A method for detecting anthocyanins

Abstract

This invention provides a method for detecting anthocyanins, belonging to the field of pigment detection technology. The method involves mixing plant petal powder with an extractant, centrifuging, collecting the supernatant, drying it under nitrogen, and redissolving it to obtain the extract. Qualitative structural analysis of the anthocyanins in the extract is performed using liquid chromatography-mass spectrometry (LC-MS). Quantitative analysis of the content of different types of anthocyanins obtained from the extract is conducted using an internal standard method. This invention detected eight anthocyanins in Rosa species, including cyanidin-3-O-glucoside (Cy3Gal) and cyanidin-3-dimethyl-glucoside (Cy3DmG), which were detected for the first time in Rosa species. The detection method of this invention features simple pretreatment, small sample volume, low solvent usage, and minimal environmental impact; fewer purification steps minimize anthocyanin loss, resulting in high extraction rate and accurate detection results.

Description

Technical Field

[0001] This invention belongs to the field of pigment detection technology, and particularly relates to a method for detecting anthocyanin glycosides. Background Technology

[0002] Anthocyanins are a class of water-soluble natural pigments widely found in plants. They possess antioxidant, antitumor, antibacterial, blood sugar-lowering, and anti-aging properties, and are widely used in the food and pharmaceutical industries. In living plants, anthocyanins mostly exist in the form of glycosides, namely anthocyanins, which belong to the flavonoid family of compounds. Furthermore, anthocyanins play a decisive role in the coloration of angiosperm petals; the color of 88% of angiosperm petals is determined by the type and content of anthocyanins, exhibiting a range of colors from orange-yellow to red, pink, purple, and blue. Anthocyanins are mainly derived from anthocyanin aglycones. Currently, over 700 anthocyanins have been isolated and identified, primarily derived from six anthocyanin aglycones: cyanidin (Cy), pelargonidin (Pg), delphinidin (Dp), peonidin (Pn), petunidin (Pt), and malvidin (Mv). Anthocyanins exhibit wide structural variation in nature, mainly reflected in differences in the number of hydroxyl groups, the number of sugar moieties, the structure of the sugar, and the position of the organic acid attached to the glycoside. To date, the main anthocyanin aglycones detected are glucose, rhamnose, xylose, galactose, and arabinose, as well as disaccharides or trisaccharides composed of them. The most common are 3-monoglucosides, 5-diglucosides, 3,5-disaccharides, and 3,7-disaccharides. These glycosides can be further modified by glycosylation, methylation, acylation, etc., to form a wide variety of anthocyanins. Glycosylation involves modifying the hydroxyl group of the 2-phenylbenzopyran cation with monosaccharides, disaccharides, or trisaccharides. Acylation involves further modifying the hydroxyl group of the glycoside with coumaric acid, caffeic acid, ferulic acid, etc., after glycosylation. These structural modifications increase the stability of anthocyanins in plant cells.

[0003] The genus *Rosa* L. is rich in plant resources, comprising approximately 200 species, widely distributed in temperate to subtropical regions of Asia, Europe, North Africa, and North America, and is one of the world's most famous ornamental plants. Roses, wild roses, and flowering roses all belong to the genus *Rosa*, boasting a rich variety of flower colors and pleasant fragrances. Many species' petals are rich in polysaccharides, polyphenols, and flavonoids, possessing antioxidant, antibacterial, sedative, hypnotic, and lipid-regulating properties, making them suitable for use in the pharmaceutical, chemical, and food industries. In recent years, research on *Rosa* plants has received increasing attention. For example, fresh rose petals can be used to extract aromatic essential oils for edible and cosmetic applications, and can be used to make pastry fillings, rose wine, and rose syrup. Dried rosebuds can be used to make tea. The fruits of some roses and wild roses are rich in vitamin C, glucose, fructose, sucrose, citric acid, malic acid, and carotene. Anthocyanins, as important flavonoids in the petals of Rosa species, together with flavonols and carotenoids, constitute the pigmentary basis for the diversity of flower colors in Rosa species. The detection and extraction of anthocyanins and other secondary metabolites in Rosa species play a crucial role in the development and utilization of the plant's value.

