CN101156604A - Extract of Baiqian and its preparation method and application - Google Patents

Abstract

The invention discloses a Radix Radix Radix Extract and a preparation method thereof. The Radix Rhizome extract of the present invention is prepared according to the following method: extract Rhizoma Rhizome with acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L), and the extract obtained is washed with chloroform Extraction is carried out, and the chloroform layer is collected to obtain the extract of Baiqian. The Radix Phytophthora extract prepared by the method of the invention has high non-selective antifeedant activity to the Lepidoptera pests. The insecticide prepared by using the Radix Radix Radix Extract of the invention has the advantages of environmental protection, low toxicity and harmlessness, etc., and meets the goal of sustainable development. In order to make full use of the existing medicinal plant resources in our country to develop one or more botanical insecticides or antibacterial agents, it provides a feasible key technology, in order to expand the application range of botanical insecticides or antibacterial agents in our country, and improve the quality of botanical insecticides. Provide new technical support for the killing effect of pests or antibacterial agents.

Description

Extract of Baiqian and its preparation method and application

technical field

The present invention relates to Radix Radix Radix Extract and its preparation method and application.

Background technique

The research and utilization of botanical insecticides, fungicides and herbicides has always been valued by countries all over the world. At present, there are relatively many studies on botanical insecticides, among which pyrethrins and azadirachtin have been relatively successful. Artificially planted neem trees have spread all over the world, and the output value of azadirachtin pesticides has exceeded hundreds of millions of dollars. Neem trees are considered to be the tree of hope to solve global chemical pesticide pollution. In addition, medicinal plants such as pyrethrum have been widely used in organic production in developed countries, and their artificial planting technology and active ingredient extraction technology are being studied in depth. At present, scientists from all over the world are looking for effective plant resources in order to develop more efficient botanical insecticidal fungicides and herbicides.

Cynanchum komarovii Al.Iljinski, Cynanchumstauntonii (Decne.) Schltr.ex Levl. and C. glaucescens (Decne.) Hand.-Mazz. et al. Baiqian is used as a traditional Chinese medicine to reduce qi, clear phlegm and relieve cough. Its plant form is as follows: erect semi-shrub, 30-50cm high; cylindrical stem with slightly thin edges; opposite leaves, oblong, 3-5cm long, 4-14mm wide, wedge-shaped at the base, acuminate at the tip, and veins on the back Slightly elevated; cymes axillary, with 3-8 flowers; calyx 5 deeply lobed; corolla purple-red, radial, villous on the inside, lobes narrowly triangular; lobes of the corona elliptic, 5 deeply lobed; stamens 5, with Pistils conjoined to form stamen column, anther 2 chambers; stigma slightly convex, wrapped in anther film; apophysis solitary.

Contents of the invention

The purpose of the present invention is to provide a Radix Radix Radix Extract and its preparation method.

The Radix Radix Radix Extract provided by the present invention is prepared according to the following method: Extract Radix Rhizoma Rhizome with acid ethanol, extract the obtained extract with chloroform, collect the chloroform layer, and obtain the Radix Radix Radix Extract;

The acid ethanol is a solution composed of inorganic acid and ethanol;

The molar concentration of the inorganic acid is 12mol/L, and the molar concentration of ethanol is 17mol/L;

The inorganic acid can specifically be hydrochloric acid or sulfuric acid.

In the extraction, the ratio of the white root and acid ethanol is 1g white root: 5-20ml acid ethanol, preferably 1g white root: 5-8ml acid ethanol or 1g white root: 15-20ml acid ethanol, especially Preferably 1g Baiqian: 15ml acid ethanol;

In the above method, the extraction time is 12-96 hours, preferably 48-96 hours, especially preferably 72 hours;

In the above method, the number of extractions is 1-5 times, preferably 4 times.

In the above method, in order to improve the extraction effect, it is best to pulverize the white root before soaking, and the particle size of the obtained particles is 6-40 mesh, preferably 16 mesh.

