CN109833337B - Momordica charantia essence compound for inhibiting pest immune response and high-efficiency preparation method and application thereof - Google Patents
Momordica charantia essence compound for inhibiting pest immune response and high-efficiency preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a charantin compound for inhibiting pest immune response and an efficient preparation method and application thereof. The preparation method uses methanol to leach the balsam pear, and three types of chemical balsam pear compounds are obtained by three-stage high performance liquid chromatography separation: momordicine II, momordicine L and momordicine I. The method utilizes the multilevel liquid chromatography of the overload critical point of the column to separate the compounds, adopts three-level high performance liquid chromatography to separate, greatly shortens the total time required by the high performance liquid chromatography, has short time consumption and high efficiency in the purification process, and can directionally, efficiently and massively purify and prepare the high-purity charantin compounds. The obtained charantin compound can effectively inhibit the immune response of various agricultural pests, thereby controlling the population growth of the agricultural pests and being applied to green healthy pest prevention and control pesticides.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a charantin compound for inhibiting pest immune response, and an efficient preparation method and application thereof.
Background
Momordica charantia (Momoradicacharantia L.) belonging to Cucurbitaceae family, belonging to genus Momordica, has bitter and cold nature, and has effects of clearing away heat and toxic materials, and resisting mutation and tumor; in food processing industry and biological control, it has the functions of resisting insect, bacteria and virus. The charantin compound is safe and harmless to vertebrates, has the characteristics of green and environment-friendly raw materials, easy acquisition and the like, and is a plant secondary metabolite with development potential. In recent years, the balsam pear has become a research hotspot of numerous scholars at home and abroad, and protein compounds comprising vegetable proteins, balsam pear lectin, alpha-charantin, beta-charantin, gamma-charantin, charantin L, charantin I and charantin II, antiviral protein MAP30, charantin, balsam pear inhibitors and the like are separated and purified from balsam pear fruits and seeds.
The inventor research team is always dedicated to the purification of the charantin compounds and the separation and identification of the compounds. At present, the charantin I and the charantin II have strong antifeedant effect on diamondback moth larvae and seriously inhibit the growth and development of the diamondback moth larvae; the charantin compound can obviously inhibit the growth, development and reproductive capacity of the ostrinia nubilalis; the charantin I and the charantin L can obviously inhibit the activities of phospholipase, trehalase and the like in bodies of Asiatic corn borer larvae; the charantin I and the charantin II can obviously inhibit the activity of in vitro phenol oxidase of the ostrinia furnacalis walker; the charantin compounds can seriously damage the normal physiology of prodenia litura ovarian cells; the charantin I and the new compound charantin B have effective proliferation inhibition and necrosis-causing effects on Asiatic corn borer Ofh cells. Therefore, the charantin compounds have important significance for preventing and controlling agricultural pests.
At present, 8 charantin compounds are obtained from bitter gourd stem and leaf extracts, wherein the proportion of charantin I, charantin II and charantin L in the charantin compounds can reach 36.38%. However, the charantin compounds have low yield in purification, complicated purification steps and long time consumption. At present, the general methods for extracting the momordicin compounds comprise gradient extraction, silica gel column chromatography, high performance liquid chromatography separation and the like. Taking a method for extracting the momordicin compound from Ling ice as an example (shown in Table 1), the specific steps comprise ethanol leaching of stems and leaves of the momordicin compound, gradient extraction and silica gel column chromatography, and then single-stage high performance liquid chromatography separation is carried out on the obtained extract, so that the momordicin compound with the purity of more than 95% is finally obtained, the total time consumption of the extraction process is about 121 hours, the total extraction rate is about 100mg/kg, and the yield is about 0.83 mg/h. The gradient extraction needs various organic solvents, and the recovery rate of the charantin compounds is greatly lost in a plurality of extraction steps; silica gel column chromatography steps are complicated, cost is high, time consumption is long, and resolution ratio is not high; although the traditional high performance liquid chromatography separation method can obtain the charantin compounds with the purity of more than 95 percent, the total time consumption is long, the total extraction efficiency is low, and the traditional high performance liquid chromatography separation method is not suitable for preparing the charantin compounds in large quantities. Because a simple and efficient method for separating and purifying the momordicin compound is lacked at present, the development of the medicine with food-grade purity or a high-efficient biological insecticide by using the momordicin compound is severely restricted.
