CN116548475A

CN116548475A - Meicai extract and extraction method and application thereof

Info

Publication number: CN116548475A
Application number: CN202310521256.6A
Authority: CN
Inventors: 王琴; 段邓乐; 杨文华; 黄晟; 冯志强; 张阮冰
Original assignee: Zhongkai University of Agriculture and Engineering
Current assignee: Zhongkai University of Agriculture and Engineering
Priority date: 2023-05-09
Filing date: 2023-05-09
Publication date: 2023-08-08
Also published as: ZA202306930B

Abstract

The invention provides a preserved vegetable extract and its extraction process and use, wherein the process comprises immersing chopped preserved vegetable in ascorbic acid solution, steaming with vinegar, freezing the dried preserved vegetable at low temperature, grinding into preserved vegetable powder, immersing in ascorbic acid-acetic acid solution, stirring in microwave environment, charging anhydrous calcium chloride and dichloromethane, ultrasonic-assisted extraction, buchner funnel suction filtration, concentrating the extract to obtain primary extract, and using C ₁₈ Eluting with column, collecting eluate, concentrating to dryness, precipitating with ethanol, filtering with Buchner funnel, subjecting the filtrate to column chromatography, concentrating, dialyzing to remove salt, and lyophilizing to obtain herba Meicariae extract. The application comprises acidifying, grinding, microwave treating, ultrasonic extracting, concentrating twice, purifying, filtering, and chromatography to obtain herba Meicarii extract containing indole substancesThe Meicai extract can effectively improve the plant growth rate, inhibit plant pathogenic microorganisms and be beneficial to improving the plant growth.

Description

Meicai extract and extraction method and application thereof

Technical Field

The invention relates to the field of Meicai extraction and application, in particular to a Meicai extract and an extraction method and application thereof.

Background

The preserved vegetable is one of popular food materials in Guangdong area, and is prepared by multiple procedures of scalding, fermenting, airing, steam cooking and the like, has golden color and unique fragrance, contains rich dietary fibers, can be matched with various food materials to prepare delicious dishes, and has the effects of enhancing digestion, removing food stagnation and removing greasiness. The history of the preserved vegetable in the aspect of diet has been thousands of years, and on the basis of the history, the extract of the preserved vegetable is researched by the university of Xiangtan Wang Chao, and has oxidation resistance and antibacterial performance, and has antibacterial effects on four microorganisms of escherichia coli, bacillus subtilis, staphylococcus aureus and aspergillus niger and fresh-keeping effect on pork.

The current research on the Meicai is limited to the application of foods, the common extraction method is to crush the Meicai into powder, then heat-extracting the powder with absolute ethyl alcohol or obtain a Meicai dry alcohol extract by adopting a Soxhlet extraction method, then suction-filtering and concentrating under reduced pressure to obtain a brown extract, dissolving the extract by using distilled water, dispersing with ultrasonic assistance, sequentially carrying out fractional extraction on the obtained suspension by using two organic solvents with different polarities of petroleum ether and ethyl acetate, collecting petroleum ether phase, ethyl acetate phase extract and raffinate, and respectively carrying out spin-steaming, concentration and drying at 40 ℃ for later use. Because the traditional extraction method is too coarse, many components in the sample cannot be effectively extracted, and the main component in the extracted Meicai extract is plant polyphenol. The dried Meicai contains not only plant polyphenol but also other organic components such as indole thioglycoside degradation products, so that a novel extraction method of the Meicai extract is necessary to be researched, the method can greatly improve the retention rate of active ingredients in the Meicai extract, and the application field of the Meicai extract is favorably expanded.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a Meicai extract and an extraction method and application thereof.

