Thyme general flavone as well as extraction method and application thereof

Abstract

The invention relates to the field of natural products, in particular to thyme total flavonoids as well as an extraction method and application thereof. The invention discloses a method for extracting total flavonoids of thyme. The maximum extraction rate of the total flavonoids of thyme extracted by adopting the method is 4.2%. The functions of the thyme total flavonoids are further discussed, it is found that the thyme total flavonoids have the effect of inhibiting rhizoctonia solani growth, pot experiments prove that after the thyme total flavonoids are applied to seed soaking, the emergence rate of cotton can be increased, and the damping-off prevention effect of the cotton in the growing period reaches 90.8%; after seedling emergence of potted cotton, the thyme total flavonoids are applied, damping-off can be effectively prevented and treated, and the prevention effect reaches 84.7%. The extraction method of the thyme total flavonoids, provided by the invention, is high in extraction rate of the thyme total flavonoids and good in activity, and has a good market application prospect.

Description

Technical Field

[0001] The present invention relates to the field of natural products, and particularly to total flavonoids from thyme, an extraction method thereof, and an application thereof. Background Art

[0002] Thyme belongs to the perennial aromatic herbaceous plants of the genus Thymus in the family Lamiaceae. It is named because its plants have a strong aromatic smell during the flowering period and is an important aromatic and medicinal plant resource. Thyme contains a large amount of phenolic, flavonoid compounds and terpenoids, and has various biological activities such as antioxidant, anti-inflammatory, anti-cancer, anti-bacterial, anti-fungal, anti-viral, and immunomodulatory activities, and is regarded as a plant resource with great development and utilization potential.

[0003] The extraction methods of flavonoid compounds mainly include Soxhlet extraction method, organic solvent extraction method, microwave extraction method, ultrasonic extraction method, supercritical fluid extraction method, enzymatic extraction method, etc. Among them, the most common extraction methods are alcohol extraction method, ultrasonic extraction method and microwave extraction method.

[0004] Thyme flavonoids are bioactive substances. In order to further improve the medicinal value and market value of thyme, researchers have successively extracted its active ingredients. However, the existing extraction process of total flavonoids from thyme has the problem of low extraction rate. For example, CN117801884A discloses an extraction method for various bioactive components in thyme, including the following steps: S1, extracting essential oil: adding a mixed enzyme and distilled water dissolved with NaCl to thyme, sealing and ultrasonically treating, and then extracting thyme essential oil by steam distillation method; S2, extracting thyme polysaccharide: after extracting thyme essential oil, centrifuging to collect the residual liquid and residue, reserving the residue, concentrating the residual liquid by rotary evaporation, adding Sevage reagent and mixing and shaking, centrifuging and separating to remove proteins, obtaining a supernatant, then adding 3 times of anhydrous ethanol of the supernatant and standing overnight, collecting the precipitate and freeze-drying to obtain thyme polysaccharide; S3, extracting total flavonoids: drying the residue, adding ethanol to the residue and ultrasonically extracting total flavonoids. The extraction rate of total flavonoids from thyme by this method is 1.8%. In order to further improve the extraction rate of total flavonoids from thyme, it is necessary to optimize the extraction method of total flavonoids from thyme, so as to improve the application value of total flavonoids from thyme. Summary of the Invention

[0005] To solve the above technical problems, the present invention uses the response surface method to analyze and optimize the influencing factors in the extraction process of total flavonoids from thyme, and combines the multiple quadratic regression equation and the fitting function to develop an extraction method for total flavonoids from thyme. This method improves the extraction rate of total flavonoids from thyme and solves the technical problem of low extraction rate of total flavonoids from thyme in the prior art. In addition, the present invention further deeply studies the efficacy of the extracted total flavonoids from thyme to improve the application value of total flavonoids from thyme.

[0006] On the one hand, the present invention provides a total flavonoids of thyme, which is obtained by extracting from thyme, and the total flavonoids of thyme can inhibit the growth of Rhizoctonia solani.

[0007] Furthermore, when the concentration of the total flavonoids of thyme is 5 - 30 mg / mL, it can inhibit the growth of Rhizoctonia solani.

[0008] On the other hand, the present invention provides the application of the total flavonoids of thyme in preventing and controlling plant damping-off.

[0009] Furthermore, in the said application, the plant is cotton.

