Method for detecting crocin content in saffron crocus extract

Abstract

The invention discloses a method for detecting the content of crocin in a saffron crocus extract. The method comprises the following steps: drawing a standard curve of crocin-I and crocin-II; performing ultra-high performance liquid chromatography detection on the saffron crocus extract; the used mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is water, and the mobile phase B is acetonitrile; a gradient elution procedure is adopted for elution, the volume fraction of the mobile phase A is 80%-60% in 0-4 min, the volume fraction of the mobile phase B is 20%-40%, the volume fraction of the mobile phase A is 60%-80% in 4-4.01 min, the volume fraction of the mobile phase B is 40%-20% in 4-4.01 min, the volume fraction of the mobile phase A is 80%, and the volume fraction of the mobile phase B is 20% in 4.01-5 min; and substituting the obtained peak area into a standard curve, and calculating the concentration. According to the method, an ultra-performance liquid chromatograph is selected as a detection means for optimizing extraction conditions, the detection quantity is low, the sensitivity is high, the repeatability is good, the stability is high, and accurate qualitation and quantification can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plant natural product extracts, and in particular to a method for detecting the content of crocin in a saffron extract. Background Art

[0002] Saffron is a plant of the Iridaceae family, also known as crocus, crocus, and contains chemical components such as crocin and crocetin. The crocin in the saffron column is the main value of saffron. According to the "Pharmacopoeia of the People's Republic of China", saffron has the effects of promoting blood circulation and removing blood stasis, reducing swelling and relieving pain, relaxing meridians and activating collaterals, cooling blood and detoxifying, and relieving depression and knots. It can be used to prevent and treat diseases such as blood deficiency, vasculitis, and cerebral thrombosis.

[0003] As a precious Chinese herbal medicine, a large number of medical experiments have shown that the real active ingredient in saffron is crocin, which is highly polar and unstable. At present, the extraction process of saffron active ingredients is mainly to obtain saffron active ingredients through soaking, filtering, concentration and other operation processes. This process is affected by factors such as temperature and time, and there are problems such as low raw material utilization and low extraction rate. The main active ingredients of saffron specified in the 2020 edition of the "Chinese Pharmacopoeia" are crocin-Ⅰ and crocin-Ⅱ, and the content of crocin-Ⅰ and crocin-Ⅱ in saffron medicinal materials is determined by high performance liquid chromatography, and the sum of the two contents is ≥10.0%. In the pharmacopoeia, the mobile phase A used is acetonitrile, the mobile phase B is water, and the elution program is: 0-20 min (13% A); 20-23 min (13-23% A); 23-45 min (23-25% A); 45-50 min (25-50% A).

[0004] At present, there are many methods for extracting crocin-Ⅰ and crocin-Ⅱ from saffron, such as solvent extraction, microwave-assisted extraction, enzyme-assisted extraction, and ultrasound-assisted extraction. The extraction rate of the above methods is not high, and some methods are difficult to operate. In the long run, the cost is high. There are many analytical methods for the determination of crocin-Ⅰ and crocin-Ⅱ, such as ultraviolet absorption, thin layer chromatography, and high performance liquid chromatography. In terms of sensitivity and accuracy, HPLC is currently the most effective method. Chinese patent application document with publication number CN114324701A discloses a method for simultaneously determining crocin-1, crocin-2, crocin-3 and crocin-4 by high performance liquid chromatography with acetonitrile and 0.1% acetic acid aqueous solution as mobile phases and analyzing their contents, and investigates their elution method, column temperature, flow rate and injection volume; Chinese patent application document with publication number CN106706774A discloses a method for determining crocin-I and crocin-II in saffron medicinal materials by high performance liquid chromatography with water and methanol as mobile phases; Chinese patent application document with publication number CN110579558A discloses a method for detecting crocin-I and crocin-II by means of high performance liquid chromatography with water and 20% acetonitrile as mobile phases, and the separation degree is good, but the methods in the above documents all have the problems of too long detection time and low efficiency. Compared with high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) has better separation efficiency, faster analysis speed and higher sensitivity, which can significantly reduce analysis time and injection volume and provide better peak shape and separation. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to detect the content of crocin-I and crocin-II in saffron.