In the above method, the proportioning of Baiqian Baiqian and acid ethanol is 1g Baiqian Baiqian: 15ml acid ethanol, the extraction time is 72 hours, the number of times of extraction is 4 times, and the particle diameter of Baiqian Baiqian granules is 16 mesh.

In the above method, the white front can be the above-ground part or the underground part of the plant, and can also be the whole plant; the white front can be the old melon head Cynanchum komarovii Al.Iljinski.

The leaching temperature in the above method may be 25-35°C, and the extraction temperature may be 25-35°C.

The method also includes a purification step.

The purification step is to repeat the following method 1-2 times: dissolving the Radix Rhizoma Radix in 0.5-0.6 mol/L hydrochloric acid aqueous solution, extracting with chloroform, collecting the water layer, adding ammonia water to adjust the pH value to 9-10, Then, it was extracted with chloroform, and the chloroform layer was recovered, and the chloroform was removed.

Experiments have proved that the non-selective antifeedant rate of 0.25 mg/ml of the crude extract of Plutella xylostella prepared by the method of the present invention is 98.27% to 3rd instar larvae of diamondback moth, and the nonselective antifeedant rate to Spodoptera litura is 95.88%.

Another object of the present invention is to provide an insecticide.

In the insecticide provided by the present invention, its active ingredient is the above-mentioned Radix Rhizoma Radix Extract.

The Rhizoma radix extract prepared by the method of the invention has good insecticidal effect on Lepidoptera pests, such as Spodoptera litura.

According to the biological characteristics of the plants of the genus Radix, the present invention conducts in-depth research on the process conditions such as different parts of the plant, material particle size, extraction solvent, extraction time, extraction times, and separation technology, and finds out the extraction and preparation of its alkaloids. craft. The Baiqian extract prepared by the invention has high non-selective antifeedant activity to the lepidopteran pests. The insecticide prepared by using the Radix Radix Radix Extract of the invention has the advantages of environmental protection, low toxicity and harmlessness, etc., and meets the goal of sustainable development. The present invention provides a feasible key technology for developing one or more botanical insecticides or antibacterial agents by making full use of the existing medicinal plant resources in my country, and for expanding the application range of botanical insecticides or antibacterial agents in my country and improving plant It provides new technical support for the killing effect of sexual insecticides or antibacterial agents, and provides new technical means for the comprehensive development and utilization of such Chinese herbal medicine plants.

Description of drawings

Figure 1 is the effect of material particle size on the refined extract of Baiqian

Figure 2 is the effect of the solid-to-liquid ratio of the extract on the refined extract of Baiqian

Figure 3 is the effect of extraction time on the refined extract of Baiqian

Figure 4 is the impact of extraction times on the refined extract of Baiqian

Detailed ways

Embodiment 1, the preparation of Bai Qian extract

The white front used in this embodiment is old melon head (Cynanchum komarovii Al.Iljinski), carries out following experiment with the plant that harvests after maturity. Wherein, the aerial part of the plant includes stems, leaves, and fruits, and the underground part of the plant consists of roots.

1. Selection of extraction solvent, separation method, and plant parts

1. The influence of extraction solvent on the refined extract of Baiqian

Use 2L denatured alcohol (the volume percent concentration of ethanol is 96%), 2L acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L), 2L acid water (0.15mol/L hydrochloric acid aqueous solution) , 2L absolute ethanol four kinds of extraction solvents, respectively extract 0.2kg of the whole plant of Baiqian 6 mesh dry powder for 72 hours, extract 4 times, and mix the extracts of 4 times to obtain an extract. Dissolve the extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, remove the chloroform, and obtain white Before the crude extract, the experiment was set in triplicate.

The content of crude extract and refined extract of Radix Rhizoma Radix in extract was determined by test tube detection method. Weigh the weight of the clean triangular flask, put the chloroform extract containing the Baiqian extract in the bottle and spin dry to remove the chloroform, then weigh the total weight, and subtract the two to get the crude extract weight. The yield of crude extract=(mass of crude extract/mass of Baiqian dry powder)×100%. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and remove the chloroform to obtain It is the refined extract of Baiqian.

