A method for simultaneously detecting contents of multiple active ingredients in Ficus hirta by HPLC

Abstract

The application belongs to the technical field of natural fruit active substance detection, and discloses a method for simultaneously detecting the contents of multiple active components in green fruits based on HPLC, wherein the active components include seven main active components in green fruits, i.e., pyrogallol, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin and ellagic acid. The detection method established by the application is good and the instrument is precise, and the method has the technical advantages of good selectivity, high sensitivity and accuracy and reliability for detecting the contents of the main active components in green fruits, thereby providing a new analysis method and reliable basis for quality analysis and quality control of green fruits.

Description

Technical Field

[0001] This invention belongs to the field of detection technology of active substances in natural fruits, and relates to a method for simultaneous detection of the content of multiple active ingredients in green fruits based on HPLC. Background Technology

[0002] Canarii fructus, also known as olive or white olive, is the mature, dried fruit of the olive tree (Canarium album (Lour.) Raeusch), a member of the Burseraceae family, native to my country. It is distributed in several provinces and regions of my country, including Fujian, Sichuan, Yunnan, and Guangdong, and was among the first batch of foods listed in my country as both food and medicine. Unlike the oil olive (Olea europaea L., native to the Mediterranean and often referred to as olive by Chinese people), which is used for oil extraction, canarii fructus is a fruit with low oil content but rich in protein, vitamins, flavonoids, and polyphenols. It is also commonly used in traditional Chinese medicine. The Compendium of Materia Medica and the Chinese Pharmacopoeia record its effects of clearing the throat and promoting saliva production, relieving cough and phlegm, and detoxifying. It can be used to treat sore throat, cough, thirst and hemoptysis, enteritis and dysentery, and fish and crab poisoning. Modern pharmacological research shows that canarii fructus has anti-tumor, antioxidant, antibacterial, anti-inflammatory, antiviral, and lipid-regulating and blood sugar-lowering effects.

[0003] With the improvement of people's living standards, people are paying more and more attention to health. As a traditional Chinese medicine, green fruit is often used in medicine and cuisine. However, the edible and medicinal effects and quality of different varieties of green fruit vary, and the content and scope of its characteristic evaluation and quality identification need to be broadened.

[0004] Currently, many scholars have conducted relevant research on the extraction, separation, and identification of chemical components in *Clerodendrum trichotomum*, such as the published literature "[Exploration of the Health Benefits and Modern Applications of *Clerodendrum trichotomum*, Chinese Herbal Medicines, 2021, No. 20]". However, there are few reports on the quantitative detection of chemical components in *Clerodendrum trichotomum*. Phenolic compounds are one of the main active ingredients in *Clerodendrum trichotomum*, and are also widely recognized natural free radical scavengers with antioxidant, anti-inflammatory, hypoglycemic, hypotensive, and anti-aging effects. Han Xue et al. published "[Simultaneous Determination of Four Components in *Clerodendrum trichotomum* by RP-HPLC, Traditional Chinese Medicine, 2017, No. 4]" which used HPLC to determine four active ingredients in *Clerodendrum trichotomum*. However, the contents of two of these active ingredients, chlorogenic acid and protocatechuic acid, were relatively low, lacking representativeness for evaluating the content levels of the main active ingredients in *Clerodendrum trichotomum*. Therefore, it is necessary to further develop and establish corresponding content determination methods for the active ingredients with high content in green plums, such as pyrogallol, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid. This is of great significance for improving the quality testing and evaluation system of green plums and enhancing their edible and medicinal value. Summary of the Invention

[0005] The purpose of this invention is to provide a method for the simultaneous detection of multiple active ingredients in green plums based on HPLC. The active ingredients include seven main active ingredients in green plums: pyrogallol, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid. The detection method and instruments established in this invention are of good quality and precision, and have the technical advantages of good selectivity, high sensitivity, accuracy, and reliability in the detection of the main active ingredients in green plums.

[0006] The objective of this invention is achieved through the following technical solution: a method for simultaneously detecting the content of multiple active ingredients in green plums based on HPLC, specifically including the following steps:

[0007] The preparation of green fruit sample solution includes the following steps: removing the pits from the green fruit and vacuum drying it, crushing and sieving it, then ultrasonically extracting it with methanol as solvent to obtain filtrate, concentrating the filtrate under reduced pressure to remove the solvent and freeze-drying it under vacuum to obtain green fruit alcohol extract, and dissolving it in methanol to prepare green fruit sample solution.