[0006] The present invention solves the above technical problems through the following technical means:

[0007] A method for detecting the content of crocin in saffron extract comprises the following steps:

[0008] (1) Draw the standard curve of crocin-Ⅰ and crocin-Ⅱ;

[0009] (2) Performing ultra-high performance liquid chromatography on the saffron extract; wherein, in the ultra-high performance liquid chromatography detection process, the mobile phase used includes mobile phase A and mobile phase B; the mobile phase A is water, and the mobile phase B is acetonitrile; a gradient elution program is used for elution, specifically including: 0-4min, the volume fraction of mobile phase A is 80% to 60%, the volume fraction of mobile phase B is 20% to 40%, 4-4.01min, the volume fraction of mobile phase A is 60% to 80%, the volume fraction of mobile phase B is 40% to 20%, 4.01-5min, the volume fraction of mobile phase A is 80%, and the volume fraction of mobile phase B is 20%;

[0010] (3) Substitute the peak area obtained in (2) into the standard curve in (1) and calculate its concentration to obtain the crocin content.

[0011] Preferably, in (2), during the ultra-high performance liquid chromatography detection process, the chromatographic column used is ACQUITY UPLC BEH C18 1.7 μm.

[0012] Preferably, in (2), the flow rate of the mobile phase is 0.2 mL / min.

[0013] Preferably, in (2), during the ultra-high performance liquid chromatography detection process, the detection wavelength is 440 nm.

[0014] Preferably, in (2), during the ultra-high performance liquid chromatography detection process, the injection volume is 5 μL.

[0015] Preferably, in (2), the preparation of the saffron extract comprises using an ethanol aqueous solution as an extraction solvent, extracting the saffron in an ultrasonic cell crusher, and then diluting it with an acetonitrile aqueous solution.

[0016] Preferably, the power of the ultrasonic cell pulverizer is 400-600W. During the extraction process, it runs for 2s and shuts down for 2s, and the total working time is 5-25min.

[0017] Preferably, the extraction temperature is 5-25° C., the volume fraction of ethanol in the ethanol aqueous solution is 40-80%, and the solid-liquid ratio is 1:20-60 g / mL.

[0018] Preferably, the power of the ultrasonic cell pulverizer is 500W. During the extraction process, it runs for 2s and shuts down for 2s, and the total working time is 15min. The extraction temperature is 10°C, the volume fraction of ethanol in the ethanol aqueous solution is 50%, and the solid-liquid ratio is 1:50g / mL.

[0019] Preferably, in (1), an ultra-high performance liquid chromatograph is used to test and analyze the crocin-I and crocin-II standards according to the detection conditions in (2) and a standard curve is drawn.

[0020] Preferably, in (1), the abscissa of the standard curve corresponds to the concentrations of crocin-I and crocin-II, and the ordinate is the peak area of ​​the liquid chromatogram.

[0021] Preferably, the saffron is obtained by freeze-drying fresh saffron.

[0022] The advantages of the present invention are:

[0023] In the present invention, an ultrasonic cell crusher is selected as an auxiliary extraction method to extract saffron, and the extracts obtained under different extraction conditions are detected and analyzed by ultra-high performance liquid chromatography, so as to optimize and obtain the best extraction conditions.

[0024] The present invention selects an ultrasonic cell crusher as a means of extracting saffron. The ultrasonic cell crusher can quickly and efficiently crush the cells or tissues of saffron, improve the quality and yield of the extract, and is more time-saving and efficient than traditional physical or chemical methods; in the crushing process, the rupture of the cell wall can be reduced, and the release of toxic substances produced during the extraction process can be reduced. In addition, the heat generated by the probe during the ultrasonic crushing process is low, which can reduce the thermal damage to the effective ingredients in saffron; compared to an ultrasonic cleaning machine, the ultrasonic cell crusher concentrates all energy on the probe, which is more conducive to the crushing, emulsification and homogenization of the saffron cell wall.

