqueous emulsion, microemulsion, oil agent or sustained release agent. The type of the agriculturally and pharmaceutically acceptable carrier is not particularly limited, and any pharmaceutical carrier conventionally used in the art for preparing pesticides may be used, such as fillers (e.g., diatomaceous earth, kaolin, etc.), co-solvents (e.g., xylene, etc.), emulsifiers (e.g., calcium dodecylbenzenesulfonate, etc.), wetting agents (e.g., saponin, sodium dodecylbenzenesulfonate, etc.), dispersants (e.g., lignosulfonate, etc.), penetrants (e.g., penetrant, etc.), adhesives (e.g., starch, gelatin, etc.), stabilizers (e.g., antioxidants, etc.), and the like. In the invention, the phytophthora bacteria preferably comprise phytophthora capsici, cucumber and pumpkin, and the ring rot bacteria preferably comprise ring rot bacteria and apple ring rot bacteria.
The technical solutions 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
Pulverizing air-dried stem and leaf of Clausena lansium (3.0 kg), reflux-extracting with 95% methanol for 3 times (10L each time) for 3 hr, mixing the three methanol extractive solutions, and concentrating under reduced pressure to obtain methanol extract (97 g); fully suspending the methanol extract and 5% hydrochloric acid according to a ratio of 1:10(m: V), adding 1/3 suspension amount of ethyl acetate, fully mixing, standing for layering, separating an ethyl acetate layer, extracting the residual liquid with ethyl acetate for 2 times, combining the ethyl acetate layers, and concentrating under reduced pressure to obtain a non-alkaloid part Fr.1(54 g).
Adjusting pH of the residual water phase to 9 with ammonia water, repeatedly extracting with ethyl acetate for 5 times, mixing ethyl acetate layers, and concentrating under reduced pressure to obtain total alkaloids Fr.2(11g) of Clausena lansium. Taking a proper amount of methanol extract, Fr.1 and Fr.2 to prepare a crude extract with the mass concentration of 10mg/mL for a test liquid mother solution, and testing the bacteriostatic activity of the methanol extract, the Fr.1 and the Fr.2 on phytophthora capsici. The result shows that the total alkaloids Fr.2 of wampee have the most obvious bacteriostatic activity on phytophthora capsici.
Fr.2 with obvious activity is selected for further activity tracking separation. Subjecting Fr.2 to 200 mesh silica gel column chromatography, eluting with petroleum ether and acetone at ratio of 10:0, 10:1, 5:1, 4:1, 3:1, 2:1, 1; 1, 0:10, obtaining 33 fractions, detecting the fractions by TLC, using 6% sulfuric acid-absolute ethyl alcohol as a color development agent, and combining 9 fractions which are colored into light blue spots under heating into 9 secondary parts Fr.1.1-Fr.2.9 according to different polarity sizes. And respectively taking a proper amount of 9 secondary parts to prepare a test solution mother solution with the concentration of 3mg/mL, diluting to the required test concentration, and further performing an activity test for inhibiting phytophthora capsici. And selecting Fr.2.3 and Fr.2.5 with strong growth inhibition activity to phytophthora capsici according to the test result for further activity tracking separation.
Fr.2.3 was stirred with 60 mesh polyamide and subjected to medium pressure MCI column chromatography eluting with a gradient of methanol to water 20:80, 40:60, 60:40, 80:20, 90:10, 100:0, fractions with the same blue spot were combined into 4 subfractions Fr.2.3.1-Fr.2.3.4 according to TLC. The fraction Fr.2.3.2 containing the main spot was subjected to repeated silica gel column chromatography eluting with petroleum ether and acetone at 5:1-3:1 by TLC, and the main spot fractions were combined to give a mixture Fr.2.3.2.3. Fr.2.3.2.3 was further separated and purified several times by HPLC (HPLC: Agilent 1260; column: XDB-C18; particle size: 5 μm; column size 9.4X 250 mm; flow rate: 2 mL/min; sample introduction: manual sample introduction; mobile phase: methanol: water: 70:30-80:20) to give the compounds dictamnine (1) (19mg,6.33ppm) and lansiumamide B (2) (31mg,10.33 ppm).
