Method for extracting and detecting buckwheat photosensitizer in buckwheat

Abstract

The invention relates to the technical field of detection, in particular to a method for extracting and detecting buckwheat photosensitizer in buckwheat. According to the specific technical scheme, the method comprises the following steps: drying and crushing tartary buckwheat buds or common buckwheat buds, mixing the crushed tartary buckwheat buds or common buckwheat buds with an extracting agent, oscillating, performing ultrasonic treatment, centrifuging, taking supernate, adding methanol, dissolving, filtering, and deriving under a full-spectrum plant lamp. According to the method, the extraction method of the buckwheat photosensitizers is optimized, qualitative and quantitative analysis is carried out on the buckwheat photosensitizers by combining the high performance liquid chromatography with a DAD detector, a mass spectrometry detector, an FLD detector and the like, the accurate and feasible method for detecting the buckwheat photosensitizers in the buckwheat through the high performance liquid chromatography is established, and reference can be provided for detection of the buckwheat photosensitizers.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for extracting and detecting buckwheat phytochrome in buckwheat. Background Art

[0002] Buckwheat is an important grain crop that can be used as both medicine and food. It has important nutritional and health value. It is rich in starch, protein, minerals, vitamins, dietary fiber, trace elements, etc. In addition, it is also rich in flavonoids, phenols, peptides, steroids and other biologically active substances. It has good blood sugar, blood lipid, cholesterol, and antioxidant functions. The traditional edible part of buckwheat is the seeds. After buckwheat germinates, its nutritional content and activity are further improved. Therefore, young buckwheat plants have also been developed into products. However, the changes in its anti-nutritional factors or potential harmful components after buckwheat germinates have not received enough attention.

[0003] The photosensitivity of buckwheat (fagopyrin) in buckwheat was discovered more than 100 years ago. Due to its special molecular structure, researchers have not obtained the pure substance of buckwheat photosensitive pigment, so its physical and chemical properties, biosynthesis method and biological activity are still unclear. Excessive consumption of buckwheat sprouts, buckwheat leaves or buckwheat flowers in the sun can cause symptoms such as skin redness, swelling and itching. Buckwheat photosensitive pigment and its original form are both present in plants. Similar to hypericin and protohypericin, it is a natural fluorescent pigment with phototoxicity. It and hypericin are derivatives of naphthodianthrone. These compounds can only be converted after being dissolved in the extraction solvent, and the original buckwheat photosensitive pigment is the only plant product. 2013 et al. separated 8 fluorescent substances through chromatographic analysis. The UV-visible absorption spectra of these 8 fluorescent materials were all at 590nm and the spectral pattern remained unchanged, proving that buckwheat phytochrome is a class of substances composed of multiple compounds. They also proposed six possible structures of buckwheat phytochrome: A, B, C, D, E and F, among which A, B and C buckwheat phytochromes have the same molecular formula but different structural formulas, and D and E have the same molecular formula but different structural formulas.

[0004] Buckwheat phytochrome is generally detected by UV-visible spectrophotometry and fluorescence spectrophotometry. The maximum absorption of buckwheat phytochrome in the UV-visible spectrum is at 284, 329, 545 and 590nm, and most of the buckwheat phytochrome content is calculated based on the absorbance value at 590nm. The buckwheat phytochrome content measured by UV-visible spectrophotometry is 6 times that of HPLC. The large amount of chlorophyll and its derivatives in buckwheat extract also have an absorption wavelength of 590nm, which will affect the accuracy of UV-visible spectrophotometry. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for extracting and detecting buckwheat phytochrome in buckwheat.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] The invention discloses a method for extracting buckwheat phytochrome from buckwheat. The method comprises the following steps: drying and crushing tartary buckwheat buds, mixing them with an extractant, and then oscillating, ultrasonicating, and centrifuging them. The supernatant is taken, dissolved with methanol, filtered, and then derived under a full-spectrum plant lamp.

[0008] Preferably, the extractant is any one of methanol, acetone, tetrahydrofuran, acetic acid, anhydrous ethanol, ethanol, and a mixture of ethanol and acetone, and the volume ratio of ethanol to acetone is 1:1.

[0009] Preferably, the solid-liquid ratio of the tartary buckwheat sprouts to the extractant is 80-120:3.

[0010] Preferably, the ultrasonic temperature is 40-60° C., and the ultrasonic time is 0.5-4 h.