0.2kg of dry powder of Baiqian whole plant 6 mesh was extracted with four kinds of extraction solvents respectively. The results showed that 70.74g of extract could be obtained by extracting with industrial alcohol. The 70.74g of extract was further extracted to obtain 0.284g of crude extract Extraction, the crude extract extraction rate is 0.142%, and then can obtain 0.078g refined extract through refining; Extract with acid water, can obtain 95.31g extract, and extract obtains 0.330g crude extract through further extraction, again through Refined to obtain 0.116g of refined extract; extracted with absolute ethanol to obtain 13.73g of extract, the extract was further extracted to obtain 0.036g of crude extract, and then refined to obtain 0.012g of refined extract; extracted with acid ethanol , 105.75g extract can be obtained, the extract is further extracted to obtain 0.303g crude extract, and then refined to obtain 0.110g refined extract. It shows that when acidic water is used as the extraction solvent, although the yield of crude and refined extracts is relatively high, the soaking and extraction process is troublesome, so it is not used. When acid ethanol is used as extraction solvent, the yields of its extract, thick and refined extracts are all higher, the extraction rate of crude extract is 0.152%, the extraction rate of refined extract is 0.055%, and the process flow is simple, Therefore, it is better to use acid ethanol as the extraction solvent.

According to the following method, the effect of refined extracts of Radix radiata extracted with different extraction solvents on the antifeedant rate of Spodoptera litura was determined. Using the traditional leaf disc method, 0.25 mg of refined extracts extracted with different extraction solvents were dissolved in 100 ml of absolute ethanol into a 0.0025 mg/ml solution, and then clean cabbage leaves were selected, and the leaf discs were punched out with a puncher. The leaf discs were soaked in the above-mentioned refined extract dissolved in ethanol for 1 second, then taken out, placed on a clean table to dry naturally, and then placed in a 9cm petri dish with moisturizing filter paper; The leaves of cabbage were used as the control, and 4 leaves per dish were put into 1 head of the third instar Spodoptera litura. After 24h and 48h, the remaining leaf area was measured with a leaf area measuring instrument, and the feeding leaf area and antifeeding rate were calculated accordingly. Selective food refusal rate=(control group feeding leaf area-treatment group feeding leaf area)÷(treatment group feeding leaf area+control group feeding leaf area). The experiment was repeated 3 times. The results are shown in Table 1, and the data in Table 1 are the mean ± standard deviation of three repetitions.

Table 1 The effect of different extraction solvents on the antifeedant rate of Spodoptera litura

  Extraction solvent  Feed refusal rate (%)X±SE   Industrial Alcohol Acid Glycolic Acid Water Anhydrous Ethanol 67.38±24.3492.83±6.1271.01±13.9927.06±30.15

The results showed that when acid ethanol was used as the extraction solvent, the refined extract of Rhizoma alba had a greater effect on the antifeedant rate of Spodoptera litura, so it is better to use acid ethanol as the extraction solvent.

2. The effect of separation technology on the content of the refined extract of Baiqian

With 2L of acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L) leaching 0.2kg Baiqian whole plant 6 mesh dry powder 72 hours, leaching 4 times, the leaching solution of 4 times mix. Divide the extract into two parts, add 1L of 0.5-0.6mol/L hydrochloric acid aqueous solution to the above extract to dissolve one part, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, and then use Extract with chloroform, recover the chloroform layer, spin dry to remove chloroform, and obtain the crude extract of Baiqian. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform , to obtain the refined extract of Baiqian, as a result, 0.137g of the refined extract was obtained. Another part of the extract was centrifuged at a speed of 10000r/min for 10 minutes in a high-speed centrifuge to collect the precipitate, dissolved in chloroform, filtered and spin-dried in chloroform to obtain 0.080 g of refined extract. The experiment was repeated three times.