[0025] The present invention selects ultra-high performance liquid chromatograph as a detection means for optimizing extraction conditions. The performance of the UPLC system is more outstanding than that of traditional or optimized HPLC, making the working efficiency of the chromatographic system higher, the linear speed, flow rate, and pressure resistance range wider, and its detection amount is low, the sensitivity is high, the repeatability is good, and the stability is high, and it can accurately perform qualitative and quantitative analysis. The advantages of selecting water and acetonitrile as the mobile phase are that crocin is a class of water-soluble carotenoid compounds, which is formed by the combination of crocetin and two molecules of gentiobiose, so it has a large polarity, and the polar force of water molecules can promote the interaction between crocin and the stationary phase, thereby helping to improve the selectivity and separation effect of chromatography; acetonitrile has good solubility and can produce strong interactions with many organic compounds, thereby quickly eluting crocin from the solid phase column, accelerating the analysis speed; it can effectively enrich crocin, increase its concentration, and enhance detection sensitivity. Acetonitrile as a mobile phase can also help maintain the balance of the chromatographic column and improve the accuracy and repeatability of chromatographic separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is an isocratic elution chromatogram with 20% ACN as the mobile phase in Example 4 of the present invention;

[0027] Figure 2This is an isocratic elution chromatogram with 30% ACN as the mobile phase in Example 4 of the present invention;

[0028] Figure 3 This is an isocratic elution chromatogram with 40% ACN as the mobile phase in Example 4 of the present invention;

[0029] Figure 4 This is a chromatogram with water-acetonitrile as the mobile phase in Example 4 of the present invention;

[0030] Figure 5 This is a chromatogram of Example 4 of the present invention using 0.1% formic acid water-methanol as the mobile phase;

[0031] Figure 6 This is a chromatogram using water-methanol as the mobile phase in Example 4 of the present invention;

[0032] Figure 7 This is a chromatogram of Example 4 of the present invention using 10 mM ammonium acetate buffer-acetonitrile as the mobile phase;

[0033] Figure 8 is an ultra-high performance liquid chromatogram of crocin-Ⅰ at different concentrations in Example 1 of the present invention;

[0034] Fig. 9 is the fitting straight line of crocin-Ⅰ in Example 1 of the present invention;

[0035] Fig.10 is an ultra-high performance liquid chromatogram of crocin-II at different concentrations in Example 2 of the present invention;

[0036] Fig.11 is the fitting straight line of crocin-II in Example 2 of the present invention;

[0037] Fig.12 The effect of ultrasonic extraction temperature on the extraction rate of crocin in Example 3 of the present invention;

[0038] Fig.13 The effect of the power of the ultrasonic cell pulverizer on the extraction rate of crocin in Example 3 of the present invention;

[0039] Fig.14 The effect of the total working time of the ultrasonic cell pulverizer on the extraction rate of crocin in Example 3 of the present invention;

[0040] Fig.15 The effect of ethanol concentration on the extraction rate of crocin in Example 3 of the present invention;

[0041] Fig.16 The effect of the solid-liquid ratio on the crocin extraction rate in Example 3 of the present invention;

[0042] Fig.17This is a schematic diagram of ultrasonic cell pulverizer extraction in Example 3 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0045] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0046] Example 1

[0047] A method for obtaining a fitting straight line of a crocin-Ⅰ standard solution comprises the following steps:

[0048] Weigh 0.0049g of crocin-Ⅰ, add 5mL of 20% ACN aqueous solution with a volume fraction by pipette to prepare a 1mmol / L crocin-Ⅰ standard solution; then take 10, 20, 30, 40, and 50μL to prepare 1mL of standard solution, with concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05mmol / L, respectively, as the test solution; use ultra-high performance liquid chromatography (ACQUITY UPLC, Waters), use ACQUITY UPLC BEH C18 1.7μm chromatographic column, water as mobile phase A, acetonitrile as mobile phase B (elution program is shown in Table 1), flow rate is 0.2mL / min, detection wavelength is 440nm, injection volume is 5μL, analyze and detect crocin-I, and obtain ultra-high performance liquid chromatograms of different gradient concentrations, as shown in Figure 1. Figure 8 The measured Area and concentration were linearly fitted to obtain the fitting line: y = 1641420x + 346.8 (R2 = 0.999), such as Fig. 9 shown.