[0008] The green fruit sample solution to be tested was analyzed using high-performance liquid chromatography (HPLC). The HPLC analysis included gradient elution of the sample and determination of the peak area of ​​the dominant peak of the eluent. The chromatographic conditions for the HPLC analysis were as follows: stationary phase: a column packed with octadecyl-bonded silica gel; mobile phase A: 0.1% aqueous acetic acid solution (v / v); mobile phase B: 100% acetonitrile solution (v / v); flow rate: 0.5 mL-1.5 mL / min; detection wavelength: 200-280 nm; injection volume: 5- 20 μL; Column temperature: 30-40℃; The gradient elution program is as follows: Time: 0-6.5 min, mobile phase A: 99%, mobile phase B: 1%; Time: 6.5-7 min, mobile phase A: 99%→92%, mobile phase B: 1%→8%; Time: 7-13 min, mobile phase A: 92%, mobile phase B: 8%; Time: 13-20 min, mobile phase A: 92%→88%, mobile phase B: 8%→12%; Time: 20-54 min, mobile phase A: 88%→83%, mobile phase B: 12%→17%.

[0009] The contents of the above seven active ingredients in green plums were calculated using the external standard method. It should be noted that the specific steps for calculating the contents of the above seven active ingredients in green plums using the external standard method are as follows: Standards for pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid were prepared. Standards of different concentrations were analyzed using high-performance liquid chromatography (HPLC). Standard curves were plotted with the injection concentration as the abscissa and the peak area as the ordinate to determine the linear regression equations for pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid. The contents of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid in the green plum sample to be tested were calculated using the linear regression equations.

[0010] Specifically, the linear regression equation is as follows:

[0011] Pyrogallic acid: Y1 = 1959X1 - 7591.3, R 2 =0.9989;

[0012] Gallic acid: Y2 = 19158X2 - 62150, R 2 =0.9991;

[0013] Epicatechin: Y3 = 1404.2X3 - 4660.5, R 2 =0.9995;

[0014] Ethyl gallate: Y4 = 6384.8X4 - 34204, R 2 =0.9979;

[0015] p-Coumaric acid: Y5 = 8222.5X5 - 8701.3, R 2 =0.9991;

[0016] Table of gallic catechins: Y6 = 5154.5X6 - 5202.1, R 2 =0.9996;

[0017] Ellagic acid: Y7 = 24927X7 - 132172, R 2 =0.9993.

[0018] Preferably, in the operation steps of preparing the green fruit sample solution, the specific operation steps of ultrasonic extraction using methanol as solvent are as follows: ultrasonic extraction is performed using a methanol aqueous solution with a volume concentration of 70% to 95%, the ultrasonic power is 300-500W, the temperature is 30-50℃, the extraction time is 10-60min, the solid-liquid mass-volume ratio of ultrasonic extraction is 1:10 to 1:25 and the unit is g / mL, after extraction twice, the filtrates are filtered and combined.

[0019] More preferably, in the step of preparing the green fruit sample solution, the volume concentration of the methanol aqueous solution is 70%.

[0020] More preferably, in the step of preparing the green fruit sample solution, the ultrasonic extraction temperature is 45℃, the ultrasonic time is 30min, the ultrasonic power is 400W, and the solid-liquid ratio of the ultrasonic extraction is 1:20.

[0021] More preferably, the chromatographic conditions for analysis using the high-performance liquid chromatograph are as follows: flow rate: 1 mL / min; detection wavelength: 254 nm; injection volume: 10 μL; column temperature: 30 °C.

[0022] Beneficial effects:

[0023] The present invention validated the established method for simultaneous detection of multiple active ingredients in green fruit based on HPLC, including specificity experiments, precision experiments, stability experiments, repeatability experiments, and sample recovery experiments. The validation results show that the detection method established by the present invention is good and the instrument is precise. Under the established detection conditions, it is feasible to simultaneously detect the content of multiple major active ingredients in the sample.