Fr.2.5 is subjected to medium-pressure RP-18 column chromatography, the eluent is methanol, water is 20:80, 40:60, 60:40, 80:20 and 100:0, detection is carried out by TLC, and 5 subfractions Fr.2.5.1-Fr.2.5.5 are combined. Then, the mixture containing the main spot was subjected to repeated silica gel column chromatography using Fr.2.5.3 containing the main spot, eluting with petroleum ether and acetone at a ratio of 5:1 to 3: 1. The compound 6-methoxy carbazolyl-3-carboxylic acid methyl ester (3) (21mg,7.00ppm) and carbazolyl-3-carboxylic acid methyl ester (4) (24mg, 8.00ppm) were obtained by separation and purification by HPLC (HPLC: Agilent 1260; chromatographic column: XDB-C18; particle size: 5 μm; column size 9.4X 250 mm; flow rate: 2 mL/min; sample injection manner: manual sample injection; mobile phase: methanol: water: 60:40-70:30) for a plurality of times. The specific flow chart of the above separation method is shown in FIG. 1.
By passing1H-NMR、13The structures of the compounds were identified by C-NMR and MS, and the spectral data for identifying the structures of the compounds dictamnine (1), lantumimide B (2), 6-methoxy carbazolyl-3-carboxylic acid methyl ester (3) and carbazolyl-3-carboxylic acid methyl ester (4) are as follows:
dictamarine (1) white solid, C12H9NO2.ESI-MS(pos.)m/z：222[M+Na]+.1H NMR (400MHz,acetone-d6)δH:8.24(d,J＝8.0Hz,1H,H-5),7.91(d,J＝8.0Hz,1H,H-8), 7.87(br.s,1H,H-2′),7.70(t,J＝8.0Hz,1H,H-7),7.46(t,J＝8.0Hz,1H,H-6),7.38 (br.s,1H,H-3′),4.50(s,3H,H-OCH3).13C NMR(100MHz,acetone-d6)δC:164.7(s, C-2),157.5(s,C-4),146.6(s,C-9),144.9(d,C-2′),130.2(d,C-7),128.6(d,C-8), 124.5(d,C-6),123.2(d,C-5),119.4(s,C-10),106.1(d,C-3′),104.4(s,C-3),59.9(q, C-OCH3).
Lansiumamide B (2) as a pale yellow powder, ESI-MS (pos.) M/z 286[ M + Na ]]+.1H NMR (400MHz,CDCl3)δH:7.59(1H,d,J＝15.8Hz,H-8′),7.05-7.42(10H,m),6.87(1H,d, J＝15.8Hz,H-7′),6.44(1H,d,J＝8.0Hz,H-8),6.18(1H,d,J＝8.0Hz,H-7),3.08 (3H,s,N-CH3).13C NMR(100MHz,acetone-d6)δC:167.9(s,C-9′),140.4(d,C-7′), 129.9(s,C-1),129.4(d,C-2,6),129.2(d,C-2′,6′),128.5(s,C-4),128.3(t,C-8),128.1 (s,C-4′),125.3(s,C-1′),117.0(d,C-8′),115.3(d,C-3′,5′),114.9(d,C-3,5),112.6(t, C-7).
6-Methoxycarbazole-3-carboxylic acid methyl ester (3) as pale yellow powder, ESI-MS (pos.) M/z 278 [ M + Na ]]+.H NMR(acetone-d6)δH:10.33(s,1H,NH),8.93(1H,brs,H-4),8.27(1H,dd, J＝7.8,1.8Hz,H-2),7.70(1H,d,J＝1.8Hz,H-5),7.48(1H,d,J＝7.8Hz,H-1),7.42 (1H,d,J＝7.8Hz,H-8),7.18(1H,dd,J＝7.8,1.8Hz,H-7),4.03(3H,s,3-COOCH3), 3.96(3H,s,6-OCH3).13C NMR(100MHz,acetone-d6)δC:167.7(s,3-COOCH3), 155.4(s,C-6),142.3(s,C-1a),137.0(s,C-8a),127.5(d,C-2),124.4(s,C-5a),123.6(s, C-4a),123.4(d,C-4),121.2(s,C-3),116.7(d,C-7),112.8(d,C-8),111.4(d,C-1), 103.8(d,C-5),56.1(q,6-OCH3),51.8(q,3-COOCH3).