In order to achieve the above object, the present invention adopts the following technical measures:

an extraction method of Meicai extract comprises the following steps:

step 1: cutting dried preserved vegetable into pieces, washing the pieces twice with distilled water, soaking and washing the pieces with 3% ascorbic acid solution for 15-60 s, and exposing the dried preserved vegetable to acetic acid vapor at 45-60 ℃ for steaming and smoking for 5-60 min;

step 2: freezing the fumigated dried preserved vegetable in a low-temperature environment of-120 ℃ to-196 ℃ for 18-24 hours, taking out, grinding the dried preserved vegetable into preserved vegetable powder at low temperature, soaking the preserved vegetable powder in an ascorbic acid-acetic acid solution, and stirring the preserved vegetable powder in a microwave environment of 300 MHz-300 kMHz for 1-5 hours;

step 3: adding anhydrous calcium chloride and dichloromethane with the same volume as that of an ascorbic acid-acetic acid solution, extracting for 1-3 hours with the aid of ultrasonic waves, carrying out suction filtration on a Buchner funnel, and washing filter residues with the dichloromethane to obtain an extracting solution;

step 4: placing the extract into a vacuum pressure-reducing concentrator for first concentration, recovering dichloromethane, raising the concentration temperature for second concentration to obtain extract, and air-drying with nitrogen to obtain primary extract;

step 5: activation of C with 15mL of methanol ₁₈ The column was equilibrated with deionized water and the primary extract was prepared with petroleum ether: ethyl acetate (V: v=9.1:0.9) was fully dissolved, poured into the sample after 3 washes, the leacheate was discarded, and petroleum ether was used: eluting with ethyl acetate (V: V=2:8), collecting eluate, concentrating to dry, precipitating with 95% ethanol for 5-10 hr, and vacuum filtering with Buchner funnel to obtain ethanol solution of extract;

step 6: filtering the ethanol solution of the obtained extract with microporous membrane with pore diameter of 0.45 μm, subjecting the filtrate to column chromatography, eluting with 0.1mol/L NaCl, collecting eluate in eluting peak region, concentrating, dialyzing to remove salt, and lyophilizing to obtain herba Meicariae extract.

In the step 1, the preserved vegetable is slightly acidified by 3% of ascorbic acid solution and acetic acid vapor before extraction, so that oxidation resistance of the preserved vegetable is improved, infection of microorganisms to the preserved vegetable can be reduced, in addition, indole substances (such as 7-oxindole, 3-diindole methane and 3-indolylacetonitrile) and indole thioglycoside degradation intermediate products are contained in indole thioglycoside degradation products, the indole thioglycoside degradation intermediate products are unstable, extraction difficulty is high, ascorbate and indole-3-methanol can be generated under the action of water and ascorbic acid, and under the conditions of heating and weak acid, indole-3-methanol can generate stable dimer 3, 3-diindole methane and other trimers, so that extraction and recovery are facilitated.

In the step 1, the fumigation temperature of the preserved vegetable is 45-60 ℃, so that indole substances are not destroyed due to the fact that the temperature is too high, and condensation of indole thioglycoside degradation intermediate products can be promoted to generate dimers and trimers; the use of fumigation rather than blanching can reduce the loss of degradation products of indolyl thioglycoside.

Specifically, the preserved vegetable can be paved in a steamer according to a single layer, 36-40% of acetic acid aqueous solution is arranged in the steamer below the steamer, the acetic acid evaporation is accelerated by heating at 45-60 ℃, the evaporated acetic acid can be adhered to the surface of the preserved vegetable, and the indole-group sulfatides degradation intermediate products of the preserved vegetable are promoted to be condensed to generate dimers and trimers.

Further, the preserved vegetable in the step 1 is prepared by the following steps:

step A: scalding potherb mustard in 3% ascorbic acid water solution at 40-60 ℃ for 5-10 s, taking out, airing for 0.5-2 h, and repeating the steps for three times;

and (B) step (B): putting the blanched potherb mustard in a steamer, steaming for 3-15 min by using steam, transferring the potherb mustard into a closed container, fermenting for 4-8 h, and airing the fermented potherb mustard in the sun for 1-5 days;

step C: and D, steaming the potherb mustard obtained in the step B for 50-80 min by using steam in a steamer, airing the potherb mustard in the sun for 1-2 days, and repeating the step three times.

The time for scalding potherb mustard each time is 5-10 s, the time is short, the conditions are acidic, the temperature is 40-60 ℃, the fibrous tissue and enzyme activity of potherb mustard can be prevented from being completely destroyed by scalding the potherb mustard for a single time, endogenous myrosinase of potherb mustard is activated, thioglycoside can be degraded, indole thioglycoside degradation products are generated, reaction conditions and sufficient time are provided for the thioglycoside degradation of potherb mustard by scalding the potherb mustard for a plurality of times, and indole thioglycoside degradation intermediate products and ascorbic acid generate indole dimers and trimers.