[0010] In addition, the present invention also provides a method for preventing and controlling plant damping-off, which uses the total flavonoids of thyme to soak plant seeds before sowing or apply the total flavonoids of thyme to plants after emergence.

[0011] In addition, the present invention also provides the application of the total flavonoids of thyme in increasing the emergence rate of cotton.

[0012] In addition, the present invention also provides a method for extracting the total flavonoids of thyme, and the method comprises the following steps:

[0013] Step 1: Using the aerial part of thyme as raw material, crushing it to obtain thyme crude powder;

[0014] Step 2: Putting the thyme crude powder into a Soxhlet extractor, defatting and decoloring it with petroleum ether to obtain thyme powder;

[0015] Step 3: Soaking the thyme powder in an extraction solvent, performing ultrasonic extraction, filtering, collecting the extract, subjecting the extract to sterile filtration, concentrating the filtrate and then freeze-drying to obtain a powder, which is the total flavonoids of thyme.

[0016] Furthermore, in the said extraction method, the extraction solvent in Step 3 is methanol, and the concentration of the methanol is 70% - 90% by volume percentage; the ultrasonic power of the ultrasonic extraction is 300W - 450W; the time of the ultrasonic extraction is 30min - 50min; the mass-to-volume ratio of the thyme powder to the extraction solvent is 1:10 - 20 g / mL.

[0017] Furthermore, in the said extraction method, the concentration of the methanol is 78.5%; the ultrasonic power of the ultrasonic is 450W; the time of the ultrasonic-assisted extraction is 38min; the mass-to-volume ratio of the thyme powder to the extraction solvent is 1:15.5 g / mL.

[0018] Finally, the present invention also provides the total flavonoids of thyme prepared by the said extraction method, and the extraction rate of the total flavonoids of thyme is greater than 2%.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] The present invention uses the response surface method in combination with traditional methods to explore the extraction process of total flavonoids from thyme. Through a series of studies, a method for extracting total flavonoids from thyme is developed. The extraction rate of total flavonoids extracted by this method is higher than 2%, and the highest extraction rate reaches 4.2%. It overcomes the technical problem of the low extraction rate of total flavonoids from thyme at present. In addition, the present invention further conducts functional research on the extracted total flavonoids from thyme and finds that the total flavonoids from thyme can inhibit the growth of Rhizoctonia solani. When the concentration of total flavonoids from thyme is 30 mg / mL, its inhibition rate on Rhizoctonia solani reaches 82.6%; cotton pot experiments prove that after soaking cotton seeds with total flavonoids from thyme and then sowing, the emergence rate is 94.0%, which is much higher than the emergence rate of the control group, indicating that soaking seeds with total flavonoids from thyme can improve the emergence rate of plants; further statistical analysis of the control effect of Rhizoctonia solani on cotton during the growth period shows that after soaking cotton seeds with total flavonoids from thyme, the control effect of Rhizoctonia solani on cotton during the growth period reaches 90.8%, indicating that soaking cotton seeds with total flavonoids from thyme can effectively control Rhizoctonia solani on cotton; in cotton pot experiments, only irrigating the roots of cotton with total flavonoids from thyme after cotton emergence can also improve the control effect of Rhizoctonia solani on cotton during the growth period, and its control effect reaches 84.7%. Description of the Drawings

[0021] Figure 1 It is the standard curve of rutin.

[0022] Figure 2 It is the result graph of the influence of the extraction solvent on the extraction rate of total flavonoids from thyme.

[0023] Figure 3 It is the result graph of the influence of the ultrasonic time on the extraction rate of total flavonoids from thyme.

[0024] Figure 4 It is the result graph of the influence of the ultrasonic power on the extraction rate of total flavonoids from thyme.

[0025] Figure 5 It is the result graph of the influence of the methanol concentration on the extraction rate of total flavonoids from thyme.

[0026] Figure 6 It is the result graph of the influence of the solid-liquid ratio on the extraction rate of total flavonoids from thyme.

[0027] Figure 7 It is the interaction influence graph of the liquid-solid ratio and the methanol concentration in the response surface experiment of the total flavonoids from thyme extraction process.

[0028] Figure 8 It is the interaction influence graph of the methanol concentration and the ultrasonic time in the response surface experiment of the total flavonoids from thyme extraction process.

