Preparation process and application of celandine alkaloid for preventing and treating plant diseases

Abstract

The invention discloses a preparation process and application of celandine alkaloid for preventing and treating plant diseases, on the basis of a single factor test, polynomial fitting regression is carried out by adopting a Box-Behnken response surface method and taking oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, ultrasonic time and extraction temperature as influence factors to obtain a regression equation, and the regression equation is used as a regression equation to obtain the celandine alkaloid for preventing and treating the plant diseases. The optimal total alkaloid extraction method is as follows: the oxalic acid concentration is 0.6%, the liquid-material ratio is 17: 1, the ethanol concentration is 70%, the ultrasonic time is 47min, the extraction temperature is 52 DEG C, and the extraction yield is 9.20 mg / mL. According to the method, strong acid is replaced with weak acid in the extraction process of the celandine total alkaloids, corrosion to equipment in the production process is reduced, production safety is improved, environmental safety is improved, the extraction process is optimized through a response surface method, and the product yield is increased. An indoor biological assay test finds that the celandine total alkaloid has good bacteriostatic activity and plant virus resisting activity, and has the potential of being further developed into a botanical fungicide and an antiviral agent.

Description

Technical Field

[0001] The invention belongs to the technical field of plant secondary metabolite extraction, and particularly relates to a preparation process and application of chelidonium alkaloids for preventing and treating plant diseases. Background Art

[0002] Alkaloids are a class of nitrogen-containing alkaline organic compounds that are widely present in organisms. Most of them are complex ring structures and are generally in the form of colorless crystals or powders. They are usually insoluble or poorly soluble in water. A large number of studies have shown that alkaloid compounds are important active ingredients in many traditional Chinese medicines, with antioxidant, anti-inflammatory, antiviral and other activities, and have great potential in the fields of medicine, chemical industry, agriculture, etc.

[0003] Greater celandine (Chelidonium majus L.) is a plant of the genus Greater celandine in the family Papaveraceae. It is also known as Babujin, Tuhuanglian, Dihuanglian, and Duanchangcao. It was first recorded in the "Jiuhuang Bencao". It tastes bitter, is cool in nature, and is poisonous. The whole plant can be used as medicine, and has pharmacological activities such as analgesia, cough relief, diuresis, and detoxification. Studies have shown that its alkaloids have antibacterial, antiviral, and insecticidal activities, laying the foundation for the development of new environmentally friendly botanical pesticides.

[0004] In the common extraction method of total alkaloids of chelidonium, hydrochloric acid is often used to assist extraction, so that the alkaloids react with acid to generate salts that are dissolved in water and then reduced with alkali to increase the extraction rate. However, the use of strong acid water in production will corrode the equipment and pollute the environment, increasing costs.

[0005] Due to the excessive use of chemical pesticides and other reasons, the occurrence of excessive pesticide residues in agricultural products is common, and resistant strains continue to appear in the field, and the problem of difficult control of plant viruses in the field is becoming increasingly serious. Non-polluting pesticides with the characteristics of low residue, easy degradation, environmental friendliness, and multiple targets are one of the important means of preventing and controlling plant diseases and are the key to achieving sustainable agricultural development. Botanical pesticides are an important part of biological pesticides. Summary of the invention

[0006] The present invention provides a preparation process and application of chelidonium alkaloids for preventing and controlling plant diseases, uses oxalic acid instead of hydrochloric acid to assist in extracting total alkaloids from chelidonium, and applies it to preventing and controlling agricultural pathogens and plant viruses, exploring its research and development potential. The present invention uses chelidonium as a raw material, uses ultrasonic extraction, and on the basis of a single factor experiment, uses the Box-Behnken response surface method, takes the effects of five factors, oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, ultrasonic time, and extraction temperature, on the total alkaloid yield as parameters, performs polynomial fitting regression analysis, and obtains the best extraction method.

[0007] In order to achieve the above technical objectives, the technical solutions adopted by the present invention include:

[0008] A preparation process of chelidonium alkaloids for preventing and treating plant diseases, the preparation process comprising: crushing chelidonium, adding ethanol to perform heating ultrasonic extraction, filtering, and spinning the filtrate to dryness; adding oxalic acid solution to re-dissolve, performing heating ultrasonic extraction, filtering, taking the filtrate to adjust the pH to alkaline, standing, and then extracting with dichloromethane and spinning to dryness to obtain total chelidonium alkaloids.

[0009] Optionally, the volume concentration of the ethanol is 55% to 95%; the mass concentration of the oxalic acid solution is 0.1% to 0.9%; the liquid-to-solid ratio of ethanol to celandine is (10 to 30) mL: 1 g;

[0010] The ultrasonic power of the heating ultrasonic extraction is 40kHz, the ultrasonic time is 30-90min, and the ultrasonic extraction temperature is 30-70℃.

[0011] Optionally, the mass concentration of the oxalic acid solution is 0.3%, 0.5% or 0.7%.

[0012] Optionally, the liquid-to-solid ratio of ethanol to celandine is 15 mL:1 g.

[0013] Optionally, the volume concentration of the ethanol is 65%.

[0014] Optionally, the ultrasonic time is 45 min and the ultrasonic extraction temperature is 50°C.

[0015] Optionally, the volume concentration of the ethanol is 70%; the mass concentration of the oxalic acid solution is 0.6%; the liquid-to-solid ratio of ethanol to celandine is 17 mL: 1 g;

[0016] The ultrasonic extraction time of the heating ultrasonic extraction is 47 minutes, and the ultrasonic extraction temperature is 52°C.

[0017] Optionally, the preparation process specifically includes:

[0018] The celandine was crushed and passed through a 40-mesh sieve. 70% ethanol with a volume concentration of 17:1 mL / g was added to the obtained celandine powder. Ultrasonic extraction was performed at 52°C for 47 minutes. The mixture was filtered and the filtrate was spin-dried. A 0.6% oxalic acid aqueous solution with a liquid-to-solid ratio of 50:1 mL / g was added and ultrasonic extraction was performed at 50°C for 30 minutes. The mixture was filtered and the filtrate was taken and the pH value of the filtrate was adjusted to 11 with a 10% NaOH solution. The mixture was allowed to stand and extracted with dichloromethane for 3 times. The dichloromethane layers were combined, the dichloromethane was recovered, and the mixture was spin-dried to obtain the total alkaloids of celandine.