In the step 2, the dried preserved vegetable can be frozen for 18-24 hours in a liquid nitrogen environment at low temperature.

Further, in the step 2, an ascorbic acid-acetic acid solution was prepared by dissolving 10g of ascorbic acid in 100mL of deionized water, and then adding 100mg of EDTA and 30mL of glacial acetic acid and shaking.

Further, in the step 2, the mixed solution of the Meicai powder and the ascorbic acid-acetic acid is transferred to an XH-100A+ microwave catalytic synthesis extractor, and stirred for 1-5 h in a microwave environment of 300 MHz-300 kMHz. The ascorbic acid-acetic acid solution is subjected to strong oscillation under the action of a microwave field, so that hydrogen bonds among the Meicaria powder molecules are loosened, a cell fiber structure is broken down, the dissolution of components to be extracted and the permeation of solution molecules are accelerated, and meanwhile, the indole thioglycoside degradation intermediate products are fully subjected to condensation reaction.

Further, in the step 2, the volume ratio of the Meicai powder to the ascorbic acid-acetic acid solution is 1:10-50.

Further, in the step 3, the volume ratio of the anhydrous calcium chloride to the dichloromethane is 1:2-3.

Further, in the step 4, the temperature of the first concentration is 39 ℃ and the concentration pressure is 45-50 kPa; the temperature of the secondary concentration is 95-100 ℃, and the concentration pressure is 0.05-0.075 Mpa.

Further, in the step 6, the dialysis bag has a molecular weight cut-off of 3500Da.

Further, in the above-mentioned step 6, column chromatography was performed using a DEAE-52 column or a Sephadex G-50 column, eluting with 0.1mol/L NaCl at a flow rate of 2mL/min.

Further, the Meicai extract is obtained by adopting the extracting method of the Meicai extract.

Further, the application of the Meicai extract is as follows:

(1) Regulating the growth rate of plants;

(2) Preparing a formulation for regulating plant growth rate;

(3) Inhibiting plant pathogenic microorganisms;

(4) The preparation method is used for preparing the preparation for inhibiting plant pathogenic microorganisms.

Further, the invention provides the application of the Meicai extract in regulating the plant growth rate:

the using mode is as follows: the method of spraying is adopted: dissolving the Meicai extract in 50-200 times of water, spraying the Meicai extract on the soil where plants are planted, and uniformly spraying the Meicai extract for 1 time every 2-3 months; compared with watering, the plant growth rate is improved by more than 30 percent.

The second mode of use is: the method adopts water culture: dissolving the Meicai extract in 50-200 times of water to obtain Meicai extract solution, soaking the seeds in water with the water temperature of 20 ℃ for 10 minutes, and then soaking the seeds in the Meicai extract solution for culture. The seeds can be rice and wheat crop seeds (such as wheat, rice, barley, tartary buckwheat, sorghum, oat, etc.), bean seeds (such as soybean, broad bean, mung bean, red bean, peanut, etc.), rhizome seeds (such as sweet potato vine, potato tuber, onion, etc.). Compared with the traditional water culture medium, the preserved vegetable extract solution is used as the culture medium, the germination rate of seeds is improved by more than 20%, and the root system growth is more developed.

And the using mode is three: the method of foliage spraying is adopted: the Meicai extract is dissolved in 50-200 times of water to obtain Meicai extract solution, and the Meicai extract solution is sprayed on the leaf surfaces and the branches of crops, so that the crops grow more luxuriantly.

Further, the present invention provides the use of a Meicai extract for the preparation of a formulation for regulating plant growth rate: the preparation also comprises auxiliary materials, wherein the auxiliary materials can be common auxiliary materials in fertilizers or common auxiliary materials in the process of preparing plant growth promoters.

The specific auxiliary materials can be at least one selected from the following materials: grass ash, organic fertilizer, humic acid, glucose, potash fertilizer, nitrogenous fertilizer and phosphate fertilizer.

Further, the preparation can be prepared into powder, granule, capsule, liquid, tablet, pill, paste, emulsion, film, etc.