Table 1:
polyphenol Oxidase (PPO) has the function of blackening and resisting invading pathogenic microorganisms, and is an important component of insect immune response. The primary phenoloxidase cascade activation system is ubiquitous in all invertebrates and is one of the most important humoral immune systems of insects. The external immunostimulation can induce the protein at the upstream of the cascade reaction to shear and activate the precursor of the prophenoloxidase activating enzyme, and the activated prophenoloxidase activating enzyme can shear and activate inactive prophenoloxidase into active prophenoloxidase and finally generate cytotoxin substances to oxidize foreign matters. The inhibition of peroxidase activity can result in abolishment of insect defense mechanism or abnormal softening of organism, and both can result in prevention and control of plant diseases and insect pests, so that Phenol Oxidase (PO) serving as a target of a novel pesticide has a new and reasonable action mode, and the purpose of preventing and controlling the pests is achieved by inhibiting the activity of Phenol Oxidase (PO) to enable the pests to lose normal defense capability or soften abnormally.
The inventor finds that momordicin I and momordicin II can obviously inhibit the activity of isolated phenol oxidase of Asiatic corn borer, but how momordicin I and momordicin II inhibit the activity of Phenol Oxidase (PO) at the level of transcription and translation is not clear, so that the development and application of momordicin pesticides targeting a primary cascade activation system of pest phenol oxidase are restricted.
Disclosure of Invention
The invention aims to overcome the defect that the prior art lacks a simple and efficient method for separating and purifying a charantin compound, and provides a method for efficiently and rapidly preparing the charantin compound and application thereof (the flow is shown in figure 1).
The first purpose of the invention is to provide a method for preparing the charantin compound with high efficiency and high speed.
The second purpose of the invention is to provide the application of one or more of the charantin compounds in the aspects of insect disinfestation and/or preparation of insecticides.
The third purpose of the invention is to provide the application of one or more of the momordicin compounds in the inhibition of immune response, the inhibition of phenoloxidase and/or the inhibition of prophenoloxidase activating enzyme, and/or the preparation of inhibitors of immune response, prophenoloxidase and/or the inhibition of prophenoloxidase activating enzyme.
It is a fourth object of the present invention to provide a pesticide, an immune response suppressant, a phenol oxidizing enzyme and/or a prophenoloxidase activating enzyme inhibitor.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for preparing momordicin compounds comprises extracting fructus Momordicae Charantiae with methanol, and separating by three-stage high performance liquid chromatography.
Preferably, the preparation method comprises the following steps:
s1, drying fresh bitter gourd stems and leaves, and grinding into powder;
s2, leaching the dry powder of the bitter gourd stem leaves with methanol, filtering and concentrating the leaching liquor to obtain a crude extract;
s3, performing primary high performance liquid chromatography separation on the crude extract, respectively collecting effluent liquid in 18-26 min time periods and effluent liquid in 30-42 min time periods, and concentrating to obtain a primary separated matter 1 and a primary separated matter 2;
s4, respectively carrying out secondary high performance liquid chromatography separation on the primary separator 1 and the primary separator 2, respectively collecting effluent liquid of the primary separator 1 in a period of 18-22 min and effluent liquid of the primary separator 1 in a period of 24-26min, drying and concentrating to obtain a secondary separator 1-1 and a secondary separator 1-2, collecting effluent liquid of the primary separator 2 in a period of 33-37 min, drying and concentrating to obtain a secondary separator 2-1;
s5, respectively carrying out three-stage high performance liquid chromatography separation on the secondary isolate 1-1, the secondary isolate 1-2 and the secondary isolate 2-1, respectively collecting effluent liquid in 20-24 min time periods, 23-26 min time periods and 34-37 min time periods, drying and concentrating to respectively obtain momordicin II, momordicin L and momordicin I.
Preferably, the parameters of the high performance liquid chromatography separation are: the mobile phase is 80% methanol, the column temperature is 28 ℃, the flow rate is 2.5mL/min, the separation column is Agilent XB-C18, and the detection wavelength is 210 nm.
Preferably, the leaching of the balsam pear stem and leaf dry powder by using methanol is to leach the balsam pear stem and leaf dry powder by using 80-100% methanol for 16-26 hours.
More preferably, the extraction rate can be obviously improved by leaching the balsam pear stem and leaf dry powder with 100% methanol for 24 h.
Preferably, the dosage ratio of the methanol to the balsam pear stem and leaf dry powder is 1: 5-10 (g: mL).