Carbazolyl-3-carboxylic acid methyl ester (4) pale yellow powder, C15H13NO3.ESI-MS(pos.)m/z:248 [M+Na]+.1H NMR(500MHz,acetone-d6)δH:10.74(s,1H,NH),8.83(br.s,1H,H-4), 8.25(d,J＝7.6Hz,1H,H-5),8.09(dd,J＝8.5,1.1Hz,1H,H-2),7.59(overlapped,1H, H-8),7.57(m,1H,H-1),7.46(t,J＝7.6Hz,1H,H-7),7.27(t,J＝7.6Hz,1H,H-6), 3.92(s,3H,OCH3).13C NMR(125MHz,acetone-d6)δC:168.1(s,COOCH3),143.8(s, C-1a),141.6(s,C-8a),127.9(d,C-2),127.4(d,C-7),124.1(s,C-4a),123.8(s,C-5a), 123.3(d,C-4),121.9(s,C-3),121.4(d,C-5),120.8(d,C-6),112.3(d,C-1),111.1(d, C-8),52.1(q,OCH3).
Example 2
Pulverizing air-dried stem and leaf of Clausena lansium (5.0 kg), reflux-extracting with 99% methanol for 2 times (16L each time) for 2 hr, mixing the three methanol extractive solutions, and concentrating under reduced pressure to obtain methanol extract; fully suspending the methanol extract and 4% hydrochloric acid according to a ratio of 1:8 (m: V), adding 1/3 suspension amount of ethyl acetate, fully mixing, standing for layering, separating an ethyl acetate layer, extracting the residual liquid with ethyl acetate for 2 times, combining the ethyl acetate layers, and concentrating under reduced pressure to obtain a non-alkaloid part Fr.1.
Adjusting pH of the residual water phase to 10 with ammonia water, repeatedly extracting with ethyl acetate for 4 times, mixing ethyl acetate layers, and concentrating under reduced pressure to obtain total alkaloids Fr.2 of Clausena lansium. And (3) preparing a crude extract with the mass concentration of 10mg/mL by taking a proper amount of methanol extract, Fr.1 and Fr.2, and testing the antibacterial activity of the methanol extract, the Fr.1 and the Fr.2 on phytophthora capsici and pyricularia pyricularis. The result shows that the total alkaloids Fr.2 of the wampee have the most obvious bacteriostatic activity on the verticillium dahliae and phytophthora capsici.
Fr.2 with obvious activity is selected for further activity tracking separation. Subjecting Fr.2 to 300 mesh silica gel column chromatography, eluting with petroleum ether and acetone at ratio of 10:0, 10:1, 5:1, 4:1, 3:1, 2:1, 1; 1, 0:10, obtaining 33 fractions, detecting the fractions by TLC, using 8% sulfuric acid-absolute ethyl alcohol as a color development agent, combining the fractions which are colored into light blue spots under heating into 9 secondary parts Fr.1.1-Fr.2.9 according to different polarity sizes. And respectively taking a proper amount of 9 secondary parts to prepare a test solution mother solution with the concentration of 3mg/mL, diluting to the required test concentration, and further performing an activity test for inhibiting phytophthora capsici. And selecting Fr.2.3 and Fr.2.5 with strong growth inhibition activity to phytophthora capsici according to the test result for further activity tracking separation.
Fr.2.3 was sampled with 60 mesh polyamide and chromatographed on medium pressure MCI column with gradient elution of methanol: water 20:80, 40:60, 60:40, 80:20, 90:10, 100:0, fractions with the same blue spots were combined into 4 subfractions Fr.2.3.1-Fr.2.3.4 according to TLC detection. The fraction Fr.2.3.2 containing the main spot was subjected to repeated silica gel column chromatography eluting with petroleum ether and acetone at 5:1-3:1 by TLC, and the main spot fractions were combined to give a mixture Fr.2.3.2.3. Fr.2.3.2.3 was further separated and purified several times by HPLC (HPLC: Agilent 1260; chromatography column: XDB-C18; particle size: 5 μm; column size 9.4X 250 mm; flow rate: 2 mL/min; sample injection manner: manual sample injection; mobile phase: methanol: water: 70:30-80:20) to give the compounds dictamnine (1) and lantumimide B (2).