Further, the invention provides the use of Meicai extract for inhibiting phytopathogenic microorganisms:

the using mode is as follows: dissolving the Meicai extract in 50-200 times of water, irrigating the solution on the soil surface, and coating a transparent film on the soil surface, wherein the inhibition rate of pathogenic microorganisms in the soil reaches more than 65% by adopting the method;

the second mode of use is: after the Meicai extract is crushed into powder, the Meicai extract is uniformly spread on the surface of soil, 30kg of Meicai extract is spread on 1 mu of land, then the soil is turned over, water is used for irrigating until the soil is permeated, and the pathogenic microorganism inhibition rate in the soil reaches more than 55%.

And the using mode is three: dissolving the Meicai extract in 50-200 times of water to obtain Meicai extract solution, and spraying the Meicai extract solution on the leaf surfaces and the branches of crops.

Further, the present invention provides the use of a Meicai extract for the preparation of a formulation for inhibiting phytopathogenic microorganisms: the preparation also comprises auxiliary materials, wherein the auxiliary materials can be common auxiliary materials in sterilizing agents.

The invention has the beneficial effects that:

the invention provides a Meicai extract, an extraction method and application thereof, wherein the content of indole substances in the Meicai extract obtained through acidification, grinding, microwave treatment, ultrasonic extraction, twice concentration, purification, filtration and chromatography is greatly improved.

Drawings

FIG. 1 is a high performance liquid chromatogram of 3-indoleacetonitrile;

FIG. 2 is a high performance liquid chromatogram of Meicai extract of example 1;

FIG. 3 is a high performance liquid chromatogram of Meicai extract of example 2;

FIG. 4 is a high performance liquid chromatogram of Meicai extract of example 3;

FIG. 5 is a high performance liquid chromatogram of the Meicai extract of comparative example 1;

FIG. 6 is a high performance liquid chromatogram of the Meicai extract of comparative example 2;

FIG. 7 is a graph comparing colonies from each group of dishes in an antimicrobial test;

FIG. 8 is a graph showing comparison of vigor of soybean seedlings in various planting pots on the second to fourth days in the growth rate test.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the examples herein, an ascorbic acid-acetic acid solution was prepared by dissolving 10g of ascorbic acid in 100mL of deionized water, and then adding 100mg of EDTA and 30mL of glacial acetic acid and shaking.

Microwave catalytic synthesis extraction instrument: the extraction instrument is a computer microwave catalytic synthesis extraction instrument of the swan XH-100AXH-100A provided by Beijing swan technology development limited company.

Grinding at low temperature: the JXFSPRP-I cryogenic mill provided by Shanghai Jijing letter industry Co., ltd was used.

Ultralow temperature industrial refrigerator: TF-B60-1500L deep freezer seafood preservation box experimental industry freezer provided by Hangzhou Ai Pu instrument and equipment limited company.

Example 1:

an extraction method of Meicai extract comprises the following steps:

step 1: cutting dried Meicai into pieces, washing with distilled water twice, soaking in 3% ascorbic acid solution for 15s, and steaming the dried Meicai in 60 deg.C acetic acid vapor for 20min;

step 2: freezing the fumigated dried Meicai at low temperature in liquid nitrogen environment at-196 ℃ for 18 hours, taking out, grinding into Meicai powder at low temperature, soaking the Meicai powder in ascorbic acid-acetic acid solution with the volume ratio of the Meicai powder to the ascorbic acid-acetic acid solution being 1:30, transferring the mixed solution to a microwave catalytic synthesis extraction instrument, and stirring for 2 hours in a 500MHz microwave environment;

step 3: adding anhydrous calcium chloride and dichloromethane with the same volume as that of an ascorbic acid-acetic acid solution, wherein the volume ratio of the anhydrous calcium chloride to the dichloromethane is 1:2, extracting for 2 hours with the aid of ultrasonic waves, carrying out suction filtration on a Buchner funnel, and washing filter residues with the dichloromethane to obtain an extracting solution;

step 4: placing the extract into a vacuum pressure-reducing concentrator for primary concentration, wherein the primary concentration temperature is 39 ℃, the concentration pressure is 45kPa, recovering dichloromethane, then raising the concentration temperature for secondary concentration, the secondary concentration temperature is 95 ℃, the concentration pressure is 0.05Mpa, and then air-drying with nitrogen to obtain primary extract;