More preferably, the dosage ratio of the methanol to the balsam pear stem and leaf dry powder is 1:5(g: mL), so that the extraction rate can be obviously improved.
The invention utilizes the multilevel liquid chromatography separation of the overload critical point of the column to obtain the compound, and utilizes the overload critical point of the column to determine the optimal HPLC sample volume.
Therefore, preferably, the volume ratio of the loading amount of the first-stage high performance liquid chromatography to the methanol in the leaching solution is 1-3: 2-10; the volume ratio of the sample loading amount of the secondary high performance liquid chromatography to the primary isolate 1 or the primary isolate 2 is 1-3: 2-10; the volume ratio of the sample loading amount of the three-level high performance liquid chromatography to the second-level isolate 1-1, the second-level isolate 1-2 or the second-level isolate 2-1 is 1-3: 2 to 10.
More preferably, the volume ratio of the loading amount of the first-stage high performance liquid chromatography to the methanol in the leaching solution is 2: 5.
more preferably, the volume ratio of the sample loading of the second stage high performance liquid chromatography to the first fraction 1 or the first fraction 2 is 2: 5.
more preferably, the volume ratio of the loading amount of the three-stage high performance liquid chromatography to the second isolate 1-1, the second isolate 1-2 or the second isolate 2-1 is 2: 5.
preferably, in step S3, first-stage isolate 1 and first-stage isolate 2 are each concentrated to 1 mL.
Preferably, in step S4, secondary isolate 1-1, secondary isolate 1-2 and secondary isolate 2-1 are each concentrated to 1 mL.
Preferably, in step S2, the filtration is filtration with a 0.22 μm filter membrane.
Further, most preferably, the method comprises the following steps:
s1, drying fresh bitter gourd stems and leaves, and grinding into powder;
s2, leaching the dry balsam pear stem and leaf powder for 24 hours by using 100% methanol, and filtering and concentrating the leaching liquor by using a 0.22 mu m filter membrane to obtain a crude extract, wherein the dosage ratio of the methanol to the dry balsam pear stem and leaf powder is 1:5(g: mL);
s3, performing primary high performance liquid chromatography separation on the crude extract, respectively collecting effluent liquid in 18-26 min time periods and 30-42 min time periods, and concentrating to obtain a primary separated substance 1 and a primary separated substance 2, wherein the volume ratio of the loading amount of the primary high performance liquid chromatography to the methanol in the leaching solution is 2: 5;
s4, respectively carrying out secondary high performance liquid chromatography separation on the primary separator 1 and the primary separator 2, respectively collecting effluent liquid in 18-22 min time periods and 24-26min time periods for the primary separator 1, drying and concentrating to obtain a secondary separator 1-1 and a secondary separator 1-2, collecting effluent liquid in 33-37 min time periods for the primary separator 2, drying and concentrating to obtain a secondary separator 2-1, wherein the volume ratio of the sample loading amount of the secondary high performance liquid chromatography to the primary separator 1 or the primary separator 2 is 2: 5;
s5, respectively carrying out three-stage high performance liquid chromatography separation on the second-stage isolate 1-1, the second-stage isolate 1-2 and the second-stage isolate 2-1, respectively collecting effluent liquid in 20-24 min time periods, 23-26 min time periods and 34-37 min time periods, drying and concentrating to respectively obtain momordicin II, momordicin L and momordicin I, wherein the volume ratio of the sample loading amount of the three-stage high performance liquid chromatography to the volume ratio of the second-stage isolate 1-1, the second-stage isolate 1-2 or the second-stage isolate 2-1 is 2: 5;
wherein, the parameters of the high performance liquid chromatography separation are as follows: the mobile phase is 80% methanol, the column temperature is 28 ℃, the flow rate is 2.5mL/min, the separation column is Agilent XB-C18, and the detection wavelength is 210 nm.
One or more of the charantin compounds prepared by the preparation method also belong to the protection scope of the invention.
Accordingly, the invention also claims the following: one or more of the momordicin compounds are applied to disinsection and/or preparation of pesticides. (ii) a
The application of one or more compounds in Momordica charantia L.can inhibit immune reaction, phenol oxidase and/or prophenoloxidase activating enzyme, and/or prepare inhibitor for immune reaction, phenol oxidase and/or prophenoloxidase activating enzyme.