[0019] Optionally, the preparation process specifically includes:

[0020] 1) putting the celandine into a grinder and grinding it to obtain celandine powder;

[0021] 2) Using ethanol as the extraction solvent to perform ultrasonic extraction on celandine powder, and determining the optimal values ​​of oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, ultrasonic time and extraction temperature by single factor experiment with the total alkaloid yield of celandine as the index;

[0022] 3) According to the single factor experimental test results, the Box-Behnken response surface method was used, with oxalic acid concentration A, liquid-to-solid ratio B, ethanol concentration C, ultrasonic time D and extraction temperature E as independent variables, and total alkaloid yield Y as an indicator, to perform polynomial fitting regression and obtain a regression equation; the regression equation is:

[0023] Y=9.02+0.2031A+0.2775B+1.10C+0.0544D+0.3644E-0.0675AB+0.0200AC-0.2550AD-0.0750AE-0.1100BC +0.3075BD-0.1650BE+0.0675CD-0.2850CE+0.2275DE-0.5829A2-0.2854B2-1.02C2-0.6046D2-0.4713E2;

[0024] Where Y is the total alkaloid yield, the independent variable A is the oxalic acid concentration, B is the liquid-to-solid ratio, C is the ethanol concentration, D is the ultrasonic time, and E is the extraction temperature;

[0025] 4) calculating the regression equation to obtain the optimal extraction conditions of chelidonium alkaloids;

[0026] 5) Under the optimal extraction method conditions, ultrasonic extraction is performed on chelidonium to obtain total alkaloids of chelidonium.

[0027] The chelidonium alkaloids prepared by the preparation process of the chelidonium alkaloids for preventing and controlling plant diseases of the present invention are used for preparing drugs for preventing and controlling plant diseases, wherein the plant diseases are caused by the following pathogens, including:

[0028] Phytophthora capsici, Fusarium graminearum Schw, Gaeumannomyces graminis, Colletotrichum gloeosporioides, Rhizoctonia solani, Botrytis cinerea, Alternaria alternata, Sclerotiniasclerotiorum, Colletotrichum gloeosporioides, Valsamali, Physalospora piricola, Botrytis cinerea, Exserohilum turcicums, Fusarium solani, Colletotrichum orbiculare, Fusarium wilt oxysporum), tomato wilt pathogen (Fusarium oxysporum), cotton Verticillium dahliae (Verticillium dahliae), cucumber mosaic virus (cucumbermosaic virus, CMV) and potato virus Y (potato virus Y, PVY) at least one of.

[0029] Compared with the existing extraction technology, the technical solution of the present invention has the following beneficial effects:

[0030] The invention provides a method for optimizing oxalic acid-assisted extraction of total alkaloids in chelandia based on response surface methodology. Chelandia is used as a raw material, an acid extraction and alkali precipitation method is adopted, oxalic acid is used instead of commonly used hydrochloric acid to assist in the extraction of total alkaloids in chelandia, corrosion to extraction equipment and pollution to the environment are reduced, and based on this process, an ultrasonic extraction method is used to explore the influence of five factors, namely, oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, ultrasonic time and extraction temperature, on the total alkaloid yield, and on the basis of a single factor experiment, a five-factor three-level analysis is performed using the Box-Behnken response surface method, and polynomial fitting regression analysis and verification are performed to obtain an optimal method for optimizing oxalic acid-assisted extraction of total alkaloids in chelandia based on the response surface method.

[0031] In another aspect, the present invention provides a use of the total alkaloids of chelidonium as a pollution-free pesticide for preventing and controlling agricultural pathogens and plant viruses.

[0032] The agricultural pathogens and plant viruses of the present invention are not limited to pepper blight (Phytophthoracapsici), wheat fusarium (Fusarium graminearum Schw), wheat take-all pathogen (Gaeumannomycesgraminis), apple anthracnose (Colletotrichum gloeosporioides), rice sheath blight (Rhizoctonia solani), tomato gray mold (Botrytis cinerea), tobacco brown spot pathogen (Alternariaalternata), rapeseed sclerotinia (Sclerotinia sclerotiorum), mango anthracnose (Colletotrichumgloeosporioides), apple rot pathogen (Valsa mali), apple ring rot pathogen (Physalosporapiricola), strawberry gray mold (Botrytis cinerea), corn leaf blight pathogen (Exserohilum turcicums), potato dry rot pathogen (Fusarium solani), cucumber anthracnose pathogen (Colletotrichum orbiculare, Fusarium oxysporum, Fusarium oxysporum, Verticillium dahliae, cucumber mosaic virus (CMV), and potato virus Y (PVY). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0034] Figure 1 is the standard curve of chelerythrine;

[0035] Figure 2 This is the effect of oxalic acid concentration on the yield of total alkaloids from chelidonium;

[0036] Figure 3 This is the effect of liquid-solid ratio on the total alkaloid yield of chelidonium;

[0037] Figure 4 This is the effect of ethanol concentration on the yield of total alkaloids from chelidonium;

[0038] Figure 5This is the effect of ultrasound time on the total alkaloid yield of chelidonium;

[0039] Figure 6 This is the effect of extraction temperature on the yield of total alkaloids from chelidonium;

[0040] Figure 7 This is a diagram showing the protective effect of chelidonium alkaloids against wheat take-all disease;

[0041] Figure 8 This is a diagram showing the therapeutic effect of chelidonium alkaloids on wheat take-all disease;

[0042] Fig. 9 The protective activity of chelidonium alkaloids on Chenopodium album (anti-CMV);

[0043] Fig.10 The therapeutic activity of chelidonium alkaloids on Chenopodium album (anti-CMV). DETAILED DESCRIPTION

[0044] The following detailed description of the present invention is exemplary and does not limit the scope of the present invention. Based on the embodiments of the present invention, for ordinary technicians in the field, all uncreative improvements should be considered as the protection scope of the present invention without departing from the present invention.

