A cicada slough processing method and a cicada slough quality detection method

Abstract

The present invention discloses a method for producing cicada sloughs, comprising the steps of: (1) washing live cicada nymphs with water; and (2) returning the washed cicada nymphs to a tree for eclosion, separating the exuviae after eclosion, collecting the exuviae, and drying the exuviae to obtain the cicada sloughs. The present invention screens out a new method for producing cicada sloughs through a large number of experiments and evaluates the quality of the cicada sloughs using a UPLC-Q-TOF-MS / MS method. The results show that washing the exuviae before eclosion can reduce the loss of the active ingredient acetyl dopamine polymers and reduce the total ash content and acid-insoluble ash content to achieve the purpose of removing sediment.

Description

Technical Field

[0001] The present invention relates to a processing method of traditional Chinese medicine, in particular to a processing method of cicada slough and a quality detection method of cicada slough.

[0002] Background

[0003] Cicada molts are the skins shed by nymphs of the black cicada Cryptotympana atrata during eclosion. They are an important medicinal resource. Their medicinal properties: They are slightly oval and curved, approximately 3.5 cm long and 2 cm wide. Their surface is yellowish-brown, translucent, and shiny. [Nature and Flavor] Sweet, cold. They enter the Lung and Liver meridians. [Functions and Indications] Dispel wind and heat, relieve sore throats, clear rashes, remove cataracts, and relieve spasms. They are used for wind-heat colds, sore throats, hoarseness, measles that has not yet cleared up, itchy urticaria, red eyes with cataracts, convulsions, and tetanus.

[0004] Cicadas belong to the genus Cicadidae, family Cicadidae, order Hemiptera, class Insecta. They are insects with incomplete metamorphosis and their growth goes through three stages: egg, nymph, and adult.

[0005] Wild cicadas have a long natural growth cycle, typically completing a generation in three to five years. To meet market demand, cicada farming has become increasingly popular. This involves cultivating cicadas in selected forests and artificially controlling the hatching of eggs to shorten their growth cycle, improve breeding efficiency, and thus increase production.

[0006] Therefore, there are two ways to obtain cicada shells. One is to harvest them after wild nymphs molt, and the other is to harvest them after artificially cultivated nymphs molt.

[0007] At present, the processing method of cicada slough in the prior art is to collect cicada slough from tree trunks, remove soil and impurities after collection, dry them in the sun, and then pack them in a dry place to prevent them from being crushed and damp.

[0008] The 2020 edition of the Chinese Pharmacopoeia stipulates that cicada sloughs must be free of mud and sand after harvest. The usual method is to wash the sloughs harvested after the nymphs molt. While this method can remove mud and sand, it also results in a significant loss of active ingredients and the sloughs are fragile, affecting their quality. Summary of the Invention

[0009] Purpose of the Invention: The present invention aims to provide a high-quality processing method for cicada sloughs that removes sediment while minimizing the loss of active ingredients, ensuring quality and enhancing the medicinal value of cicada sloughs. Another purpose of the present invention is to provide a quality testing method for cicada sloughs.

[0010] Technical solution: To achieve the above objectives, the technical solution adopted by the present invention is:

[0011] A method for producing cicada sloughs comprises the following steps:

[0012] (1) Take a live cicada nymph and wash it with water;

[0013] (2) The washed cicada nymphs are placed back on the tree to eclode, and the exuviae are separated and collected after eclosion. The exuviae are dried to obtain the cicada exuviae.

[0014] Preferably, the cicada slough production method of the present invention further includes a step of capturing cicada nymphs, and the capturing method is as follows: in a woodland where cicadas are artificially bred, first wrap a circle of 2-10 cm wide plastic tape around a tree trunk at 80-150 cm, and capture the cicada nymphs when they crawl to the tree trunk under the plastic tape; wherein, the capturing time can be from mid-to-late June to mid-August each year, preferably from 6 pm to 12 midnight, and preferably after rain.

[0015] Preferably, in the cicada slough production method of the present invention, the washing with water in step (1) comprises placing the captured cicada nymphs into a container filled with clean water, gently washing them with clean water for multiple times, pouring out the water containing mud and sand and replacing it with new water each time until the water is clear.

[0016] Preferably, in the cicada slough production method of the present invention, in step (2), the washed cicada nymphs are placed back on the tree, and the nymphs crawl along the tree trunk for a while before becoming still and beginning to eclode. The eclosion process lasts about 90 to 150 minutes. After the nymphs have completed their molting, the cicada sloughs are collected. The drying step is to place the cicada sloughs in a ventilated and dry place to dry naturally.

