Method for separating, detecting and identifying flavonoid components in cocoa

Abstract

The invention relates to the field of biological detection, in particular to a method for separating, detecting and identifying flavonoid components in cocoa. The invention provides a method for high-throughput identification of flavonoid substances in cocoa. The method comprises the following steps: 1) extracting the flavonoid substances in the cocoa by using an ultrasonic extraction technology; (2) detecting flavonoid components by using an ultra-high performance liquid chromatography-orbitrap mass spectrometry technology; 3) establishing a standard map of cocoa flavonoid components; and 4) carrying out first-step operation and second-step operation on cocoa to be identified, and then comparing the cocoa with the standard map established in the third step so as to finish the separation and identification of the flavonoid substances. According to the method for identifying cocoa flavonoid substances, the flavonoid substances in cocoa can be rapidly and efficiently extracted and detected, and the influence of isomeride ion interference is reduced.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to methods for the separation, detection and identification of flavonoid components in cocoa. Background Technology

[0002] Cocoa (Theobroma cacao L.), an evergreen shrub or small tree belonging to the genus Theobroma in the Malvaceae family (Malvaceae), is native to the Amazon River basin in South America and is a native species of the American rainforest. Since the late 20th century, the Chinese cocoa market has shown rapid growth, with an average annual growth rate consistently above 10%, far exceeding the world average growth rate of 3%, making it an important consumer market for cocoa and related products. Cocoa is rich in cocoa butter, polyphenolic compounds, proteins, and dietary fiber, making it an important raw material for chocolate, pastries, candies, and other foods. Due to its high polyphenol content, cocoa has attracted increasing attention in nutrition and pharmacology. It shows promising potential as an antioxidant, providing cardioprotection, neuroprotection, and chemoprevention.

[0003] Flavonoids have a wide range of health-promoting effects and are indispensable components in various nutritional foods, pharmaceuticals, medicines, and cosmetics. This is because they possess powerful antioxidant, anti-inflammatory, antimutagenic, antibacterial, anticancer, vasoactive, free radical scavenging, and other medicinal properties, as well as the ability to regulate the function of essential cellular enzymes. Previous studies have shown that cocoa is rich in phenolic substances, and its antioxidant capacity is stronger than that of similar species. However, current identification of cocoa flavonoid components has a limited coverage. Existing analytical methods can accurately identify and quantify only a limited number of flavonoid species in a single test, and many trace or structurally similar components are difficult to capture comprehensively, resulting in an insufficient overall understanding of the complex cocoa flavonoid system. Secondly, isomers are difficult to distinguish effectively. Flavonoids contain a wide variety of structural or stereoisomers, such as aglycones and glycosides. Their retention times and mass spectrometric characteristics are very similar in conventional chromatography-mass spectrometry analysis, and there is a lack of efficient separation and identification methods. In addition, the detection process is time-consuming. From sample pretreatment to chromatographic separation and even data analysis, existing methods often require a lot of time, which is difficult to meet the needs of high-throughput screening and limits research efficiency and application promotion.

[0004] Traditional methods for determining cocoa flavonoids typically rely on chromatographic techniques, which are often cumbersome, time-consuming, and unsuitable for rapid screening of large-scale samples. The low throughput of traditional methods limits their application in quality control, breeding screening, and large-scale sample analysis, becoming a bottleneck restricting in-depth research and industrial standardization of cocoa flavonoids. This high-throughput determination technology, combining efficient sample pretreatment, parallel detection workflows, and a rapid data analysis system, can simultaneously perform qualitative and quantitative analysis of multiple flavonoid components in hundreds of samples within a given time. This method not only significantly improves detection efficiency but also effectively reduces the cost per sample while maintaining high sensitivity and accuracy, meeting the dual requirements of large-scale screening and precise analysis. Currently, there is a lack of systematic and large-scale research on high-throughput determination technology for cocoa flavonoids. Therefore, this technology will fill this technological gap, aiming to establish a method for rapid identification of cocoa flavonoid components based on high-resolution mass spectrometry data. It also establishes standard spectra, providing strong data support for the utilization of cocoa germplasm resources and offering theoretical basis and practical value for research on cocoa in food, pharmaceuticals, and health products. Summary of the Invention