[0045] The present invention provides a method for optimizing oxalic acid-assisted extraction of total alkaloids from celandine based on response surface methodology, comprising the following steps:

[0046] 1) putting the celandine into a grinder and grinding it to obtain celandine powder;

[0047] 2) Using ethanol as the extraction solvent to perform ultrasonic extraction on celandine powder, and determining the optimal values ​​of oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, ultrasonic time and extraction temperature by single factor experiment with the total alkaloid yield of celandine as the index;

[0048] 3) Based on the results of the single factor experiment, the Box-Behnken response surface methodology was used to analyze the relationship between oxalic acid concentration (A),

[0049] Liquid-to-solid ratio (B), ethanol concentration (C), ultrasonic time (D) and extraction temperature (E) were used as independent variables, and total alkaloid yield (Y) was used as an indicator. Polynomial fitting regression was performed to obtain the regression equation.

[0050] 4) calculating the regression equation to obtain the optimal extraction conditions of chelidonium alkaloids;

[0051] 5) Under the optimal extraction method conditions, ultrasonic extraction is performed on chelidonium to obtain total alkaloids of chelidonium.

[0052] As a preferred solution, the regression equation is:

[0053] Y=9.02+0.2031A+0.2775B+1.10C+0.0544D+0.3644E-0.0675AB+0.0200AC-0.2550AD -0.0750AE-0.1100BC+0.3075BD-0.1650BE+0.0675CD-0.2850CE+0.2275DE-0.5829A 2 -0.2854B 2 -1.02C 2 -0.6046D 2 -0.4713E 2 ;

[0054] Where Y is the total alkaloid yield, the independent variable A is the oxalic acid concentration, B is the liquid-to-solid ratio, C is the ethanol concentration, D is the ultrasonic time, and E is the extraction temperature.

[0055] According to the above regression equation, it can be seen that ethanol concentration (C) has the greatest impact on the total alkaloid yield. The quadratic term of ethanol concentration (C2), the interaction between liquid-to-solid ratio and ultrasonic time (BD), and the interaction between ethanol concentration and extraction temperature (CE) also have a great influence on the total alkaloid yield.

[0056] As a preferred solution, the extraction method of the single factor test is: crush and sieve the celandine, add ethanol, heat and ultrasonically extract, filter, spin-dry the filtrate, add oxalic acid water and ultrasonically extract at 50°C for 30 minutes, filter, take the filtrate and adjust the pH to 11 with 10% NaOH solution, let it stand, put it in a separatory funnel, extract it with dichloromethane, spin-dry, and obtain the total alkaloids of celandine.

[0057] As a preferred solution, in step 1), the celandine is crushed and then passed through a No. 3 sieve (40 meshes).

[0058] As a preferred solution, the oxalic acid concentration is 0.1% to 0.9%. When the oxalic acid concentration is 0.1% to 0.5%, as the oxalic acid concentration increases, the total alkaloid yield also increases. When the oxalic acid concentration is 0.5%, the yield reaches the maximum value. After that, the increase in oxalic acid concentration reduces the yield. This may be due to the excessively high acid concentration, which causes the alkaloids to decompose or precipitate, thereby affecting the extraction yield.

[0059] As a preferred solution, the liquid-to-solid ratio of ethanol to celandine during ultrasonic extraction is (10-30) mL:1 g. As the liquid-to-solid ratio increases, the total alkaloid yield gradually increases, because as the amount of ethanol increases, the contact area between celandine powder and ethanol gradually increases. When the liquid-to-solid ratio is 15 mL:1 g, the surface of celandine powder is completely coated with ethanol, and further increasing the amount of ethanol will increase the dissolution of flavonoids and other substances, affecting the extraction of alkaloids.

[0060] As a preferred solution, the ethanol concentration is 55% to 95%. When the ethanol concentration is 65%, the total alkaloid yield is the highest, which may be because when the ethanol concentration is 65%, its polarity is close to that of the total alkaloids of chelidonium, which is conducive to the dissolution of the total alkaloids.

[0061] As a preferred solution, the ultrasonic time is 30 to 90 minutes. Ultrasonic waves assist the dispersion and dissolution of substances in the solvent, thereby achieving rapid and efficient extraction and separation of substances. When the ultrasonic time is 45 minutes, the total alkaloid yield reaches the highest. When the ultrasonic time is too long, the dissolution of other substances may increase or the structure of the alkaloids may be destroyed.

[0062] As a preferred solution, the ultrasonic extraction temperature is 30-70° C. As the extraction temperature increases, the total alkaloid yield increases accordingly, and when the temperature is 50° C., it is suitable for the dissolution of total alkaloids.

[0063] As a preferred solution, it is characterized in that the optimal extraction method conditions are oxalic acid concentration of 0.6%, liquid-to-solid ratio of 17mL:1g, ethanol concentration of 70%, ultrasonic time of 47min, and extraction temperature of 52°C.

[0064] Embodiment 1:

[0065] Accurately weigh 3 mg of chelerythrine and dilute it to volume in a 25 mL volumetric flask with methanol to obtain a 120 μg / mL reference solution.

[0066] Precisely measure 0.5, 1, 1.5, 2, 2.5, and 3 mL in a 10 mL volumetric flask, dilute to the mark with methanol, shake well, use methanol as a blank control, and measure the absorbance at 288 nm. Draw a standard curve with the concentration of the chelerythrine reference substance (x, μg / mL) as the abscissa and the absorbance as the ordinate (y). The standard curve equation y=0.0187x+0.023 is obtained, and R2 is 0.9966, indicating that the concentration of the chelerythrine solution is in the range of 6 to 36 μg / mL, and the linear relationship between the concentration and the absorbance is good, such as Figure 1 shown.