[0011] Preferably, the derivatization time is 0.5 to 4 hours.

[0012] Correspondingly, a method for detecting buckwheat phytochrome is provided, wherein the buckwheat phytochrome in the sample extract is characterized and identified by electrospray mass spectrometry, and the mass spectrometry conditions are as follows: liquid chromatography-mass spectrometry, ESI positive ion mode, flow rate 0.3 mL / min, column temperature 35°C, mobile phase A: 0.1% formic acid aqueous solution, v / v; mobile phase B: acetonitrile, 0-5 min, 90% B; capillary voltage 135 V, capillary temperature 300°C, mass scanning range m / z: 600-800 Da.

[0013] Preferably, the chromatographic conditions are chromatographic column C 18 , mobile phase A liquid: acetonitrile solution, B liquid: any one of 0.1% trifluoroacetic acid water, 0.1% formic acid water, 0.1% acetic acid water, 0.2% phosphoric acid water, 10mmol / L ammonium acetate water, primary water, 6mmol / L disodium hydrogen phosphate aqueous solution, pH=3.0, gradient change: 0-5min: 40%-30% B; 5-10min: 30%-10% B; 10-15min: 10%-0% B; 15-20min: 0% B; 20-23min: 0%-40% B; injection volume: 10μL, flow rate: 1.0mL / min, DAD detection wavelength 590±2nm; FLD: excitation wavelength: 330nm, emission wavelength: 590nm.

[0014] The present invention has the following beneficial effects:

[0015] 1. The present invention optimizes the extraction method of buckwheat phytochrome, and conducts qualitative and quantitative analysis on it by high performance liquid chromatography combined with DAD detector, mass spectrometry detector, FLD detector, etc., and establishes an accurate and feasible high performance liquid chromatography method for detecting buckwheat phytochrome in buckwheat, which can provide a reference for the detection of buckwheat phytochrome.

[0016] 2. The present invention establishes a high performance liquid chromatography-DAD and FLD determination method for buckwheat phytochrome in buckwheat, optimizes the determination method for buckwheat phytochrome in buckwheat, and compares the effects of different extractants, solid-liquid ratios, ultrasonic temperature, ultrasonic time, and exposure time on buckwheat phytochrome. Currently, it can be determined that at least 3 derivatives of buckwheat phytochrome exist. The method has a low detection limit, accurate quantitative determination, high sensitivity, and good repeatability. The technical indicators can meet the requirements of daily buckwheat analysis and detection, and can provide a reliable detection method basis for the determination of buckwheat phytochrome in buckwheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electrospray mass spectrum of 90% acetone extract in positive ion mode;

[0018] Figure 2 is the UV-vis spectrum;

[0019] Figure 3 is a high performance liquid chromatogram;

[0020] Figure 4 It is the UV-vis spectrum of three peaks in the sample extract;

[0021] Figure 5 This is a comparison chart of the absorption intensity of hypericin UV-visible and fluorescence;

[0022] Figure 6 Chromatograms of phytochrome and hypericin in different aqueous phases; (A) 0.1% trifluoroacetic acid water; (B) 0.1% formic acid water; (C) 0.1% acetic acid water; (D) 0.2% phosphoric acid water; (E) 10mmol / L ammonium acetate water; (F) primary water; (G) 6mmol / L disodium hydrogen phosphate (pH=3.0);

[0023] Figure 7 The results of phytochrome content extracted with different extractants;

[0024] Figure 8 Effects of extraction conditions on the content of buckwheat phytochrome; (A) solid-liquid ratio; (B) ultrasonic temperature; (C) ultrasonic time; (D) exposure time. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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.

[0026] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0027] 1. The present invention discloses a method for extracting buckwheat phytochrome from buckwheat, wherein bitter buckwheat buds or sweet buckwheat buds are dried, crushed, mixed with an extractant, and then oscillated, ultrasonicated, and centrifuged. The supernatant is taken, dissolved with methanol, filtered, and then derivatized under a full-spectrum plant lamp for 0.5 to 4 hours. The extractant is any one of methanol, acetone, tetrahydrofuran, acetic acid, anhydrous ethanol, ethanol, and a mixture of ethanol and acetone, and the volume ratio of ethanol to acetone is 1:1. The solid-liquid ratio of bitter buckwheat buds to the extractant is 80 to 120:3. The ultrasonic temperature is 40 to 60°C, and the ultrasonic time is 0.5 to 4 hours.

