Method for rapidly detecting content of phenolic acid components in kok-saghyz
Through the high-performance liquid chromatograph combined with the extract volume method, the problem of difficult to quickly and accurately detect the content of phenolic acid components in rubber grass in the existing technology is solved, and efficient and simple detection operations are achieved, reducing equipment costs and maintenance are achieved. need.
Patent Information
- Application Number
- CN202411718780.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-28
- Publication Date
- 2025-02-21
AI Technical Summary
The prior art is difficult to quickly and accurately detect the content of phenolic acid components in rubber grass, and the existing methods are complex in operation and high equipment maintenance costs, so it is impossible to achieve high-throughput rapid detection.
The high-performance liquid chromatograph combined with the extraction liquid volume concentration method is used to accurately weigh phenolic acid standards, dissolve and volume concentration, and establish a standard curve; then the rubber grass sample is extracted and filtered, and the sample is injected into the high-performance liquid chromatogram The content of phenolic acid components is calculated according to the standard curve.
It has achieved rapid and accurate detection of the content of various phenolic acid components in rubber grass, with simple operation, low equipment cost and low maintenance, filling the industry's technological gap.
Smart Images
Figure CN119492831A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of rubber compounds, and more specifically, to a method for rapidly detecting the content of phenolic acid components in rubber grass. Background Art
[0002] Rubber grass, also known as Russian dandelion, is a perennial herbaceous plant of the genus Taraxacum in the Asteraceae family. The roots of rubber grass contain 2.89% to 27.89% natural rubber and 25% to 40% inulin, which can be used as a raw material for rubber products. Rubber grass is considered to be an economic crop that can replace the Brazilian rubber tree in the production of natural rubber and a model plant for scientific research. Due to its wide growth range, high natural rubber content, short growth cycle, and relatively simple genome, rubber grass is considered to be the most likely economic crop to replace the production of natural rubber.
[0003] The drug components in rubber grass are very complex. Currently, there are a few literatures that focus on the methods of drug components in rubber grass, mainly using ultra-high performance liquid chromatography tandem mass spectrometry to qualitatively identify the drug components in rubber grass. The existing methods use large instruments, which have high operation and equipment maintenance costs, complex operation procedures, and are qualitative analysis methods that cannot achieve high-throughput rapid detection of the content of drug components in rubber grass. So far, no methods or standards for quantitative analysis of drug components in rubber grass have been reported. Summary of the invention
[0004] In order to further improve the detection rate and accuracy of the content of phenolic acid components in rubber grass, the present application provides a method for quickly detecting the content of phenolic acid components in rubber grass.
[0005] The present application provides a method for rapidly detecting the content of phenolic acid components in rubber grass, comprising the following steps:
[0006] S1. Accurately weigh the phenolic acid component standard and put it into a 10 mL volumetric flask, add a certain amount of extract to dissolve it, shake it well and dissolve it completely, and then use the extract to make up to volume to obtain a phenolic acid component standard mixed solution;
[0007] S2. Measure different volumes of the mixed solution of the phenolic acid component standard and inject it into the high performance liquid chromatograph, and perform the test according to the preset chromatographic conditions. After the test is completed, obtain the chromatogram of the standard of each target component, integrate the chromatogram to obtain the chromatographic peak area of the standard of each component, perform linear regression analysis with the injection volume as the abscissa (X, μg) and the peak area as the ordinate (Y, μV·s), prepare the standard curve, and establish the linear regression equation;
[0008] S3, drying the rubber grass and grinding it into powder, adding an extracting solution to the powder, extracting the target component according to a preset extraction condition, and filtering to obtain a sample solution to be tested;
[0009] S4, filtering the sample solution to be tested through an organic phase microporous filter membrane, injecting the sample solution to be tested into a high performance liquid chromatograph, testing according to preset chromatographic conditions, obtaining a chromatogram of the sample to be tested, and integrating to obtain the peak area of each target component;
[0010] S5. Substitute the peak area of each target component into the standard curve, calculate the injection mass of the actual sample, and calculate the content of each phenolic acid component in the sample to be tested.
[0011] Furthermore, in step S1, phenolic acid components include but are not limited to protocatechuic acid, protocatechuic aldehyde, monocaffeoyltartaric acid, chlorogenic acid, caffeic acid, chicoric acid, and ferulic acid, and at least 5 phenolic acid standards with different injection volumes are used, the injection volume is 5 to 50 μL, and the corresponding injection amount is 0.01 to 2 μg.
