CN1709065A - Bitter gourd leaf insect-resistant extract and its preparation and application - Google Patents

Abstract

The present invention relates to a bitter melon leaf insect-resisting extract, its preparation method and application. It is characterized by that it utilizes bitter melon leaf as raw material, and uses deionized water, ethyl ether or acetone as extraction solvent, and adopts the processes of grinding, standing still, oscillation, filtering and constant volume extraction so as to obtain the green liquor containing protein. The ratio of bitter melon leaf and solvent is 1g/1ml. Said product can obtain good insect-resisting and insect-killing effect.

Description

Bitter gourd leaf insect-resistant extract and its preparation and application

Technical field:

The invention belongs to a new type of insect-resistant agent for agriculture, and relates to an insect-resistant extract from bitter gourd waste leaves and its preparation and application. In particular, it relates to a water-soluble insect-resistant extract of bitter gourd leaves using deionized water, ether or acetone as an extraction solvent, its preparation and its application in insect resistance and insecticide.

Background technique:

Insect pests reduce the yield of crops in a large amount, so it is of great significance to develop new pollution-free insecticides. Although the extensive use of chemical pesticides today can control the occurrence and development of agricultural pests in a timely manner, it has brought many disadvantages, such as the perennial use of chemical pesticides, which makes pests resistant to pesticides, and the residue of pesticides makes the pollution of farmland and agricultural products more and more serious. Human beings have brought great potential threats, causing great harm to human health and the environment, especially the vegetables and plants that people eat directly, are more likely to be directly victimized by chemical pesticides; and the cultivation of insect-resistant transgenic plants is expected to avoid pesticides. pests and reduce the losses caused by insect pests to agriculture, but the long cycle and high cost make the progress in this area relatively lagging behind. Therefore, there is an urgent need for the development of biopesticides, the development and screening of biocontrol agents.

Bitter melon is a kind of natural vegetable crop, which is not only edible, but also plays an important role in medicine, agriculture and basic biological research. However, the leaves of bitter gourd are regarded as waste and have not been developed and utilized. So far, there is no research report on the development of biological insecticides using bitter gourd leaves as raw materials.

Contents of the invention

The problem that the present invention solves:

The invention uses waste bitter gourd leaves to provide a new type of insect-resistant biological pesticide with low cost, strong insect resistance, wide insect resistance spectrum, simple and convenient preparation method and water solubility. It overcomes the hazards of chemical pesticide toxicity, and lays the foundation for the development of new biopesticides and their industrialization.

Technical solutions

Bitter melon leaf anti-insect extract, which is a green liquid containing protein, which uses the waste-bitter melon (Momordica charantia L.) leaves as a resource, adds an extraction solvent, grinds, extracts by standing, shakes, filters and extracts at a constant volume ;

Properties of this insect-resistant extract:

Solubility: the extract is water-soluble and can be mixed with water in any ratio;

Anti-insect spectrum: It has broad-spectrum anti-insect effect and insecticidal effect, especially effective against pests with chewing mouthparts.

Wherein, the extraction solvent is deionized water, ether (≥99%) or acetone (≥95%). When deionized water is used as the extraction solvent, the extract contains proteins with molecular weights of 22KD, 23KD, 24KD, 25KD, 28KD and 35KD; 38KD, 40KD, 65KD and 66KD proteins; when acetone is used as the extraction solvent, the extract contains proteins with molecular weights of 31KD, 32KD and 33KD. The anti-insect extract has good stability at 0-40°C.

The preparation method of the anti-insect extract of bitter gourd leaves is to use the waste-bitter gourd leaves as a resource, add an extraction solvent, grind, stand for extraction, vibrate, filter and extract at a constant volume. The specific method is as follows:

Use fresh balsam pear leaves or dry powder of balsam pear leaves, the ratio of balsam pear leaves to extraction solvent is 1g: 1ml, control the pH to 6.5-7.0, and grind within the range of temperature 0-4°C, then use static extraction for 0.5h, and use 20r /min, vibrate for 8-10 hours, filter, and then dilute to 2 times the amount of extraction solvent with natural water to prepare the anti-insect extract stock solution of balsam pear leaves.

It should be noted:

The extraction solvent is selected from deionized water, diethyl ether (≥99%) or acetone (≥95%).