A pesticide, immunoreaction inhibitor, phenol oxidase and/or prophenoloxidase activating enzyme inhibitor contains one or more compounds selected from Momordica charantia.
Compared with the prior art, the invention has the following beneficial effects:
the invention closely combines the modern pharmacological technology and the new chemical separation technology to establish a plurality of screening modes so as to discover more new active chemical components and lay a foundation for the application of the active chemical components. The method utilizes the multilevel liquid chromatography of the overload critical point of the column to separate the compounds, adopts three-level high performance liquid chromatography to separate, obtains the charantin compounds with the purity of more than 99 percent, improves the yield by 100 percent compared with the prior extraction method, shortens the time to 50 hours, greatly shortens the total time required by the high performance liquid chromatography to separate, has short time consumption and high efficiency in the purification process, and can directionally, efficiently and massively purify and prepare the high-purity charantin compounds. The obtained charantin compound can effectively inhibit the immune response of various agricultural pests, thereby controlling the population growth of the agricultural pests and being applied to green healthy pest prevention and control pesticides.
Drawings
FIG. 1 is a flow chart of a process for preparing momordicin.
FIG. 2 is a first-order high performance liquid chromatography separation chart of a momordicin crude extract.
FIG. 3 is a two-stage high performance liquid chromatography separation chart of a momordicin crude extract.
FIG. 4 is a three-stage HPLC separation chart of a crude extract of momordicin.
FIG. 5 shows qRT-PCR detection of the prodelphiniis phenoloxidase activating enzyme PAP gene after injection.
FIG. 6 Activity assay of phenol oxidase by Plutella xylostella and Spodoptera litura.
FIG. 7 shows the mortality of plutella xylostella and prodenia litura.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 preparation of momordicin compounds
Firstly, sample treatment
1. 1kg of aged stems and leaves of balsam pear is taken, cleaned, dried and ground into powder to obtain about 100g of dry powder of the stems and leaves of the balsam pear (the time is about 1 hour).
2. Adding 500mL of 100% methanol to extract dry stem and leaf powder of Momordica charantia, collecting the extract after 24h, filtering the extract with 0.22 μm filter membrane, and concentrating to obtain 5mL of momordicin crude extract (which takes about 26 h).
Second, HPLC
The HPLC conditions were as follows: mobile phase (80% methanol), column temperature (28 ℃), flow rate (2.5mL/min), separation column (Agilent XB-C18), detection wavelength (210nm), flow chart is shown in fig. 1.
1. First-stage high performance liquid chromatography
(1) Operation of
And (3) performing first-stage high performance liquid chromatography separation on the charantin crude extract by taking 300 mu L as a sample amount, collecting the effluent liquid in 18-26 min time period and 30-42 min time period respectively, and concentrating (about 10 h).
(2) Results
Performing primary high performance liquid chromatography to obtain 1mL of primary isolate 1 and 1mL of primary isolate 2, wherein HPLC chromatogram is shown in FIG. 2, and the primary isolate 1 and the primary isolate 2 are both light green liquid.
2. Two-stage high performance liquid chromatography
(1) Operation of
Performing secondary high performance liquid chromatography separation on the primary isolate 1 by taking 200 mu L as a sample amount, respectively collecting the effluent liquid in 18-22 min time period and 24-26min time period, drying and concentrating (about 4h is consumed); and (3) carrying out secondary high performance liquid chromatography separation on the primary isolate 2 by taking 200 mu L as a sample amount, collecting effluent liquid in a period of 33-37 min, and drying and concentrating (about 4 h).
And performing liquid mass identification and nuclear magnetic identification on the secondary isolate 1-1, the secondary isolate 1-2 and the secondary isolate 2-1.
(2) Results
Performing secondary high performance liquid chromatography to obtain about 240mg of secondary isolate 1-1 and about 130mg of secondary isolate 1-2, and HPLC chromatogram shown in FIG. 3; obtaining about 240mg of second-stage isolate 2-1, and the HPLC chromatogram is shown in figure 3; the secondary isolate 1-1, the secondary isolate 1-2 and the secondary isolate 2-1 are all white crystals.
And determining that the secondary isolate 1-1, the secondary isolate 1-2 and the secondary isolate 2-1 are charantin II, charantin L and charantin I respectively through nuclear magnetic identification.