The results showed that 0.137±0.0024g (mean±standard deviation) refined extract was obtained by extraction, and 0.080±0.0015g (mean±standard deviation) refined extract was obtained by centrifugation. It shows that when two methods of extraction and centrifugation are used for extraction, the content of the refined extract of Radix Rhizoma Radix obtained by the extraction method is much greater than that obtained by the centrifugal method. After extraction and centrifugation, detect the content of the extract in the remaining liquid with bioactive component chromogen (iodine-potassium iodide and bismuth potassium iodide), the results show that the extract of Baiqian is almost completely extracted by the extraction method; The centrifugation method is not complete for the extraction of the white extract, and there are still a lot of extract residues. It cannot be completely extracted by adjusting the speed of the centrifuge and extending the centrifugation time.

According to the method of step 1, the antifeedant rate of the refined extract obtained by extraction and centrifugation to Spodoptera litura was determined. The experiment was repeated three times. Table 2 shows the effect of the refined extract of 0.0025mg/ml on the selective antifeedant rate of Spodoptera litura.

Table 2 The effect of different separation techniques on the antifeedant rate of Spodoptera litura refined extracts

Extraction Method  Feed refusal rate (%)X±SE Extraction 89.70±8.439 Centrifugal 71.01±9.345

The results showed that the refined extract of A. litura obtained by the extraction method had a great influence on the antifeedant rate of Spodoptera litura, so it is better to use the extraction method to separate the refined extract of A. litura.

3. The effect of the extraction part on the antifeedant activity of Spodoptera litura

Use 0.5L of acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L) to leach respectively 0.05kg of white front ground and underground part 6 mesh dry powder, leaching 4 times, each time 72 hours, Mix the 4 extractions, add 1 L of 0.5-0.6 mol/L hydrochloric acid aqueous solution to the above extraction solution, extract with chloroform, collect the water layer, add ammonia water to adjust its pH value to 9-10, and then extract with chloroform, The chloroform layer was recovered and spin-dried to remove chloroform to obtain the crude extract of Baiqian. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform , to obtain the refined extract of Baiqian, and the experiment was repeated three times. The results showed that 0.05kg of Baiqian underground part 6 mesh dry powder was extracted with acid ethanol to obtain 20.718g of extract, further extracted to obtain 0.280g of crude extract, and then refined to obtain 0.125g of refined extract; 0.05kg of Baiqian aboveground part The 6-mesh dry powder was extracted by the same method to obtain 19.263g of extract, further extracted to obtain 0.120g of crude extract, and then refined to obtain 0.066g of refined extract.

According to the method of step 1, the effects of the refined extracts from the extracts obtained from the above-ground parts of Baiqian and the extracts obtained from the underground parts on the antifeedant rate of Spodoptera litura were respectively determined. The experiment was repeated three times. The effect of 0.0025mg/ml refined extract on the selective antifeedant rate of Spodoptera litura is shown in Table 3.

Table 3 Effects of materials from different parts on the antifeedant rate of Spodoptera litura

  Sites  Feed refusal rate (%)X±SE   Underground part Aboveground part 90.72±7.4087.94±9.10

The results showed that the refined extract content of the underground part of Baiqian was higher than that of the aerial part, which had a greater impact on the antifeedant rate of Spodoptera litura, so the underground part of Baiqian was selected as the experimental material.

2. Orthogonal experiment to determine the influence of various factors on the content of the refined extract of Baiqian

1. The effect of the solid-liquid ratio of Baiqian dry powder and extraction solvent on the refined extract

Use 0.25L, 0.4L, 0.5L, 0.75L and 1L of acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L) to extract 0.05kg of the 10-mesh dry powder of the underground part of Baiqian, Extract for 24 hours, extract once, add 1L of 0.5-0.6mol/L hydrochloric acid aqueous solution to the extract, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, and recover the chloroform layer , spin-dried to remove chloroform, and obtained the crude extract of Baiqian. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform , to obtain the refined extract of Baiqian, the quality of the refined extract is respectively 0.070g, 0.071g, 0.054g, 0.079g and 0.067g. The experiment was repeated three times. The results are shown in Figure 2. The results show that under different solid-to-liquid ratios, the content of the refined extract of Radix Rhizoma Radix has certain fluctuations. When the ratio of 1g Radix Radix Radix: 15ml of acid ethanol is used for extraction, the content of the refined extract is the highest, as shown in Fig. Values in 2 are the average of three replicates.