step 5: activation of C with 15mL of methanol ₁₈ The column was equilibrated with deionized water and the primary extract was prepared with petroleum ether: ethyl acetate (V: v=9.1:0.9) was fully dissolved, poured into the sample after 3 washes, the leacheate was discarded, and petroleum ether was used: eluting with ethyl acetate (V: v=2:8), collecting eluate, concentrating to dryness, precipitating with 95% ethanol for 6 hr, and vacuum filtering with buchner funnel to obtain ethanol solution of extract;

step 6: filtering the ethanol solution of the obtained extract with microporous membrane with pore diameter of 0.45 μm, subjecting the filtrate to column chromatography with Sephadex G-50 column chromatography, eluting with 0.1mol/L NaCl at flow rate of 2mL/min, collecting eluate in eluting peak region, concentrating, dialyzing with dialysis bag with molecular weight cut-off of 3500Da to remove salt, and lyophilizing to obtain herba Meicariae extract.

In this example, the Meicai in step 1 was prepared by the following steps:

step A: scalding potherb mustard in 3% ascorbic acid water solution at 50deg.C for 8s, taking out, airing for 1 hr, and repeating the steps for three times;

and (B) step (B): steaming and boiling potherb mustard in a steamer for 8min, transferring the potherb mustard to a closed container for fermentation for 8h, and airing the potherb mustard in sunlight for 3 days;

step C: and D, steaming the potherb mustard obtained in the step B in steam in a steamer for 60min, airing the potherb mustard in sunlight for 2 days, and repeating the step three times.

Example 2

An extraction method of Meicai extract comprises the following steps:

step 1: cutting dried Meicai into pieces, washing with distilled water twice, soaking in 3% ascorbic acid solution for 30s, and steaming the dried Meicai in 45 deg.C acetic acid vapor for 60min;

step 2: freezing the fumigated dried Meicai in an ultralow-temperature industrial refrigerator at the low temperature of-120 ℃ for 24 hours, taking out the dried Meicai, grinding the dried Meicai into Meicai powder at the low temperature, soaking the Meicai powder in an ascorbic acid-acetic acid solution, enabling the volume ratio of the Meicai powder to the ascorbic acid-acetic acid solution to be 1:20, transferring the mixed solution to a microwave catalytic synthesis extraction instrument, and stirring the mixed solution in a microwave environment of 300MHz for 3 hours;

step 3: adding anhydrous calcium chloride and dichloromethane with the same volume as that of an ascorbic acid-acetic acid solution, wherein the volume ratio of the anhydrous calcium chloride to the dichloromethane is 1:3, extracting for 1.5 hours with the assistance of ultrasonic waves, carrying out suction filtration on a Buchner funnel, and washing filter residues with the dichloromethane to obtain an extracting solution;

step 4: placing the extract into a vacuum pressure-reducing concentrator for primary concentration, wherein the primary concentration temperature is 39 ℃, the concentration pressure is 48kPa, recovering dichloromethane, then raising the concentration temperature for secondary concentration, wherein the secondary concentration temperature is 95 ℃, the concentration pressure is 0.06Mpa, and then air-drying with nitrogen to obtain primary extract;

step 5: activation of C with 15mL of methanol ₁₈ The column was equilibrated with deionized water and the primary extract was prepared with petroleum ether: acetic acid ethyl ester(V: v=9.1:0.9) was fully dissolved, poured into the sample in 3 washes, the leacheate was discarded, and petroleum ether was used: eluting with ethyl acetate (V: v=2:8), collecting eluate, concentrating to dryness, precipitating with 95% ethanol for 5 hr, and vacuum filtering with buchner funnel to obtain ethanol solution of extract;

In this example, the Meicai in step 1 was prepared by the following steps:

step A: scalding cleaned potherb mustard in 3% ascorbic acid water solution at 60deg.C for 5s, taking out, airing for 1.5 hr, and repeating the steps for three times;

and (B) step (B): steaming and boiling potherb mustard in a steamer for 15min, transferring the potherb mustard to a closed container for fermentation for 6h, and airing the potherb mustard in sunlight for 2 days;

step C: and D, steaming the potherb mustard obtained in the step B for 50min by using steam in a steamer, airing the potherb mustard in sunlight for 1 day, and repeating the step three times.