[0004] Pretreatment and detection processes significantly impact the rapid extraction and qualitative / quantitative analysis of anthocyanins. Previous studies have primarily employed column chromatography or solid-phase extraction for anthocyanin extraction and purification, which are costly, require sophisticated experimental procedures, and are time-consuming. Furthermore, some anthocyanins are lost during extraction, affecting the results. Currently, there are few reports on methods for the extraction and detection of anthocyanins in Rosa petals. Establishing rapid extraction and detection methods for Rosa petals not only provides a reference for the genetic improvement of flower color in Rosa species but also offers important guidance for developing and utilizing the practical value of Rosa plants. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for detecting anthocyanins, so as to solve the problems of high extraction cost, high experimental operation requirements, complex experimental steps, long time consumption, and large loss of anthocyanins in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for detecting anthocyanins, comprising the following steps:

[0008] (1) After mixing the plant petal powder with the extractant, centrifuge, collect the supernatant, dry it with nitrogen, and redissolve it to obtain the extract;

[0009] (2) The anthocyanin glycosides in the extract were qualitatively analyzed by liquid chromatography-mass spectrometry.

[0010] (3) The content of different types of anthocyanins obtained from the extract was quantitatively analyzed by internal standard method;

[0011] The extractant is a mixture of methanol and formic acid.

[0012] Preferably, the volume ratio of methanol to formic acid in the extractant is 7-12:1.

[0013] Preferably, the extractant further includes an internal standard, which is paeoniflorin 3-glucoside, and the concentration of the internal standard in the extractant is 2-5 μg / mL.

[0014] Preferably, the mass-to-volume ratio of the plant petal powder to the extract is 0.2-0.8g: 2-6mL, and the mass-to-volume ratio of the plant petal powder to the extract during redissolution is 0.2-0.8g: 0.8-1.5mL.

[0015] Preferably, in step (2), the conditions for liquid chromatography detection are as follows:

[0016] Column type: ACQUITYUPLCCSH C18;

[0017] Column parameters: 2.1 mm × 100 mm, 1.7 μm;

[0018] Mobile phase A: a mixture of acetonitrile and formic acid in a volume ratio of 95:5;

[0019] Mobile phase B: a mixture of water and formic acid in a volume ratio of 95:5;

[0020] The gradient elution program is as follows: the initial volume ratio of mobile phase A is 2.5%, mobile phase A increases from 2.5% to 10% within 5 min, then increases from 10% to 25% within 20 min, holds for 5 min, and then decreases from 25% to 2.5% within 30 min;

[0021] Flow rate: 0.05-0.2 mL / min;

[0022] Column temperature: 20-30℃;

[0023] Autosampler temperature: 2-6℃;

[0024] Injection volume: 0.5-1.5 μL.

[0025] Preferably, in step (2), the conditions for mass spectrometry detection are as follows:

[0026] The mass spectrometer was set to positive ion mode.

[0027] Ion source: ESI source;

[0028] Capillary voltage: 1.5-2.5kV;

[0029] Ion source temperature: 80-120℃;

[0030] Desolvation gas temperature: 250-350℃;

[0031] Desolventizing gas flow rate: 700-900 L / h;

[0032] Conical orifice airflow: 40-60 L / h;

[0033] Mass spectrometry acquisition mode: Fast DDA mode;

[0034] Analysis mode: Resolution mode;

[0035] Precursor ion collection range: 200 m / z - 850 m / z;

[0036] Femtoion collection range: 50 m / z - 850 m / z;

[0037] A maximum of five daughter ions can be selected for qualitative analysis.

[0038] The collision energy range is 10V-80V;

[0039] Spectral conditions: The wavelength range of the diode array detector is 190nm-800nm.

[0040] Preferably, in the quantitative analysis of step (3), the quantitative method is to calculate the content using a standard curve regression equation formula with the peak area of ​​the anthocyanin glycoside in the ultraviolet absorption chromatogram at 535 nm as the ordinate and the concentration of the internal standard as the abscissa. The correlation of the standard curve is 1>R. 2 >0.999;

[0041] The linear range is 1-1000 μg / mL;

[0042] Quantitative range: 0-1 μg / mL.

[0043] Preferably, the temperature for the mixed extraction is 2-6℃, and the extraction time is 8-15h.