[0005] In view of this, the present invention provides a method for the separation, detection, and identification of flavonoid components in cocoa. The present invention provides a high-throughput method for identifying flavonoids in cocoa, comprising the following steps: 1) extracting flavonoids from cocoa using ultrasonic extraction technology; 2) detecting flavonoid components using ultra-high performance liquid chromatography-orbit trap mass spectrometry; 3) establishing a standard spectrum of cocoa flavonoid components; 4) comparing the cocoa sample to be identified after the first and second steps with the standard spectrum established in the third step, thereby completing the separation and identification of flavonoids. The method for identifying cocoa flavonoids provided by the present invention can rapidly and efficiently extract and detect flavonoids in cocoa, reducing the influence of isomer ion interference. The extraction and detection method of this application is reliable, rapid, efficient, produces clear spectra, has good repeatability, and yields reliable results. It can detect low-concentration samples and perform high-throughput analysis of complex samples, providing a technical scope for cocoa metabolomics research and representing a set of detection techniques suitable for the analysis of cocoa flavonoids.

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

[0007] This invention provides a method for separating and detecting flavonoid components in cocoa, comprising the following steps:

[0008] S1: After pretreatment of cocoa raw materials, the sample to be tested is obtained, and the separated components are obtained by ultra-high performance liquid chromatography.

[0009] S2: High-resolution mass spectrometry is used to detect the separated components and obtain the detection results;

[0010] Chromatographic conditions include:

[0011] Mobile phase A and mobile phase B: Mobile phase A comprises 0.03~0.10% V / V aqueous acetic acid solution; mobile phase B comprises 0.02~0.08% V / V acetic acid acetonitrile solution;

[0012] The elution gradients included: 0→5 min, 2% B; 5→15 min, 2%→98% B; 15→17.1 min, 98%→2% B; 17.1→20 min, 2% B.

[0013] In some embodiments of the present invention, the pretreatment in the above separation and detection method includes the following steps: after alcohol extraction of cocoa raw material, sonication, purification, centrifugation, collection of supernatant, and obtaining pretreated cocoa raw material; the alcohol extraction uses methanol and / or ethanol.

[0014] In some embodiments of the present invention, the alcohol extraction in the above separation and detection method uses methanol.

[0015] In some embodiments of the present invention, the volume ratio of methanol to water in the above separation and detection method is 7:3.

[0016] In some embodiments of the present invention, the chromatographic conditions described in the separation and detection methods above include: a mobile phase flow rate of 0.2~0.4 mL / min and an injection volume of 2 μL; a column temperature of 38~42℃; and a 100 mm × 2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column.

[0017] In some embodiments of the present invention, the cocoa raw materials in the above separation and detection methods include: cocoa beans and / or cocoa powder.

[0018] In some embodiments of the present invention, the mass spectrometry conditions for the high-resolution mass spectrometry in the above separation and detection methods include: ionization method is electrospray ionization, and positive and negative ion full scan modes are adopted; the ion spray voltage for positive ion mode is 4000 V, the voltage for negative ion mode is -3500 V, and the mass spectrometry scan range is 50~1500 Da m / z.

[0019] In some embodiments of the present invention, the high-resolution mass spectrometry conditions described in the above separation and detection methods further include: a scan time of 0.2 s and a detection time of 20 min; an nebulizer temperature of 350 °C, an ion transmission tube temperature of 325 °C, and a collision energy of 20~60 V.

[0020] In some embodiments of the present invention, the chromatographic conditions in the above separation method include:

[0021] The mobile phase A and the mobile phase B: Mobile phase A comprises: 0.04% V / V aqueous acetic acid solution; Mobile phase B comprises: 0.04% V / V acetic acid acetonitrile solution;

[0022] The elution gradients included: 0→5 min, 2% B; 5→15 min, 2%→98% B; 15→17.1 min, 98%→2% B; 17.1→20 min, 2% B.

[0023] The column temperature is 40℃, and the column is a 100mm×2.1mm, 1.8μm Waters ACQUITYUPLC HSS T3 column.

[0024] The mobile phase flow rate was 0.35 mL / min, and the injection volume was 2 μL.

[0025] This invention also provides a method for constructing a standard spectrum of flavonoid components in cocoa, which constructs a standard spectrum of cocoa flavonoid components based on the mass spectrometry data obtained by the above detection method.