[0067] Embodiment 2:

[0068] In the extraction process of total alkaloids of chelidonium, hydrochloric acid is often used to react with alkaloids to generate salts that are soluble in water. After removing non-alkaloids, it is further reduced with alkali to obtain alkaloids. However, hydrochloric acid, as a strong acid, is highly corrosive and can damage the equipment when in contact with production equipment, shortening the service life of the equipment. In addition, hydrochloric acid can also produce harmful waste liquid and waste gas, which can cause harm to the environment and the human body. The present invention uses a weak acid to replace the hydrochloric acid in the original production process to reduce the adverse effects of hydrochloric acid on equipment, environment and human body in the production process. The present invention selects oxalic acid, formic acid, acetic acid, citric acid, phosphoric acid and tartaric acid 6 kinds of weak acids to replace hydrochloric acid to extract chelidonium alkaloids, so as to select a suitable weak acid for application.

[0069] Determination of the yield of total alkaloids of chelidonium: Take 10g of the powder of this product (passed through No. 3 sieve, 40 mesh), accurately weigh, place in a conical flask, add 250mL of 95% ethanol, wait for the ultrasonic temperature to rise to 50°C, start ultrasonic extraction for 60min, filter, evaporate the filtrate to dryness, add 500mL of 0.5% acid aqueous solution, wait for the ultrasonic temperature to rise to 50°C, start ultrasonic extraction for 30min, take the filtrate and adjust the pH to 11 with 10% NaOH solution, let stand for 30min, place in a separating funnel, extract with dichloromethane 3 times, 200mL each time, combine the dichloromethane layers, recover dichloromethane, and obtain total alkaloids of chelidonium, dissolve and transfer with methanol in batches and make up to 50mL volumetric flask. Take 1mL from it and place it in a 50mL volumetric flask, add methanol to make up, use methanol as a blank control, measure the absorbance at 288nm, and calculate the yield of total alkaloids according to the standard curve of chelidonium red alkaloid and the following formula.

[0070] Total alkaloid yield (mg / g) = (C·V) / 1000M;

[0071] Where C is the total alkaloid mass concentration (μg / mL); V is the total alkaloid volume (mL); M is the powder mass (g).

[0072] Table 1 Effect of different types of weak acids replacing hydrochloric acid on the extraction rate

[0073]

[0074] Through the above screening, it can be seen that when oxalic acid is used instead of hydrochloric acid, the yield of total alkaloids from chelidonium is higher, so oxalic acid was selected for single factor and response surface experiments.

[0075] Embodiment 3:

[0076] In this example, five factors, namely, oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, ultrasonic time and extraction temperature, were set at five levels respectively to investigate the influence of each factor on the yield of total alkaloids from chelidonium.

[0077] Effect of oxalic acid concentration on the yield of total alkaloids: Take 10g of the powder (passed through a No. 3 sieve, 40 mesh), weigh it accurately, put it in a conical flask, add 250mL of 95% ethanol, and wait until the ultrasonic temperature rises to 50℃, start ultrasonication.

[0078] Ultrasonic extraction was performed for 60 minutes, filtered, and the filtrate was evaporated to dryness. 500 mL of aqueous oxalic acid solution with oxalic acid concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9% were added. When the ultrasonic temperature rises to 50°C, ultrasonic extraction was started for 30 minutes. The filtrate was taken and the pH was adjusted to 11 with 10% NaOH solution. It was allowed to stand for 30 minutes and placed in a separatory funnel. It was extracted with dichloromethane for 3 times, 200 mL each time. The dichloromethane layers were combined and the dichloromethane was recovered to obtain the total alkaloids of chelerythrine. The solution was transferred and diluted to a 50 mL volumetric flask with methanol in batches. Then 1 mL was taken from it and placed in a 50 mL volumetric flask, and methanol was added to make up the volume. Methanol was used as a blank control. The absorbance was measured at 288 nm, and the total alkaloid yield was calculated according to the chelerythrine standard curve and the formula shown in Example 2. Figure 2 As shown, when the oxalic acid concentration is 0.3%, 0.5%, and 0.7%, the total alkaloid yield is higher, and 0.5% is the optimal oxalic acid concentration.

[0079] Effect of liquid-to-solid ratio on the yield of total alkaloids: Take 10g of the powder of this product (passed through No. 3 sieve, 40 mesh), weigh it accurately, place it in a conical flask, add 95% ethanol with liquid-to-solid ratios of 10:1, 15:1, 20:1, 25:1, and 30:1mL / g, respectively, and start ultrasonic extraction when the ultrasonic temperature rises to 50℃ for 60min. Filter, evaporate the filtrate to dryness, add 500mL of 0.5% oxalic acid aqueous solution, and start ultrasonic extraction when the ultrasonic temperature rises to 50℃ for 30min. Take the filtrate and adjust the pH to 11 with 10% NaOH solution, let it stand for 30min, place it in a separatory funnel, extract it with dichloromethane for 3 times, 200mL each time, combine the dichloromethane layers, recover the dichloromethane, and obtain the total alkaloids of chelidonium, dissolve it in methanol in batches, transfer it, and make up to the volume in a 50mL volumetric flask. Take 1 mL of the solution and put it in a 50 mL volumetric flask, add methanol to make up the volume, use methanol as a blank control, and measure the absorbance at 288 nm.

[0080] The total alkaloid yield was calculated using the standard curve of chelerythrine and the formula shown in Example 2. Figure 3 As shown, when the liquid-to-solid ratio was 15:1 g / mL, the total alkaloid yield was the highest.