[0024] The method for simultaneous detection of multiple active ingredients in green fruit based on HPLC established in this invention has good selectivity, high sensitivity, accuracy and reliability, providing a new analytical method and reliable basis for the quality analysis and quality control of green fruit. Attached Figure Description

[0025] Figure 1 It is the standard curve of pyrogallol;

[0026] Figure 2 It is the standard curve of gallic acid;

[0027] Figure 3 It is the standard curve of epicatechin;

[0028] Figure 4 It is the standard curve of ethyl gallate;

[0029] Figure 5 This is the standard curve for coumaric acid;

[0030] Figure 6 This is the standard curve of gallic acid catechin;

[0031] Figure 7 It is the ellagic acid standard curve;

[0032] Figure 8 This is the chromatogram of the test sample solution;

[0033] Figure 9 This is the chromatogram for Comparative Example 1;

[0034] Figure 10 This is the chromatogram for Comparative Example 2;

[0035] Figure 11 This is the chromatogram of the reference solution;

[0036] Figure 12 This is a blank solvent chromatogram. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0038] I. Sources of Materials and Reagents

[0039] Pyrogallic acid standard: purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity: 99%;

[0040] Gallic acid standard: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity: 99%;

[0041] Epicatechin standard: purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity: 98%;

[0042] Ethyl gallate standard: purchased from Shanghai Maclean Biochemical Technology Co., Ltd., purity: 98%;

[0043] p-Coumaric acid standard: purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity: 98%;

[0044] Epigallocatechin standard: purchased from Shanghai Maclean Biochemical Technology Co., Ltd., purity: 98%;

[0045] Ellagic acid standard: purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity: 98%;

[0046] Raw material: Mature olives purchased from olive orchards in Minqing County, Fuzhou City, Fujian Province; variety: sandalwood olive.

[0047] Acetic acid: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., HPLC grade, purity: 99.9%;

[0048] 100% acetonitrile solution: purchased from Anhui Tiandi High Purity Solvent Co., Ltd., HPLC grade.

[0049] II. Instruments

[0050] The liquid chromatograph is a SHIMADZU LC-20A, manufactured by Shimadzu Enterprise Management Co., Ltd.

[0051] Example 1

[0052] This embodiment provides a specific method for the simultaneous detection of multiple active ingredients in green plums based on HPLC. The operation steps are as follows:

[0053] Step S1: Take an appropriate amount of green fruit sample, remove the pits from the green fruit and vacuum dry it. After pulverizing and sieving, add 70% methanol aqueous solution for ultrasonic extraction. The ultrasonic power is 400W, the temperature is 45℃, the extraction time is 30min, and the solid-liquid ratio of ultrasonic extraction is 1:20. After extraction twice, filter and combine the filtrates. After removing the pits, place it in a vacuum drying oven to dry to constant weight, pulverize and sieve. Accurately weigh the fine powder, add methanol solution for ultrasonic extraction twice, filter and combine the filtrates, concentrate under reduced pressure to remove the solvent, and freeze dry under vacuum to obtain the green fruit alcohol extract. After dissolving in methanol, it becomes the green fruit sample solution M2 to be tested.

[0054] Step S2: The green fruit sample solution to be tested was filtered through a 0.22 μm filter and then sent to a high-performance liquid chromatograph (HPLC) for analysis. The chromatographic conditions for the HPLC analysis were as follows: stationary phase: octadecyl-bonded silica column; mobile phase A: 0.1% acetic acid aqueous solution; mobile phase B: 100% acetonitrile solution; flow rate: 1 mL / min; detection wavelength: 254 nm; injection volume: 10 μL; column temperature: 30℃. The sample was subjected to gradient elution, and the peak area of ​​the dominant peak of the eluent was determined. The gradient elution program is shown in Table 1, and the corresponding chromatograms obtained are shown below. Figure 8 As shown.