The use of the anti-insect extract of bitter gourd leaves is in the application of insect resistance and insecticide. The anti-insect extract can be widely used in biological control of vegetable pests, and is particularly suitable for biological control of chewing mouthparts pests. Its use method is: dilute the anti-insect extract stock solution of bitter gourd leaves with water to make the concentration 30-50%, and spray it on the crops. good.

Beneficial effect:

The invention adopts waste-bitter melon leaves as resources to develop a novel biological insecticide. It has strong insect resistance, broad insect resistance spectrum, and good stability at 0-40°C. The stock solution obtained after extraction can be used directly, or it can be made into a concentrated solution, and it can also be processed into a powder, which is convenient for storage and transportation. The preparation method of the insecticide is simple, fast and low in cost. This new insect-resistant biological agent can be dissolved in water at any ratio, is convenient to use, and has remarkable insect-resistant and insecticidal effects. The bitter melon leaf ether insect-resistant extract stock solution was prepared into different concentrations (30%, 40%, 50%), and after it was used to treat the larvae of diamondback moth (Plutella xylostella L.), the larvae of diamondback moth were stunted, unresponsive, and curled. , its curling degree increases with the increase of treatment concentration, and its curling rate is respectively 47.5%, 49.1%, 77.5%; Mortality rate (including curling rate) is respectively 79%, 79.6%, 90-100%. The acetone insect-resistant extract stock solution of bitter melon leaves was prepared into seed concentrations (30%, 40%, 50%) respectively, and after using them to treat Plutella xylostella larvae, the curling rates of test insects were 37.1%, 40.0%, 42.0% respectively; (Curl rate included) were 60.0%, 74.0%, and 78.0%, respectively. After the anti-insect extract stock solution of bitter melon leaf deionized water was prepared into different concentrations (30%, 40%, 50%) to deal with diamondback moth larvae, the curling rates of test insects were 30%, 35%, and 53% respectively; Curl rate) were 30%, 40%, and 70%, respectively. The bitter melon leaf ether anti-insect extract stock solution was prepared to a concentration of 50%, and after it was used to treat cabbage caterpillar (Artogela (pieris) rapae Linnaeus), the curling rate of the test insect was 42.5%; the mortality rate (including the curling rate) was 72.5%. The stock solution of the acetone anti-insect extract was prepared to a concentration of 50%, and after it was used to treat the cabbage worm, the curl rate of the test insect was 35.5%; the mortality rate (including the curl rate) was 70.5%. The deionized water insect-resistant extract stock solution of bitter gourd leaves was prepared to a concentration of 50%, and after it was used to treat cabbage caterpillars, the curling rate of the test insects was 40%; the mortality rate (including the curling rate) was 68%. The anti-insect extraction stock solution of bitter melon leaves with ether is prepared at a concentration of 50%. After treating Pieris rapae with it, the curling rate of the pupal stage is 20% and the mortality rate is 40%. Prepare a 50% acetone anti-insect extract from bitter gourd leaves, and use it to treat cabbage caterpillars, the curling rate of pupae is 20%; the mortality rate is 40%. The deionized water insect-resistant extract of bitter gourd leaves with a concentration of 50% is prepared, and after the cabbage caterpillar is treated, the pupal curl rate is 20% and the mortality rate is 80%. The above experiments prove that the extract has anti-insect and insect-killing effects, and when the concentration is 45-50%, the anti-insect and insect-killing effects are better when sprayed on crops, and can be widely used in the control of vegetable pests. The product not only overcomes the hazards of chemical pesticide toxicity, but also adopts bitter gourd waste as a resource, which has very low cost and is easy to prepare, thus laying the foundation for the development of new insecticides for biological control of pests. The extract has broad development potential and application prospects in biological control of pests, production of non-polluting crops and vegetables, etc., and has great advantages in future development and application. The invention will realize the industrialization of the biological control of plant pests and the research and development of biological pesticides, has important theoretical significance and application value, and has both economic benefits and social benefits.