[0026] This invention also provides a high-throughput identification method for flavonoid components in cocoa, comprising the following steps:

[0027] S1: Obtain the results of the above separation and detection methods;

[0028] S2: Compare the detection results with the standard spectrum obtained by the above construction method to obtain the identification results.

[0029] The present invention also provides a method for preparing flavonoid monomers, wherein the above separation and detection methods are used to separate cocoa raw materials to obtain the flavonoid monomers.

[0030] In some embodiments of the present invention, in the above preparation method, the flavonoid monomers include: kaempferol 3-neohesperidin, 6-methoxykaempferol 3-O-galactoside, kaempferol 3-O-arabinoside, kaempferol 3,7-rhamnoside, kaempferol 3-arabinofuranoside, quercetin 7-O-glucoside, quercetin 2''-O-arabinoside, quercetin 4'-O-galactoside, Quercetin-3-D-xyloside, Tamarind, Isorhamnetin-3-O-glucoside, Isorhamnetin, 6-hydroxykaempferol, Kaempferol-3-O-rutin glycoside, Paclitaxel 3-rhamnoside, (2S,3S)-(-)-glucosidoside, (2S,3S)-dihydroquercetin 3-O-α-L-rhamnoside, Apigenin 7-glucoside, Apigenin 5-O-neohesperidin, Hesperidin 7-Rhamnose, Biflavonoids, Luteolin 7-Glucuronide, Luteolin-5-O-Glucose, Vitexin 2''-O-Rhamnose, Apigenin 7-O-Neohesperidin, Apigenin 7-O-Rutinoside, (+)-Catechin, (+)-Gallicatechin, (-)-Epigallicatechin, (-)-Epigallicatechin, Catechin 3-Rhamnose, Catechin 7-Xyloside, (- (-)-Catechin gallate, (-)-Epicatechin gallate, Proanthocyanidin B1, senna-7-O-glucoside, high senna-7-O-glucoside, kaempferol-3-O-robinoside, kaempferol-3-O-galactoside, apigenin 5-O-glucoside, pinocembrin chalcone, naringin dihydrochalcone, amygdalin B, mesoprolin, and acebotin.

[0031] The beneficial effects of this invention include:

[0032] (1) The method of the present invention can shorten the extraction time. In previous studies, the extraction solvent for catechins and epicatechin was methanol, and the solvent for proanthocyanidins was hexane and acetone, which required two extractions and a total extraction time of 2 hours. However, this method can extract and detect catechins, epicatechins and proanthocyanidins at the same time, and the time is only 40 minutes.

[0033] (2) In previous studies, it was necessary to prepare one extract of cocoa sample, while this method only requires 0.1 g, saving manpower and resources;

[0034] (3) Simplify the detection method. In previous studies, catechins and epicatechins were detected using reversed-phase HPLC technology, while proanthocyanidins were detected using normal-phase HPLC-ESI / MS technology. However, this method uses UHPLC-Orbitrap-MS to detect them simultaneously, and the results are reliable.

[0035] (4) The method of the present invention provides for the first time eight flavonoid compounds applicable to the identification method in cocoa, enriching the sources of compounds. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 This shows the positive ion pattern spectrum of the cocoa flavonoid components in Example 1;

[0038] Figure 2 This shows the negative ion pattern spectrum of the cocoa flavonoid components in Example 1;

[0039] Figure 3 Structural diagrams of compounds identified from cocoa (Formulas 1 to 8);

[0040] Figure 4 Structural diagrams of compounds identified from cocoa (Formulas 9 to 16);

[0041] Figure 5 Structural diagrams of compounds identified from cocoa (Formulas 17 to 25);

[0042] Figure 6 Structural diagrams of compounds identified from cocoa (Formulas 26 to 34);

[0043] Figure 7 Structural diagrams of compounds identified from cocoa (Formulas 35 to 43);

[0044] Figure 8 The structural diagrams of compounds identified from cocoa are shown (Formulas 44 to 45). Detailed Implementation

[0045] This invention discloses a method for the separation, detection and identification of flavonoid components in cocoa.

[0046] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0047] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0048] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0049] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0050] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0051] This invention provides a method for detecting flavonoid components in cocoa, which can effectively separate and identify flavonoid components in cocoa seeds.

[0052] The first aspect of the present invention is to provide a method for detecting cocoa flavonoids, using cocoa seeds as the detection target and employing high performance liquid chromatography-orbit trap mass spectrometry for determination.