[0044] Preferably, the centrifugation speed is 3500-4500 rpm and the centrifugation time is 15-30 min.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] This invention establishes a rapid extraction and qualitative / quantitative detection method for anthocyanins in the petals of Rosa species. The method of this invention features simple pretreatment, requires small sample volumes, little solvent, and has minimal environmental impact; the purification steps minimize anthocyanin loss, resulting in high extraction rates and accurate detection results.

[0047] This invention detected eight anthocyanin glycosides in Rosa species, namely cyanidin-3-(caffeoyl)-glucoside (Cy3CafG), cyanidin-3-O-galactoside (Cy3Gal), paeoniflorin-3-(caffeoyl)-glucoside (Pn3CafG), cyanidin-3-(cis-caffeoyl)-dimethylglucoside (Cy3(cis-Caf)DmG), and cyanidin-3-(trans-caffeoyl)-dimethylglucoside (Cy3(tr)DmG). The study identified three compounds in Rosa spp.: cis-Caf (Cy3DmG), cyanidin-3-dimethyl-glucoside (Cy3DmG), paeoniflorin-3-(cis-caffeoyl)-rutin (Pn3(cis-Caf)Ru), and paeoniflorin-3-(trans-caffeoyl)-rutin (Pn3(trans-Caf)Ru). Two of these compounds, cyanidin-3-O-glucoside (Cy3Gal) and cyanidin-3-dimethyl-glucoside (Cy3DmG), were detected for the first time in Rosa spp. This study provides a solid experimental foundation for the genetic improvement of anthocyanin glycosides in Rosa spp. petals and also lays the groundwork for the extraction, quantitative detection, and utilization of flavonoids from Rosa spp. petals. Attached Figure Description

[0048] Figure 1 Images of the flowers of 8 species of Rosa genus;

[0049] Figure 2 Ultraviolet spectra of petals from eight different color series of Rosa species acquired by liquid chromatography-time-of-flight mass spectrometry at a wavelength of 535 nm. Figure 2 The retention time of anthocyanins in Georgia was 6.58 min (chromatogram of anthocyanins). Figure 2 The chromatogram of anthocyanins in Damask rose with a retention time of 9.71 min (i) Figure 2 (j) of the middle;

[0050] Figure 3The diagrams show the primary and secondary ion sequences of eight anthocyanin glycosides: (a) Primary ion sequence of cyanidin-3-(caffeoyl)-glucoside (Cy3CafG); (b) Secondary ion sequence of cyanidin-3-(caffeoyl)-glucoside (Cy3CafG); (c) Primary ion sequence of cyanidin-3-O-galactoside (Cy3Gal); (d) Secondary ion sequence of cyanidin-3-O-galactoside (Cy3Gal); (e) Secondary ion sequence of paeoniflorin-3-(caffeoyl)-glucoside. f: Primary ion diagram of peonidin-3-(caffeoyl)-glucoside (Pn3CafG); g: Primary ion diagram of cyanidin-3-(cis-caffeoyl)-dimethylglucoside (Cy3(cis-Caf)DmG); h: Secondary ion diagram of cyanidin-3-(cis-caffeoyl)-dimethylglucoside (Cy3(cis-Caf)DmG); i: Secondary ion diagram of cyanidin-3-(trans-caffeoyl)-dimethylglucoside (Cy3(cis-Caf)DmG); j: Primary ion diagram of cyanidin-3-(trans-Caf)-dimethyl glucoside (Cy3(trans-Caf)DmG); k: Primary ion diagram of cyanidin-3-dimethyl-glucoside (Cy3DmG); l: Secondary ion diagram of cyanidin-3-dimethyl-glucoside (Cy3DmG); m: Primary ion diagram of paeonol-3-(cis-Caf)-dimethyl-glucoside (Cy3DmG); Primary ion diagram of acyl-3-(cis-caffeoyl)-rutin (Pn3(cis-Caf)Ru); secondary ion diagram of n: paeoniflorin-3-(cis-caffeoyl)-rutin (Pn3(cis-Caf)Ru); primary ion diagram of o: paeoniflorin-3-(trans-caffeoyl)-rutin (Pn3(trans-Caf)Ru); secondary ion diagram of paeoniflorin-3-(trans-caffeoyl)-rutin (Pn3(trans-Caf)Ru).