[0017] In the method of the present invention, the cicada nymphs are wild or artificially cultivated, preferably artificially cultivated.

[0018] The definitions of the relevant terms are as follows:

[0019] Cicada nymph: The black cicada in this growth stage between hatching from eggs and metamorphosis into an adult.

[0020] Trees: Parasitic tree species of cicadas include willows, elms, apple trees, pear trees, etc.

[0021] Emergence: The process of cicada transforming from larva to adult.

[0022] Molting: During the process of molting into an adult, the cicada nymph leaves behind the outer shell and becomes an adult.

[0023] Washing: Wash with clean water.

[0024] The destination of cicada nymphs after molting: they stay on the tree and wait for their wings to dry before flying away on their own, or they are temporarily placed in water and can be eaten.

[0025] The method of the present invention is obtained through screening, and the screening process is as follows:

[0026] Trial groups:

[0027] Group 1: cicada sloughs (R-SXT) produced by the method of the present invention: artificially cultured nymphs were captured, washed before molting, and collected after molting;

[0028] Group 2: Artificial nymph unwashed cicada molt (R-WSX): the nymphs were not washed before and after molting;

[0029] Group 3: Washed cicada sloughs after wild nymph molt (Y-TSX): The cicada sloughs collected after molting were repeatedly washed with clean water until the water was clear;

[0030] Group 4: Unwashed cicada sloughs from wild nymphs (Y-WSX): cicada sloughs collected after wild nymphs molted and were not washed during the entire process.

[0031] The four groups of cicada sloughs produced by the above different methods were tested for active ingredient content and ash content. The test results showed that the method of the present invention, that is, washing the artificially cultivated cicada nymphs before molting, can not only reduce the loss of the active ingredient acetyldopamine polymer in the cicada slough medicinal material, but also significantly reduce the total ash content and the acid-insoluble ash content. Specific test results and statistical analysis are shown in Example 2.

[0032] In order to detect the quality of the cicada slough product of the present invention, the present invention further develops a new detection method, which adopts liquid chromatography-mass spectrometry to detect the content of acetyldopamine polymer components in the cicada slough product.

[0033] The detection method of the present invention comprises the following steps:

[0034] (1) Preparation of test solution: Weigh cicada slough powder, add methanol, place in a stoppered test tube, weigh to determine the weight, perform ultrasonic extraction, weigh again, make up the loss with methanol, centrifuge the extract, take the supernatant, dilute with methanol and make up to volume;

[0035] (2) Preparation of mixed reference solution: Accurately weigh the reference substances of acetyl dopamine dimer A and B, and add methanol to prepare reference substance stock solutions A and B, respectively; then, take appropriate amounts of reference substance stock solutions A and B, mix them, and add methanol to make up to volume to prepare a mixed reference solution;

[0036] (3) The test solution of step (1) and the mixed reference solution of step (2) were respectively injected into UPLC-Q-TOF-MS / MS for analysis, and the peak areas were recorded. The total dimer, dimer side chain isomer, trimer, trimer side chain isomer, tetramer, and pentamer in each sample of cicada slough were semi-quantitated based on the absolute quantitative results of acetyl dopamine A. The calculation formula is as follows: A1 / A0=C1 / C0;

[0037] Wherein A1 is the peak area of ​​acetyl dopamine dimer A, A0 is the peak area of ​​the analyte, C1 is the concentration of acetyl dopamine dimer A, and C0 is the concentration of the analyte.

[0038] As a preferred embodiment, the preferred detection method of the present invention comprises the following steps:

[0039] Step (1) Preparation of the test solution: Weigh 1 g of cicada slough powder, add 10 mL of 80% methanol and place it in a stoppered test tube, weigh it, extract it ultrasonically at 400W and 25kHz for 1 hour, weigh it again, make up the loss with 80% methanol, centrifuge the extract at 13000 rpm for 10 minutes, take 1 mL of the supernatant, dilute it with 80% methanol and make up to 10 mL, and obtain the product.

[0040] Step (2) Preparation of reference solution: Accurately weigh 1 mg of each of the reference substances of acetyl dopamine dimer A and B, add 1 mL of methanol to prepare 1 mg / mL reference substance stock solutions A and B, respectively; then take 200 μL of each reference substance stock solution A and B, add methanol to 1 mL, and prepare mixed reference substance stock solutions A and B with a concentration of 200 μg / mL, and finally dilute stepwise to 10 μg / mL to obtain mixed reference substance solutions.