[0053] The chromatographic column was a 100mm × 2.1mm, 1.8μm Waters ACQUITY UPLC HSS T3 column;

[0054] Column temperature: 38~42℃;

[0055] Volumetric flow rate: 0.2~0.4 mL / min;

[0056] Mobile phase: Mobile phase A is an aqueous solution of acetic acid with a volume concentration of 0.03~0.10%; Mobile phase B is an acetic acid-acetonitrile solution with a volume concentration of 0.02~0.08%.

[0057] The elution gradients were as follows: 0 min, 2% B; 5 min, 2% B; 15 min, 98% B; 17.1–20 min, 2% B.

[0058] Preferably, before performing the determination using ultra-high performance liquid chromatography-orbit trap mass spectrometry, the target substance is extracted with alcohol, preferably methanol and / or ethanol.

[0059] Preferably, the column temperature is 40°C.

[0060] Preferably, the volumetric flow rate is 0.25~0.35 mL / min, more preferably 0.35 mL / min.

[0061] Preferably, the mobile phase A is an aqueous solution of acetic acid with a volume concentration of 0.03~0.10%, more preferably an aqueous solution of acetic acid with a volume concentration of 0.04%.

[0062] Preferably, the mobile phase B is an acetonitrile acetic acid solution with a volume concentration of 0.02~0.08%, more preferably an acetonitrile acetic acid solution with a volume concentration of 0.04%.

[0063] Preferably, the mass spectrometry conditions of the mass spectrometer include: full-scan electrospray ionization, positive / negative ion mode, ion spray voltage (IS) of 4000 V for positive ion mode and -3500 V for negative ion mode, and a scan range of 50~1500 Da.

[0064] Preferably, the mass spectrometry conditions of the mass spectrometer also include: a scan time of 0.2 s, a detection time of 20 min; an nebulizer temperature of 350 ℃, an ion transmission tube temperature of 325 ℃, and a collision voltage of 20~60 V.

[0065] The detection method of the present invention can identify 45 compounds from cocoa (see Table 1).

[0066] The second aspect of this invention is to provide a method for separating cocoa flavonoids, using cocoa seeds as the separation target, and employing ultra-high performance liquid chromatography (UHPLC) for separation. The UHPLC conditions are as follows:

[0067] The chromatographic column was a 100mm × 2.1mm, 1.8μm Waters ACQUITY UPLC HSS T3 column;

[0068] Column temperature: 38 to 42℃;

[0069] Volumetric flow rate: 0.2 to 0.4 mL / min;

[0070] Mobile phase: Mobile phase A is an aqueous solution of acetic acid with a volume concentration of 0.03~0.10%; Mobile phase B is an acetic acid-acetonitrile solution with a volume concentration of 0.02~0.08%.

[0071] The elution gradients were as follows: 0 min, 2% B; 5 min, 2% B; 15 min, 98% B; 17.1–20 min, 2% B.

[0072] Preferably, before performing the determination using ultra-high performance liquid chromatography-orbit trap mass spectrometry, the target substance is extracted with alcohol, preferably methanol and / or ethanol.

[0073] Preferably, the column temperature is 40°C.

[0074] Preferably, the volumetric flow rate is 0.25~0.35 mL / min, more preferably 0.35 mL / min.

[0075] Preferably, the mobile phase A is an aqueous solution of acetic acid with a volume concentration of 0.03~0.10%, more preferably an aqueous solution of acetic acid with a volume concentration of 0.04%.

[0076] Preferably, the mobile phase B is an acetonitrile acetic acid solution with a volume concentration of 0.02 to 0.08%, more preferably an acetonitrile acetic acid solution with a volume concentration of 0.04%.

[0077] Using the separation method of the present invention, 45 compounds can be separated from cocoa.