2. The influence of extraction time on the extraction of refined extracts

Use 0.5L acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L) leaching 0.05kg of 10 mesh dry powder of the underground part of Baiqian, the leaching time is set to 12h, 24h, 48h, 72h and 96h, extract once, and compare the content of the refined extract under different extraction times. Add 1L of 0.5-0.6mol/L hydrochloric acid aqueous solution to the above extract, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, spin dry to remove chloroform , to obtain the crude extract of Baiqian. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform , to obtain the refined extract of Baiqian, and the experiment was repeated three times. The results are shown in Figure 3. The results show that the extraction time has a greater impact on the content of the refined extract, and the content of the refined extract increases with the prolongation of the extraction time. After 72 hours of extraction, the content of the refined extract is the highest.

3. The influence of extraction times on the refined extract

Use 0.5L acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L) to extract 0.05kg of dry powder of 10 mesh in the underground part of Baiqian, extract for 24h, extract once, twice, three times times, 4 times and 5 times, and compare the content of the refined extract when the extraction times are different. Add 1L of 0.5-0.6mol/L hydrochloric acid aqueous solution to the above extract, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform. The crude extract of Baiqian was obtained. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform , to obtain the refined extract of Baiqian, and the experiment was repeated three times. The results are shown in Figure 4, the results show that with the increase of the number of times of extraction, the content of refined extracts in the extract of Baiqian is greatly improved, and the content of refined extracts increases with the increase of times of extraction, but in the extraction After the fifth extraction, the obtained refined extract decreased. It is speculated that the extraction times were too many, and the time was too long, which destroyed the components, resulting in a decrease in the refined extract content. When leaching for 4 times, the refined extract content in the leaching liquid reaches the highest.

4. The effect of particle size of Baiqian dry powder on the refined extract

Use 0.5L of acid ethanol (wherein the molar concentration of hydrochloric acid is 12mol/L, and the molar concentration of ethanol is 17mol/L) leaching each 0.05kg of dry powder of 6 mesh, 10 mesh, 16 mesh, 20 mesh and 40 mesh of the underground part of Baiqian, Extract for 24 hours, extract once, and compare the content of refined extract in the extract when the particle size of Baiqian dry powder is different. Add 1L of 0.5-0.6mol/L hydrochloric acid aqueous solution to the above extract, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform. The crude extract of Baiqian was obtained. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform , to obtain the refined extract of Baiqian, and the experiment was repeated three times. The results are shown in Figure 1. The results show that the influence of the dry powder particle size of Radix Rhizoma Rhizoma Rhizome on the content of refined extract is restricted by the above factors. When the 16-mesh Baiqian dry powder is extracted, the refined extract content in the extract is the highest.

5. Orthogonal experiment to determine better process conditions

According to the above single factor test results, L ₉ (3 ⁴ ) orthogonal table was designed, and the test results were analyzed to determine the best extraction conditions. The results are shown in Table 6, Table 7 and Table 8. Among them, A represents the particle size of Baiqian dry powder, B represents the solid-to-liquid ratio, C represents the extraction time, and D represents the number of extractions.

Table 6 Range analysis of multi-factor horizontal orthogonal results

Table 7 Factor variable table

factor variable level and its value The number of observations (N) A 1 (6 mesh) 2 (10 mesh) 3 (16 mesh) 333 B 1(1:8)2(1:10)3(1:15) 333 C 1(24h)2(48h)3(72h) 333 D. 1(2 times) 2(3 times) 3(4 times) 333

Note: The data in the brackets of the horizontal column is the value of the level.