[0029] Figure 9It is the interaction influence diagram of liquid-solid ratio and ultrasonic time in the response surface experiment of the total flavonoids extraction process from thyme.

[0030] Figure 10 It is the result diagram of the inhibitory effect of total flavonoids from thyme on Rhizoctonia solani. Specific implementation manners

[0031] Next, the technical solutions of the present invention will be described in conjunction with embodiments. However, the present invention is not limited to the following embodiments.

[0032] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings, but the described embodiments shall not be construed as limiting the present invention.

[0033] In the following embodiments, unless otherwise specified, the experimental methods and detection methods are all conventional methods; unless otherwise specified, the reagents and materials can be purchased on the market, and the percentages of the solvents are all volume percentages.

[0034] Embodiment 1

[0035] This embodiment is to evaluate the influence of different influencing factors on the extraction rate of total flavonoids from thyme.

[0036] Raw materials: Fresh thyme (above-ground part) is washed, naturally air-dried at room temperature, crushed and sieved (60 mesh), and the material passing through the sieve is the original thyme powder.

[0037] Drawing of the rutin standard curve:

[0038] The total flavonoids were determined by the aluminum nitrate-sodium nitrite colorimetric method, using rutin as the standard substance. Weigh 20 mg of rutin standard into a 10 mL volumetric flask, dissolve rutin completely by heating in a water bath with 70% ethanol, and make up the volume to 10 mL to obtain a standard rutin solution of 2.0 mg / mL for standby. Dilute it in a brown bottle to obtain seven concentration gradients. Respectively take 0.00, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00 mL of the rutin standard solution into 10 mL stoppered test tubes, then add 70% ethanol to make up the volume to 3 mL, add 0.5 mL of 5% sodium nitrite solution, shake well to mix evenly, let stand at room temperature for 6 min, then add 0.50 mL of 10% aluminum nitrate solution, shake well to mix evenly, let stand at room temperature for 6 min, finally add 4 mL of 4% sodium hydroxide solution and add 2 mL of 70% ethanol to make the total volume of the reaction system reach 10 mL, let stand at room temperature for 15 min. After the reaction is completed, measure the absorbance at 510 nm. The determination is carried out according to the above test method. Each reaction is measured in triplicate, and the average of the three measurements is taken.

[0039] According to the determination results of rutin standard, with the concentration of rutin (mg / mL) as the abscissa and the absorbance value at 510 nm as the ordinate, the standard curve of rutin was plotted. The standard curve is as shown in Figure 1 shown. The standard curve equation is: y = 6.345x - 0.002789, and the correlation coefficient R 2 = 0.9985. The content (%) of total flavonoids in thyme was calculated using the following formula, which is the extraction rate (%) of total flavonoids in thyme.

[0040] Content (%) of total flavonoids in thyme = (C × D) / M × 100%

[0041] Where: C is the mass concentration (mg / mL) calculated by substituting the absorbance into the standard curve; D is the dilution factor; M is the weight (mg) of the thyme sample taken.

[0042] (1) Influence of different solvents on the extraction rate of total flavonoids in thyme

[0043] The original thyme powder was defatted and decolorized with petroleum ether in a Soxhlet extractor under reflux conditions for 5 - 10 h, filtered, and the solid phase was collected and dried at room temperature to prevent the residue of petroleum ether, obtaining thyme powder.

[0044] Weighed the thyme powder. According to the solid-liquid ratio of 1:15 (g / mL), the active ingredients in thyme were extracted with 4 solvents with different polarities, namely methanol, ethanol, ethyl acetate, and petroleum ether, using ultrasonic-assisted extraction method. Placed in an ultrasonic cleaner, under the power of 300 W, ultrasonicated continuously for 40 min, filtered, and the filtrate was retained. The filter residue was poured back into a new extraction solvent, and the three filtrates were combined after filtration. The filtrate was concentrated and a part of the clarified liquid was taken to measure the extraction rate of total flavonoids (i.e., the content % of total flavonoids in thyme).

[0045] The test results are as shown in Figure 2 shown. The total flavonoid content in the extracts of petroleum ether and ethyl acetate is relatively low. The extraction rates of total flavonoids in thyme with ethanol and methanol solutions are significantly higher than those of petroleum ether and ethyl acetate, and the total flavonoid content in methanol is greater than that in ethanol, which is in line with the characteristic that flavonoid substances are mainly dissolved in medium and high polarity solvents.