[0051] Figure 4 Analysis of the differences in the types and contents of anthocyanins in the petals of eight Rosa species;

[0052] Figure 5 This figure shows the types and percentages of anthocyanins in the petals of eight Rosa species. Detailed Implementation

[0053] This invention provides a method for detecting anthocyanins, comprising the following steps:

[0054] (1) After mixing the plant petal powder with the extractant, centrifuge, collect the supernatant, dry it with nitrogen, and redissolve it to obtain the extract;

[0055] (2) The anthocyanin glycosides in the extract were qualitatively analyzed by liquid chromatography-mass spectrometry.

[0056] (3) The content of different types of anthocyanins obtained from the extract was quantitatively analyzed by internal standard method;

[0057] In this invention, plant petal powder is mixed with an extractant, centrifuged, the supernatant is collected, dried under nitrogen, and redissolved to obtain an extract. The plant petal powder is obtained by cryogenically grinding fresh plant petals in liquid nitrogen. The extractant is preferably a mixture of methanol and formic acid. The volume ratio of methanol to formic acid in the extractant is preferably 7-12:1, more preferably 8-11:1. The extractant also includes an internal standard, preferably paeoniflorin 3-glucoside, and the concentration of the internal standard in the extractant is preferably 2-5 μg / mL, more preferably 2.5-4 μg / mL. The mass-to-volume ratio of plant petal powder to extractant is preferably 0.2-0.8 g: 2-6 mL, more preferably 0.3-0.7 g: 3-5 mL. The preferred extraction temperature is 2-6℃, more preferably 3-5℃; the preferred extraction time is 8-15h, more preferably 9-14h; the preferred centrifugation speed is 3500-4500rpm, more preferably 3800-4200rpm; the preferred centrifugation time is 15-30min, more preferably 18-28min; the preferred mass-to-volume ratio of plant petal powder to extractant during redissolution is 0.2-0.8g:0.8-1.5mL, more preferably 0.3-0.7g:0.9-1.4mL; after redissolution, the mixture is filtered, and the pore size of the organic phase filter membrane is preferably 0.2-0.4μm, more preferably 0.22μm.

[0058] In this invention, the qualitative structural analysis of anthocyanin glycosides in the extract is performed using a combination of liquid chromatography and mass spectrometry; the conditions for the liquid chromatography detection are as follows:

[0059] Column type: ACQUITYUPLCCSH C18;

[0060] Column parameters: 2.1 mm × 100 mm, 1.7 μm;

[0061] Mobile phase A: a mixture of acetonitrile and formic acid in a volume ratio of 95:5;

[0062] Mobile phase B: a mixture of water and formic acid in a volume ratio of 95:5;

[0063] The gradient elution program is as follows: the initial volume ratio of mobile phase A is 2.5%, mobile phase A increases from 2.5% to 10% within 5 min, then increases from 10% to 25% within 20 min, holds for 5 min, and then decreases from 25% to 2.5% within 30 min;

[0064] Flow rate: 0.05-0.2 mL / min, preferably 0.1-0.18 mL / min;

[0065] Column temperature: 20-30℃, preferably 22-28℃;

[0066] Automatic sampler temperature: 2-6℃, preferably 3-5℃;

[0067] Injection volume: 0.5-1.5 μL, preferably 0.8-1.2 μL.

[0068] The conditions for the mass spectrometry detection are as follows:

[0069] The mass spectrometer was set to positive ion mode.

[0070] Ion source: ESI source;

[0071] Capillary voltage: 1.5-2.5kV, preferably 1.8-2.4kV;

[0072] Ion source temperature: 80-120℃, preferably 90-110℃;

[0073] Desolvation gas temperature: 250-350℃, preferably 280-320℃;

[0074] The solvent removal gas flow rate is 700-900 L / h, and the preferred solvent removal gas flow rate is 750-850 L / h.

[0075] Conical orifice airflow: 40-60 L / h, preferably 45-55 L / h;

[0076] Mass spectrometry acquisition mode: Fast DDA mode;

[0077] Analysis mode: Resolution mode;

[0078] Precursor ion collection range: 200 m / z - 850 m / z;

[0079] Femtoion collection range: 50 m / z - 850 m / z;

[0080] A maximum of five daughter ions can be selected for qualitative analysis.

[0081] The collision energy range is 10V-80V;

[0082] Spectral conditions: The wavelength range of the diode array detector is 190nm-800nm.