[0012] Further, in step S1, the extracting solution is selected from one or more of a mixture of acid, alcohol, and water, preferably, at least one of formic acid, methanol, and water, more preferably, a mixture of formic acid, methanol, and water, and most preferably, the extracting solution comprises formic acid: methanol: aqueous solution = 5:47.5:47.5 in a volume ratio.
[0013] Furthermore, in step S2, the chromatographic conditions include: wavelength, column temperature, mobile phase, mobile phase elution program, pH value; the wavelength is 260-320 nm, preferably 300-320 nm; the column temperature is 25-45° C., preferably 30-40° C.
[0014] Furthermore, the mobile phase is a mixed mobile phase of an organic reagent, water, an inorganic acid, and a buffer salt, wherein the organic reagent is preferably methanol and acetonitrile, the acid is preferably formic acid and phosphoric acid, the buffer salt is preferably potassium dihydrogen phosphate, and the organic reagent, acid, and buffer salt are all chromatographic grade; more preferably, the mobile phase is methanol-potassium dihydrogen phosphate-phosphoric acid-aqueous solution; most preferably, the mobile phase includes phase A and phase B, phase A is methanol, phase B is 0.05 mol / L potassium dihydrogen phosphate solution, and the pH value of the mobile phase is 2-4, preferably 2-3.
[0015] Furthermore, the mobile phase program adopts a gradient elution program, and the ratio of phase A to phase B is defined as the proportion of the organic phase. Within 0 to 22 minutes, the proportion of the organic phase is 10% to 20%, within 23 to 33 minutes, the proportion of the organic phase is 20% to 30%, and within 34 to 40 minutes, the proportion of the organic phase is 10% to 20%.
[0016] Furthermore, the high performance liquid chromatograph includes an automatic sample injector, a column oven, a C18 reverse phase chromatographic column, an ultraviolet visible detector, and an online recording software system.
[0017] Further, step S3 comprises: fully drying the freshly picked rubber grass in an oven to dry the water to a constant weight, crushing the dried rubber grass into a complete powder using a crusher or grinder, placing it in a sealed plastic bag, and storing it in a desiccator at room temperature until it is extracted and used;
[0018] Weigh the rubber grass sample powder to be tested, put it into a 100mL round-bottom flask, add a certain volume of extracting solution, install the condenser, and extract the phenolic acid target components according to the set extraction temperature, extraction time, extraction method and other conditions. After the extraction is completed, take out the round-bottom flask and cool it to room temperature, then weigh the mass, add the extracting solution to make up the mass, shake well and let it stand for 10 minutes, take the supernatant and filter it with a 0.22μm microporous filter membrane to obtain the sample solution to be tested.
[0019] Further, in step S3, the mass of the powder is 0.1 to 1.0 g;
[0020] The volume of the extract is calculated based on the mass of the rubber grass powder. For every gram of rubber grass powder, the volume of the extract added is 10 to 100 mL, preferably 30 to 80 mL.
[0021] Furthermore, in step S3, the preset extraction conditions include extraction temperature, extraction time and extraction method.
[0022] Furthermore, the extraction temperature is 20 to 100° C., preferably 40 to 90° C.; the extraction time is 10 to 120 min, preferably 10 to 60 min.
[0023] Furthermore, the extraction method is ultrasonic extraction and water bath extraction, preferably water bath extraction.
[0024] Further, step S4 includes: using an automatic sampler to inject the sample solution to be tested into a high performance liquid chromatograph, with an injection volume of 20 μL, testing according to the set chromatographic conditions to obtain a chromatogram of the sample to be tested, and integrating the chromatographic peaks of each phenolic acid component in the sample chromatogram against the retention time of the standard to obtain the peak area of each phenolic acid target component.