3. Three-stage high performance liquid chromatography
(1) Operation of
Dissolving the charantin II, the charantin L and the charantin I obtained by the secondary separation in 1mL of 100% methanol, respectively carrying out three-stage high performance liquid chromatography separation on the charantin II, the charantin L and the charantin I solution obtained by the secondary separation by taking 200 mu L as a sample amount, collecting effluent liquid in 20-24 min time periods, 23-26 min time periods and 34-37 min time periods, and drying and concentrating (about 4h is consumed).
(2) Results
Separating by three-stage high performance liquid chromatography to obtain momordicin II about 200mg, and obtaining HPLC chromatogram as shown in FIG. 4; momordica charantia L about 100mg, HPLC chromatogram is shown in FIG. 7; momordica charantia I about 200mg, HPLC chromatogram is shown in FIG. 4.
4. Liquid quality identification
(1) Operation of
Dissolving momordicin II, momordicin L and momordicin I obtained by three-stage high performance liquid chromatography in 1mL of 100% methanol, and performing liquid quality identification.
(2) Results
The liquid quality identification result shows that: the purity of the charantin II, the charantin L and the charantin I obtained by three-stage high performance liquid chromatography separation is more than 99%, the time consumed in the whole purification process is shortened by 50h, and the yield is improved by 30% compared with the previous yield.
The time consumption and yield of the preparation of the momordica charantia compounds in this example are shown in table 2.
Table 2:
EXAMPLE 2 determination of overload Critical Point for first-order HPLC separation column
One, operation
Determining overload critical points of a first-stage HPLC separation column of the charantin crude extract, and performing first-stage high performance liquid chromatography separation tests on the charantin crude extracts with the sample volumes of 100 mu L, 200 mu L, 300 mu L and 400 mu L respectively. Other parameters were the same as "1, one-stage high performance liquid chromatography separation" in example 1.
Second, result in
Determining the crude extract of momordicin at the concentration according to the ratio of the peak area to the sample amount, wherein the sample amount of 300 mu L is the overload critical point of the column, and the optimal sample amount is separated by one-stage high performance liquid chromatography (about 2 h).
EXAMPLE 3 determination of the overload Critical Point of the two-stage HPLC separation column
One, operation
And (3) determining overload critical points of the first-stage isolate 1 and the first-stage isolate 2 by using the first-stage isolate 1 and the first-stage isolate 2 with the sample volumes of 100 mu L, 200 mu L and 300 mu L respectively to perform a two-stage high performance liquid chromatography separation test. Other parameters were the same as "2, two-stage high performance liquid chromatography separation" in example 1.
Second, result in
And determining the first-stage isolate 1 and the first-stage isolate 2 under the concentration according to the ratio of the peak area to the sample volume, wherein the sample volume of 200 mu L is the overload critical point of the column and is the optimal sample volume for the separation of the second-stage high performance liquid chromatography (about 2 h).
EXAMPLE 4 determination of the overload Critical Point of the three-stage HPLC separation column
One, operation
Dissolving charantin II, charantin L and charantin I obtained by secondary separation in 1mL of 100% methanol, determining overload critical point of three-stage HPLC separation column, and performing three-stage high performance liquid chromatography separation test with charantin II, charantin L and charantin I solution obtained by secondary separation with sample amount of 100 muL, 200 muL and 300 muL respectively. Other parameters were the same as those of "3, three-stage high performance liquid chromatography separation" in example 1.
Second, result in
Determining the charantin II, charantin L and charantin I solution under the concentration according to the ratio of the peak area to the sample volume, wherein the sample volume of 200 mu L is the column overload critical point and is the optimal sample volume for three-stage high performance liquid chromatography separation (about 2 h).
Example 5 Momordica charantia compounds have oxidase inhibiting effect
Firstly, experimental steps
1. Preparing charantin II, charantin L and charantin I solutions (100 mu g/mL) with different concentrations respectively.
2. Selecting the plutella xylostella larvae of the first day of 4 years, injecting 1 mu L of charantin I, charantin II and charantin L solution with the same concentration into each group of 100 larvae, and injecting 0.5% DMSO as a control. Sampling was randomly carried out 6, 12, 24, 36, 48 and 60 hours after feeding with normal feed, and 10 samples were taken each time. Pricking first gastropod of polypide, extracting hemolymph of polypide, centrifuging at 4 deg.C for 3min at 500g, collecting blood cells after centrifugation, extracting total RNA of blood cells, and performing qRT-PCR detection according to primer of Plutella xylostella phenol oxidase proactivating enzyme (PxPAP 1).