[0078] A third aspect of the present invention is to provide a method for preparing a compound, said compound being kaempferol 3-neohesperidin, and / or 6-methoxykaempferol 3-O-galactoside, and / or kaempferol 3-O-arabinoside, and / or kaempferol 3,7-rhamnoside, and / or kaempferol 3-arabinofuranoside, and / or quercetin 7-O-glucoside, and / or quercetin 2''-O-arabinoside, and / or quercetin 4'-O-galactoside, and / or quercetin-3-D-xyloside. And / or tamarind, and / or isorhamnetin-3-O-glucoside, and / or isorhamnetin, and / or 6-hydroxykaempferol, and / or kaempferol-3-O-rutin glycoside, and / or paclitaxel 3-rhamnoside, and / or (2S,3S)-(-)-glucosidoside, and / or (2S,3S)-dihydroquercetin 3-O-α-L-rhamnoside, and / or apigenin 7-glucoside, and / or apigenin 5-O-neohesperidin, and / or hesperidin 7-rhamnoside, and / or biflavonoids, And / or luteolin 7-glucuronide, and / or luteolin-5-O-glucose, and / or vitexin 2''-O-rhamnoside, and / or apigenin 7-O-neohesperidin, and / or apigenin 7-O-rutin, and / or (+)-catechin, and / or (+)-gallicatechin, and / or (-)-epigallocatechin, and / or (-)-epigallicatechin, and / or (-)-epicatechin, and / or catechin 3-rhamnoside, and / or catechin 7-xyloside, and / or (-)-catechin gallate And / or (-)-epicatechin gallic acid, and / or proanthocyanidin B1, and / or senna-7-O-glucoside, and / or homosenna-7-O-glucoside, and / or kaempferol-3-O-robinoside, and / or kaempferol-3-O-galactoside, and / or apigenin 5-O-glucoside, and / or pinocembrin chalcone, and / or naringin dihydrochalcone and / or amygdalin B and / or mesoproline and / or acebotin, were separated from cocoa by ultra-high performance liquid chromatography.

[0079] The conditions for ultra-high performance liquid chromatography are:

[0080] The chromatographic column was a 100mm × 2.1mm, 1.8μm Waters ACQUITY UPLC HSS T3 column;

[0081] Column temperature: 38~42℃;

[0082] Volumetric flow rate: 0.2~0.4 mL / min;

[0083] Mobile phase: Mobile phase A is an aqueous solution of acetic acid with a volume concentration of 0.03~0.10%; Mobile phase B is an acetic acid-acetonitrile solution with a volume concentration of 0.02~0.08%.

[0084] The elution gradients were as follows: 0 min, 2% B; 5 min, 2% B; 15 min, 98% B; 17.1–20 min, 2% B.

[0085] Preferably, before performing the determination using ultra-high performance liquid chromatography-orbit trap mass spectrometry, the target substance is extracted with alcohol, preferably methanol and / or ethanol.

[0086] Preferably, the column temperature is 40°C.

[0087] Preferably, the volumetric flow rate is 0.25~0.35 mL / min, more preferably 0.35 mL / min.

[0088] Preferably, the mobile phase A is an aqueous solution of acetic acid with a volume concentration of 0.03~0.10%, more preferably an aqueous solution of acetic acid with a volume concentration of 0.04%.

[0089] Preferably, the mobile phase B is an acetonitrile acetic acid solution with a volume concentration of 0.02~0.08%, more preferably an acetonitrile acetic acid solution with a volume concentration of 0.04%.

[0090] The materials for this invention were collected as follows: the test materials were selected from cocoa germplasm resources harvested from the cocoa germplasm resource nursery of the Spice and Beverage Research Institute of the Chinese Academy of Tropical Agricultural Sciences. The cocoa beans were frozen in liquid nitrogen and then stored in a -80°C refrigerator for the determination of flavonoid components.

[0091] The main instruments and equipment used in this invention experiment are as follows:

[0092] The Vanquish Flex ultra-high performance liquid chromatography (UPLC) system was purchased from Thermo Fisher Scientific, USA; the Orbitrap Exploris 480 ultra-high resolution mass spectrometer was purchased from Thermo Fisher Scientific, USA, and the mass spectrometer was equipped with a heated electrospray ionization (HESI) ion source; the Waters ACQUITY UPLC HSS T3 (100mm × 2.1mm, 1.8μm) was purchased from Waters Corporation, USA.

[0093] In both Example 1 and Comparative Example 1 of this invention, the raw materials and reagents used can be purchased from the market.

[0094] The cocoa beans used in this invention are from the cocoa germplasm resource nursery of the Institute of Flavors and Beverages, Chinese Academy of Tropical Sciences.