Example 3

An extraction method of Meicai extract comprises the following steps:

step 1: cutting dried Meicai into pieces, washing with distilled water twice, soaking in 3% ascorbic acid solution for 60s, and steaming the dried Meicai in acetic acid vapor at 50deg.C for 30min;

step 2: freezing the fumigated dried Meicai at low temperature in a liquid nitrogen environment at-196 ℃ for 20 hours, taking out the dried Meicai, grinding the dried Meicai into Meicai powder at low temperature, soaking the Meicai powder in an ascorbic acid-acetic acid solution, wherein the volume ratio of the Meicai powder to the ascorbic acid-acetic acid solution is 1:10, transferring the mixed solution to a microwave catalytic synthesis extraction instrument, and stirring the mixed solution in a microwave environment at 800MHz for 1 hour;

step 3: adding anhydrous calcium chloride and dichloromethane with the same volume as that of an ascorbic acid-acetic acid solution, wherein the volume ratio of the anhydrous calcium chloride to the dichloromethane is 1:2, extracting for 3 hours with the aid of ultrasonic waves, carrying out suction filtration on a Buchner funnel, and washing filter residues with the dichloromethane to obtain an extracting solution;

step 4: placing the extract into a vacuum pressure-reducing concentrator for primary concentration, wherein the primary concentration temperature is 39 ℃, the concentration pressure is 50kPa, after recovering dichloromethane, raising the concentration temperature for secondary concentration, the secondary concentration temperature is 100 ℃, the concentration pressure is 0.075Mpa, and after obtaining extract extractum, air-drying with nitrogen to obtain a primary extract;

step 5: activation of C with 15mL of methanol ₁₈ The column was equilibrated with deionized water and the primary extract was prepared with petroleum ether: ethyl acetate (V: v=9.1:0.9) was fully dissolved, poured into the sample after 3 washes, the leacheate was discarded, and petroleum ether was used: eluting with ethyl acetate (V: v=2:8), collecting eluate, concentrating to dryness, precipitating with 95% ethanol for 10 hr, and vacuum filtering with buchner funnel to obtain ethanol solution of extract;

step 6: filtering the ethanol solution of the obtained extract with microporous membrane with pore diameter of 0.45 μm, subjecting the filtrate to column chromatography with DEAE-52 column chromatography, eluting with 0.1mol/L NaCl at flow rate of 2mL/min, collecting eluate in eluting peak region, concentrating, dialyzing with dialysis bag with molecular weight cut-off of 3500Da to remove salt, and lyophilizing to obtain Meicai extract.

In this example, the Meicai in step 1 was prepared by the following steps:

step A: scalding potherb mustard in 3% ascorbic acid water solution at 40deg.C for 10s, taking out, airing for 2 hr, and repeating the steps for three times;

and (B) step (B): steaming and boiling potherb mustard in a steamer for 15min, transferring the potherb mustard to a closed container for fermentation for 4h, and airing the potherb mustard in sunlight for 1 day;

step C: and D, steaming the potherb mustard obtained in the step B for 70min by using steam in a steamer, airing the potherb mustard in sunlight for 2 days, and repeating the step three times.

Comparative example 1

An extraction method of Meicai extract comprises the following steps:

(1) Pulverizing dried preserved vegetable into powder by high-speed universal pulverizer;

(2) Adding absolute ethyl alcohol according to a feed liquid ratio of 1:5 (w/v), oscillating and leaching for 4 hours in a constant-temperature water bath at 60 ℃, carrying out suction filtration, repeatedly extracting filter residues according to the conditions, combining the two filtrates after suction filtration, and carrying out reduced pressure rotary steaming at 40 ℃ to obtain a brown dried vegetable ethanol extract;

(3) Adding a certain amount of distilled water, performing ultrasonic assisted dispersion, extracting the obtained suspension with dichloromethane, collecting organic phase extract, and concentrating and drying the organic phase extract at 40deg.C to obtain herba Meicariae extract.

The preserved vegetable in the step 1 is specifically prepared by the following steps:

step A: scalding potherb mustard in water at 95-100 ℃ for 10min, taking out, transferring into a closed container, fermenting for 12h, and airing the potherb mustard in sunlight for 5 days;

and (B) step (B): and D, steaming the potherb mustard obtained in the step A in steam in a steamer for 70min, and airing the potherb mustard in sunlight for 5 days.