[0028] 2. A method for detecting buckwheat phytochrome, characterizing and identifying the buckwheat phytochrome in the sample extract (the extract extracted by the above method) by electrospray mass spectrometry, mass spectrometry conditions: liquid chromatography-mass spectrometry, ESI positive ion mode, flow rate 0.3 mL / min, column temperature 35°C, mobile phase A: 0.1% formic acid aqueous solution, v / v; mobile phase B: acetonitrile, 0-5 min, 90% B; capillary voltage 135 V, capillary temperature 300°C, mass scanning range m / z: 600-800 Da.

[0029] Chromatographic conditions: Thermo Fisher C 18 (250mm×4.6mm, 5μm), mobile phase A liquid: acetonitrile solution, B liquid: any one of 0.1% trifluoroacetic acid water, 0.1% formic acid water, 0.1% acetic acid water, 0.2% phosphoric acid water, 10mmol / L ammonium acetate water, primary water, 6mmol / L disodium hydrogen phosphate (pH=3.0), gradient change: 0-5min: 40%-30% B; 5-10min: 30%-10% B; 10-15min: 10%-0% B; 15-20min: 0% B; 20-23min: 0%-40% B; injection volume: 10μL, flow rate: 1.0mL / min, DAD detection wavelength 590±2nm; FLD: excitation wavelength: 330nm, emission wavelength: 590nm.

[0030] The present invention will be further described below in conjunction with specific embodiments.

[0031] In the following examples, the bitter buckwheat buds (Xiqiao No. 1, Miqiao No. 1, Chuanqiao No. 1, Chengqiao No. 1, Chuanqiao No. 2) and sweet buckwheat buds (Honghua Buckwheat, Yuqiao, Qinqiao No. 3, Yanqiao) from the experimental base of Chengdu University were naturally grown and harvested for 12 days, dried at 60°C, crushed, passed through a 40-mesh sieve, and frozen at -18°C for later use; 4 products with added buckwheat buds or plants: 3% bitter buckwheat flour noodles, 3% bitter buckwheat shaqima, whole plant bitter buckwheat tea (bagged), and whole plant bitter buckwheat tea (canned) were all purchased from supermarkets; hypericin standard HPLC (≥98%): Sichuan Weikeqi Biotechnology Co., Ltd.

[0032] Equipment: UltiMate 3000 high performance liquid chromatograph equipped with a diode array detector (DAD) and a fluorescence detector (FLD); 1290 Infinity II-6470 liquid chromatograph-mass spectrometer (LCMSMS) equipped with an electrospray mass spectrometer; N-EVAP11250 24-position water bath nitrogen blowdown instrument; full-spectrum plant light 0.3m / 10W Meixin Lighting.

[0033] Example 1

[0034] The extraction process of buckwheat phytochrome is as follows:

[0035] 100 mg of sample was added with 3 ml of 90% acetone and then shaken for 10 min, ultrasonicated at 45°C for 1 h, and centrifuged at 4000 r for 10 min. 1 mL of supernatant was taken, nitrogen blown, and dissolved with 1 mL of methanol through a 0.22 μm microporous filter membrane into a 1.5 mL transparent HPLC bottle, and derivatized under a full-spectrum plant lamp for 1 h as the sample test solution.

[0036] A standard curve was drawn with hypericin concentration as the horizontal axis and peak area as the vertical axis to calculate the buckwheat phytochrome content.

[0037] Seven extractants (methanol, 90% acetone, 80% tetrahydrofuran, 60% acetic acid, anhydrous ethanol, 90% ethanol, ethanol: acetone = 1: 1) were selected for investigation. At the same time, the effects of different solid-liquid ratios (80: 3, 90: 3, 100: 3, 110: 3, 120: 3), ultrasonic temperature (40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃), ultrasonic time (0.5h, 1h, 2h, 3h, 4h) and exposure time (0.5h, 1h, 2h, 3h, 4h) on the extraction of buckwheat phytochrome were investigated to optimize the optimal extraction conditions.

[0038] The test was performed according to the above-mentioned test method 2.