3. Selecting 5-year-old first-day larvae of prodenia litura, injecting three charantin I, charantin II and charantin L solutions with the same concentration into each group with 100 heads, injecting 10 mu L of each head, and injecting 0.5% DMSO as a control. Samples were taken randomly 6, 12, 24, 36, 48, 60h after feeding with normal feed, 5 at a time. Piercing the first gastropod of the polypide, extracting hemolymph of the polypide, centrifuging for 3min at 4 ℃ and 500g, centrifuging to collect blood cells, extracting total RNA of the blood cells, carrying out qRT-PCR detection according to a prodenia litura phenoloxidase prolidase (SlPPAE1) specific primer,
4. and (3) extracting the total RNA of the blood cells of the prodenia litura and the diamond back moth by using a Trizol method and synthesizing a first chain of cDNA. 4 pairs of fluorescent quantitative PCR primers are respectively designed according to gene sequences of small and medium plutella xylostella PAP1(JQ581597) and RPS13(NM _001305523) and PPAE1(AY677082) and Actin (DQ494753) in GenBank, the primer sequences are shown in Table 3, and the transcription level of the prophenoloxidase activating enzyme PAP gene is detected by using real-time fluorescent quantitative PCR.
Table 3:
second, experimental results
As can be seen from FIG. 5, three momordin compounds of plutella xylostella have inhibitory effect on plutella xylostella PAP 1. The momordicin I has the strongest inhibition activity on PAP1, the transcription of PAP1 gene is inhibited all the time within 6-60 hours, and compared with momordicin II and momordicin L, the momordicin I has the best inhibition effect and reaches the level of obvious difference. After the plutella xylostella is injected with the charantin II and the charantin L, the two reach a remarkable difference level in 24 hours, and the inhibition effects of the charantin II and the charantin L on the plutella xylostella PAP1 are consistent between 6 hours, 12 hours, 36 hours, 48 hours and 60 hours.
The results are shown in FIG. 5. The three momordins have inhibitory effect on PPAE1 of prodenia litura. The momordicin I has the strongest inhibition activity on PAP, the transcription of PPAE1 gene is inhibited within 6-60 h, and compared with momordicin II and momordicin L, the momordicin I has the best inhibition effect and reaches the level of obvious difference. The prodenia litura achieves a remarkable difference level after injecting the charantin II and the charantin L for 24 and 36 hours, and the inhibition effects of the charantin II and the charantin L on the prodenia litura PPAE1 are consistent between 6, 12, 48 and 60 hours.
The charantin I, the charantin II and the charantin L have strong inhibition effects on PAP of two pests, and the inhibition effect of the charantin on PAP1 of plutella xylostella is stronger than that of PPAE1 of prodenia litura. The charantin compounds (charantin II, charantin L and charantin I) further inhibit the activation of prophenoloxidase by inhibiting the initiation of PAP in vivo, and effectively inhibit immune reaction, thereby leading pests to die.
Example 6 biological Activity assay
Firstly, experimental steps
And (3) carrying out biological activity determination on the three compounds (charantin II, charantin L and charantin I).
Preparing the three purified compounds into 5 liquid medicines with the concentrations of 2.5, 5, 10, 20 and 40mg/ml by DMSO% respectively, and preparing the liquid medicines according to the following artificial feed: the liquid medicine is 5: 1 is prepared from 0.05%, 0.010%, and 0.20%The mixed feed with the concentration of 0.40 percent and 0.80 percent is used for feeding plutella xylostella and prodenia litura, and each group has 100 heads. Randomly selecting 0, 12, 24, 36, 48 and 60 parts of the feed respectively, dissecting, puncturing the first ventral poda of the polypide, extracting the blood lymph of the prodenia litura (5 times) and the diamond back moth (10 times) and uniformly mixing with the anticoagulant according to the proportion of 1:1, centrifuging for 3min at 4 ℃ and 500g, respectively collecting the precipitate (blood cells) and the supernatant (blood plasma) after centrifuging, and placing the blood cells and the blood plasma in a refrigerator at-80 ℃ for later use. While 0.5% DMSO was added to diamond back moth and prodenia litura as controls, 60 heads per group. Adding 200 μ L16 mM L-levodopa into each group of plasma, and measuring OD within 4min with enzyme labeling instrument490The change in nm was measured every 30 s.