[0081] Effect of ethanol concentration on the yield of total alkaloids: Take 10g of the powder of this product (passed through No. 3 sieve, 40 mesh), accurately weigh it, place it in a conical flask, add 250mL of ethanol solution with concentrations of 55%, 65%, 75%, 85%, and 95%, respectively, wait for the ultrasonic temperature to rise to 50°C, start ultrasonic extraction for 60min, filter, evaporate the filtrate to dryness, add 500mL of 0.5% oxalic acid aqueous solution, wait for the ultrasonic temperature to rise to 50°C, start ultrasonic extraction for 30min, take the filtrate and adjust the pH to 11 with 10% NaOH solution, let it stand for 30min, place it in a separating funnel, extract it with dichloromethane 3 times, 200mL each time, combine the dichloromethane layers, recover the dichloromethane, and obtain the total alkaloids of chelidonium, dissolve it in methanol in batches, transfer it, and make up to the volume in a 50mL volumetric flask. Then take 1 mL of the solution into a 50 mL volumetric flask, add methanol to make up the amount, use methanol as a blank control, measure the absorbance at 288 nm, and calculate the total alkaloid yield based on the chelerythrine standard curve and the formula shown in Example 2. Figure 4 As shown, when the ethanol concentration was 65%, the total alkaloid yield was the highest.

[0082] Effect of ultrasonic time (ultrasonic power of 40kHz) on the yield of total alkaloids: Take 10g of the powder of this product (passed through No. 3 sieve, 40 mesh), accurately weigh it, put it in a conical flask, add 250mL of 95% ethanol, and start ultrasonic extraction when the ultrasonic temperature rises to 50°C for 30, 45, 60, 75, and 90min respectively. Filter, evaporate the filtrate to dryness, add 500mL of 0.5% oxalic acid aqueous solution, and start ultrasonic extraction when the ultrasonic temperature rises to 50°C for 30min. Take the filtrate and adjust the pH to 11 with 10% NaOH solution, let it stand for 30min, put it in a separating funnel, extract it with dichloromethane 3 times, 200mL each time, combine the dichloromethane layers, recover the dichloromethane, and obtain the total alkaloids of chelidonium, dissolve it in methanol in batches, transfer it, and make up to the volume in a 50mL volumetric flask. Then take 1 mL of the solution into a 50 mL volumetric flask, add methanol to make up the amount, use methanol as a blank control, measure the absorbance at 288 nm, and calculate the total alkaloid yield based on the chelerythrine standard curve and the formula shown in Example 2. Figure 5 As shown, the total alkaloid yield was the highest when the ultrasonic time was 45 min.

[0083] Effect of extraction temperature on the yield of total alkaloids: Take 10g of the powder of this product (passed through No. 3 sieve, 40 mesh), accurately weigh it, place it in a conical flask, add 250mL of 95% ethanol, the extraction temperatures are 30, 40, 50, 60, and 70°C, ultrasonic extraction is carried out for 60min, filtered, the filtrate is evaporated to dryness, 500mL of 0.5% oxalic acid aqueous solution is added, and ultrasonic extraction is started when the ultrasonic temperature rises to 50°C for 30min. The filtrate is taken and the pH is adjusted to 11 with 10% NaOH solution, allowed to stand for 30min, placed in a separatory funnel, extracted 3 times with dichloromethane, 200mL each time, the dichloromethane layers are combined, and the dichloromethane is recovered to obtain the total alkaloids of chelidonium, which are dissolved and transferred with methanol in batches and fixed to a 50mL volumetric flask. Then take 1 mL of the solution into a 50 mL volumetric flask, add methanol to make up the amount, use methanol as a blank control, measure the absorbance at 288 nm, and calculate the total alkaloid yield based on the chelerythrine standard curve and the formula shown in Example 2. Figure 6 As shown in the figure, when the extraction temperature is 50℃, the total alkaloid yield is the highest.

[0084] Embodiment 4:

[0085] On the basis of single factor experiment, Box-Behnken response surface methodology was used, with oxalic acid concentration (A), liquid-to-solid ratio (B), ethanol concentration (C), ultrasonic time (D) and extraction temperature (E) as influencing factors and total alkaloid yield (Y) as evaluation index. 43 groups of experiments were designed and analyzed using Design Expert 13 software. The response surface analysis factors and levels are shown in Table 2. The response surface experiment design and results of total alkaloid extraction from Chelidonium truncatum are shown in Table 3.

[0086] Table 2 Response surface analysis factors and levels

[0087]

[0088] Table 3 Response surface design and results of total alkaloid extraction of chelidonium

[0089]

[0090]

[0091]

[0092] Regression model establishment and variance analysis

[0093] The above experimental results were analyzed by variance analysis using Design Expert 13 software. The results of variance analysis are shown in Table 4.

[0094] Table 4 Analysis of variance of response surface test results for total alkaloid extraction of chelidonium

[0095]

[0096] Note: Significant difference, *P<0.05; extremely significant difference, **P<0.01.

[0097] Taking the total alkaloid extraction rate (Y) as the index, and the five factors of oxalic acid concentration (A), liquid-to-solid ratio (B), ethanol concentration (C), ultrasonic time (D) and extraction temperature (E) as independent variables, polynomial fitting regression was performed, and the regression equation was obtained as follows:

[0098] Y=9.02+0.2031A+0.2775B+1.10C+0.0544D+0.3644E-0.0675AB+0.0200AC-0.2550AD -0.0750AE-0.1100BC+0.3075BD-0.1650BE+0.0675CD-0.2850CE+0.2275DE-0.5829A 2 -0.2854B 2 -1.02C 2 -0.6046D 2 -0.4713E 2 ;

[0099] From the results of variance analysis in the above table, it can be seen that the total model P<0.0001, indicating that the response surface model has significant differences; the lack of fit term P=0.0591>0.05, indicating that the model has a good fit and the model is established. From the F value and P value, it can be seen that oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, extraction temperature, the interaction between liquid-to-solid ratio and ultrasonic time, the interaction between ethanol concentration and extraction temperature, and the quadratic terms of each factor have significant effects on the total alkaloid yield, and ethanol concentration has the most significant effect on it.