Table 8 variance analysis table

As can be seen from the data in the above table, factors A, B, C, and D all have a significant impact on the content of the refined extract, and the primary and secondary order of the influence of the four factors on the refined extract is B>A>C>D. The best extraction condition is B ₁ A ₃ C ₃ D ₂ .

Example 2, the influence of the Radix Rhizome extract prepared by the present invention on the non-selective antifeedant activity of Lepidoptera pests

1. Preparation of Radix Radix Extract

1) After leaching 0.2 kg of Baiqian underground part 16-mesh dry powder with 2L acid ethanol (wherein the molar concentration of hydrochloric acid is 12 mol/L, and the molar concentration of ethanol is 17 mol/L), after 72 hours, collect the extract and residue respectively; The extraction solution is the first extraction solution with a volume of 1.5-1.6L.

2) The residue collected in step 1) was leached for 72 hours with 2L acid ethanol (wherein the molar concentration of hydrochloric acid was 12mol/L, and the molar concentration of ethanol was 17mol/L), and the leachate and residue were collected respectively; The liquid is the second extraction liquid with a volume of 1.6-1.8L.

3) The residue collected in step 2) was leached with 2L acid ethanol (wherein the molar concentration of hydrochloric acid was 12mol/L, and the molar concentration of ethanol was 17mol/L) for 72 hours, and the leachate and residue were collected respectively; The liquid is the third extraction liquid, with a volume of 1.7-1.8L.

4) The residue collected in step 3) was leached for 72 hours with 2L acid ethanol (wherein the molar concentration of hydrochloric acid was 12mol/L, and the molar concentration of ethanol was 17mol/L), and the leachate and residue were collected respectively; The liquid is the fourth extraction liquid with a volume of 1.8-1.9L.

5) After mixing the above four extracts, spin dry, add 2.5L of 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, and then use chloroform to Extract, recover the chloroform layer, spin dry to remove the chloroform, and obtain the crude extract of Baiqian. Dissolve the above crude extract in 0.5-0.6mol/L hydrochloric acid aqueous solution, extract with chloroform, collect the water layer, add ammonia water to adjust the pH value to 9-10, then extract with chloroform, recover the chloroform layer, and spin dry to remove the chloroform , to obtain the refined extract of Baiqian, and the experiment was repeated three times. The results showed that 0.2kg Baiqian underground part 16 mesh dry powder obtained a total of 0.382±0.0041g (mean±standard deviation) crude extract. The crude extract was purified to obtain a total of 0.187±0.0038 g (mean±standard deviation) of refined extract.

2. The effect of the crude extract of white front on the biological activity of Spodoptera litura

According to the following method, the influence of the crude extract prepared in step 1 on the non-selective antifeedant activity of Spodoptera litura was determined. Using the traditional leaf disk method, the crude extract extracted in step 1 was dissolved in absolute ethanol into solutions of different concentrations as shown in Table 9, and then the clean cabbage leaves were selected, and the leaf disk was punched out with a puncher, and the leaves The dish was soaked in the above-mentioned crude extracts of different concentrations for 1 sec, then taken out, placed on a clean table to dry naturally, and then the leaf dish was placed in a 9cm petri dish with moisturizing filter paper; Cabbage leaves were used as controls. With 4 leaves per dish, put 1 each of the third instar and the fifth instar Spodoptera litura. Each treatment was repeated 10 times. After 24h and 48h, the remaining leaf area was measured with a leaf area measuring instrument, and the feeding leaf area and antifeeding rate were calculated accordingly. Non-selective food refusal rate=(control group feeding leaf area-treatment group feeding leaf area)÷control group feeding leaf area. The measurement results are shown in Table 9. Area represents the area eaten up in the activity assay.