[0039] The test results are as follows:

[0040] 1. The electrospray mass spectrometer of buckwheat extract in positive ion mode is as follows Figure 1 As shown, combined with the prior art ( BE, D, Friedrich M, et al. Isolation, analysis and structures of phototoxic fagopyrins from buckwheat [J]. Food Chemistry, 2014, 143: 432-439.) The theoretical values ​​of the three quasi-molecular ion peaks of original buckwheat phytochrome, buckwheat phytochrome F, buckwheat phytochrome D and / or E were 673.25, 671.24 and 657.22, respectively. Combined with the retention times of HPLC, which were 11.58 min, 12.02 min and 12.60 min, respectively, the three substances were determined to be original buckwheat phytochrome, buckwheat phytochrome F, buckwheat phytochrome D and / or E.

[0041] 2. Pretreatment and on-machine detection were performed according to the chromatographic conditions of the above extraction method 1 and detection method 2. The retention time of buckwheat phytochrome was about 12 minutes, and the retention time of hypericin was 15.48 minutes. The peaks were symmetrical and well separated. The representative spectra of buckwheat phytochrome in hypericin standard solution and buckwheat samples are shown in Figure 2 and Figure 3 .Depend on Figure 2 It is known that the UV-vis spectra of hypericin and buckwheat phytochrome are basically the same, and the maximum absorption wavelengths are 591.00nm and 548.00nm. Figure 3 It is known that there are three peaks in the sample, with retention times of 11.58min, 12.02min and 12.60min respectively. The UV-vis absorption spectra of the three peaks in the sample all have maximum absorption wavelengths of 548nm and 591nm ( Figure 4 ). It is speculated that these peaks are original buckwheat phytochrome, buckwheat phytochrome and its derivatives. The total amount of the three peaks is taken as the buckwheat phytochrome content in the present invention.

[0042] Example 2 Optimization of chromatographic conditions

[0043] Buckwheat phytochrome and hypericin have absorption peaks under UV-visible and fluorescence detection ( Figure 5 ), the absorption intensity of the fluorescence detector is more than 6,000 times that of the UV detector. Both buckwheat phytochrome and hypericin have good fluorescence spectrum characteristics, making it possible to establish a highly sensitive buckwheat phytochrome detection method. DAD spectrum can check the absorption spectrum of buckwheat phytochrome and hypericin to confirm whether it is the target peak, so two detectors, DAD and FLD, are selected, DAD for qualitative analysis and FLD for quantitative analysis.

[0044] Optimization of chromatographic column conditions: Comparison of C 18 Chromatographic column (4.6×250mm, 5μm), C 18The separation effect of the chromatographic column (4.6×150mm, 5μm). From the experimental results, the 150mm chromatographic column cannot separate buckwheat phytochrome and its derivatives, and the 250mm chromatographic column has better separation effect, so C is selected. 18 The chromatographic column (4.6×250mm, 5μm) was further optimized. The effects of methanol and acetonitrile as organic phases on the separation effect were compared. The results showed that acetonitrile had an earlier elution time and a better peak shape than methanol, so acetonitrile was selected as the organic phase.

[0045] The separation effects of 7 aqueous phases were compared: 0.1% trifluoroacetic acid water, 0.1% formic acid water, 0.1% acetic acid water, 0.2% phosphoric acid water, 10mmol / L ammonium acetate water, primary water, and 6mmol / L disodium hydrogen phosphate (pH=3.0). The results showed that 0.2% phosphoric acid water ( Figure 6 D), 0.1% trifluoroacetic acid water ( Figure 6 A), 0.1% acetic acid water ( Figure 6 When C) was used as the water phase, three chromatographic peaks appeared in Hypericum, which might be due to the decomposition or transformation of hypericin under acidic conditions, and the chromatographic peaks of the sample and the blank overlapped; primary water ( Figure 6 F) When used as the aqueous phase, hypericin showed three chromatographic peaks, while the sample showed only one peak; 10mmol / L ammonium acetate water ( Figure 6 E) When used as the aqueous phase, hypericin showed a chromatographic peak, but the sample did not show a peak; 0.1% formic acid water ( Figure 6 B) When the aqueous phase was used, three chromatographic peaks appeared for hypericin and two chromatographic peaks appeared for the sample; 6mmol / L disodium hydrogen phosphate (pH=3.0) ( Figure 6 G) When used as the aqueous phase, the chromatographic peaks of hypericin all showed one peak, and the sample also showed three peaks. Hypericin and the photosensitive pigment in the sample were well separated, so 6 mmol / L disodium hydrogen phosphate (pH = 3.0) was selected as the aqueous phase.