Comparative example 2

The present comparative example differs from the above comparative example 1 in that the preparation method of the preserved vegetable is different, wherein the preserved vegetable obtained in the step 1 of preparing the preserved vegetable of example 1 is used.

Performance testing

Test for measuring effective ingredient in extract

The test method comprises the following steps: and (3) determining the types and the contents of the compounds contained in the dried Meicai extract by adopting an Agilent 1200 type high performance liquid chromatograph. Each sample was prepared with chromatographic grade anhydrous methanol to give a sample solution of 5mg/ml, the standard was prepared to give a standard sample solution of 0.2mg/ml, and each sample was filtered with a membrane filter having a pore size of 0.45. Mu.m, before sample introduction. The chromatographic column is C ₁₈ A chromatographic column. Mobile phase: methanol-4% acetic acid (35:65), flow rate of L ml/min, column temperature of 40deg.C, sample injection amount of 10 μl, detection wavelength of 261nm, and various samples were obtained by comparisonThe number of peaks present in the sample and determining whether a peak of the standard is present.

Standard solution: placing 0.02g of 3-indoleacetonitrile into a 20ml volumetric flask, adding a mobile phase to dissolve and dilute to a scale, shaking uniformly to prepare a 1.0mg/ml 3-indoleacetonitrile solution, placing 1ml of 3-indoleacetonitrile solution into a 100ml volumetric flask, adding the mobile phase to dissolve and dilute to the scale, and preparing a 20 mu g/ml standard substance solution;

sample solution: taking 0.02g of Meicai extract prepared in examples 1-3 and comparative examples 1-2, placing in a 20ml volumetric flask, adding mobile phase to dissolve and dilute to scale, shaking uniformly to prepare 1.0mg/ml extract solution, placing 1ml extract solution in a 100ml volumetric flask, adding mobile phase to dissolve and dilute to scale, and preparing 20 mug/ml standard solution;

test results: reference is made to fig. 2-6.

From the figures, the samples of examples 1 to 3 have a certain difference in the number of HPLC peaks, retention time, and response value from those of comparative examples 1 to 2. The effective peak numbers and peak response values of examples 1 to 3 are more than those of comparative examples 1 and 2, and it is proved that the Meicai extracts obtained in examples 1 to 3 of the present application have more effective components than those of comparative examples 1 and 2, whereas comparative example 1 has the least peak numbers and low peak response values. 3-indoleacetonitrile was used as a standard (chromatogram refer to FIG. 1), and the presence or absence of 3-indoleacetonitrile in each sample was identified based on the retention time at which the peak occurred, and it was found that 3-indoleacetonitrile was present in each of examples 1 to 3 and comparative example 2, but the peak response value of comparative example 2 was lower than that of examples 1 to 3.

Antibacterial test

The test method comprises the following steps:

(1) Selecting brown colloid of a cabbage rotting part, inoculating to 50ml of LB liquid medium, and culturing at 25deg.C for 24 hr to obtain bacterial suspension;

(2) Adding 1ml of bacterial suspension into 19ml of sterilized normal saline for dilution, placing on a shaking table for shaking for 10min at 200r/min, and subpackaging into 6 test tubes, wherein each test tube contains 1.5ml of bacterial suspension, and the tube walls of the 6 test tubes are respectively marked with 1-6;

(3) Respectively labeling 7-12 on the covers of 6 culture dishes, respectively adding 20ml of melted agar culture medium into each culture dish, sterilizing at 121 ℃ for 20min, cooling and solidifying, and placing on an ultra-clean workbench;

(4) Taking 1g of Meicai extract of each of examples 1-3 and comparative examples 1-2, respectively dissolving in 100ml of deionized water, obtaining samples 1-5 at a concentration of 10mg/ml, respectively adding 0.5ml of samples 1-5 into test tubes marked with 1-5, respectively adding 0.5ml of deionized water into test tubes marked with 6, and shaking uniformly;

(5) Respectively taking 1ml of sample liquid from test tubes with the reference numbers of 1-6, pouring the sample liquid into culture dishes with the reference numbers of 7-12 correspondingly, and oscillating the culture dishes to uniformly cover the sample liquid on the surface of the agar culture medium;

(6) The dishes were placed in an incubator at 25℃for 48 hours, the number of colonies in each dish was counted, and the antibacterial rate was calculated.