[0046] (2) Influence of different ultrasonic times on the extraction rate of total flavonoids in thyme

[0047] Using methanol as the extraction solvent, different groups were set according to the ultrasonic time in Table 1, and other conditions were the same as those in the extraction conditions of (1) Influence of different solvents on the extraction rate of total flavonoids in thyme in this example.

[0048] Table 1 Design table of process optimization factors with methanol as the extraction solvent

[0049]

[0050]

[0051] The test results are as Figure 3 shown. With other conditions unchanged, the extraction amount of total flavonoids from thyme shows an upward trend within the ultrasonic time of 10 - 40 min. When the ultrasonic time exceeds 40 min, the extraction amount of total flavonoids decreases, and the extraction effect of total flavonoids is the best at 40 min of ultrasonic treatment. This may be because as the ultrasonic time prolongs, total flavonoids from thyme can be fully extracted into the extraction solvent. When the ultrasonic time reaches a certain value, the overlong ultrasonic time will cause partial oxidation of flavonoid compounds with antioxidant properties, thus reducing the extraction amount.

[0052] (3) Influence of different ultrasonic powers on the extraction rate of total flavonoids from thyme

[0053] Fix the solid - liquid ratio at 1:15 (g / mL), the extraction time at 40 min, and the methanol volume fraction at 100%. Extract thyme with ultrasonic powers of 300 W, 350 W, 400 W, 450 W, and 500 W as shown in Table 1, and determine the extraction rate of total flavonoids from thyme.

[0054] The test results are as Figure 4 shown. With other single - factor extraction conditions unchanged, the extraction amount of total flavonoids from thyme gradually increases as the ultrasonic power increases from 300 - 450 W. When the ultrasonic power exceeds 450 W, the extraction amount of total flavonoids shows a downward trend, and the extraction effect is the best at 450 W.

[0055] (4) Influence of different methanol concentrations on the extraction rate of total flavonoids from thyme

[0056] Fix the ultrasonic power at 500 W, the extraction time at 40 min, and the solid - liquid ratio at 1:15 (g / mL). Extract thyme with methanol concentrations of 60%, 70%, 80%, 90%, and 100% as shown in Table 1, and determine the extraction rate of total flavonoids from thyme.

[0057] The test results are as Figure 5 shown. When the methanol concentration is 60% - 80%, the extraction amount of total flavonoids shows an upward trend; when the methanol concentration is higher than 80%, the extraction amount of total flavonoids shows a downward trend, and the extraction rate of total flavonoids is the highest at 80% methanol concentration. This may be because the higher the methanol concentration, the more the leaching amount of alcohol - soluble impurities, pigments, and components with strong lipophilicity will increase, resulting in a decrease in the extraction rate of total flavonoid compounds.

[0058] (5) Influence of different solid - liquid ratios on the extraction rate of total flavonoids from thyme

[0059] The ultrasonic power was fixed at 500 W, the extraction time was 40 min, the methanol volume fraction was 100%, and the solid-liquid ratio was as shown in Table 1, namely 1:5 (g / mL), 1:10 (g / mL), 1:15 (g / mL), 1:20 (g / mL), and 1:25 (g / mL) respectively. Thyme was extracted, and the extraction rate of total flavonoids in thyme was determined.

[0060] The test results are as Figure 6 shown. The highest value was reached when the solid-liquid ratio was 1:15 (g / mL). As the volume of the solvent increased, the extraction amount of total flavonoids decreased.

[0061] Example 2

[0062] This example was to optimize the extraction process of total flavonoids in thyme by response surface test.

[0063] According to the results of single-factor tests, the ultrasonic-assisted extraction process was optimized using the ultrasonic extraction step method in Example 1. With other factors fixed, single-factor tests were carried out on the methanol concentration, solid-liquid ratio, and ultrasonic time for the ultrasonic-assisted extraction of total flavonoids in thyme to optimize the extraction process conditions. A response surface test design with three factors and three levels was carried out. The test design is shown in Table 2, and the test results are shown in Tables 3 and 4.