[0041] The chromatographic conditions of UPLC-Q-TOF-MS / MS in step (3) are as follows: using Waters ACQUITY UPLC SYSTEM to analyze the small molecule components in cicada shells; diode array detector; automatic sampler; chromatographic column: ACQUITY HSS C 18 Column, specifications: 100 mm × 2.1 mm, Φ = 1.8 μm; mobile phase: phase A: 0.1% formic acid in water - phase B: 0.1% formic acid in acetonitrile; elution gradient: 0-10 min, 15-50% B; 10-10.5 min, 50-95% B; 10.5-12.5 min, 95% B; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL;

[0042] Mass spectrometry conditions: A Synapt G2-S Q-TOF Mass Spectrometer system was used to obtain mass spectral information of the compounds, with an electrospray ion source, positive ion scan mode, mass scan range m / z 50-1500, capillary voltage 3000 V, cone voltage 40 V, ion source temperature 120°C, desolvation temperature 450°C, desolvation gas flow rate 800 L / h, cone gas flow rate 50 L / h, MS collision energy: 6 eV, and MS / MS collision energy: 30-60 V. MassLynx software was used for analysis.

[0043] The detection method of the present invention can simultaneously identify 39 acetyl dopamine polymer compounds in cicada sloughs, including 4 dimers; 1 dimer side chain isomer; 8 trimers; 7 trimer side chain isomers; 10 tetramers; and 9 pentamers. This method can effectively evaluate the quality of cicada sloughs.

[0044] Through extensive experimental screening, the present invention proposes a new method for processing cicada sloughs, in which the nymphs are washed before molting, and then processed through the steps of capture, washing, molting, and drying. The cicada sloughs produced by the present invention are tested using a dedicated detection method, achieving good technical results. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the BPI graph of artificial nymph water washing before molting (R-SXT) in positive ion mode.

[0046] Figure 2 This is the BPI graph of artificial nymph unwashed cicada shell (R-WSX) in positive ion mode.

[0047] Figure 3 This is the BPI graph of the washed cicada shell (Y-TSX) after wild nymph molting in positive ion mode.

[0048] Figure 4 This is the BPI graph of the unwashed cicada shell (Y-WSX) of wild nymphs in positive ion mode. Specific embodiments

[0049] The present invention will be further described below with reference to specific examples, but these examples should not be construed as limiting the present invention.

[0050] Example 1

[0051] A method for processing cicada sloughs comprises the following steps:

[0052] (1) Capture

[0053] The best time to catch cicadas is from mid-to-late June to mid-August. The peak period for nymphs to emerge is after rain. In artificial cicada-breeding forests, wrap a 5-cm-wide plastic tape around a tree trunk 100-130 cm high to prevent the nymphs from climbing too high after emerging, making them easier to catch. Then, using a flashlight, search the tree trunks between 6:00 PM and 12:00 AM for nymphs. Place captured nymphs directly into a container of clean water.

[0054] (2) Washing

[0055] Rinse gently with clean water several times, pouring out the water containing mud and sand and replacing it with new water each time until the water is clear.

[0056] (3) Shedding

[0057] After washing, place the nymphs back on the tree. They will crawl along the trunk for a while before settling down and beginning to eclode. The eclosion process takes about 90 to 150 minutes. Collect the cicada shells after the nymphs have finished molting.

[0058] (4) Drying

[0059] Place the cicada shells in a ventilated and dry place to dry naturally.

[0060] Example 2 Quality evaluation of cicada shells obtained by different treatment methods

[0061] 1. Analysis and content determination of small molecule components

[0062] The cicada shell samples were divided into four groups, namely, cicada shells washed before molting of artificial nymphs (R-SXT): artificially cultured nymphs were captured, washed before molting, and collected after molting; cicada shells not washed from artificial nymphs (R-WSX): the nymphs were not washed before and after molting; cicada shells washed after molting of wild nymphs (Y-TSX): the cicada shells collected after molting were repeatedly washed with clean water until the water was clear; cicada shells not washed from wild nymphs (Y-WSX): the cicada shells collected after molting of wild nymphs were not washed during the whole process.

[0063] Sample preparation: Four groups of samples were placed in a ventilated and cool place at room temperature to dry naturally for one week, crushed, passed through a No. 3 sieve, and set aside.