Test results: refer to fig. 7.

As can be seen from the figure, the culture dishes with reference number 12 do not contain the Meicai extract, the colonies in the culture medium are densely distributed, the colonies in the culture dishes with reference number 10 and reference number 11 are far more than the culture dishes with reference number 7 to 9, although the colonies in the culture dishes with reference number 12 are less than the culture dishes with reference number 12, and the colony count in each culture dish is calculated, and the results are shown in the following table 1, and it can be seen that the Meicai extracts of examples 1 to 3 have antibacterial effects:

table 1 shows the colony count of each dish in the bacteriostasis test

Component (A)	Colony count	Antibacterial rate
			Culture dish 7 (sample 1)	14	95.3％
Culture dish 8 (sample 2)	12	95.9％
			Culture dish 9 (sample 3)	19	93.5％
Culture dish 10 (sample 4)	193	34.8％
			Culture dish 11 (sample 5)	149	49.6％
Culture dish 12 (deionized water)	296	0％

Growth Rate test

The test method comprises the following steps:

(1) Sub-packaging pure plant type culture substrates provided by Nanjing perennial root flower plant garden limited company into 4 planting pots, wherein each planting pot contains 200g of culture substrates, and respectively planting 20 peas into the 4 planting pots;

(2) 100g of the Meicai extracts of the examples 1-2 and the comparative examples 1-2 are respectively dissolved in 1000ml of water to obtain water culture agents 1-4, 4 planting pots are distributed in a shape of a 'field' and are divided into two layers, 50ml of the water culture agents 4 are irrigated by the planting pot on the right of the upper layer, 50ml of the water culture agents 3 are irrigated by the planting pot on the left, 50ml of the water culture agents 1 are irrigated by the planting pot on the right of the lower layer, 50ml of the water culture agents 2 are irrigated by the planting pot on the left, 1 time a day is irrigated, and the growth vigor of bean seedlings in the planting pots on the second day, the third day and the fourth day is compared.

Test results: refer to fig. 8.

From the graph, in the planting pot irrigated by the water culture agent 1 and the water culture agent 2, the germination rate and the growth vigor of peas are obviously better than those of peas irrigated by the water culture agent 3 and the water culture agent 4 in the same growth time and growth environment, so that the preserved vegetable extract has obvious effect on regulating the plant growth rate.

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.

Claims

1. The extraction method of the Meicai extract is characterized by comprising the following steps of:

2. The method for extracting a Meicai extract according to claim 1, wherein the Meicai in step 1 is prepared by:

3. The method according to claim 1, wherein the ascorbic acid-acetic acid solution in step 2 is prepared by dissolving 10g of ascorbic acid in 100mL of deionized water, and adding 100mg of EDTA and 30mL of glacial acetic acid and shaking.

4. The method according to claim 1, wherein in the step 2, the volume ratio of the Meicai powder to the ascorbic acid-acetic acid solution is 1:10-50.

5. The method according to claim 1, wherein in the step 3, the volume ratio of anhydrous calcium chloride to dichloromethane is 1:2-3.

6. The method according to claim 1, wherein in the step 4, the temperature of the first concentration is 39 ℃ and the concentration pressure is 45 to 50kPa; the temperature of the secondary concentration is 95-100 ℃, and the concentration pressure is 0.05-0.075 Mpa.

7. The method according to claim 1, wherein in the step 5, the alcohol concentration of the alcohol precipitation is 60% -80%, and the alcohol precipitation temperature is 2-6 ℃.

8. The method according to claim 1, wherein in the step 6, the dialysis bag has a molecular weight cut-off of 3500Da.

9. A potentilla anserina extract characterized in that it is obtained by the extraction method of the potentilla anserina extract according to any one of claims 1 to 8.

10. Use of a Meicai extract according to claim 9 in any of the following:

(1) Regulating the growth rate of plants;

(2) Preparing a formulation for regulating plant growth rate;

(3) Inhibiting plant pathogenic microorganisms;