[0100] Response surface analysis

[0101] The effect of the interaction between factors on the extraction rate of total alkaloids from chelidonium can be analyzed by response surface and contour map. The steeper the slope of the response surface of the interaction between two factors, the greater the curvature of the contour line, and the more its shape tends to be elliptical, the stronger the interaction between the two factors, and the more significant the effect on the total alkaloid yield. All factors have an effect on the total alkaloid yield, among which the effect of ethanol concentration is the most significant. The interaction between liquid-to-solid ratio and ultrasonic time, and ethanol concentration and extraction temperature all have a significant effect on the total alkaloid yield of chelidonium.

[0102] The optimal process conditions for the extraction of total alkaloids from chelidonium were obtained by response surface analysis. Considering the experimental operation, the extraction process of total alkaloids from chelidonium was modified to 0.6% oxalic acid concentration, 17:1 liquid-to-solid ratio, 70% ethanol concentration, 47min ultrasonic time, and 52℃ extraction temperature. That is, the optimal method for the extraction of total alkaloids from chelidonium is as follows: crush chelidonium, pass through No. 3 sieve, add 70% ethanol with a liquid-to-solid ratio of 17:1mL / g to the obtained chelidonium powder, ultrasonically extract for 47min at 52℃, filter, spin dry the filtrate, add 0.6% oxalic acid aqueous solution with a liquid-to-solid ratio of 50:1mL / g, ultrasonically extract for 30min at 50℃, filter, take the filtrate and adjust the pH to 11 with 10% NaOH solution, let it stand, put it in a separatory funnel, extract it with dichloromethane for 3 times, combine the dichloromethane layers, recover the dichloromethane, spin dry, and obtain total alkaloids from chelidonium. Under this condition, the validation experiment showed that the total alkaloid yield of chelidonium was 9.20 mg / g, which was close to the model prediction value (9.28 mg / g), indicating that the regression equation obtained by the response surface design of chelidonium total alkaloid extraction was reliable. This study provides support for the development of chelidonium total alkaloid extraction using chelidonium as raw material.

[0103] Embodiment 4:

[0104] In this embodiment, the total alkaloids of chelidonium extracted by the above method are used to determine the inhibitory activity of the total alkaloids against pathogens in a dish and in a potted plant efficacy test by using the mycelium growth rate method. The present invention does not limit the application of the total alkaloids of chelidonium to other pathogens.

[0105] In-dish antibacterial activity assay:

[0106] The mycelial growth rate method was used to determine the inhibitory activity of total alkaloids of chelidonium against pathogens. In the clean bench, the quantitative mother solution of total alkaloids of chelidonium was added to the culture medium cooled to 50-55°C to prepare a drug-containing plate. At the same time, an equal amount of sterile water was added as a blank control, and each treatment was repeated 3 times. After the test strain was activated, a bacterial cake with a diameter of 5 mm was punched at the periphery of the circular colony with a hole puncher. The bacterial cake was inoculated into the center of the culture medium with an inoculation needle and inverted for culture. When the diameter of the blank colony grew to 80%-90% of the diameter of the culture dish, the cross method was used to measure the diameter (mm) of each colony twice, and the average value was used to represent the size of the colony. The data were substituted into the growth inhibition rate formula to calculate the inhibition rate. The toxicity regression equation, EC50 and correlation coefficient were obtained by SPSS23.0 statistical analysis based on the concentration and inhibition rate.

[0107]

[0108] Potted plant efficacy test:

[0109] Potted efficacy test of chelidonium alkaloids against wheat take-all disease: potted seedlings, about 15-20 wheat seeds per pot, when the wheat in the culture pot grows to the 2-leaf stage, the potted efficacy of chelidonium alkaloid solution against wheat take-all disease is determined by root irrigation method. Protective effect test: 10mL chelidonium alkaloid solution is poured at 3cm away from the rhizome, and pathogens are inoculated 24h later; therapeutic effect test: pathogens are inoculated first, and chelidonium alkaloid solution is poured 24h later. Six pots of wheat are treated with each agent in the whole test, and the whole test is repeated three times. The treated wheat seedlings are placed in an artificial incubator with a temperature of 25℃, a relative humidity of 50-80%, and a photoperiod of 11:13h. The disease index and efficacy are calculated after 30d.

[0110] The incidence of wheat take-all disease is divided into the following 7 levels according to the standards:

[0111] Level 0: no disease;

[0112] Level 1: The blackened root area accounts for less than 5% of the total root area;

[0113] Level 2: The blackened root area accounts for 6-10% of the total root area;

[0114] Level 3: The blackened root area accounts for 11-25% of the total root area;

[0115] Level 4: The blackened root area accounts for 26-40% of the total root area;

[0116] Level 5: The blackened root area accounts for 41-65% of the total root area;

[0117] Level 6: The blackened root area accounts for 66-100% of the total root area.

[0118]

[0119] Where T is the DI of the chelidonium alkaloid-treated group, and C is the DI of the control group.

[0120] Potted efficacy test of total alkaloids of chelidonium against pepper blight: pepper seeds were planted in seedling trays and placed in an artificial incubator, moisturized at 25℃ and 12h:12h photoperiod. When the seedlings grew to 4-6 true leaves, plants with consistent growth were transplanted and continued to be cultivated in the same environment. The potted efficacy of chelidonium alkaloid solution against pepper blight was determined by root irrigation method. Protective effect test: 10mL chelidonium alkaloid solution was poured at 3cm away from the rhizome, and pathogens were inoculated 24h later; therapeutic effect test: pathogens were inoculated first, and chelidonium alkaloid solution was poured 24h later. Each treatment was repeated 3 times, and 6 pots of plants were used for each repetition. After 24h, water was applied appropriately to ensure that the soil moisture was nearly saturated, and the temperature was kept at 25-28℃. The disease was investigated after 14d.

[0121] The incidence of pepper phytophthora is divided into the following 6 levels according to the standards:

[0122] Level 0: The plant is healthy, with no disease spots or water-soaked rot at the root base;

[0123] Level 1: The plant surface is healthy, with slight lesions at the root base, but no water-soaked rot;

[0124] Level 3: The plant is unhealthy, with diseased spots at the root base and slight water-soaked rot;

[0125] Level 5: The plant wilts, with large lesions at the root base and obvious water-soaked rot;

[0126] Level 7: The plant wilts and most of the root base rots;

[0127] Level 9: The plant dies and the root base is completely rotten.