[0049] Table 1

[0050] Time (min) Mobile phase A (%) Mobile phase B (%) 0～4 80→60 20→40 4～4.01 60→80 40→20 4.01～5 80 20

[0051] Note: The chromatographic column is ACQUITY UPLC BEH C18 1.7μm; the flow rate is 0.2mL / min; the detection wavelength is 440nm; the injection volume is 5μL, and the percentages in the table are volume fractions.

[0052] Example 2

[0053] A method for obtaining a fitting straight line of a crocin-II standard solution comprises the following steps:

[0054] Take 0.0041g of crocin-II, add 5mL of 20% ACN aqueous solution with a volume fraction by a pipette to prepare a 1mmol / L crocin-II standard solution; then take 10, 20, 30, 40, and 50μL to prepare 1mL of standard solution with concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05mmol / L, respectively, as the test solution; use an ultra-high performance liquid chromatograph (ACQUITY UPLC, Waters), use an ACQUITY UPLC BEH C18 1.7μm chromatographic column, use water as mobile phase A, acetonitrile as mobile phase B (the elution program is shown in Table 1), the flow rate is 0.2mL / min, the detection wavelength is 440nm, the injection volume is 5μL, analyze and detect crocin-II, and obtain ultra-high performance liquid chromatograms of different gradient concentrations, as shown in FIG. Fig.10 The measured Area and concentration were linearly fitted to obtain the fitting straight line: y = 861540x + 1733.2 (R2 = 0.999), such as Fig.11 shown.

[0055] Example 3

[0056] A method for efficiently extracting crocin from saffron and detecting its content using an ultra-high performance liquid chromatography, the steps are as follows:

[0057] Extraction temperature optimization

[0058] Weigh 5 portions of 0.1 g dry saffron powder and place them in a beaker. Add 5 mL of 50% ethanol aqueous solution and extract at 5, 10, 15, 20, and 25 ° C using an ultrasonic cell crusher. The schematic diagram is as follows: Fig.17 As shown, the power is 450W, the total working time is 20min, the ultrasonic on time is 2s, the off time is 2s, and the extract is obtained, and centrifuged; 5μL of the supernatant is removed, diluted to 1mL with a 20% ACN aqueous solution by volume fraction, filtered with a 0.22μm organic nylon filter head, and the solution to be tested is obtained. The ultra-high performance liquid chromatography is used for detection, and the detection conditions are the same as those in Example 1. The extraction rate is shown in the figure Fig.12 .

[0059] Ultrasonic power optimization

[0060] Weigh 5 portions of 0.1 g dry saffron powder, add 5 mL of 50% ethanol aqueous solution, extract at 10 ° C using an ultrasonic cell crusher, the power is 400, 450, 500, 550, 600 W, the total working time is 20 min, the ultrasonic on time is 2 s, the off time is 2 s, and the extract is obtained. Centrifuge; take 5 μL of the supernatant, dilute it to 1 mL with 20% ACN aqueous solution, filter it with a 0.22 μm organic nylon filter head, obtain a test solution, and detect it using an ultra-high performance liquid chromatograph. The detection conditions are the same as in Example 1. The extraction rate is shown in the figure. Fig.13 .

[0061] Optimization of total working time

[0062] Weigh 5 portions of 0.1 g dry saffron powder, add 5 mL of 50% ethanol aqueous solution, extract at 10 ° C using an ultrasonic cell crusher, the total working time is 5, 10, 15, 20, 25 min, the power is 500 W, the ultrasonic on time is 2 s, the off time is 2 s, and the extract is obtained. Centrifuge; take 5 μL of the supernatant, dilute it to 1 mL with 20% ACN aqueous solution, filter it with a 0.22 μm organic nylon filter head, obtain a test solution, and detect it using an ultra-high performance liquid chromatograph. The detection conditions are the same as in Example 1. The extraction rate is shown in the figure. Fig.14 .

[0063] Optimization of ethanol concentration

[0064] Weigh 5 portions of 0.1 g dry saffron powder, add 5 mL of 40, 50, 60, 70, 80% ethanol aqueous solution, respectively, extract at 10 ° C using an ultrasonic cell crusher, the power is 500 W, the total working time is 15 min, the ultrasonic on time: 2 s, the off time: 2 s, to obtain an extract, centrifuge; remove 5 μL of the supernatant, dilute to 1 mL with a 20% ACN aqueous solution, filter with a 0.22 μm organic nylon filter to obtain a test solution, and detect using an ultra-high performance liquid chromatograph. The detection conditions are the same as in Example 1. The extraction rate is shown in the figure. Fig.15 .