Description of drawings:

Figure 1: Two-dimensional electrophoresis of proteins contained in the anti-insect extract of bitter gourd leaves with ether as the extraction solvent;

Figure 2: Two-dimensional electrophoresis of proteins contained in the anti-insect extract of bitter gourd leaves using acetone as the extraction solvent;

Figure 3: Two-dimensional electrophoresis of proteins contained in the anti-insect extract of bitter gourd leaves using deionized water as the extraction solvent;

Figure 4: Effect diagram of treating Pieris rapae with bitter melon leaf insect-resistant extract whose extraction solvent is ether;

Figure 5: Effect diagram of treating Pieris rapae with anti-insect extract from bitter gourd leaves whose extraction solvent is acetone;

Figure 6: Effect diagram of treating Pieris rapae with anti-insect extract from bitter gourd leaves using deionized water as the extraction solvent.

Detailed ways

The substantive features and practicability of the present invention are embodied by the following examples, but the scope of the present invention should not be limited thereto.

Example 1:

The method for preparing the anti-insect extract of bitter gourd leaves by using the waste—the leaves of bitter gourd as resources, comprises the following steps:

·Using diethyl ether as the extraction solvent, take 50g of fresh bitter gourd leaves, 50ml of diethyl ether (≥99%) according to the ratio of 1g:1ml, grind under the conditions of pH 7 and temperature 0°C, extract by standing for 0.5h, and use gyration Oscillate at 20r/min for 8-10h to filter, then dilute to 100ml with natural water as stock solution for later use.

·Performance testing:

The anti-insect extract prepared above was taken for detection. Figure 1 shows the two-dimensional electrophoresis of the proteins contained in the anti-insect extract of bitter gourd leaves using ether as the extraction solvent. After the leaves of bitter gourd were extracted with ether, 2D-SDS-PAGE was used for protein detection. Between PI 5.0-7.0, seven protein spots with molecular weights of 26KD, 27KD, 29KD, 38KD, 40KD, 65KD and 66KD were detected, as shown in Figure 1 indicated by the middle arrow. The above shows that the extract obtained in this example contains proteins with molecular weights of 26KD, 27KD, 29KD, 38KD, 40KD, 65KD and 66KD. The extract is a dark green water-soluble liquid, which can be concentrated or diluted, and can be mixed with water in any ratio. Its anti-insect effect is still stable when stored at 0-40°C for one year.

Application experiment 1-2:

Dilute the bitter melon leaf ether insect-resistant extract stock solution prepared in Example 1 with water to make the concentration 50% (preparation method: take 50ml bitter melon leaf ether insect-resistant extract stock solution, add 50ml water), smear it on the front of the cabbage leaf, and let it dry After drying, the test insects were reared. The second-instar larvae of diamondback moth (Plutella xylostella L.) were used as the total test insect source, and three replicates were set up. The food intake of the test worms decreased on the first day, the worm body was unresponsive on the second day, the worm body curled up on the third day, and died on the fourth day. The curling rate was 77.5% on the fifth day, and the mortality rate was 100%. Apply the same method to the front of cabbage leaves, dry and feed the first-instar cabbage caterpillars. The food intake of the test insects is reduced on the second day, and the response is slow. On the third day, curling occurs, and death occurs on the fourth day. The curling rate is 42.5 on the sixth day. %, the mortality rate is 72.5%, and its curling rate is 20% and the mortality rate is 40% at the pupal stage.

Application experiment 3:

Dilute the bitter melon leaf ether insect-resistant extract stock solution prepared in Example 1 with water to make the concentration 40% (preparation method: take 40ml bitter melon leaf ether insect-resistant extract stock solution, add 60ml water), smear it on the front of the cabbage leaf, and let it dry After drying, the test insects were reared. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set up. The food intake of the test insects decreased on the first day, the worms were unresponsive on the second day, curled up on the third day, died on the fourth day, the rate of curling was 49.1% on the fifth day, and the mortality rate was 79.6%.

Application experiment 4:

Dilute the bitter melon leaf ether insect-resistant extract stock solution prepared in Example 1 with water so that its concentration is 30% (preparation method: take 30ml bitter melon leaf ether insect-resistant extract stock solution, add 70ml water), smear it on the front of the cabbage leaf, and let it dry in the air. After drying, the test insects were reared. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set up. On the second day, the food intake of the test insects decreased, on the third day the worms were unresponsive, curled up on the fourth day, died on the fifth day, the curling rate was 47.5% on the sixth day, and the mortality rate was 79%.