[0064] UPLC-Q-TOF-MS / MS technology was used to characterize and determine the content of small molecular components in cicada shells.

[0065] Preparation of the test solution: Weigh 1 g of cicada slough powder, add 10 mL of 80% methanol and place it in a stoppered test tube, weigh it, extract it by ultrasound (400 W, 25 kHz) for 1 hour, weigh it again, make up the loss with 80% methanol, centrifuge the extract (13000 rpm, 10 min), take 1 mL of the supernatant, dilute it with 80% methanol and make up to 10 mL.

[0066] Preparation of reference solution: Accurately weigh 1 mg each of acetyl dopamine dimer A and B reference substances and add 1 mL of methanol to prepare 1 mg / mL reference substance stock solutions A and B, respectively. Then, take 200 μL of each reference substance stock solution and add methanol to 1 mL to prepare a mixed reference substance stock solution with a concentration of 200 μg / mL. Finally, dilute the solution in series to 10 μg / mL to obtain the mixed reference substance solution.

[0067] Chromatographic conditions: A Waters ACQUITY UPLC SYSTEM was used to analyze small molecules in cicada sloughs. Diode array detector; autosampler; chromatographic column: ACQUITY HSS C18 column (100 mm × 2.1 mm, Φ = 1.8 μm); mobile phase: 0.1% formic acid in water (A) - 0.1% formic acid in acetonitrile (B); elution gradient: 0-10 min, 15-50% B; 10-10.5 min, 50-95% B; 10.5-12.5 min, 95% B; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0068] Mass spectrometry conditions: Mass spectrometry was acquired using a Synapt G2-S Q-TOF Mass Spectrometer system (Waters MS Technologies, UK). Electrospray ionization source; positive ion scan mode; mass scan range m / z 50–1500; capillary voltage 3000 V, cone voltage 40 V; source temperature 120°C; desolvation temperature 450°C; desolvation gas flow rate 800 L / h; cone gas flow rate 50 L / h; MS collision energy: 6 eV; MS / MS collision energy: 30–60 V. Analysis was performed using MassLynx software (version 4.1, Waters, USA).

[0069] Take the above test solution, inject and analyze according to the above chromatographic and mass spectrometric conditions, record the peak area, and semi-quantitate the total dimer, dimer side chain isomer, trimer, trimer side chain isomer, tetramer, and pentamer in each sample cicada shell based on the absolute quantitative results of acetyl dopamine A. The calculation formula is as follows: A1 / A0=C1 / C0

[0070] Wherein A1 is the peak area of ​​acetyl dopamine dimer A, A0 is the peak area of ​​the analyte, C1 is the concentration of acetyl dopamine dimer A, and C0 is the concentration of the analyte.

[0071] 2. Determination of total ash and acid-insoluble ash

[0072] The determination of total ash and acid-insoluble ash shall refer to the 2020 edition of the Chinese Pharmacopoeia, Part IV, under "2302 Ash Determination Method", as follows:

[0073] 2.2.1 Total ash determination method

[0074] Four groups of test samples, including artificial nymph cicada sloughs washed before molting (R-SXT), artificial nymph cicada sloughs unwashed (R-WSX), wild nymph cicada sloughs washed after molting (Y-TSX), and wild nymph cicada sloughs unwashed (Y-WSX), were crushed to pass through a No. 2 sieve and mixed evenly. 3 g of each of the four groups of test samples were placed in a crucible heated to a constant weight, weighed, and slowly heated until completely carbonized. The temperature was then gradually raised to 500-600°C to completely ash and reach a constant weight. Based on the weight of the residue, the total ash content (%) in the test sample was calculated.

[0075] 2.2.2 Determination of acid-insoluble ash

[0076] Take the ash obtained above and carefully add approximately 10 mL of dilute hydrochloric acid to a crucible. Cover the crucible with a watch glass and heat in a water bath for 10 minutes. Rinse the watch glass with 5 mL of hot water, adding the washings to the crucible. Filter through ash-free filter paper. Rinse the residue in the crucible with water on the filter paper until the washings show no chloride reaction. Transfer the filter residue and filter paper to the same crucible, dry, and ignite to constant weight. Calculate the acid-insoluble ash content (%) in the sample based on the weight of the residue.

[0077] 3. Experimental results

[0078] 3.1. Appearance Description

[0079] The cicada shells washed with water before artificial nymph molting are brown and look cleaner.