[0083] In this invention, the internal standard method is used to quantitatively analyze the content of different types of anthocyanins obtained from the extract. The quantitative analysis involves calculating the content using a standard curve regression equation formula with the peak area of ​​the anthocyanin's UV absorption chromatogram at 535 nm as the ordinate and the concentration of the internal standard as the abscissa. The correlation of the standard curve is 1>R. 2 >0.999;

[0084] The linear range is 1-1000 μg / mL;

[0085] Quantitative range: 0-1 μg / mL.

[0086] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0087] Instruments: HPLC-QTOF / PDA (Waters ACQUITY UPLC I-Class-Xevo G2-XSQTOF / PDA eLambda Detector); 8 species of Rosa species: collected from the Nankou Experimental Base and Nanbu Experimental Base of the Chinese Academy of Agricultural Sciences.

[0088] Example 1

[0089] The petals of eight Rosa species (details of the eight Rosa species are shown in Table 1, and images of the flowers of the eight Rosa species are shown in the image below) are listed. Figure 1 (As shown) After freezing in liquid nitrogen, the petal powder was ground. 0.5 g of the petal powder was weighed into a 15 mL brown glass centrifuge tube, and 4 mL of a methanol / formic acid (9:1, v / v) solution containing 15 μg of paeonidin-3-glucoside chloride was added. The extract was shaken well and extracted at 4 °C for 12 h. The 15 mL glass centrifuge tube was then centrifuged at 3900 rpm for 20 min at 4 °C. The supernatant was transferred to a new 15 mL brown glass centrifuge tube, placed on ice, and dried with nitrogen. The dried sample was redissolved in 200 μL of methanol / formic acid (9:1, v / v) solution and placed on ice. The solution was then filtered through a 0.22 μm organic phase filter and placed in a brown sample vial for instrumental analysis.

[0090] Table 18 Detailed Information on Rosa Species

[0091]

[0092]

[0093] Chromatographic conditions:

[0094] The chromatographic column used was an ACQUITY UPLC CSH C18 (2.1 mm × 100 mm id, 1.7 μm). Mobile phase A was acetonitrile / formic acid (95:5, v / v), and mobile phase B was water / formic acid (95:5, v / v). The flow rate was 0.15 mL / min. The initial volume fraction of mobile phase A was 2.5%. Mobile phase A was increased from 2.5% to 10% over 5 min, then increased from 10% to 25% over 20 min, held for 5 min, and then decreased from 25% to 2.5% over 30 min. The column oven temperature was maintained at 25 °C, and the autosampler temperature was maintained at 4 °C. The injection volume was 1 μL.

[0095] Mass spectrometry conditions:

[0096] The mass spectrometry was performed in positive ion mode. The ESI ion source parameters were as follows: capillary voltage, 2.0 kV; ion source temperature, 100℃; desolvation gas temperature, 300℃; desolvation gas flow rate, 800 L / h; cone gas flow rate, 50 L / h. Mass spectrometry acquisition was performed in Fast DDA mode. The analysis mode was resolution mode. The precursor ion acquisition range was 200 m / z to 850 m / z. The daughter ion acquisition range was 50 m / z to 850 m / z. A maximum of 5 daughter ions were selected for qualitative analysis. The collision energy range was 10 V to 80 V. Spectroscopic conditions: diode array detector wavelength range was 190 nm to 800 nm. The primary and secondary ion information is shown in Table 2, and the primary and secondary ion chromatograms are shown in... Figure 3 As shown.

[0097] Table 2 Information on primary and secondary ions

[0098]

[0099]

[0100] Experimental results: as shown in Table 3 and Figure 2 , Figure 4 , 5 As shown.

[0101] Table 3. Types and contents of anthocyanin glycosides detected in 8 species of Rosa species.