Fr.2.5 is subjected to medium-pressure RP-18 column chromatography, the eluent is methanol, water is 20:80, 40:60, 60:40, 80:20 and 100:0, detection is carried out by TLC, and 5 subfractions Fr.2.5.1-Fr.2.5.5 are combined. Then, the mixture containing the main spot was subjected to repeated silica gel column chromatography using Fr.2.5.3 containing the main spot, eluting with petroleum ether and acetone at a ratio of 5:1 to 3: 1. Separating and purifying by HPLC (HPLC: Agilent 1260; chromatographic column: XDB-C18; particle diameter: 5 μm; column specification 9.4 × 250 mm; flow rate: 2 mL/min; sample injection manner: manual sample injection; mobile phase: methanol: water ═ 60:40-70:30) for multiple times to obtain the compound 6-methoxy carbazol-3-carboxylic acid methyl ester (3) and carbazol-3-carboxylic acid methyl ester (4).
Example 3
A crude methanol extract (methanol extract) of a wampee plant obtained in example 1 was prepared as a mother liquor at a concentration of 10g/L using acetone as a solvent, and a total alkaloid fraction Fr.2, an non-alkaloid fraction Fr.1, and roughly separated fractions Fr.2.3 and Fr.2.5 obtained in example 1 were prepared as mother liquors at a concentration of 3mg/mL, respectively. The above mother solutions were added to the thawed PDA medium (about 50 deg.C), mixed well and poured into petri dishes to prepare toxic media with concentration gradients of 1000, 500, 250, 125 and 62.5. mu.g/mL. The control was made by mixing the same volume of acetone medium. Using a puncher to punch and take the phytophthora capsici cakes with the diameter of 0.6cm for test, respectively inoculating the cakes to the centers of a toxic medium and a control medium, enabling the side with hyphae to face downwards, placing one cake in each culture dish, repeating the treatment for three times, placing the cakes in a constant-temperature incubator at 25 ℃ for culture for 4 days, and measuring the colony diameter (cm) by adopting a cross method. The bacteriostatic rate was calculated according to the following formula.
Figure RE-GDA0003346408320000101
And converting the inhibition percentage into an inhibition ratio value according to a biometric ratio value conversion table. Calculating toxicity regression linear equation and effective intermediate concentration EC of methanol crude extract, total alkaloid fraction, non-alkaloid fraction and crude separated fraction (Fr.2.3, Fr.2.5) by using logarithm of concentration set in test as abscissa and inhibiting rate value as ordinate50. The results are shown in Table 1.
TABLE 1 results of growth inhibition of Phytophthora capsici by different groups
Figure RE-GDA0003346408320000111
As can be seen from Table 1, the concentrations of the methanol crude extracts, the total alkaloids and the crude separated parts of the clausena lansium stems and leaves have higher linear correlation with the inhibition activity on the growth of phytophthora capsici leonian, the total alkaloids have higher inhibition activity on the phytophthora capsici leonian, and the non-alkaloid has no inhibition activity.
Example 4
The alkaloids dictamnine (1), lantumimide B (2), 6-methoxy carbazolyl-3-carboxylic acid methyl ester (3) and carbazolyl-3-carboxylic acid methyl ester (4) separated in example 1 were prepared into mother liquor with concentration of 1mg/mL respectively using acetone as solvent, and the mother liquor was added to the melted PDA culture medium (about 50 ℃) and mixed uniformly, and poured into a culture dish to prepare a toxic medium with concentration of 200. mu.g/mL. The control was made by mixing the same volume of chlorothalonil medium. Using a puncher to punch test pyricularia piricola (B.dothidea) and phytophthora capsici (P.capsicii) fungus cakes with the diameter of 0.6cm respectively, inoculating the fungus cakes into the centers of a toxic medium and a control medium respectively, enabling one side with hyphae to face downwards, placing one fungus cake on each culture dish, repeating the treatment for three times, placing the culture dishes in a constant-temperature incubator at 25 ℃ for culturing for 4 days, and measuring the diameter (cm) of a bacterial colony by adopting a cross method. The inhibition rate was calculated according to the formula of example 3. The results are shown in Table 2.