Example 2:

Acetone (≥99%) was used as the extraction solvent and shaken for 10 h, and the others were the same as in Example 1. After the leaves of bitter gourd were extracted with acetone, 2D-SDS-PAGE was used for protein detection. Between PI 6.5-7.5, three protein spots with molecular weights of 31KD, 32KD, and 33KD were detected, as shown by the arrows in Figure 2. The above shows that the extract obtained in this example contains proteins with molecular weights of 31KD, 32KD, and 33KD, and the extract is a light green water-soluble liquid. Others are with embodiment 1.

Application Lab 5-6:

The bitter melon leaf acetone anti-insect extract stoste prepared in Example 2 is diluted with water so that its concentration is 50%

(i.e. take 50ml of bitter melon leaf acetone anti-insect extract stock solution, add 50ml of water), smear it on the front of cabbage leaves, dry and raise test insects. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set up. The food intake of the test insects decreased on the first day, the response of the worms was slow on the second day, the worms curled up on the third day, and died on the fourth day. The curling rate was 42.0% on the fifth day, and the mortality rate was 78.0%. Apply the same method above to the front of the cabbage leaves, and raise the first-instar cabbage caterpillars after drying. On the second day, the food intake of the test insects decreased, and the response was slow. On the third day, curls appeared, and on the fourth day, death occurred. On the sixth day, the rate of curling was 35.5%, and the mortality rate was 70.5%.

Application experiment 7:

Dilute the bitter melon leaf acetone insect-resistant extract stock solution prepared in Example 2 with water to make the concentration 40% (that is, take 40ml bitter melon leaf acetone insect-resistant extract stock solution and add 60ml water), apply it on the front of the cabbage leaf, and dry it Rearing test insects. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set up. The food intake of the test insects decreased on the first day, the worms were unresponsive on the second day, curled up on the third day, died on the fourth day, the rate of curling was 40.0% on the fifth day, and the mortality rate was 74.0%.

Application Experiment 8

Dilute the bitter melon leaf acetone insect-resistant extract stock solution prepared in Example 2 with water to make the concentration 30% (that is, take 30ml of bitter melon leaf acetone insect-resistant extract stock solution and add 70ml of water), smear it on the front of the cabbage leaf, and dry it Rearing test insects. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set for each. On the second day, the food intake of the test insects decreased, on the third day the worms were unresponsive, on the fourth day they curled up, on the fifth day they died, on the sixth day the rate of curling was 42.0%, and the mortality rate was 78%.

Example 3:

Adopt deionized water as extraction solvent, grind at pH 6.8, 4 ℃, other are the same as embodiment 1. After the bitter gourd leaves were extracted with deionized water, 2D-SDS-PAGE was used for protein detection. Between PI 6.5-7.5, six protein spots with molecular weights of 22KD, 23KD, 24KD, 25KD, 28KD and 35KD were detected, as shown in Figure 3 indicated by the middle arrow. The above shows that the extract obtained in this example contains proteins with molecular weights of 22KD, 23KD, 24KD, 25KD, 28KD and 35KD. The extract is a light green water-soluble liquid.

Applied Labs 9-10:

Dilute the bitter gourd leaf deionized water insect-resistant extract stock solution prepared in Example 3 with water to make the concentration 50% (that is, take 50ml of bitter gourd leaf deionized water insect-resistant extract stock solution and add 50ml of water), and smear it on the front of cabbage leaves , and reared the test insects after drying. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set up. The food intake of the test insects decreased on the first day, the worm body was unresponsive on the second day, the worm body curled up on the third day, and died on the fourth day, the curling rate was 53% on the fifth day, and the mortality rate was 70%. Apply the same method above to the front of cabbage leaves, and feed the first-instar cabbage caterpillars after drying. The food intake decreases on the second day and the response is slow. Curls appear on the third day, death occurs on the fourth day, and the curling rate is 40% on the sixth day. , The mortality rate was 68%.