[0080] 3.2 Analysis and content determination of small molecule components

[0081] according to Figures 1 to 4 A total of 39 acetyl dopamine aggregates were identified from the base peak ion (BPI) chromatograms of four cicada slough samples, including four dimers, one dimer side chain isomer, eight trimers, seven trimer side chain isomers, ten tetramers, and nine pentamers. The composition of acetyl dopamine aggregates was largely consistent across the four cicada slough samples. Detailed information, including retention time, molecular weight, molecular formula, and fragment ions, is provided in Table 1.

[0082] Table 1 Identification of small molecule compounds in cicada slough

[0083]

[0084]

[0085]

[0086]

[0087] According to the test results in Table 2, the total acetyl dopamine polymer content in the artificial nymph molting cicada shell sample was 7.68 mg / g, in the artificial nymph unwashed cicada shell sample was 6.26 mg / g, in the wild nymph molting cicada shell sample was 5.06 mg / g, and in the wild nymph unwashed cicada shell sample was 6.08 mg / g. Among them, there was no significant difference in the total acetyl dopamine polymer content and the total amount of each acetyl dopamine polymer in the artificial nymph unwashed (R-WSX) and the wild nymph unwashed cicada shell (Y-WSX), indicating that the quality of the two is comparable. Compared with the nymph washed after molting (Y-TSX) and the nymph washed before molting (R-SXT), the total acetyl dopamine polymer content was significantly reduced ( * p<0.05), it is speculated that the content of acetyl dopamine aggregates increased relatively because washing before molting reduced the content of sediment and other substances, and washing before molting only washed the outer surface of the cicada shell, so the loss of components was relatively small. The total acetyl dopamine aggregate content of wild cicada shells washed after molting was significantly reduced compared with the wild cicada shells not washed ( ** p < 0.01), indicating that washing after molting causes excessive loss of acetyl dopamine aggregates. Therefore, washing before nymphal molting is a reliable method to reduce the loss of active ingredients in cicada sloughs and ensure their quality. See Table 2.

[0088] Table 2 Total amount of acetyl dopamine polymers in cicada shell samples ( n=3)

[0089]

[0090]

[0091] Note: Compared with Y-TSX, *** : p<0.001, ** : p<0.01, * : p<0.05; compared with R-SXT, # : p < 0.05. 3.3 Determination results of total ash and acid-insoluble ash

[0092] Washing the nymphs before or after molting reduced the amount of sediment, and the total ash and acid-insoluble ash contents were lower than those in the unwashed cicada slough samples. The total ash and acid-insoluble ash contents of the artificial nymph molting cicada slough samples (R-SXT) were significantly lower than those of the artificial nymph molting cicada slough samples (R-WSX). ** p<0.01); the total ash and acid-insoluble ash contents of wild nymph molt cicada slough samples (Y-TSX) were significantly lower than those of wild nymph molt cicada slough samples (Y-WSX). ** p<0.01, *p<0.05). Therefore, washing cicada shells before molting can effectively reduce sediment content and improve the quality of cicada shells. See Table 3.

[0093] Table 3 Determination of total ash and acid insoluble ash of cicada shells ( n=3)

[0094] Group Total ash / % Acid insoluble ash / % R-SXT 23.37±1.36 19.69±1.27 R-WSX <![CDATA[31.04±1.70 ** ]]> <![CDATA[26.18±1.61 ** ]]> Y-TSX 22.95±1.44 19.55±1.23 Y-WSX <![CDATA[30.01±2.17 ** ]]> <![CDATA[25.52±2.17 * ]]>

[0095] Note: Compared with R-SXT, ** : p<0.01; compared with Y-TSX, * :p<0.05.

[0096] The above experimental results show that the cicada slough processing method provided by the present invention, that is, washing the nymphs before molting, is obtained by capturing, washing, molting, drying, etc., and the quality of the processed cicada slough is evaluated by UPLC-Q-TOF-MS / MS. It is found that washing before molting can not only reduce the content loss of the effective ingredient acetyldopamine polymers, but also significantly reduce the total ash and acid-insoluble ash to achieve the purpose of removing mud and sand, indicating that this method can improve the quality of cicada sloughs and ensure the quality of cicada sloughs. It is a simple, reliable and popular processing method.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for producing cicada sloughs, characterized in that: The steps include: (1) In a forest where cicadas are artificially cultivated, a 2-10 cm wide plastic tape is wrapped around a tree trunk at 80-150 cm. When the cicada nymphs crawl onto the tree trunk under the plastic tape, they are captured. The capture period is from mid-to-late June to mid-August each year. (2) Place the captured cicada nymphs in a container of clean water and gently rinse them several times with clean water. Pour out the water containing mud and sand and replace it with new water each time until the water is clear. (3) After washing, the nymphs of the cicada are put back on the tree to eclode. The eclosion process takes 90 to 150 minutes. After eclosion, the exuviae are separated and collected. The exuviae are placed in a ventilated and dry place to dry naturally.