[0055] Table 1 Gradient elution program

[0056] Time (min) A% (0.1% acetic acid aqueous solution) B% (100% acetonitrile solution) 0~6.5min 99％ 1％ 6.5–7 min 99％→92％ 1％→8％ 7–13 min 92％ 8％ 13-20 min 92％→88％ 8％→12％ 20–54 min 88％→83％ 12％→17％

[0057] Step S3: Obtain standards for pyrogallol, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid. Analyze the standards of different concentrations using high-performance liquid chromatography (HPLC). The chromatographic conditions for HPLC analysis are the same as in step S2. Specifically, accurately weigh 5.0 mg of pyrogallic acid standard, 4.0 mg of gallic acid standard, 5.0 mg of epicatechin standard, 5.0 mg of ethyl gallate standard, 1.0 mg of p-coumaric acid standard, 2.0 mg of epigallocatechin standard, and 5.0 mg of ellagic acid standard. Dissolve them in methanol and dilute to volume in a 50 mL volumetric flask to obtain the stock solution. Then, take a portion of the stock solution and dilute it sequentially to different concentrations. Filter the solution through a 0.22 μm microporous membrane. Inject 10 μL of the filtrate for each analysis and record the peak area. Plot a standard curve with peak area (Y) as the ordinate and standard concentration (X, in μg / mL) as the abscissa. The regression equations for the pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid standards are as follows:

[0058] The determined linear regression equation is as follows: Pyrogallic acid: Y1 = 1959X1 - 7591.3, R 2 =0.9989, the corresponding standard curve is as follows Figure 1 As shown; Gallic acid: Y2=19158X2-62150, R 2 =0.9991, the corresponding standard curve is as follows Figure 2 As shown; epicatechin: Y3 = 1404.2X3 - 4660.5, R 2 =0.9995, the corresponding standard curve is as follows Figure 3 As shown; Ethyl gallate: Y4 = 6384.8X4 - 34204, R 2 =0.9979, the corresponding standard curve is as follows Figure 4 As shown; for coumaric acid: Y5 = 8222.5X5 - 8701.3, R 2 =0.9991, the corresponding standard curve is as follows Figure 5 As shown; Table: Gallic catechin: Y6 = 5154.5X6 - 5202.1, R 2 =0.9996, the corresponding standard curve is as follows Figure 6 As shown; Ellagic acid: Y7=24927X7-132172, R 2 =0.9993, the corresponding standard curve is as follows Figure 7As shown, the linear relationship between pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid is good. The contents of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid in the tested green fruit samples were calculated using linear regression equations to be 20.95 mg / g, 54.77 mg / g, 48.83 mg / g, 30.78 mg / g, 9.80 mg / g, 23.21 mg / g, and 22.19 mg / g, respectively.

[0059] Comparative Example 1

[0060] The only difference between Comparative Example 1 and Example 1 is that the specific steps of gradient elution are shown in Table 2; all other steps are identical. The corresponding chromatograms obtained are shown below. Figure 9 As shown.

[0061] Table 2

[0062] Time (min) A% (0.1% acetic acid aqueous solution) B% (100% acetonitrile solution) 0~6min 99％ 1％ 6–6.5 min 99％→93％ 1％→7％ 6.5–15 min 93％ 7％ 15–19 min 93％→88％ 7％→12％ 19–54 min 88％→83％ 12％→17％

[0063] Compared to Example 1, such as Figure 9 As shown, the chromatogram peak shape and separation effect in Comparative Example 1 were both unsatisfactory.

[0064] Comparative Example 2

[0065] The only difference between Comparative Example 2 and Example 1 is that the specific steps of the gradient elution are shown in Table 3; all other operations are exactly the same. The corresponding chromatograms obtained are shown below. Figure 10 As shown.

[0066] Table 3

[0067] Time (min) A% (0.1% acetic acid aqueous solution) B% (100% acetonitrile solution) 0~6.5min 99％ 1％ 6.5–7.5 min 99％→92％ 1％→8％ 7.5–12 min 92→90％ 8％→10％ 12-20 min 90％→87％ 10％→13％ 20–50 min 87％→81％ 13％→19％ 50–54 min 81％→75％ 19％→25％

[0068] Compared to Example 1, such as Figure 10 As shown, the peak shape and separation effect of the chromatogram in Comparative Example 2 were both unsatisfactory.

[0069] Test Example 1: Specificity Test

[0070] Accurately transfer pyrogallol (50 μg / mL), gallic acid (40 μg / mL), epicatechin (50 μg / mL), ethyl gallate (50 μg / mL), p-coumaric acid (10 μg / mL), epigallocatechin (20 μg / mL), and ellagic acid (50 μg / mL) to a mixture as reference solution M1. Then, take a blank negative solution, i.e., a 70% methanol aqueous solution, as a blank control solution M3. After filtering through a 0.22 μm filter, the corresponding chromatograms were obtained by detection according to the chromatographic conditions of step S2 in Example 1.