[0025] In summary, this application has the following beneficial effects:
[0026] 1. Compared with the existing detection technology, the test method provided by the present invention has an HPLC test process of 34 to 40 minutes for unknown samples, is simple to operate and efficient, and can achieve efficient, rapid and accurate detection of the content of various phenolic acid components in rubber grass samples, filling the technical gap in the industry. At the same time, the high-performance liquid chromatograph used in the test method provided by the present invention is a general-purpose equipment, which is simple and convenient to operate, and has low equipment cost and maintenance;
[0027] 2. The extract of the present invention is composed of the following components in a volume ratio of formic acid: methanol: aqueous solution = 5:47.5:47.5. The extract adopts the above-mentioned component ratio. On the one hand, it can improve the separation degree and chromatographic peak shape: in chromatographic analysis, the mixed use of formic acid and methanol can improve the separation degree of phenolic substances and avoid the tailing phenomenon of chromatographic peaks. It may be because phenolic substances contain phenolic hydroxyl and carboxyl groups, which are easily ionized in aqueous solution, and adding a small amount of acidic reagents (such as formic acid) can inhibit the ionization of phenolic substances, thereby improving the separation effect; on the other hand, it can optimize the chromatographic conditions: when using formic acid aqueous solution and methanol as the mobile phase for gradient elution, the baseline has a smaller drift, and a better separation effect is obtained, thereby improving the resolution and accuracy of chromatographic analysis;
[0028] 3. This application uses a combination of potassium dihydrogen phosphate solution and methanol as an eluent, which can provide a stable pH environment, increase ionic strength, improve chromatographic separation effect, and is suitable for the analysis of phenolic acid compounds under acidic conditions, and has good stability and safety, as follows: First, potassium dihydrogen phosphate can increase the pH value of the solution, so that it remains stable within a certain range. In liquid chromatography analysis, the pH value has a significant effect on the retention behavior of the compound, and the separation conditions can be optimized by adjusting the pH value; second, potassium dihydrogen phosphate can increase the ionic strength of the solution and increase the retention time of the ion pair sample. This is particularly important for improving the chromatographic separation effect, especially for the analysis of ionic compounds; third, the combination of potassium dihydrogen phosphate and methanol can provide better separation effect. Methanol can form a gradient elution with the potassium dihydrogen phosphate solution in the aqueous phase, which helps to improve the separation and peak shape; fourth, the use of potassium dihydrogen phosphate solution adjusted to pH 2.9 can effectively analyze phenolic compounds under acidic conditions; fifth, in liquid chromatography analysis, the use of potassium dihydrogen phosphate and methanol as mobile phases can improve the sensitivity and selectivity of the analysis, thereby improving the accuracy of the quantification of phenolic acid compounds. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 is an HPLC chromatogram of six phenolic acid standards in an exemplary embodiment of the present application;
[0030] Figure 2 It is a HPLC chromatogram of phenolic acid components in 5# rubber grass sample in an exemplary embodiment of the present application.
[0031] Explanation of the accompanying drawings: 1. protocatechuic acid; 2. monocaffeoyltartaric acid; 3. chlorogenic acid; 4. caffeic acid; 5. chicoric acid; 6. ferulic acid. DETAILED DESCRIPTION
[0032] The present application is further described in detail with reference to the following examples. It should be noted that if no specific conditions are specified in the following examples, the conventional conditions or the conditions recommended by the manufacturer are followed, and the raw materials used in the following examples can be obtained from common commercial sources unless otherwise specified.
[0033] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.
[0034] In this application, "first aspect", "second aspect", "third aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.
[0035] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.
[0036] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.
[0037] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
[0038] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those generally understood by those skilled in the art to which this application belongs. The terms used in this application and in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used in this application includes any and all combinations of one or more related listed items. The term "multiple" in this application means at least two, such as two, three, etc., unless otherwise clearly and specifically limited.
[0039] The high performance liquid chromatograph used in this application is Model 1525 produced by Waters Corporation of the United States.
[0040] The raw materials of this application: rubber grass samples are provided by Heilongjiang Academy of Sciences and Xinjiang Academy of Agricultural Sciences and are commercially available products.
[0041] Example 1
[0042] A method for rapidly detecting the content of phenolic acid components in rubber grass specifically comprises the following steps:
[0043] (1) Preparation of standard samples
[0044] Use a precision weighing instrument to weigh 5.000 mg of monocaffeoyltartaric acid reference substance, 1.245 mg of chlorogenic acid reference substance, 1.332 mg of caffeic acid reference substance, 10.015 mg of chicoric acid reference substance, 1.336 mg of protocatechuic acid reference substance, and 0.215 mg of ferulic acid reference substance, respectively, place them in the same 5 mL volumetric flask, add the extract to the scale, shake well, and obtain the mixed reference substance stock solution.
[0045] Accurately pipette 0.5 mL of the mixed reference stock solution into a 10 mL volumetric flask, add the extract to make up to the mark, shake well, and use it as the mixed standard solution. The concentrations of the standards are: monocaffeoyltartaric acid 0.0500 mg / mL, chlorogenic acid 0.0125 mg / mL, caffeic acid 0.0133 mg / mL, chicoric acid 0.1002 mg / mL, protocatechuic acid 0.0134 mg / mL, and ferulic acid 0.0022 mg / mL.