[0064] Table 2 Factor levels and coding table of the test design

[0065]

[0066] Table 3 Results of the response surface test

[0067]

[0068]

[0069] Table 4 Variance analysis of the regression model for the yield of total flavonoids in thyme

[0070]

[0071]

[0072] Note: * indicates that the P value < 0.05, and ** indicates that the P value < 0.01

[0073] The results of the interactive effect of the solid-liquid ratio and methanol concentration in the response surface test of the total flavonoid extraction process in thyme are as Figure 7 shown. The solid-liquid ratio (mL / g) was 10 - 20, the methanol concentration was 70% - 90%, and the extraction rate of total flavonoids in thyme was between 3.4% and 4.0%.

[0074] The results of the interactive effect of the methanol concentration and ultrasonic time in the response surface test of the total flavonoid extraction process in thyme are as Figure 8As shown, the ultrasonic time is 30 - 50 min, the methanol concentration is 70% - 90%, and the extraction rate of total flavonoids from thyme is between 3.3% and 3.9%.

[0075] The results of the interaction between the liquid - solid ratio and ultrasonic time in the response surface experiment of the total flavonoids extraction process from thyme are as Figure 9 shown. The liquid - solid ratio (mL / g) is 10 - 20, the ultrasonic time is 30 - 50 min, and the extraction rate of total flavonoids from thyme is between 3.6% and 4.2%.

[0076] The results of the experiment were analyzed by the Design - Expert software program for quadratic regression response surface analysis, and a multiple quadratic response surface regression model Y = 4.214 - 0.131A + 0.055B - 0.084C - 0.090AB + 0.0978AC + 0.125BC - 0.468A2 - 0.226B2 - 0.223C2 was established. The variance and significance of the model were analyzed. The F - value = 33.21, the P - value < 0.0001, P - value = 0.0001 < 0.001, and all P - values were less than 0.05, indicating that the model was significant. The lack - of - fit term value of the flavonoid extraction amount model was 0.5198, and the lack - of - fit term was greater than 0.05, indicating that the lack of fit was not significant and the model was reliable. The reliability of the model was analyzed. The multiple correlation coefficient R 2 represents the percentage of the model response value derived from the variables. The higher the percentage, the more reliable the model. The multiple correlation coefficient R 2 of the model was 97.71%, indicating that 97.71% of the response values of this model were derived from the variables. The coefficient of variation represents the precision of the model. The lower the CV value, the more reliable the model. The coefficient of variation of the model was 2.07%, and the CV value was relatively low. Adeq Precision (usually recommended to be greater than 4, indicating that the model has a good signal - to - noise ratio and can effectively predict the response variable) is a measure of the signal - to - noise ratio, which measures the ability of the model to predict the response variable. The signal - to - noise ratio of the model was 14.3367, and the CV values were all > 4. In summary, the model designed by the Design - Expert software had a high reliability and could be used to predict the extraction process parameters of flavonoid extraction amount and polyphenol extraction amount.

[0077] By comparing the F - values of each factor, it can be concluded that the order of the influence degree on the flavonoid extraction rate is A: methanol concentration > C: ultrasonic time > B: liquid - solid ratio. The interaction factor BC has the greatest influence on the flavonoid extraction rate, and AB has the least influence.

[0078] The regression model was optimized using DesignExpert software. According to the actual test conditions, the extraction process parameters were adjusted to: solid-liquid ratio of 1:15.5 g / mL, methanol concentration of 78.5%, and ultrasonic time of 38 min. To verify the effectiveness of the model, an extraction verification test was carried out under these conditions, and the extraction rate of total flavonoids from thyme was 4.21 ± 0.29%, compared with the theoretical value of 4.24%. The relative error was within the allowable range, indicating that the solid-liquid ratio of 1:15.5 g / mL, methanol concentration of 78.5%, and ultrasonic time of 38 min were the optimal extraction process conditions.

[0079] Example 3

[0080] This example was a test for the inhibitory effect of total flavonoids from thyme on Rhizoctonia solani.

[0081] Raw materials: Fresh thyme (above-ground part) was washed and naturally air-dried at room temperature, then crushed and sieved (60 mesh), and the material passing through the sieve was the original thyme powder.

[0082] Preparation of the mother liquor of total flavonoids from thyme: Total flavonoids from thyme were extracted according to the optimal process provided in Example 2. Taking 10 g of thyme powder as an example, on the basis of an ultrasonic power of 450 w, the solid-liquid ratio of 1:15.5 g / mL, methanol concentration of 78.5%, and ultrasonic time of 38 min were selected to extract total flavonoids from thyme. After filtration, the filtrates were combined after three extractions and rotary evaporation was carried out. The mother liquor of total flavonoids from thyme was prepared with 1% DMSO and made up to a constant volume for standby (1 g of dry thyme powder was made up to a constant volume with 1 mL of 1% DMSO solution) for the antibacterial effect test.