Application Lab 11:

Dilute the bitter gourd leaf deionized water insect-resistant extract stock solution prepared in Example 3 with water to make its concentration 40% (that is, take 40ml of bitter gourd leaf deionized water insect-resistant extract stock solution and add 60ml of water), and smear it on the front of cabbage leaves , and reared the test insects after drying. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set up. The food intake of the test insects decreased on the first day, the worms were unresponsive on the second day, curled up on the third day, died on the fourth day, curled up to 35% on the fifth day, and died 40%.

Application Lab 12:

Dilute the bitter gourd leaf deionized water insect-resistant extract stock solution prepared in Example 3 with water to make the concentration 30% (i.e. take 30ml of bitter gourd leaf deionized water insect-resistant extract stock solution and add 70ml of water), and smear it on the front of cabbage leaves , and reared the test insects after drying. The second instar larvae of Plutella xylostella were used as the total test insect source, and three replicates were set up. On the second day, the food intake of the test insects decreased, on the third day, the worm body was unresponsive, on the fourth day, curling occurred, and on the fifth day, death occurred. On the sixth day, the curling rate was 30%, and the mortality rate was 30%.

The above experiments show that the anti-insect extracts of balsam pear prepared by three kinds of solvents all have anti-insect and insecticidal effects; % Among the three concentrations, the anti-insect effect is better when the concentration is 50%. In conclusion, bitter gourd leaf extract has strong resistance to pests and is a good new agricultural biological insecticide.

Claims (7)

1. momordica charantic leaf insect-resisting extract, it is characterized in that it is to utilize discarded object---balsam pear (Momordicacharantia L.) blade is a resource, add to extract solvent, leaves standstill the green liquid that contains protein that extraction, vibration, filtration and constant volume extract by grinding, adopting;

The characteristic of this insect-resisting extract:

Dissolubility: extract has water-soluble, and can mix with water with any ratio;

Pest-resistant spectrum: pest-resistant effect and insecticidal effect, particularly effective to the insect of biting mouthparts with wide spectrum.

2. according to the described momordica charantic leaf insect-resisting extract of claim 1, it is characterized in that extracting solvent is deionized water, ether (〉=99%) or acetone (〉=95%); When extracting solvent employing deionized water, contain the protein that molecular weight is 22KD, 23KD, 24KD, 25KD, 28KD and 35KD in the extract; When extracting solvent employing ether, contain the protein that molecular weight is 26KD, 27KD, 29KD, 38KD, 40KD, 65KD and 66KD in the insect-resisting extract; When extracting solvent employing acetone, contain the protein that molecular weight is 31KD, 32KD and 33KD in the extract.

3. the preparation method of the described momordica charantic leaf insect-resisting extract of claim 1, it is characterized in that it is to utilize discarded object---balsam pear (Momordica charantia L.) blade is a resource, add to extract solvent, leave standstill by grinding, adopting that extraction, vibration, filtration and constant volume extract, concrete grammar is as follows:

With fresh momordica charantic leaf or momordica charantic leaf dry powder, momordica charantic leaf is 1g: 1ml with the ratio that extracts solvent, control pH grinds in 6.5-7.0, temperature 0-4 ℃ scope, employing is left standstill and is extracted 0.5h, and with behind the 20r/min vibration 8-10h, filter, be settled to 2 times extraction quantity of solvent again with nature water, promptly make momordica charantic leaf insect-resisting extract stoste.

4. according to the preparation method of the described momordica charantic leaf insect-resisting extract of claim 3, it is characterized in that extracting solvent and be selected from deionized water, ether (〉=99%) or acetone (〉=95%).

5. the application of the described momordica charantic leaf insect-resisting extract of claim 1 aspect pest-resistant and desinsection, it is characterized in that this insect-resisting extract is used for the biological control of vegetable-crop pest-insect, it makes usage be: with momordica charantic leaf insect-resisting extract stoste dilute with water, making its concentration is 30%-50%, is sprayed on the crop.

According to the described momordica charantic leaf insect-resisting extract of claim 5 in the application aspect pest-resistant and the desinsection, it is characterized in that being used for the biological control of pests with chewing mouthparts.

7, according to the described momordica charantic leaf insect-resisting extract of claim 5 in the application aspect pest-resistant and the desinsection, it is characterized in that with momordica charantic leaf insect-resisting extract stoste dilute with water, making its concentration is 45%-50%, is sprayed on the crop.

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