[0128] The protective effect is calculated according to the following formula:

[0129]

[0130] Where T is the DI of the chelidonium alkaloid-treated group, and C is the DI of the control group.

[0131] Table 5 In vitro activity of chelidonium alkaloids against tested pathogens

[0132]

[0133]

[0134] Note: The concentration of chelidonium alkaloids in the table is 200 mg / L; the number of repetitions for each pathogen treatment is 3, and the table shows the average value of 3 repetitions.

[0135] Table 6 Indoor toxicity test of chelidonium alkaloids against 7 pathogens

[0136]

[0137] Note: EC50 of pathogens was determined using methanol extracts of 5 concentrations; the number of repetitions for each pathogen and each concentration was 3, and the table shows the average of 3 repetitions.

[0138] The results of indoor toxicity test showed that the EC of chelidonium alkaloids against wheat take-all pathogen, rice sheath blight pathogen and mango anthracnose pathogen was 50 All were below 200 mg / L, namely 132.370 mg / L, 149.442 mg / L and 148.522 mg / L.

[0139] Table 7 Potted activity of chelidonium alkaloids against wheat take-all disease

[0140]

[0141]

[0142] Note: Each pesticide treatment experiment in the table was repeated 3 times; in the control effect column, numbers in the same column with different lowercase letters indicate significant differences at the 0.05 level of variance analysis (LSD method). The same applies to the following tables.

[0143] Table 8 Potted activity of chelidonium alkaloids against pepper phytophthora

[0144]

[0145] Note: Each pesticide treatment experiment in the table was repeated 3 times; in the control effect column, numbers in the same column with different lowercase letters indicate significant differences at the 0.05 level of variance analysis (LSD method). The same applies to the following tables.

[0146] From Table 7, Figure 7 and Figure 8 It can be seen that the protective effect of chelidonium alkaloids on wheat take-all disease at different concentrations is better than the therapeutic effect. At a concentration of 2000 mg / L, chelidonium alkaloids have a higher protective effect on wheat take-all disease, with a prevention rate of 68.34%, which is equivalent to the control agent 2500 mg / L Pseudomonas fluorescens.

[0147] As shown in Table 8, at different concentrations, the protective effect of chelidonium alkaloids on pepper phytophthora is better than the therapeutic effect. At a concentration of 2000 mg / L, it has good protective and therapeutic effects, with the prevention rates being 75.01% and 64.32% respectively, which is better than the control agent 2800 mg / L mancozeb.

[0148] Based on this, it was shown that the total alkaloids of Greater celandine had good antibacterial activity and had the potential to be further developed as plant-based fungicides.

[0149] Embodiment 5:

[0150] This example uses the total alkaloids of chelidonium extracted by the above method to carry out antiviral potted experiments. The activity of total alkaloids of chelidonium against cucumber mosaic virus (CMV) and potato virus Y (PVY) is taken as an example below, but the present invention does not limit the application of total alkaloids of chelidonium against other viruses. The determination method is as follows:

[0151] The total alkaloids of chelidonium were dissolved with N,N-dimethylacetamide to prepare a 5×102 mg / mL stock solution, which was then diluted with a 0.5% Tween 80 aqueous solution to a desired concentration. The 8% Ningnanmycin aqueous solution used was a commercially available product.

[0152] In vivo protective activity: Select 6-7 leaf stage Amaranthaceae with consistent growth, cut off the excess leaves, and keep 3-4 leaves of similar size for each Amaranthaceae plant for testing. Use a brush to apply the prepared agent on the right half of the leaf, and apply the corresponding solvent on the left half of the leaf as a control. After 1 day, use diamond abrasive to rub the virus liquid on the leaves, dry it, and rinse the leaves with clean water. Repeat three times for each treatment of 3 plants. Record the number of lesions after 3-5 days and calculate the protective effect.

[0153] In vivo therapeutic activity: Select 6-7 leaf stage Amaranthus quinoa with consistent growth, remove excess leaves, inoculate the virus solution on the leaves with corundum, rinse the leaves with clean water after drying, and apply the prepared agent on the right half of the leaves with a brush after the leaves are dried, and apply the corresponding solvent on the left half of the leaves as a control. Treat 3 plants each time and repeat three times. Record the number of lesions after 3-5 days and calculate the prevention effect.

[0154] The inhibition rate formula is:

[0155]

[0156] Table 9 Protective activity of chelidonium alkaloids on Chenopodium album (anti-CMV)

[0157]

[0158] Note: Each pesticide treatment experiment in the table was repeated 3 times; in the control effect column, numbers in the same column with different lowercase letters indicate significant differences at the 0.05 level of variance analysis (LSD method). The same applies to the following tables.

[0159] Table 10 Therapeutic activity of chelidonium alkaloids on Chenopodium album (anti-CMV)

[0160]

[0161]

[0162] Note: Each pesticide treatment experiment in the table was repeated 3 times; in the control effect column, numbers in the same column with different lowercase letters indicate significant differences at the 0.05 level of variance analysis (LSD method). The same applies to the following tables.

[0163] Table 11 Protective activity of chelidonium alkaloids on Chenopodium album (anti-PVY)

[0164]

[0165] Note: Each pesticide treatment experiment in the table was repeated 3 times; in the control effect column, numbers in the same column with different lowercase letters indicate significant differences at the 0.05 level of variance analysis (LSD method). The same applies to the following tables.

[0166] Table 12 Therapeutic activity of chelidonium alkaloids on Chenopodium album (anti-PVY)

[0167]

[0168] Note: Each pesticide treatment experiment in the table was repeated 3 times; in the control effect column, numbers in the same column with different lowercase letters indicate significant differences at the 0.05 level of variance analysis (LSD method). The same applies to the following tables.