2. The method according to claim 1, characterized in that The steps for capturing cicada nymphs are: In the woods where cicadas are artificially bred, first wrap a circle of 5 cm wide plastic tape around the tree trunk at 100 to 130 cm. When the cicada nymphs crawl to the tree trunk under the plastic tape, catch them; the catching time is from 6 pm to 12 am.

3. The method according to claim 2, characterized in that The cicada nymphs are caught after the rain.

4. The method according to claim 1, wherein In the method, the cicada nymphs are artificially cultured.

5. The method according to claim 1, wherein The produced cicada slough is detected by the following steps: (1) Preparation of test solution: Weigh the cicada slough powder, add methanol and place it in a stoppered test tube, weigh it to determine the weight, perform ultrasonic extraction, weigh it again, make up the loss with methanol, centrifuge the extract, take the supernatant, dilute it with methanol and make up to the volume, and obtain the solution; (2) Preparation of mixed reference solution: Accurately weigh the reference substances of acetyl dopamine dimer A and B, and add methanol to prepare reference substance stock solutions A and B respectively; then take appropriate amounts of reference substance stock solutions A and B, mix them, and add methanol to make up to the volume to prepare the mixed reference solution; (3) The test solution of step (1) and the mixed reference solution of step (2) were injected into UPLC-Q-TOF-MS / MS for analysis, and the peak areas were recorded. The total dimer, dimer side chain isomer, trimer, trimer side chain isomer, tetramer, and pentamer in each sample of cicada slough were semi-quantitated based on the absolute quantitative results of acetyl dopamine A. The calculation formula is as follows: A1 / A0=C1 / C0; Wherein A1 is the peak area of ​​acetyl dopamine dimer A, A0 is the peak area of ​​the analyte, C1 is the concentration of acetyl dopamine dimer A, and C0 is the concentration of the analyte.

6. The method according to claim 5, characterized in that The detection steps are as follows: Step (1) Preparation of test solution: Weigh 1 g of cicada slough powder, add 10 mL of 80% methanol and place in a stoppered test tube, weigh the solution, extract by ultrasonication at 400 W and 25 kHz for 1 h, weigh the solution again, make up the loss with 80% methanol, centrifuge the extract at 13,000 rpm for 10 min, take 1 mL of the supernatant, dilute with 80% methanol and make up to 10 mL, and obtain the solution; Step (2) Preparation of reference solution: Accurately weigh 1 mg of each of the reference substances of acetyl dopamine dimer A and B, and add 1 mL of methanol to prepare 1 mg / mL reference substance stock solutions A and B, respectively; then take 200 μL of each reference substance stock solution A and B, add methanol to make up to 1 mL, and prepare mixed reference substance stock solutions A and B with a concentration of 200 μg / mL, and finally dilute stepwise to 10 μg / mL to obtain mixed reference substance solutions; The chromatographic conditions of UPLC-Q-TOF-MS / MS in step (3) are as follows: using Waters ACQUITY UPLC SYSTEM to analyze the small molecule components in cicada slough; diode array detector; automatic sampler; Column: ACQUITY HSS C 18 column, specifications: 100 mm × 2.1 mm, Φ=1.8 μm; Mobile phase: Phase A: 0.1% formic acid in water - Phase B: 0.1% formic acid in acetonitrile; elution gradient: 0-10 min, 15-50% B; 10-10.5 min, 50-95% B; 10.5-12.5 min, 95% B; flow rate: 0.4 mL / min; column temperature: 40°C; injection volume: 2 μL; Mass spectrometry conditions: Synapt G2-S Q-TOF Mass Spectrometer system was used to obtain mass spectrometry information of the compounds, electrospray ionization source; positive ion scan mode; mass scan range m / z 50-1500; capillary voltage 3000 V, cone voltage 40 V; ion source temperature 120 °C; desolvation temperature 450 °C; desolvation gas flow rate 800 L / h; cone gas flow rate 50 L / h; MS collision energy: 6 eV; MS / MS collision energy: 30-60 V; analyzed using MassLynx software.

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