[0095] The present invention will be further illustrated below with reference to the embodiments:

[0096] Example 1

[0097] The method for preparing the sample of this invention is as follows:

[0098] Cocoa beans, after being flash-frozen in liquid nitrogen, were freeze-dried in a vacuum freeze dryer. The freeze-dried cocoa beans were then ground into powder in a grinder. 0.1 g of the powder was weighed using a 0.1 g electronic balance and transferred to a 2 mL centrifuge tube. 1 mL of flavonoid extract (volume ratio: methanol:water 70:30) was added, and the mixture was then ultrasonically extracted in a 20°C water bath for 30 min. The extracted flavonoid extract was purified by centrifuging at 4°C for 10 min using a centrifuge (12000 rpm). The supernatant was then filtered through a 0.22 μm microporous membrane before analysis.

[0099] The ultra-high performance liquid chromatography (UHPLC) detection conditions are as follows:

[0100] Sample temperature 4℃; column temperature 40℃; flow rate 0.35 mL / min; injection volume 2 μL; mobile phase gradient, where mobile phase A was 0.04% acetic acid aqueous solution; mobile phase B was 0.04% acetic acid and acetonitrile: 0 min, 2% B; 5 min, 2% B; 15 min, 98% B; 17.1~20 min, 2% B. (Acetic acid and acetonitrile were 100% chromatographic grade, and water was 18.2 M deionized water).

[0101] Orbitrap Exploris 480 ultra-high resolution mass spectrometry detection conditions:

[0102] The ion source mode was electrospray ionization; full-scan electrospray ionization, positive / negative ion mode; primary MS full-scan resolution set to 60000 (FWHM at m / z 200), scan range 50~1500 Da; ion spray voltage (IS): positive ion mode 4000 V, negative ion mode -3500 V; nebulizer temperature: 350℃; ion transmission tube temperature: 325℃; secondary dd-MS. 2 The resolution was set to 15000, the collision voltage was 20~60 V, the scanning time was 0.2s, and the detection time was 20 min.

[0103] This invention utilizes ultrasonic extraction technology to extract flavonoids and ultra-high performance liquid chromatography-orbit trap mass spectrometry (UHPLC-MS / MS) to detect flavonoid components, establishing a standard spectrum of cocoa flavonoid components. The spectrum is clear, reproducible, and reliable, providing fundamental theoretical research for the development and utilization of cocoa germplasm resources. The complete spectrum of cocoa flavonoids established in this invention is shown in Table 1: 45 flavonoids were identified from cocoa. Figures 3 to 8 The numbers below the structural formulas of the compounds in the figure correspond to No. in Table 1. They include 19 flavonols, 12 flavones, 9 flavanols, 2 flavanones, and 3 chalcones. Among them, 8 flavonoids (numbers 38-45 in Table 1) are provided for the first time and are applicable to the identification method in cocoa.

[0104] Table 1. Full spectrum of cocoa flavonoids

[0105]

[0106]

[0107]

[0108] Comparative Example 1

[0109] For comparison, 1 g of the same cocoa powder was added to 5 mL of n-hexane, shaken on a shaker at 30 °C for 1 h, centrifuged at 3000 rpm for 15 min, the supernatant was discarded, and then 20 mL of extraction buffer (acetone:water:acetic acid = 70:29.5:0.5) was added. The mixture was sonicated at 50 °C for 30 min, centrifuged at 4000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm microporous membrane before being used for detection by normal phase HPLC-ESI / MS.

[0110] Comparative Example 2

[0111] Take 1 g of the same cocoa powder, add 5 mL of n-hexane, shake on a shaker at 30 °C for 1 h, centrifuge at 3000 rpm for 15 min, discard the supernatant, add 20 mL of extraction solution (methanol:water = 80:2:0), sonicate at 50 °C for 30 min, centrifuge at 4000 rpm for 15 min, filter the supernatant using a 0.22 μm microporous membrane, and then use it for detection by reversed-phase HPLC-ESI / MS.

[0112] Example of effect

[0113] With the same extraction rate, the ultrasonic extraction method in this example reduced the extraction time by more than 74% and the sample volume by 90% compared with Comparative Example 1 and Comparative Example 2.