[0046] In order to further achieve the separation effect of phytochrome in buckwheat sample solution, the mobile phase gradient was adjusted. The results are as follows (see Figure 6 G): Mobile phase A liquid: acetonitrile solution, B liquid: 6mmol / L disodium hydrogen phosphate aqueous solution (pH=3.0), determine the gradient change as follows: 0-5min: 40%-30% B; 5-10min: 30%-10% B; 10-15min: 10%-0% B; 15-20min: 0% B; 20-23min: 0%-40% B; injection volume: 10μL, flow rate: 1.0mL / min.

[0047] Example 3 Effect of extraction conditions on buckwheat phytochrome

[0048] According to the above extraction method 1 and detection method 2, pretreatment and on-machine detection were performed, and the peak area of ​​buckwheat phytochrome in the sample test solution was recorded. The results were calculated according to the hypericin standard curve method, and the effects of different extractants on extracting buckwheat phytochrome were compared ( Figure 7 ). The results showed that the content of buckwheat phytochrome in the extracts of 90% acetone and 80% tetrahydrofuran was high. Tetrahydrofuran is highly toxic and has a pungent odor, so 90% acetone was selected as the extraction agent in subsequent experiments.

[0049] Depend on Figure 8 It can be seen that the solid-liquid ratio does not have a particularly large effect on the extraction of photosensitin. At 100:3 (mg:ml), the photosensitin content reaches the highest. The increase in the solid-liquid ratio does not increase the photosensitin extraction rate, but instead slightly decreases. The ultrasonic temperature is between 40 and 60°C, and the photosensitin content increases with increasing temperature. However, when the temperature exceeds a certain level, photosensitin may decompose or transform. It should also be noted that the boiling point of acetone is 53°C. When the ultrasonic temperature exceeds 50°C, acetone evaporates quickly and the volume of the extractant is difficult to control during the experiment. When the ultrasonic time is within 0.5 to 4 hours, the photosensitin content first increases sharply and then tends to equilibrium, indicating that after the ultrasonic time is greater than 1 hour, it has little effect on the photosensitin extraction amount, but instead has a downward trend. This may be because the ultrasonic time is too long and the photosensitin decomposes or transforms. When the exposure time is within 0.5 to 4 hours, the photosensitin content has the same trend as the ultrasonic time, which first increases sharply and then tends to equilibrium. In summary, the optimal extraction conditions were selected as follows: solid-liquid ratio 100:3 (mg:ml), ultrasonic temperature 48°C, ultrasonic time 1h, and exposure time 1h.

[0050] Example 4 Methodological Investigation

[0051] 1. Solution preparation

[0052] Hypericin standard solution: 5.00 mg (accurate to 0.01 mg) in a 10 mL volumetric flask, dissolve with methanol and dilute to the mark, mix well, prepare 500 μg / mL hypericin stock solution, and store at 4 ° C. Take 1 mL of hypericin stock solution and dilute to 10 mL with methanol to obtain a 50 μg / mL hypericin standard working solution, and dilute it step by step before use. This solution is used for linearity, detection limit, quantitation limit, precision and other experiments.

[0053] 2. Preparation of standard curve

[0054] The 50 μg / mL hypericin standard working solution was diluted stepwise to prepare a series of 0, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, and 25 μg / mL hypericin standard solutions, respectively.

[0055] 3. Precision experiment

[0056] Take the hypericin standard solution (5 μg / ml), inject it 6 times continuously, and calculate the RSD value of the hypericin peak area.

[0057] 4. Stability test

[0058] Take the Xiqiao No. 1 test solution and sample it every 1 hour within 5 hours, store it away from light, record the peak area, and calculate the RSD value of the buckwheat phytochrome peak area.

[0059] 5. Repeatability Experiment

[0060] The above-mentioned extraction method 1 weighed 6 samples of Xiqiao No. 1 buckwheat seedlings to prepare sample test solutions. The peak area of ​​buckwheat phytochrome in the sample test solutions was recorded under the chromatographic conditions in the above-mentioned detection method 2. The peak area was calculated according to the hypericin standard curve method, and the RSD of the peak area was calculated 6 times.