Table 9 The impact of Baiqian extract on the non-selective antifeedant activity of Lepidoptera pests

Extract concentration (mg/ml) 3rd instar Extract concentration (mg/ml) 5th instar Area (mm ² ) Feed refusal rate% Area (mm ² ) Feed refusal rate% 0.250.10.050.0250.01 Control (CK) 3.00±1.5510.40±2.6215.00±4.5940.60±4.3055.60±5.4372.80±5.05 95.88Aa85.71abAB79.40bB44.23cC23.63dD- 0.50.250.10.050.01 control (CK) 27.40±2.5066.00±4.69216.80±9.00393.60±12.27549.40±21.99687.00±22.93 96.01aA90.40bB68.44cC42.71dD20.03eE-

The data in the table are the mean ± SE of 10 repetitions; the data in the column of "antifeeding rate%" are marked with different letters, indicating that there is a significant difference at the 5% level by the LSD method, and the uppercase letters indicate that there is a limit difference at the 1% level.

The results showed that the crude extract prepared in Step 1 had strong non-selective antifeedant activity against Spodoptera litura larvae, and the antifeedant activity increased with the concentration of the crude extract, and the antifeedant activity against 3rd instar larvae Stronger than 5th instar larvae. The crude extract with a concentration of 0.5 mg/ml has a feed repelling rate of over 95% for the 5th instar larvae of Spodoptera litura; for the 3rd instar larvae, the feed repelling rate is over 95% when the concentration is 0.25 mg/ml. When the concentration is 0.05mg/ml, the rate of refusal to feed at the 5th age is only 42.71%, while the rate of refusal at the 3rd age has reached 79.40%. At a low concentration (0.01mg/ml), the antifeeding rates of the 3rd and 5th instar larvae are almost the same, and this concentration has no obvious antifeeding effect on the 3rd and 5th instar larvae.

3. The effect of the crude extract of Plutella xylostella on the biological activity of Plutella xylostella

The effect of the crude extract prepared in step 1 on the biological activity of Plutella xylostella was determined according to the following method.

(1) Determination of selective antifeedant activity

Fresh, flat and uniform cabbage leaves were punched into leaf discs with a puncher, soaked in ethanol dilutions of crude extracts of different concentrations for 1 second, and after the leaf discs were dried, put them into a petri dish with a diameter of 9 cm ( Inner pad filter paper for moisturizing), each dish is placed into each 4 leaves of treatment and control (absolute ethanol), arranged relatively. Put 10 3rd instar larvae of Plutella xylostella in each dish, and each concentration was repeated 3 times. After 24 hours, the remaining leaf area was measured with graph paper, and the feeding leaf area and antifeeding rate were calculated. The measurement results are shown in Table 10.

The impact of table 10 Baiqian crude extract on the selective antifeedant activity of diamondback moth

Concentration (mg/ml)  Feed refusal rate (%)X±SE 0.250.10.050.0250.01 98.2517±0.0897a94.8166±0.2355a85.7121±0.5178b63.5122±1.078c49.8034±1.279d

The data in the table are the mean ± SE of 3 repetitions.

The results showed that the crude extract in the Radix radix extract prepared by the method of Example 1 had strong antifeedant activity to the third instar larvae of Plutella xylostella xylostella, and the selective antifeedant activity increased with the increase of the concentration of the crude extract. The crude extracts with concentrations of 0.25mg/ml, 0.1mg/ml, 0.05mg/ml, 0.025mg/ml, and 0.01mg/ml had selective antifeedant rates of 98.25%, 94.81%, 85.71%, 63.51%, and 49.80%.

(2) Determination of non-selective antifeedant activity

Fresh, flat and uniform cabbage leaves were punched into leaf disks with a puncher, soaked in ethanol dilutions of different concentrations of crude extracts for 1 sec, and after the leaf disks were dried, put them into a 9cm-diameter petri dish ( Inner pad filter paper for moisturizing), each dish is placed into each 4 leaves of treatment and control (absolute ethanol), arranged relatively. Put 10 3rd instar larvae of Plutella xylostella in each dish, and each concentration was repeated 3 times. After 24 hours, the remaining leaf area was measured with graph paper. Calculate the feeding leaf area and the feeding refusal rate. The measurement results are shown in Table 11.