[0169] From Table 9-12 and Figure 9-10 It can be seen that the total alkaloids of chelidonium at a dosage of 5 mg / mL have a protective and therapeutic effect on CMV comparable to that of ningnanmycin, and a therapeutic effect on PVY comparable to that of ningnanmycin, and a good protective effect. Therefore, the total alkaloids of chelidonium have the potential to be further developed as a plant-derived antiviral agent.

[0170] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims

1. A process for preparing chelidonium alkaloids for preventing and controlling plant diseases, characterized in that: The preparation process includes: After the celandine is crushed, ethanol is added for heating and ultrasonic extraction, filtered, and the filtrate is spin-dried; After adding oxalic acid solution to dissolve, perform heating ultrasonic extraction, filter, take the filtrate to adjust the pH to alkaline, let it stand, extract with dichloromethane and spin dry, so as to obtain the total alkaloids of chelidonium.

2. The preparation process of chelidonium alkaloids for preventing and controlling plant diseases according to claim 1, characterized in that: The volume concentration of the ethanol is 55% to 95%; the mass concentration of the oxalic acid solution is 0.1% to 0.9%; the liquid-to-solid ratio of ethanol to celandine is (10 to 30) mL: 1 g; The ultrasonic power of the heating ultrasonic extraction is 40kHz, the ultrasonic time is 30-90min, and the ultrasonic extraction temperature is 30-70℃.

3. The preparation process of chelidonium alkaloids for preventing and controlling plant diseases according to claim 1 or 2, characterized in that: The mass concentration of the oxalic acid solution is 0.3%, 0.5% or 0.7%.

4. The preparation process of chelidonium alkaloids for preventing and controlling plant diseases according to claim 1 or 2, characterized in that: The liquid-to-solid ratio of the ethanol to the celandine is 15 mL:1 g.

5. The preparation process of chelidonium alkaloids for preventing and controlling plant diseases according to claim 1 or 2, characterized in that: The volume concentration of the ethanol is 65%.

6. The preparation process of chelidonium alkaloids for preventing and controlling plant diseases according to claim 1 or 2, characterized in that: The ultrasonic time is 45 minutes, and the ultrasonic extraction temperature is 50°C.

7. The preparation process of chelidonium alkaloids for preventing and controlling plant diseases according to claim 1 or 2, characterized in that: The volume concentration of the ethanol is 70%; the mass concentration of the oxalic acid solution is 0.6%; the liquid-to-solid ratio of ethanol to celandine is 17mL:1g; The ultrasonic extraction time of the heating ultrasonic extraction is 47 minutes, and the ultrasonic extraction temperature is 52°C.

8. The process for preparing chelidonium alkaloids for preventing and controlling plant diseases according to claim 1 or 2, characterized in that: The preparation process specifically comprises: The celandine was crushed and passed through a 40-mesh sieve. 70% ethanol with a volume concentration of 17:1 mL / g was added to the obtained celandine powder. Ultrasonic extraction was performed at 52°C for 47 minutes. The mixture was filtered and the filtrate was spin-dried. A 0.6% oxalic acid aqueous solution with a liquid-to-solid ratio of 50:1 mL / g was added and ultrasonic extraction was performed at 50°C for 30 minutes. The mixture was filtered and the filtrate was taken and the pH value of the filtrate was adjusted to 11 with a 10% NaOH solution. The mixture was allowed to stand and extracted with dichloromethane for 3 times. The dichloromethane layers were combined, the dichloromethane was recovered, and the mixture was spin-dried to obtain the total alkaloids of celandine.

9. The process for preparing chelidonium alkaloids for preventing and controlling plant diseases according to claim 1 or 2, characterized in that: The preparation process specifically comprises: 1) putting the celandine into a grinder and grinding it to obtain celandine powder; 2) Using ethanol as the extraction solvent to perform ultrasonic extraction on celandine powder, and determining the optimal values ​​of oxalic acid concentration, liquid-to-solid ratio, ethanol concentration, ultrasonic time and extraction temperature by single factor experiment with the total alkaloid yield of celandine as the index; 3) According to the single factor experimental test results, the Box-Behnken response surface method was used, with oxalic acid concentration A, liquid-to-solid ratio B, ethanol concentration C, ultrasonic time D and extraction temperature E as independent variables, and total alkaloid yield Y as an indicator, to perform polynomial fitting regression and obtain a regression equation; the regression equation is: Y=9.02+0.2031A+0.2775B+1.10C+0.0544D+0.3644E-0.0675AB+0.0200AC-0.2550AD-0.0750AE-0.1100BC +0.3075BD-0.1650BE+0.0675CD-0.2850CE+0.2275DE-0.5829A2-0.2854B2-1.02C2-0.6046D2-0.4713E2; Where Y is the total alkaloid yield, the independent variable A is the oxalic acid concentration, B is the liquid-to-solid ratio, C is the ethanol concentration, D is the ultrasonic time, and E is the extraction temperature; 4) calculating the regression equation to obtain the optimal extraction conditions of chelidonium alkaloids; 5) Under the optimal extraction method conditions, ultrasonic extraction is performed on chelidonium to obtain total alkaloids of chelidonium.

10. Use of the chelidonium alkaloids prepared by the process for preparing chelidonium alkaloids for preventing and controlling plant diseases according to any one of claims 1 to 9 for preparing drugs for preventing and controlling plant diseases, wherein the plant diseases are caused by the following pathogens, including: Phytophthora capsici, Fusarium graminearum Schw, Gaeumannomyces graminis, Colletotrichum gloeosporioides, Rhizoctonia solani, Botrytis cinerea, Alternaria alternata, Sclerotiniasclerotiorum, Colletotrichum gloeosporioides, Valsamali, Physalospora piricola, Botrytis cinerea, Exserohilum turcicums, Fusarium solani, Colletotrichum orbiculare, Fusarium wilt oxysporum), tomato wilt pathogen (Fusarium oxysporum), cotton Verticillium dahliae (Verticillium dahliae), cucumber mosaic virus (cucumbermosaic virus, CMV) and potato virus Y (potato virus Y, PVY) at least one of.

Images