[0071] like Figure 8 The chromatogram of the green plum sample solution M2 extracted in Example 1 shows that the separation of each component is ideal, with a resolution greater than 1.5; Figure 11 The chromatogram of reference solution M1 is shown below. Figure 12 The image shows a blank solvent chromatogram. (Comparison is needed.) Figure 8 , Figure 11 and Figure 12 The chromatograms show that the blank solvent did not interfere with the determination of the main substance, indicating that the method has good specificity.

[0072] Test Example 2: Precision Test

[0073] Take the mixed reference solution M1, inject it 6 times repeatedly, with an injection volume of 10 μL, and determine it according to the chromatographic conditions of Example 1. Record the peak area and retention time of each peak. Use gallic acid (peak 2) as the internal reference peak and calculate the RSD of the relative peak area and the RSD of the relative retention time of the remaining peaks.

[0074] The experimental results are shown in Table 4. The results show that the RSD (%) of the relative retention time of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin and ellagic acid are all <0.5%, and the RSD (%) of the relative peak area is all less than 3%, indicating that the instrument has good precision.

[0075] Table 4 Precision Test Results

[0076]

[0077] Test Example 3: Stability Test

[0078] Mixed reference solution M1 was injected in six batches at 0, 3, 6, 9, 12, and 24 hours, corresponding to batches 1 to 6 in Table 5, with an injection volume of 10 μL. The peak areas were measured according to the chromatographic conditions of Example 1, and the peak areas were recorded. Gallic acid (peak 2) was used as the internal reference peak, and the RSD of the relative peak area and the RSD of the relative retention time of the other peaks were calculated.

[0079] The experimental results are shown in Table 5. The results show that the relative retention time (RSD) of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin and ellagic acid is all <0.5%, and the relative peak area (RSD) is all less than 3%, indicating that the solution has good stability within 24 hours and can meet the determination requirements.

[0080] Table 5. Results of stability tests

[0081]

[0082] Test Example 4: Repeatability Test

[0083] Take 10 mg of the ethanol extract powder from Example 1, precisely stabilize it, and dilute it to 10 mL with methanol. Filter the solution through a 0.22 μm filter to obtain the sample solution. Prepare six parallel sample solutions corresponding to Sample 1 to Sample 6 in Table 6, with an injection volume of 10 μL. Determine the chromatographic conditions according to Example 1, using gallic acid (peak 2) as the internal reference peak, and calculate the relative peak area RSD and relative retention time RSD of the remaining peaks.

[0084] The experimental results are shown in Table 6. The results show that the relative retention time (RSD) of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin and ellagic acid is all <0.5%, and the relative peak area (RSD) is all less than 3%, indicating good repeatability.

[0085] Table 6 Results of Repeatability Tests

[0086]

[0087] Test Example 5: Spiking Recovery Test

[0088] Accurately weigh 1.0 mg of pyrogallic acid, 2.5 mg of gallic acid, 2.5 mg of epicatechin, 1.5 mg of ethyl gallate, 0.5 mg of p-coumaric acid, 1.0 mg of epigallocatechin, and 1.0 mg of ellagic acid standards and place them in seven conical flasks. Accurately add 0.05 g of the known content of the pine olive extract powder from Example 1 to each flask. The contents of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid are 1.05 mg, 2.74 mg, 2.44 mg, 1.54 mg, 0.49 mg, 1.16 mg, and 1.11 mg, respectively. Add methanol to a final volume of 100 mL. Inject 10 μL of the sample and determine the chromatographic conditions according to Example 1. Record the peak area and calculate the recovery rate and RSD according to formula (1).

[0089]

[0090] The experimental results are shown in Table 7. The results show that the recoveries of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid are all between 95% and 105%, with average recoveries of 99.43%, 99.11%, 99.25%, 99.25%, 100.07%, 99.79%, and 99.83%, respectively. The RSDs are all less than 2.5%, indicating that the method established in this invention is reliable and meets the requirements for content determination.

[0091] Table 7 Results of the recovery test

[0092] .