[0114] Table 2

[0115]

[0116] 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 separating and detecting flavonoid components in cocoa, characterized in that, Includes the following steps: S1: After pretreatment of cocoa raw materials, the sample to be tested is obtained, and the separated components are obtained by ultra-high performance liquid chromatography. S2: High-resolution mass spectrometry is used to detect the separated components and obtain the detection results; Chromatographic conditions include: Mobile phase A and mobile phase B: Mobile phase A comprises 0.03~0.10% V / V aqueous acetic acid solution; mobile phase B comprises 0.02~0.08% V / V acetic acid acetonitrile solution; The elution gradients included: 0→5 min, 2% B; 5→15 min, 2%→98% B; 15→17.1 min, 98%→2% B; 17.1→20 min, 2% B.

2. The separation and detection method as described in claim 1, characterized in that, The pretreatment includes the following steps: after alcohol extraction of cocoa raw material, sonication, purification, centrifugation, and collection of supernatant to obtain pretreated cocoa raw material; the alcohol extraction uses methanol and / or ethanol.

3. The separation and detection method as described in claim 1 or 2, characterized in that, The chromatographic conditions include: a mobile phase flow rate of 0.2~0.4 mL / min and an injection volume of 2 μL; a column temperature of 38~42℃; and a 100 mm × 2.1 mm, 1.8 μm Waters ACQUITY UPLC HSS T3 column.

4. The separation and detection method according to any one of claims 1 to 3, characterized in that, The cocoa raw materials include: cocoa beans and / or cocoa powder.

5. The separation and detection method according to any one of claims 1 to 4, characterized in that, The high-resolution mass spectrometry conditions include: ionization method is electrospray ionization, and positive and negative ion full scan modes are adopted; the ion spray voltage is 4000 V in positive ion mode and -3500 V in negative ion mode, and the mass spectrometry scan range is 50~1500 Da m / z.

6. The separation and detection method according to any one of claims 1 to 5, characterized in that, The high-resolution mass spectrometry conditions also include: a scan time of 0.2 s and a detection time of 20 min; an nebulizer temperature of 350 ℃, an ion transmission tube temperature of 325 ℃, and a collision energy of 20~60 V.

7. A method for constructing a standard spectrum of flavonoid components in cocoa, characterized in that, Based on the mass spectrometry data obtained by any one of the detection methods described in claims 1 to 6, a standard spectrum of cocoa flavonoid components is constructed.

8. A high-throughput method for identifying flavonoid components in cocoa, characterized in that, Includes the following steps: S1: Obtain the result of the separation and detection method as described in any one of claims 1 to 6; S2: Compare the detection results with the standard spectrum obtained by the construction method as described in claim 7 to obtain the identification results.

9. A method for preparing flavonoid monomers, characterized in that, The cocoa raw material is separated using the separation and detection method described in any one of claims 1 to 6 to obtain the flavonoid monomer.

10. The preparation method according to claim 9, characterized in that, The flavonoid monomers include: kaempferol 3-neohesperidin, 6-methoxykaempferol 3-O-galactoside, kaempferol 3-O-arabinoside, kaempferol 3,7-rhamnoside, kaempferol 3-arabinofuranoside, quercetin 7-O-glucoside, quercetin 2''-O-arabinoside, quercetin 4'-O-galactoside, quercetin-3-D-xyloside, tamarind, Isorhamnetin-3-O-glucoside, isorhamnetin, 6-hydroxykaempferol, kaempferol-3-O-rutin glycoside, paclitaxel 3-rhamnoside, (2S,3S)-(-)-glucosidoside, (2S,3S)-dihydroquercetin 3-O-α-L-rhamnoside, apigenin 7-glucoside, apigenin 5-O-neohesperidin, hesperidin 7-rhamnoside, biflavonoids, Luteolin 7-glucuronide, luteolin-5-O-glucose, vitexin 2''-O-rhamnoside, apigenin 7-O-neohesperidin, apigenin 7-O-rutin, (+)-catechin, (+)-gallicatechin, (-)-epigallocatechin, (-)-epicatechin, catechin 3-rhamnoside, catechin 7-xyloside, (-)-catechin gallate, (-)-epicatechin gallic acid, proanthocyanidin B1, senna-7-O-glucosinolate, hypersenna-7-O-glucosinolate, kaempferol-3-O-robinoside, kaempferol-3-O-galactoside, apigenin 5-O-glucosinolate, pinocembrin chalcone, naringin dihydrochalcone, amygdalin B, mesopresin, and acebotin.

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