[0065] Optimization of material-liquid ratio

[0066] Weigh 5 portions of 0.1 g dry saffron powder, add 2, 3, 4, 5, 6 mL of 50% ethanol aqueous solution (solid-liquid ratio is 1:20, 1:30, 1:40, 1:50, 1:60), respectively, at 10 ° C, use an ultrasonic cell crusher for extraction, the power is 500 W, the total working time is 15 min, the ultrasonic on time is 2 s, the off time is 2 s, and the extract is obtained. Centrifuge; take 5 μL of the supernatant, dilute it to 1 mL with a volume fraction of 20% ACN aqueous solution, filter it with a 0.22 μm organic nylon filter head, obtain a test solution, and detect it using an ultra-high performance liquid chromatograph. The detection conditions are the same as in Example 1. The extraction rate is shown in the figure. Fig.16 .

[0067] In the above extraction process, the detected chromatographic peaks are integrated to obtain the peak area, and the peak area is substituted into the fitting straight lines of Example 1 and Example 2 to obtain the concentrations, and then the corresponding contents of crocin-I and crocin-II are obtained by further calculation, thereby optimizing the extraction process.

[0068] pass Figure 12-16 Taking into account the contents of saffron glycosides-Ⅰ and Ⅱ, the optimal extraction temperature is 10°C, the power of the ultrasonic cell crusher is 500W, the total working time is 15min, the ethanol concentration is 50%, and the solid-liquid ratio is 1:50, the extraction rate is the highest. Under the optimal conditions, the dry saffron powder is extracted according to the above method to obtain a test solution, which specifically includes the following steps: weigh 0.1g of dry saffron powder, add 5mL of 50% ethanol aqueous solution (solid-liquid ratio is 1:50), extract at 10°C using an ultrasonic cell crusher with a power of 500W, a total working time of 15min, ultrasonic on time: 2s, off time: 2s, to obtain an extract, centrifuge; remove 5μL of the supernatant, dilute to 1mL with a volume fraction of 20% ACN aqueous solution, filter with a 0.22μm organic nylon filter head, and obtain a test solution; use the detection conditions in Example 1 to detect the test solution using an ultra-high performance liquid chromatograph, and the results are as follows: Figure 4 As shown, the peak area is obtained by integrating the chromatographic peaks, and the peak area is substituted into the fitting lines of Example 1 and Example 2 to obtain the concentrations. Then, the corresponding contents of crocin-Ⅰ and crocin-Ⅱ are obtained by further calculation. It can be seen from the calculation that the extraction rate of crocin-Ⅰ is about 23.39%, and the extraction rate of crocin-Ⅱ is about 14.29%.

[0069] Example 4

[0070] The elution procedure and the mobile phase are changed to detect the test solution of the saffron extract, wherein the test solution is extracted according to the extraction step of Example 3 under the optimal conditions (extraction temperature is 10°C, ultrasonic cell crusher power is 500W, total working time is 15min, ethanol concentration is 50%, and solid-liquid ratio is 1:50), specifically comprising the following steps: weigh 0.1g dry saffron powder, add 5mL of 50% ethanol aqueous solution (solid-liquid ratio is 1:50), extract at 10°C using an ultrasonic cell crusher with a power of 500W, a total working time of 15min, ultrasonic on time: 2s, off time: 2s, to obtain an extract, and centrifuge; remove 5μL of the supernatant, dilute to 1mL with a 20% ACN aqueous solution by volume, and filter with a 0.22μm organic nylon filter to obtain a test solution;

[0071] (1) Selection of program. Water-acetonitrile was selected as the mobile phase, and acetonitrile solutions with volume concentrations of 20%, 30% and 40% were used for isocratic elution, and 20%-40% ACN aqueous solution was used for gradient elution according to the program in Table 1, all for 5 min. The other detection conditions were consistent with those in Example 1. The separation effect is shown in the table below.