TABLE 2 inhibitory Activity results (concentration: 200. mu.g/mL) of 4 Clausena lansium alkaloids of the invention against Ribes nigrum and Phytophthora capsici
Figure RE-GDA0003346408320000112
As can be seen from the table 2, the 4 compounds of the invention have better inhibition effect on the activity of phytophthora capsici pathogens, and the compound 1 dictamnine also has better inhibition effect on the activity of the pyricularia pyricularis pathogens.
Example 5
The compounds 1 to 4 isolated in example 1 were added to a 4% ethanol sulfate solution at pH 4, respectively, and filtered and dried to obtain sulfate compounds 1 to 4.
Example 6
The compounds 1 to 4 isolated in example 2 were added to a 4% hydrochloric acid solution at pH 4, respectively, and filtered and dried to obtain hydrochloride compounds 1 to 4.
Example 7
The compounds 1 to 4 isolated in example 1 were added to 4% tartaric acid solutions at pH 4, respectively, and filtered and dried to obtain tartrate compounds 1 to 4.
Example 8
Dissolving the compound 1-4 obtained in example 1 in 10% of xylene organic solvent, adding 5% of calcium dodecyl benzene sulfonate emulsifier and 2% of other auxiliary agents, making up 100% with xylene, stirring and mixing to prepare a homogeneous transparent oily liquid.
Example 9
The salt obtained in the example 4 is dissolved in xylene organic solvent according to the proportion of 10 percent, then 5 percent of calcium dodecyl benzene sulfonate emulsifier and 2 percent of other auxiliary agents are added, the xylene is used for complementing 100 percent, and the mixture is stirred and mixed to prepare a homogeneous transparent oily liquid.
Example 10
Mixing the compound 1-4 obtained in the example 2, a toluene organic solvent and a pesticide emulsifier 300 in a mass ratio of 10:85:5 to form a uniform and transparent oil phase, slowly adding distilled water under stirring to form a water-in-oil type emulsion, stirring and heating to rapidly convert the emulsion into the oil-in-water type emulsion, cooling to room temperature, standing and filtering to obtain the microemulsion.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (7)

1. The application of Clausena lansium alkaloid and its salt in preparing product for inhibiting Phytophthora and/or Phyllospora striolata activity is characterized in that the Clausena lansium alkaloid is dictamnine;
the phytophthora is phytophthora capsici, and the ring rot is pyricularia pyriformis.
2. The use of claim 1, wherein the salt comprises a salt of the wampee alkaloid reacted with an inorganic acid, an organic acid, an alkali metal, an alkaline earth metal, or a basic amino acid.
3. Use according to claim 1, characterized in that the product comprises a pesticide, a growth inhibitor.
4. The use according to claim 1, wherein the preparation method of the clausena lansium alkaloid comprises the following steps:
mixing and reflux-extracting stem and leaf of clausena lansium with methanol, and concentrating under reduced pressure to obtain methanol extract; performing acid extraction and alkali precipitation on the methanol extract to obtain a clausena lansium total alkaloid part and an non-alkaloid part, performing silica gel column chromatography coarse separation on the clausena lansium total alkaloid part to obtain secondary parts, performing phytophthora root rot primary bacteria inhibition activity test on the secondary parts respectively, performing TLC (thin layer chromatography) detection and activity tracking separation on the secondary parts with stronger activity, separating active alkaloids by combining MCI (methanol-supported chemical indicator) column chromatography and RP-18 (RP-18) column chromatography separation methods, and finally performing HPLC (high performance liquid chromatography) separation and purification to obtain the active clausena lansium alkaloid component.
5. The use according to claim 4, wherein the methanol is a 95-99% by volume methanol solution.
6. The use of claim 4, wherein the acid is hydrochloric acid with a volume fraction of 4-6%, and the mass-to-volume ratio of the methanol extract to the hydrochloric acid is 1:8-1: 10.
7. Use according to claim 4, wherein the alkaline precipitation is carried out by adjusting the pH to 9-10 with aqueous ammonia.
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