Table 11 The effect of the crude extract of Predella alba on the non-selective antifeedant activity of diamondback moth

Concentration (mg/ml)  Feed refusal rate (%)X±SE 0.25 98.2753±0.3128a

0.10.050.025 93.6829±0.1038b90.0357±1.0895b61.2948±3.7123c 0.01 56.7869±5.6839c

The data in the table are the mean ± SE of 3 repetitions.

The results showed that the crude extract of Plutella xylostella prepared by the method in Example 1 had strong antifeedant activity against the third instar larvae of Plutella xylostella, and the antifeedant activity increased with the increase of the concentration of the crude extract. The crude extracts with concentrations of 0.25mg/ml, 0.1mg/ml, 0.05mg/ml, 0.025mg/ml, and 0.01mg/ml have non-selective antifeedant rates of 98.27%, 93.68%, 90.04%, and 61.29%, respectively , 56.79%.

Claims (10)

1. the preparation method of a Rhizoma Cynanchi Stauntonii extract is that the leaching liquor that obtains extracts with chloroform with sour ethanol lixiviate Cynanchum glaucescens, collects chloroform layer, obtains Rhizoma Cynanchi Stauntonii extract;

The solution that described sour ethanol is made up of inorganic acid and ethanol;

The molar concentration of described inorganic acid is 12mol/L, and the molar concentration of described ethanol is 17mol/L.

2. preparation method according to claim 1 is characterized in that: described inorganic acid is hydrochloric acid or sulfuric acid;

In the described lixiviate, the proportioning of described Cynanchum glaucescens and sour ethanol is 1g Cynanchum glaucescens: a 5-20ml acid ethanol, is preferably 1g Cynanchum glaucescens: 5-8ml acid ethanol or 1g Cynanchum glaucescens: 15-20ml acid ethanol, especially is preferably 1g Cynanchum glaucescens: 15ml acid ethanol;

Described extraction time is 12-96 hour, is preferably 48-96 hour, especially is preferably 72 hours;

Described lixiviate number of times is 1-5 time, is preferably 4 times.

3. preparation method according to claim 1 and 2 is characterized in that: described Cynanchum glaucescens is pulverized before lixiviate, and the Cynanchum glaucescens particle grain size that obtains is the 6-40 order, is preferably 16 orders.

4. preparation method according to claim 3, it is characterized in that: in the described method, the proportioning of described Cynanchum glaucescens and sour ethanol is 1g Cynanchum glaucescens: a 15ml acid ethanol, and described extraction time is 72 hours, described lixiviate number of times is 4 times, and described Cynanchum glaucescens particle grain size is 16 orders.

5. according to arbitrary described preparation method among the claim 1-4, it is characterized in that: described Cynanchum glaucescens is acrial part, under ground portion or the whole plant of plant; Described Cynanchum glaucescens is Cynanchum Komarrivii AI Iijiniski Cynanchum komaroviiAl.Iljinski..

6. according to arbitrary described preparation method among the claim 1-5, it is characterized in that: the step that also comprises purifying in the described method.

7. preparation method according to claim 6, it is characterized in that: described purification step is for being repeated below method 1-2 time: described Rhizoma Cynanchi Stauntonii extract is dissolved in the 0.5-0.6mol/L aqueous hydrochloric acid solution, use chloroform extraction, collect water layer, add ammoniacal liquor adjusting pH value and be 9-10, with chloroform extraction, reclaim chloroform layer again, remove chloroform.

8. the Rhizoma Cynanchi Stauntonii extract that obtains by arbitrary described method among the claim 1-7.

9. insecticide, its active component is the described Rhizoma Cynanchi Stauntonii extract of claim 8.

10. insecticide according to claim 9 is characterized in that: the insecticidal spectrum of described insecticide is a lepidopterous insects, as prodenia litura or diamond-back moth.

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