Selecting larvae of diamondback moth of 4 th age and prodenia litura of 5 th age which can molt and have consistent growth state on the same day, wherein each sample is 100 times in a repeated way, and feeding the larvae with different bitter gourd compound mixed feeds with uniform concentration (0.40%). Each treatment was repeated 3 times. The feed with fresh momordicin is changed every day, and the feed is changed once every day. The number of test insects died within 0, 12, 24, 36, 48, 60, 72, 84h was recorded daily after treatment.
Second, experimental results
Three compounds (charantin II, charantin L and charantin I) were used to treat plutella xylostella and spodoptera litura, the results are shown in FIGS. 6 and 7. As can be seen from FIG. 6, the three compounds, momordicin I, momordicin II and momordicin L, have strong inhibitory action on phenol oxidase in spodoptera litura larvae and diamondback moth larvae, and the inhibitory effect is more obvious with the increase of the compound concentration, showing the concentration time effect. The charantin I has the strongest effect of inhibiting phenol oxidase in a worm body, the charantin II has the second time, and the charantin L also has the weakest effect on the phenol oxidase in the worm body compared with the charantin I.
Three compounds (charantin II, charantin L and charantin I) with the concentration of 0.40% are treated on diamond back moths and prodenia litura, the death rate is observed for 0-84 h, and the result is shown in figure 7. As can be seen from FIG. 7, the effect of plutella xylostella treated by the three compounds (momordicin II, momordicin L and momordicin I) is better than that of prodenia litura, and the effect of momordicin I is the best, and the death rates of the plutella xylostella and the prodenia litura are 68.89% and 54.565% respectively.
Further shows that the three compounds (charantin II, charantin L and charantin I) can effectively inhibit the immune reaction of diamond back moth and prodenia litura.
Claims (3)
1. A method for preparing momordicin compounds is characterized in that, methanol is used for leaching bitter gourd stem leaves, and three-stage high performance liquid chromatography separation is carried out; the preparation method comprises the following steps:
s1, drying fresh bitter gourd stems and leaves, and grinding into powder;
s2, leaching the dry bitter gourd stem and leaf powder with methanol, wherein the dosage ratio of the methanol to the dry bitter gourd stem and leaf powder is 1: 5-10 (g: mL), filtering and concentrating the leaching solution to obtain a crude extract;
s3, performing primary high performance liquid chromatography separation on the crude extract, collecting the effluent liquid in 18-26 min time period and 30-42 min time period respectively, and concentrating to obtain a primary isolate 1 and a primary isolate 2;
s4, respectively carrying out secondary high performance liquid chromatography separation on the primary isolate 1 and the primary isolate 2, respectively collecting the effluent liquid of the primary isolate 1 in a period of 18-22 min and 24-26min, drying and concentrating to obtain a secondary isolate 1-1 and a secondary isolate 1-2, collecting the effluent liquid of the primary isolate 2 in a period of 33-37 min, drying and concentrating to obtain a secondary isolate 2-1;
s5, respectively carrying out three-stage high performance liquid chromatography separation on the secondary isolate 1-1, the secondary isolate 1-2 and the secondary isolate 2-1, respectively collecting effluent liquid in 20-24 min time periods, 23-26 min time periods and 34-37 min time periods, drying and concentrating to respectively obtain momordicin II, momordicin L and momordicin I;
the parameters of the high performance liquid chromatography separation are as follows: the mobile phase is 80% methanol, the column temperature is 28 ℃, the flow rate is 2.5mL/min, the separation column is Agilent XB-C18, and the detection wavelength is 210 nm;
the volume ratio of the sample loading amount of the primary high performance liquid chromatography to the methanol leaching liquor is 1-3: 2-10; the volume ratio of the sample loading amount of the secondary high performance liquid chromatography to the primary isolate 1 or the primary isolate 2 is 1-3: 2-10; the volume ratio of the sample loading amount of the three-stage high performance liquid chromatography to the second-stage separator 1-1, the second-stage separator 1-2 or the second-stage separator 2-1 is 1-3: 2 to 10.
2. The method according to claim 1, wherein the concentration of methanol used in the leaching process is 80 to 100%, and the number of times of leaching is 1 to 3.
3. Use of momordicin L prepared by the preparation method of any one of claims 1 to 2 in the preparation of inhibitors of prophenoloxidase activating enzyme of plutella xylostella or prodenia litura.
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