[0061] 6. Sample recovery experiment

[0062] Using Xiqiao No. 1 as the matrix, spike recovery experiments were carried out at three content levels (0.05, 0.5, and 5.0 μg / mL) (n=6), and the peak area of ​​buckwheat phytochrome was recorded. The RSD of the six peak areas was calculated according to the hypericin standard curve method.

[0063] 7. Limit of detection and limit of quantification

[0064] The limit of detection (LOD) and limit of quantification (LOQ) were calculated using the signal-to-noise ratio (S / N), with 3 times the S / N as the LOD and 10 times the S / N as the LOQ.

[0065] 8. Sample determination

[0066] Prepare the sample test solution according to the above extraction method 1. For samples with low phytochrome content, the sample weight can be increased.

[0067] The result is:

[0068] 1) Linear regression was performed using the concentration of hypericin as the abscissa (x) and the peak area corresponding to the concentration as the ordinate (y), and the regression equation was obtained: y = 252276x-6091.3 (r = 0.9995). The detection limit and quantification limit reached 0.001 mg / g and 0.003 mg / g, respectively. The concentration and peak area of ​​hypericin had good linearity within the range of 0.00 μg / ml to 25 μg / ml.

[0069] 2) In the precision experiment, the RSD of the peak area of ​​hypericin was 0.17%, indicating that the instrument had good precision.

[0070] 3) In the stability experiment, the RSD of the peak area of ​​buckwheat phytochrome was 1.96%, indicating that the sample test solution was relatively stable within 5 hours under light-proof conditions.

[0071] 4) In the repeatability experiment, the average content of phytochrome in buckwheat No. 1 was measured to be 0.667 mg / g, with RSD = 0.95%, indicating that the method has good precision.

[0072] 5) The recovery rate was calculated according to the method under the above-mentioned sample recovery experiment (Table 1). The mean recovery rate of hypericin was between 83.7% and 100.9%, and the RSD range was between 1.13% and 1.85%. It can be seen that the recovery rate and RSD are in line with the requirements of GB / T 27404-2008 "Laboratory Quality Control Standards for Food Physical and Chemical Testing", indicating that this method is accurate and reliable for the detection of buckwheat phytochrome in buckwheat.

[0073] Table 1 Recovery and RSD results of spiked buckwheat phytochrome in samples (n=6)

[0074] Hypericin spiked amount (μg / ml) Sample photofrin content (mg / g) Average recovery rate (%) RSD(%) 0.05 0.667 83.7 1.68 0.5 0.667 88.0 1.85 5.0 0.667 100.9 1.13

[0075] 6) Results of buckwheat phytochrome determination in 5 kinds of bitter buckwheat sprouts and 4 kinds of sweet buckwheat sprouts (Tables 2 and 3). In the prior art (Jaecheol K, Krum T H. Fagopyrins in different parts of common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum) during growth [J]. Journal of Food Composition and Analysis. 2020 (86): 103354-103354.), the content of fagopyrin in the roots, stems and leaves of sweet buckwheat and tartary buckwheat is between 0.68 and 0.79 mg / g and 0.81 and 0.94 mg / g, respectively; the content of fagopyrin in the roots, stems, leaves and flowers of sweet buckwheat and tartary buckwheat is between 0.00 and 0.31 mg / g and 0.0 and 0.88 mg / g, respectively (Kentaro E, Tomohiro A, Hideji O. Development of a High-Performance Liquid Chromatography Method to Determine the Fagopyrin Content of Tartary Buckwheat (Fagopyrum tartaricum Gaertn.) and Common Buckwheat (F.esculentum Moench) [J]. Plant Prod, 2009, 12 (4): 475-480.); the maximum content of buckwheat phytochrome in the mixed leaves of bitter buckwheat and sweet buckwheat is 786 μg / g (Lu Sha. Extraction, content analysis and phototoxicity of buckwheat phytochrome [D]. Taiyuan: Shanxi University. 2019.). The phytochrome content in sweet buckwheat buds and bitter buckwheat buds was 0.201-0.459 mg / g and 0.422-0.975 mg / g, respectively, which is similar to the results reported in previous literature. Due to different varieties, measurement sites and culture methods, the phytochrome content is slightly different. The average content of buckwheat phytochrome in bitter buckwheat buds is higher than that in sweet buckwheat buds. There are also differences in the content of phytochrome between different varieties of sweet buckwheat and bitter buckwheat, indicating that genetic factors are one of the important factors affecting the content of buckwheat phytochrome.