[0046] It should be noted that the extract is prepared by the following components in a volume ratio, formic acid: methanol: aqueous solution = 5: 47.5: 47.5. The extract adopts the above component ratio. On the one hand, it can improve the separation degree and chromatographic peak shape: in chromatographic analysis, the mixed use of formic acid and methanol can improve the separation degree of phenolic substances and avoid the tailing phenomenon of chromatographic peaks. It may be because phenolic substances contain phenolic hydroxyl and carboxyl groups, which are easy to ionize in aqueous solution, and adding a small amount of acidic reagents (such as formic acid) can inhibit the ionization of phenolic substances, thereby improving the separation effect; on the other hand, it can optimize the chromatographic conditions: when using formic acid aqueous solution and methanol as the mobile phase for gradient elution, the baseline has a smaller drift, and a better separation effect is obtained, thereby improving the resolution and accuracy of chromatographic analysis.
[0047] (2) Preparation of standard curve
[0048] The liquid chromatography conditions adopted are: chromatographic column: Waters SunFire C18 column (150mm×4.6mm, 5μm); mobile phase includes phase A and phase B: phase A is methanol, phase B is 0.05mol / L potassium dihydrogen phosphate solution (phosphoric acid is added to pH 2.9), and the mobile phase gradient is shown in Table 1. The HPLC mobile phase gradient elution program in the embodiment is specifically as follows: by volume fraction, at 0 minute, the mobile phase A is 12%, and the mobile phase B is 88%; at 17 minutes, the mobile phase A is 19%, and the mobile phase B is 81%; at 22 minutes, the mobile phase A is 19%, and the mobile phase B is 81%; at 23 minutes, the mobile phase A is 30%, and the mobile phase B is 70%; at 33 minutes, the mobile phase A is 30%, and the mobile phase B is 70%; at 34 minutes, the mobile phase A is 12%, and the mobile phase B is 88%; at 40 minutes, the mobile phase A is 12%, and the mobile phase B is 88%. Flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 300 nm; injection volume: 20 μL.
[0049] Table 1 HPLC mobile phase gradient elution program in the embodiment
[0050]
[0051]
[0052] It should be noted that the combination of potassium dihydrogen phosphate solution and methanol as eluent can provide a stable pH environment, increase ionic strength, improve chromatographic separation effect, and is suitable for the analysis of phenolic acid compounds under acidic conditions, and has good stability and safety, as follows: First, potassium dihydrogen phosphate can increase the pH value of the solution, making it stable within a certain range. In liquid chromatography analysis, the pH value has a significant effect on the retention behavior of the compound, and the separation conditions can be optimized by adjusting the pH value; second, potassium dihydrogen phosphate can increase the ionic strength of the solution and increase the retention time of the ion pair sample. This is particularly important for improving the chromatographic separation effect, especially for the analysis of ionic compounds; third, the combination of potassium dihydrogen phosphate and methanol can provide better separation effect. Methanol can form a gradient elution with the potassium dihydrogen phosphate solution in the aqueous phase, which helps to improve the separation and peak shape; fourth, potassium dihydrogen phosphate solution is adjusted to pH 2.9, which can effectively analyze phenolic compounds under acidic conditions; fifth, in liquid chromatography analysis, the use of potassium dihydrogen phosphate and methanol as mobile phases can improve the sensitivity and selectivity of the analysis, thereby improving the accuracy of the quantification of phenolic acid compounds.
[0053] The mixed standard solution prepared in step (1) was weighed and injected with 5, 10, 15, 20, 25, 30, and 35 μL of the mixed standard solution using an automatic sampler of a high performance liquid chromatograph. The HPLC chromatograms of the six phenolic acid standards were obtained as follows: Figure 1 As shown, 1 is protocatechuic acid, 2 is monocaffeoyltartaric acid, 3 is chlorogenic acid, 4 is caffeic acid, 5 is chicoric acid, and 6 is ferulic acid.
[0054] Linear regression analysis was performed with the standard sample injection mass as the horizontal axis (X, μg) and the peak area as the vertical axis (Y, μV·s). The regression equation and linear correlation coefficient are shown in Table 2. The R values of the linear regression method for the six phenolic acid components were 2 The values are all greater than 0.9998, and the linear relationship of the standard curve is good, which meets the requirements of quantitative determination. The linear correlation coefficient of the standard curve equation is closer to 1, so the above standard curve can be used for quantitative calculation with high accuracy.