[0102]

[0103] The anthocyanin glycosides detected, in chronological order, were cyanidin-3-(caffeoyl)-glucoside (Cy3CafG), cyanidin-3-O-galactoside (Cy3Gal), paeoniflorin-3-(caffeoyl)-glucoside (Pn3CafG), cyanidin-3-(cis-caffeoyl)-dimethylglucoside (Cy3(cis-Caf)DmG), and cyanidin-3-(trans-caffeoyl)-dimethylglucoside (Cy3(trans-Caf)DmG). The products include cyanidin-3-dimethyl-glucoside (Cy3DmG), paeoniflorin-3-(cis-caffeoyl)-rutin (Pn3(cisCaf)Ru), and paeoniflorin-3-(trans-caffeoyl)-rutin (Pn3(trans-Caf)Ru), among which cyanidin-3-O-galactoside (Cy3Gal) and cyanidin-3-dimethyl-glucoside (Cy3DmG) are anthocyanin glycosides from Rosa species that were detected for the first time.

[0104] Depend on Figure 4 and Figure 5 It can be seen that among the eight different Rosa species, the red roses 'Yima Dangxian' and 'Tianxiang' have higher anthocyanin content, mainly Cy3CafG and Cy3Gal.

[0105] As can be seen from the above embodiments, the present invention detected eight types of anthocyanin glycosides in eight different Rosa species. The detection method of the present invention has a relatively simple pretreatment, fewer purification steps to minimize the loss of anthocyanin glycosides, high extraction rate, and accurate detection results.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting anthocyanins, characterized by, Includes the following steps: (1) After mixing the plant petal powder with the extractant, centrifuge, collect the supernatant, dry it with nitrogen, and redissolve it to obtain the extract; (2) The anthocyanin glycosides in the extract were qualitatively analyzed by liquid chromatography-mass spectrometry. (3) The content of different types of anthocyanins obtained from the extract was quantitatively analyzed by internal standard method; The extractant is a mixture of methanol and formic acid; The volume ratio of methanol to formic acid in the extractant is 7-12:1; The extractant also includes an internal standard, which is paeoniflorin 3-glucoside, and the concentration of the internal standard in the extractant is 2-5 μg / mL; The mass-to-volume ratio of the plant petal powder to the extract is 0.2-0.8g: 2-6mL, and the mass-to-volume ratio of the plant petal powder to the extract during redissolution is 0.2-0.8g: 0.8-1.5mL. The temperature for the mixed extraction is 2-6℃, and the extraction time is 8-15 hours. In step (2), the conditions for liquid chromatography detection are as follows: Column type: ACQUITYUPLCCSH C18; Column parameters: 2.1 mm × 100 mm, 1.7 μm; Mobile phase A: a mixture of acetonitrile and formic acid in a volume ratio of 95:5; Mobile phase B: a mixture of water and formic acid in a volume ratio of 95:5; The gradient elution program is as follows: the initial volume ratio of mobile phase A is 2.5%, mobile phase A increases from 2.5% to 10% within 5 min, then increases from 10% to 25% within 20 min, holds for 5 min, and then decreases from 25% to 2.5% within 30 min; Flow rate: 0.05-0.2 mL / min; Column temperature: 20-30℃; Autosampler temperature: 2-6℃; Injection volume: 0.5-1.5 μL; In step (2), the conditions for mass spectrometry detection are as follows: The mass spectrometer was set to positive ion mode. Ion source: ESI source; Capillary voltage: 1.5-2.5kV; Ion source temperature: 80-120℃; Desolvation gas temperature: 250-350℃; Desolventizing gas flow rate: 700-900 L / h; Conical orifice airflow: 40-60 L / h; Mass spectrometry acquisition mode: Fast DDA mode; Analysis mode: Resolution mode; Precursor ion collection range: 200 m / z - 850 m / z; Femtoion collection range: 50 m / z - 850 m / z; A maximum of five daughter ions can be selected for qualitative analysis. The collision energy range is 10V-80V; Spectral conditions: The wavelength range of the diode array detector is 190nm-800nm.

2. The method for detecting anthocyanins according to claim 1, characterized in that, In the quantitative analysis of step (3), the quantitative method is to calculate the content using a standard curve regression equation formula with the peak area of ​​the anthocyanin glycoside in the ultraviolet absorption chromatogram at 535 nm as the ordinate and the concentration of the internal standard as the abscissa. The correlation of the standard curve is 1>R2>0.

999. The linear range is 1-1000 μg / mL; Quantitative range: 0-1 μg / mL.

3. The method for detecting anthocyanins according to claim 1, characterized by, The centrifugation is performed at a speed of 3500-4500 rpm for 15-30 min. The centrifugation is performed at a speed of 3500-4500 rpm for 15-30 min.

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