[0072] Mobile phase: water-acetonitrile Separation effect 20% ACN Crocin-I / II were not eluted (see Figure 1 ) 30%ACN Crocin-I / II were both eluted, but the two peaks were not separated (see Figure 2 ) 40%ACN Crocin-I / II were both eluted, but the two peaks were not separated (see Figure 3 ) 20%-40% ACN Crocin-I / II were both eluted, and gradient elution had a better separation effect (see Figure 4 )

[0073] Therefore, water-acetonitrile was selected as the mobile phase and gradient elution was selected as the program (see Table 1), and the obtained chromatographic peaks had good separation effect, narrow peaks, and high detection efficiency.

[0074] (2) Selection of mobile phase. 0.1% formic acid water (volume ratio)-methanol, water-methanol, 10 mM ammonium acetate buffer (containing 0.1% acetic acid, volume ratio)-acetonitrile and water-acetonitrile gradient elution were used respectively (elution program is shown in Table 1), and the other detection conditions were consistent with Example 1, and the influence of different mobile phases on the separation effect was investigated. See the table below.

[0075]

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the content of crocin in saffron extract, characterized in that: The following steps are involved: (1) Draw the standard curve of crocin-Ⅰ and crocin-Ⅱ; (2) Performing ultra-high performance liquid chromatography on the saffron extract; wherein, in the ultra-high performance liquid chromatography detection process, the mobile phase used includes mobile phase A and mobile phase B; the mobile phase A is water, and the mobile phase B is acetonitrile; a gradient elution program is used for elution, specifically including: 0-4min, the volume fraction of mobile phase A is 80% to 60%, the volume fraction of mobile phase B is 20% to 40%, 4-4.01min, the volume fraction of mobile phase A is 60% to 80%, the volume fraction of mobile phase B is 40% to 20%, 4.01-5min, the volume fraction of mobile phase A is 80%, and the volume fraction of mobile phase B is 20%; (3) Substitute the peak area obtained in (2) into the standard curve in (1) and calculate its concentration to obtain the crocin content.

2. The method for detecting the crocin content in the saffron extract according to claim 1, characterized in that: In (2), during the ultra-high performance liquid chromatography detection process, the chromatographic column used was ACQUITY UPLC BEH C18 1.7 μm.

3. The method for detecting the crocin content in the saffron extract according to claim 1, characterized in that: In (2), the flow rate of the mobile phase is 0.2 mL / min.

4. The method for detecting the crocin content in the saffron extract according to claim 1, characterized in that: In (2), during the ultra-high performance liquid chromatography detection process, the detection wavelength is 440 nm.

5. The method for detecting the crocin content in the saffron extract according to claim 1, characterized in that: In (2), during the ultra-high performance liquid chromatography detection process, the injection volume was 5 μL.

6. The method for detecting the crocin content in the saffron extract according to claim 1, characterized in that: In (2), the preparation of the saffron extract includes using ethanol aqueous solution as the extraction solvent, extracting saffron in an ultrasonic cell crusher, and then diluting it with acetonitrile aqueous solution.

7. The method for detecting the crocin content in the saffron extract according to claim 6, characterized in that: The power of the ultrasonic cell pulverizer is 400-600W. During the extraction process, it runs for 2s and shuts down for 2s, and the total working time is 5-25min.

8. The method for detecting the crocin content in the saffron extract according to claim 6 or 7, characterized in that: The extraction temperature is 5-25° C., the volume fraction of ethanol in the ethanol aqueous solution is 40-80%, and the solid-liquid ratio is 1:20-60 g / mL.

9. The method for detecting the crocin content in the saffron extract according to any one of claims 6 to 8, characterized in that: The power of the ultrasonic cell crusher is 500W. During the extraction process, it runs for 2s and shuts down for 2s, and the total working time is 15min. The extraction temperature is 10°C, the volume fraction of ethanol in the ethanol aqueous solution is 50%, and the solid-liquid ratio is 1:50g / mL.

10. The method for detecting the crocin content in the saffron extract according to any one of claims 1 to 9, characterized in that: In (1), using ultra-high performance liquid chromatography, according to the detection conditions in (2), the crocin-I and crocin-II standards are tested and analyzed and a standard curve is drawn.

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