[0055] Table 2 Linear test range and regression equation of 6 phenolic acid standards in the embodiment
[0056]
[0057] (3) Preparation of sample solution
[0058] Place the rubber grass sample in an oven at 60°C and thoroughly dry it to constant weight. Grind it into powder with a multi-sample tissue grinder and pass it through a No. 4 sieve. Accurately weigh 0.5g (accurate to 0.0001g) and place it in a 100mL round-bottom flask. Add 25ml of the extracting solution formic acid-methanol-water (volume ratio of 5:47.5:47.5), shake well, weigh the mass, install a condenser, set the water bath temperature to 80°C, extract in a water bath for 30 minutes, take out the round-bottom flask, cool it to room temperature, weigh it again, add the extracting solution to make up the mass, shake well and let it stand for 10 minutes, take the supernatant and filter it with a 0.22μm microporous filter membrane to obtain the sample solution to be tested.
[0059] It should be noted that 10 rubber grass samples were selected, and each rubber grass sample was prepared with the above method to prepare the sample solution with the same process parameters.
[0060] (4) Test the sample
[0061] 20 μL of the prepared sample solution was injected into the HPLC autosampler and measured according to the above chromatographic conditions to obtain the HPLC spectrum of the sample. Taking sample 5# as an example, the HPLC chromatogram of phenolic acid components obtained is as follows: Figure 2 As shown. Figure 1 The retention time of each standard component was integrated to obtain the component peak areas of monocaffeoyltartaric acid, chlorogenic acid, caffeic acid, chicoric acid, protocatechuic acid, and ferulic acid.
[0062] (5) Calculation of the contents of six phenolic acid components
[0063] Substitute the peak area of the measured phenolic acid target components into the standard curve equation to obtain the injection mass of the six phenolic acid components, and then calculate the contents of the six phenolic acid components in rubber grass according to the sample mass, the concentration of the solution to be tested and the HPLC injection volume. As shown in Table 3, the determination results of the contents of the six phenolic acid components in 10 rubber grass samples.
[0064] Table 3 Determination results of the contents of 6 phenolic acids in rubber grass samples
[0065]
[0066] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A method for rapidly detecting the content of phenolic acid components in rubber grass, characterized in that: The steps include: S1. Weigh a phenolic acid component standard, add the extract to dissolve it, and obtain a phenolic acid component standard mixed solution; S2, measuring different volumes of the mixed solution of the phenolic acid component standard substance, injecting it into a high performance liquid chromatograph, testing it according to preset chromatographic conditions, obtaining a chromatogram of the standard substance of each phenolic acid target component, integrating the chromatogram to obtain the chromatographic peak area of the standard substance of each component, and preparing a standard curve; S3, drying the rubber grass and grinding it into powder, adding an extracting solution to the powder, extracting the target component according to a preset extraction condition, and filtering to obtain a sample solution to be tested; S4, filtering the sample solution to be tested through an organic phase microporous filter membrane, injecting the sample solution to be tested into a high performance liquid chromatograph, testing according to the preset chromatographic conditions, obtaining a chromatogram of the sample to be tested, and integrating to obtain the peak area of each target component; S5. Substitute the peak area of each target component into the standard curve, calculate the injection mass of the actual sample, and calculate the content of each phenolic acid component in the sample to be tested.
2. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 1, characterized in that: In step S1, the phenolic acid components include protocatechuic acid, protocatechuic aldehyde, monocaffeoyltartaric acid, chlorogenic acid, caffeic acid, chicoric acid, and ferulic acid.
3. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 1, characterized in that: In step S1, the extract comprises formic acid: methanol: aqueous solution in a volume ratio of 5:47.5:47.
5.
4. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 1, characterized in that: In step S2, the chromatographic conditions include: wavelength, column temperature, mobile phase, mobile phase program, pH value; the wavelength is 260-320 nm, and the column temperature is 25-45°C.
5. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 4, characterized in that: The mobile phase comprises phase A and phase B, phase A is methanol, phase B is 0.05 mol / L potassium dihydrogen phosphate solution, and the pH value of the mobile phase is 2-4.
6. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 5, characterized in that: The mobile phase program adopts a gradient elution program, defining the ratio of phase A to phase B as the proportion of the organic phase. Within 0 to 22 minutes, the proportion of the organic phase is 10% to 20%, within 23 to 33 minutes, the proportion of the organic phase is 20% to 30%, and within 34 to 40 minutes, the proportion of the organic phase is 10% to 20%.
7. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 1, characterized in that: In step S3, the mass of the powder is 0.1 to 1.0 g; The volume of the extract added to each gram of the powder is 10 to 100 mL.
8. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 1, characterized in that: In step S3, the preset extraction conditions include extraction temperature, extraction time and extraction method.
9. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 8, characterized in that: The extraction temperature is 20-100° C., and the extraction time is 10-120 minutes.
10. The method for rapidly detecting the content of phenolic acid components in rubber grass according to claim 8, characterized in that: The extraction method is ultrasonic extraction and water bath extraction.