[0076] Table 2 Results of determination of phytochrome content in tartary buckwheat sprouts

[0077] Serial number Product Name Measurement results (mg / g) 1 Xiqiao No.1 0.665±0.006 2 Miqiao No.1 0.519±0.009 3 Chuanqiao No.1 0.834±0.006 4 Chengqiao No.1 0.975±0.006 5 Chuanqiao No.2 0.422±0.004

[0078] Table 3 Determination results of phytochrome content in sweet buckwheat sprouts

[0079] Serial number Product Name Measurement results (mg / g) 1 Buckwheat 0.338±0.005 2 Fagopyrum truncatum 0.201±0.004 3 Qinqiao No.3 0.459±0.005 4 Salt buckwheat 0.281±0.006

[0080] 7) The phytochrome content of the four products on the market that added buckwheat sprouts or plants was less than one in 100,000 (Table 4). Due to the limited amount of buckwheat sprouts added to the products and the different processing, the buckwheat phytochrome content is already very low. The market demand is not only growing for buckwheat seeds, but also for buckwheat plants, sprouts and extracts, which has raised questions about the safety of these products. Existing studies have shown that the research on the toxicity of buckwheat phytochrome has not come up with a standard of daily intake that should not be exceeded. It is only known that it is impossible for any part of the whole buckwheat plant to cause acute poisoning in humans.

[0081] Table 4 Results of determination of phytochrome content in products with added buckwheat sprouts or plants

[0082] Serial number Product Name Measurement results (mg / g) 1 3% Buckwheat flour noodles 0.006±0.001 2 3% Buckwheat Powder Shaqima 0.006±0.001 3 Whole Buckwheat Tea (Bag) 0.003±0.000 4 Whole Buckwheat Tea (Canned) <0.003±0.000

[0083] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting buckwheat phytochrome from buckwheat, characterized in that: The bitter buckwheat buds or sweet buckwheat buds are dried, crushed, mixed with the extractant, shaken, ultrasonicated, and centrifuged; the supernatant is taken, dissolved with methanol, filtered, and then derived under a full-spectrum plant lamp.

2. The extraction method according to claim 1, characterized in that: The extractant is any one of methanol, acetone, tetrahydrofuran, acetic acid, anhydrous ethanol, ethanol, and a mixture of ethanol and acetone, and the volume ratio of ethanol to acetone is 1:

1.

3. The extraction method according to claim 1 or 2, characterized in that: The solid-liquid ratio of the tartary buckwheat sprouts to the extractant is 80-120:

3.

4. The extraction method according to claim 1, characterized in that: The ultrasonic temperature is 40-60°C, and the ultrasonic time is 0.5-4h.

5. The extraction method according to claim 1, characterized in that: The derivatization time is 0.5 to 4 hours.

6. A method for detecting buckwheat phytochrome extracted by the extraction method according to any one of claims 1 to 5, characterized in that: The buckwheat phytochrome in the sample extract was characterized and identified by electrospray ionization mass spectrometry. The mass spectrometry conditions were: liquid chromatography-mass spectrometry, ESI positive ion mode, flow rate 0.3 mL / min, column temperature 35°C, mobile phase A: 0.1% formic acid aqueous solution, v / v; mobile phase B: acetonitrile, 0-5 min, 90% B; capillary voltage 135 V, capillary temperature 300°C, mass scanning range m / z: 600-800 Da.

7. The detection method according to claim 6, characterized in that: Chromatographic conditions: Column C 18 , mobile phase A liquid: acetonitrile solution, B liquid: any one of 0.1% trifluoroacetic acid water, 0.1% formic acid water, 0.1% acetic acid water, 0.2% phosphoric acid water, 10mmol / L ammonium acetate water, primary water, 6mmol / L disodium hydrogen phosphate, pH=3.0, gradient change: 0-5min: 40%-30% B; 5-10min: 30%-10% B; 10-15min: 10%-0% B; 15-20min: 0% B; 20-23min: 0%-40% B; injection volume: 10μL, flow rate: 1.0mL / min, DAD detection wavelength 590±2nm; FLD: excitation wavelength: 330nm, emission wavelength: 590nm.

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