Claims

1. A method for simultaneous determination of multiple active ingredients in green plums based on HPLC, characterized in that, The various active ingredients include seven active ingredients: pyrogallol, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid. The method specifically includes the following steps: The preparation of green fruit sample solution includes the following steps: removing the pits from the green fruit and vacuum drying it, crushing and sieving it, then ultrasonically extracting it with a methanol aqueous solution with a volume concentration of 70%~95% to obtain a filtrate, concentrating the filtrate under reduced pressure to remove the solvent and then freeze-drying it under vacuum to obtain an alcohol extract of green fruit, which is then dissolved in methanol to prepare the green fruit sample solution. The green fruit sample solution to be tested was analyzed using high-performance liquid chromatography (HPLC). The HPLC analysis included gradient elution of the sample and determination of the peak area of ​​the dominant peak of the eluent after gradient elution. The chromatographic conditions for the HPLC analysis were as follows: stationary phase was a column packed with octadecyl-bonded silica gel; mobile phase A was a 0.1% (v / v) aqueous acetic acid solution, and mobile phase B was pure acetonitrile; flow rate was 1 mL / min; detection wavelength was 254 nm; injection volume was 5-20 μL; column temperature was 30-40 °C. The gradient elution program was as follows: time: 0-6.5 min, mobile phase A: 99%, mobile phase B: 1%; time: 6.5-7 min, mobile phase A: 99%→92%, mobile phase B: 1%→8%; time: 7-13 min, mobile phase A: 92%, mobile phase B: 8%; time: 13-20 min... min: mobile phase A: 92%→88%, mobile phase B: 8%→12%; time: 20~54 min, mobile phase A: 88%→83%, mobile phase B: 12%→17%; The contents of the above seven active ingredients in green fruit were calculated using the external standard method.

2. The method for simultaneous detection of multiple active ingredients in green plums based on HPLC according to claim 1, characterized in that, The specific steps for ultrasonic extraction using a methanol aqueous solution with a volume concentration of 70%~95% are as follows: ultrasonic extraction is performed using a methanol aqueous solution with a volume concentration of 70%~95%, with an ultrasonic power of 300-500W, a temperature of 30~50℃, an extraction time of 10~60min, and a solid-liquid mass-to-volume ratio of 1:10~1:25 in g / mL. After two extractions, the mixture is filtered and the filtrates are combined.

3. The method for simultaneous detection of multiple active ingredients in green plums based on HPLC according to claim 2, characterized in that, The volume concentration of the methanol aqueous solution is 70%.

4. The method for simultaneous detection of multiple active ingredients in green plums based on HPLC according to claim 2, characterized in that, The ultrasonic extraction temperature was 45℃, the ultrasonic time was 30 min, the ultrasonic power was 400 W, and the solid-liquid ratio of the ultrasonic extraction was 1:

20.

5. The method for simultaneous detection of multiple active ingredients in green plums based on HPLC according to claim 1, characterized in that, The chromatographic conditions for analysis using the high-performance liquid chromatograph are as follows: flow rate 1 mL / min; detection wavelength 254 nm; injection volume 10 μL; column temperature 30 °C.

6. The method for simultaneous detection of multiple active ingredients in green plums based on HPLC according to claim 1, characterized in that, The steps for calculating the content of the above seven active ingredients in green fruit using the external standard method are as follows: Prepare standards for pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid. Analyze the standards at different concentrations using high-performance liquid chromatography (HPLC). Plot standard curves with the injection concentration as the abscissa and the peak area as the ordinate to determine the linear regression equations for pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid. Calculate the content of pyrogallic acid, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid in the green fruit sample to be tested using the linear regression equations.

7. The method for simultaneous detection of multiple active ingredients in green plums based on HPLC according to claim 6, characterized in that, The linear regression equations for pyrogallol, gallic acid, epicatechin, ethyl gallate, p-coumaric acid, epigallocatechin, and ellagic acid are as follows: Pyrogallic acid: Y1=1959X1-7591.3, R 2 =0.9989; Gallic acid: Y2=19158X2-62150, R 2 =0.9991; Epicatechin: Y3 = 1404.2X3 - 4660.5, R 2 =0.9995; Ethyl gallate: Y4 = 6384.8X4 - 34204, R 2 =0.9979; p-Coumaric acid: Y5 = 8222.5X5 - 8701.3, R 2 =0.9991; Table of gallic catechin: Y6 = 5154.5X6 - 5202.1, R 2 =0.9996; Ellagic acid: Y7=24927X7-132172, R 2 =0.9993.

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