[0083] Antibacterial effect test of total flavonoids from thyme:

[0084] PDA medium containing different concentrations of total flavonoids from thyme (0 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL) was prepared with the mother liquor of total flavonoids from thyme prepared in this example, and the antibacterial test was carried out using the growth rate method.

[0085] Rhizoctonia solani was cultured using the above-prepared PDA medium with different concentrations of total flavonoids from thyme. After culturing for 3 d, the growth of Rhizoctonia solani was observed. The results were as Figure 10 shown. The colony growth of the treatment groups with different concentrations of total flavonoids from thyme was different, and the growth rate of the hyphae could be delayed to varying degrees, showing an obvious dose effect. The antibacterial effects of different concentrations of total flavonoids from thyme were 7.6%, 28.0%, 51.8%, 68.0%, 74.0%, and 82.6% respectively. By measuring the effect of total flavonoids from thyme on the mycelial growth of Rhizoctonia solani, its EC50 = 14.44 mg / mL was obtained. The calculation formula for the mycelial growth inhibition rate (%) is as follows:

[0086] Mycelial growth inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter -

[0087] diameter of the fungal disc) × 100

[0088] Example 4

[0089] This example is a test on the control effect of total flavonoids from thyme on damping-off of potted cotton.

[0090] Preparation of Rhizoctonia solani - infested soil: Clean the wheat seeds. First, soak the evenly - sized wheat seeds for 24 h, then boil them for 30 min, put them into a triangular flask, and sterilize them at 121 °C for 30 min to obtain the wheat - grain medium. Take fresh fungal discs from a PDA plate of Rhizoctonia solani that has been pre - cultured for 3 d, and inoculate them into the sterilized wheat - grain medium, 5 fungal discs per flask. Place them in a constant - temperature incubator at 25 °C and shake the flasks once in the morning and once in the evening every day. After the wheat grains are covered with visible Rhizoctonia solani mycelia, use them to prepare the Rhizoctonia solani - infested soil. Adopt soil - mixing with the fungus (mix the wheat - grain medium for culturing Rhizoctonia solani with the sterilized soil at a ratio of 0.5 wt% to obtain the Rhizoctonia solani - infested soil.

[0091] Preparation of sterilized soil: Put the soil for growing cotton into a sterilized bag, sterilize it in a pressure steam sterilizer (121 °C, 30 min), and take it out and let it cool naturally to obtain the sterilized soil.

[0092] Seed disinfection treatment: Before sowing, disinfect the cotton seeds with 2 wt% sodium hypochlorite for 15 min, and then wash them clean with pure water.

[0093] The test setup included a total flavonoids of thyme seed soaking test group, a total flavonoids of thyme root irrigation test group, a pathogen control group, and a blank control group. The total flavonoids of thyme seed soaking test group used the seeds disinfected with 2 wt% sodium hypochlorite as described above, and then soaked them in thyme total flavonoids with a concentration of 15 mg / mL for 12 h and sowed them into the soil containing Rhizoctonia solani; the total flavonoids of thyme root irrigation test group used the seeds disinfected with 2 wt% sodium hypochlorite as described above, sowed them into the soil containing Rhizoctonia solani, and when two cotyledons of the cotton seeds emerged after emergence, irrigated the roots with 15 mg / mL of total flavonoids of thyme, 50 mL per pot; the pathogen control group used the seeds disinfected with 2 wt% sodium hypochlorite as described above, and sowed them into the soil containing Rhizoctonia solani; the blank control group used the seeds disinfected with 2 wt% sodium hypochlorite as described above, and sowed them into the sterilized soil. When sowing in each group treatment, the cotton seeds were arranged with the roots down and the heads up in a 4×5 pattern, 20 seeds in each group. Except for the different treatments in each group, the rest of the management was the same, all were conventional management. After the cotton seeds emerged, the emergence rate was counted 5 - 7 days later, 10 plants were thinned out 10 days later, and the soil was kept moist. At 35 days, the disease index, incidence rate, and relative control effect were investigated and counted. The disease grading standard for Rhizoctonia solani is shown in Table 5, and the control effect results of total flavonoids of thyme on cotton Rhizoctonia solani are shown in Table 6.

[0094] Table 5 Disease grading standard for Rhizoctonia solani

[0095]

[0096] Incidence rate (%) = number of diseased plants / number of investigated plants × 100

[0097] Disease index = ∑(number of plants at disease level × representative level) / total number of plants × highest representative level value × 100

[0098] Control effect (%) = (control disease index - treatment disease index) / control disease index × 100

[0099] Table 6 Statistical results of the control effect of total flavonoids of thyme on cotton Rhizoctonia solani

[0100] Treatment Emergence rate (%) Incidence rate (%) Disease index Control efficacy (%) Soaking seeds with total flavonoids from Thymus mongolicus 94.0a 32.0b 2.7b 90.8a Irrigation roots with total flavonoids from Thymus mongolicus 89.0b 37.0b 4.5b 84.7b Pathogen control 77.0d 42.0a 29.4a - Blank control 86.0c - - -

[0101] The antibacterial effect of total flavonoids of thyme on Rhizoctonia solani of potted cotton is shown in Table 6, indicating that after soaking the seeds with total flavonoids of thyme, compared with the blank control and the pathogen group, the emergence rate of cotton increased, indicating that soaking the seeds with total flavonoids of thyme can improve the emergence rate of cotton; and the soaking treatment reduced the incidence rate of cotton Rhizoctonia solani, effectively reducing the disease degree of cotton seedlings at the seedling stage, and the control effect of the soaking treatment on cotton Rhizoctonia solani reached 90.8%.

[0102] The root irrigation treatment with total flavonoids of thyme can effectively reduce the incidence rate of cotton seedlings against Rhizoctonia solani, reduce the disease degree of cotton seedlings at the seedling stage, and the control effect of the root irrigation treatment on cotton Rhizoctonia solani is 84.7%.

[0103] As described above, the basic principles, main features, and advantages of the present invention have been better described. The above embodiments and the description are only descriptions of the preferred embodiments of the present invention. The present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A total flavonoid of thyme, characterized in that, The total flavonoids of thyme are obtained by extracting from thyme, and the total flavonoids of thyme can inhibit the growth of Rhizoctonia solani.

2. The total flavonoids of thyme according to claim 1, characterized in that, The concentration of the total flavonoids of thyme is 5-30 mg / mL.

3. Use of the total flavonoids of thyme according to claim 1 in the prevention and control of plant damping-off.

4. The application according to claim 3, wherein The plant is cotton.

5. A method for preventing and controlling plant damping-off, characterized in that, The total flavonoids of thyme according to claim 1 are used for soaking the plant seeds before sowing or applying the total flavonoids of thyme according to claim 1 to the plants after the plants emerge.

6. Use of the total flavonoids of thyme according to claim 1 in increasing the emergence rate of cotton.

7. The extraction method of total flavonoids from thyme according to claim 1, characterized in that, It includes the following steps: Step 1: Using the above-ground part of thyme as the raw material, crushing it to obtain the original thyme powder; Step 2: Putting the original thyme powder into a Soxhlet extractor, degreasing and decolorizing it with petroleum ether to obtain thyme powder; Step 3: Soaking the thyme powder in an extraction solvent, performing ultrasonic extraction, filtering, collecting the extract, subjecting the extract to sterile filtration, concentrating the filtrate and then freeze-drying to obtain a powder, which is the total flavonoids of thyme.

8. The extraction method according to claim 7, characterized in that, The extraction solvent in step 3 is methanol, and the concentration of the methanol is 70%-90% by volume percentage; the ultrasonic power of the ultrasonic extraction is 300W-450W; the time of the ultrasonic extraction is 30min-50min; the mass-volume ratio of the thyme powder to the extraction solvent is 1:10-20 g / mL.

9. The extraction method according to claim 8, wherein The concentration of the methanol is 78.5%; the ultrasonic power of the ultrasonic extraction is 450W; the time of the ultrasonic extraction is 38min; the mass-volume ratio of the thyme powder to the extraction solvent is 1:15.5 g / mL. The total flavonoids of thyme prepared by the extraction method according to any one of claims 7 to 9, characterized in that, The extraction rate of the total flavonoids of thyme is greater than 2%.

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