Method for simultaneously determining contents of three tannin substances in rapeseeds

Abstract

The invention provides a method for simultaneously determining the contents of three tannin substances in rapeseeds, and belongs to the technical field of chemical detection. The method provided by the invention comprises the following steps: soaking rapeseeds in a mixed solvent, then carrying out ultrasonic treatment and centrifugation, and filtering supernate obtained by centrifugation to obtain liquid to be detected; detecting the liquid to be detected by adopting HPLC (High Performance Liquid Chromatography); the mixed solvent is a mixed solution of methanol and a phosphoric acid aqueous solution in a volume ratio of 1: 1; the mass concentration of the phosphoric acid aqueous solution is 1%. Methanol and phosphoric acid are used as extracting solutions, so that the stability of detected components is facilitated. According to the method, the content detection result of the three tannin substances (gallic acid, catechin and tannic acid) in the rapeseeds is good, meanwhile, time is saved, and operation steps are simple and convenient.

Description

Technical Field

[0001] The invention relates to the technical field of chemical detection, and in particular to a method for simultaneously determining the contents of three tannin substances in rapeseed. Background Art

[0002] Tannins are a class of compounds with multiple hydroxyl groups in their molecular structure. The name of tannins comes from the dark precipitate they form with metal ions, which is called "tannin". Tannins are widely found in plants, especially trees and fruits, and they play an important role in the physiological functions and protective mechanisms of plants. They include small molecule phenolic compounds, such as anthocyanins, catechins, quercetin, gallic acid, tannic acid, arbutin and other natural phenols.

[0003] Gallic acid has multiple biological activities such as anti-inflammatory, anti-mutagenic, antioxidant, and anti-free radical. Gallic acid also has anti-tumor effects and can inhibit the metastasis of mast cell tumors, thereby prolonging survival. It is also a relatively suitable candidate drug for killing trypanosomiasis. It has a protective effect on the liver and can resist the physiological and biochemical changes of the liver induced by carbon tetrachloride. It can induce endothelium-dependent contraction and endothelium-dependent relaxation by inhibiting the production of endothelial NO.

[0004] Catechins are natural oil antioxidants that can remove free radicals produced by the human body to protect cell membranes and delay aging. Catechins can inhibit human pathogens (such as botulism) without harming the reproduction of beneficial bacteria (such as lactic acid bacteria); they can inhibit bacteria that cause human skin diseases and have a good effect on the treatment of eczema. Some studies have shown that catechins can also inhibit blood pressure (can reduce diastolic and systolic blood pressure) and blood sugar (inhibit glycolytic enzymes), reduce blood cholesterol and low-density lipoprotein (LDL), and increase the amount of high-density lipoprotein (HDL).

[0005] Tannic acid not only has antibacterial, antiviral, anti-inflammatory, and antioxidant effects, and has a certain inhibitory effect on cancer and tumors, but also has physiological activities such as lowering blood sugar, blood pressure, and lipids.

[0006] In the prior art, the commonly used methods for detecting tannins are GC, Kjeldahl nitrogen determination and Soxhlet extraction. The GC method is usually used to analyze fatty acids and other volatile components in rapeseed. When determining tannins, the tannins need to be converted into more volatile derivatives in combination with a derivatization step to make them suitable for gas chromatography separation. The Kjeldahl nitrogen determination and Soxhlet extraction methods have complex steps and are time-consuming. Therefore, it is necessary to provide a simple method for simultaneously determining the content of three tannins. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a method for simultaneously determining the contents of three tannins in rapeseed. The method provided by the present invention has good detection results for the contents of three tannins (gallic acid, catechins and tannic acid) in rapeseed, while saving time and having simple operation steps.

[0008] In order to achieve the above object, the present invention provides the following technical solution: a method for simultaneously determining the contents of three tannin substances in rapeseed, comprising the following steps:

[0009] The rapeseed is soaked in a mixed solvent, then subjected to ultrasound and centrifugation, and the supernatant obtained by centrifugation is filtered to obtain a test solution;

[0010] Detecting the liquid to be tested by HPLC;

[0011] The mixed solvent is a mixed solution of methanol and phosphoric acid aqueous solution in a volume ratio of 1:1; the mass concentration of the phosphoric acid aqueous solution is 0.1%.

[0012] In the present invention, if the extracting solution (i.e., the mixed solvent) is replaced with other extracting solutions, such as replacing methanol with ethanol and replacing phosphoric acid with acetic acid, then during the extraction process of gallic acid, catechins and tannic acid, these three tannin substances cannot be extracted simultaneously, and the extraction rate will also be reduced.

[0013] In some embodiments of the present invention, the tannin substances are gallic acid, catechin and tannic acid.

[0014] In some embodiments of the present invention, the mass volume ratio of the rapeseed to the mixed solvent is 100 mg:10 mL.

[0015] In the present invention, if the amount of the mixed solvent used is reduced, the extraction rate of the three tannins in rapeseed will be reduced.

[0016] In some embodiments of the present invention, the soaking time is 20-24 hours.

[0017] In some embodiments of the present invention, the frequency of the ultrasound is 40 KHz and the duration is 60 min.

[0018] In the present invention, as the ultrasonic time increases, the extraction rate of the three tannins gradually increases, but when the ultrasonic time is too long (more than 60 minutes), the extraction rate of the tannins will be reduced. The present invention also verifies that replacing the ultrasonic method with an oscillation method, and the results show that the oscillation method greatly reduces the extraction rate of the tannins.

[0019] In some embodiments of the present invention, the centrifugation condition is: centrifugation at 2500 r / min at 4°C for 30 min.

[0020] In some embodiments of the present invention, the filtration is performed using a 0.45 μm filter membrane.

[0021] In some embodiments of the present invention, the HPLC parameters are: mobile phase A is acetonitrile, mobile phase B is 0.1wt% phosphoric acid solution, the flow rate of the mobile phase is 1mL / min, the elution method is gradient elution, the chromatographic column is NovaAtomC18, the column temperature of the chromatographic column is 25°C, and the injection volume is 20μL.

[0022] In some embodiments of the present invention, the gradient elution is: 0-10 min, the volume of mobile phase B is maintained at 87.5%; 10-14 min, the volume of mobile phase B is reduced from 87.5% to 80.0%; 14.0-14.5 min, the volume of mobile phase B is maintained at 80.0%; 14.5-16 min, the volume of mobile phase B is increased from 80.0% to 95%.

[0023] The present invention discloses the following technical effects:

[0024] The present invention uses methanol and phosphoric acid as extracting liquid, which is beneficial to the stability of the components to be tested and can simultaneously extract three tannin substances.

[0025] The method provided by the present invention is simple, time-saving, and also ensures accuracy. Experiments have shown that the present invention has good results for detecting the contents of three tannin substances (gallic acid, catechins, and tannic acid) in rapeseed, while saving time and having simple operation steps. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Room temperature in the present invention means a temperature of 20-30°C.

[0032] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0033] The rapeseed samples used in the examples are two varieties, "Fengyou" and "Sanyue", both of which can be obtained through commercial channels.

[0034] Unless otherwise specified, all reagents used in the examples were of analytical grade, where water was grade III water as specified in GB / T 6682; acetonitrile was of chromatographic grade; and phosphoric acid was of analytical grade.

[0035] The instruments used in the examples are shown in Table 1.

[0036] Table 1 Experimental instruments and equipment

[0037] Instrument Name Instrument Information Instrument brand Ultra-high performance liquid chromatography Vanquish UHPLC ThermoFisher Ultrasonic Cleaner F-100SD FuYangInstrument Desktop high speed centrifuge TGL-20bR AntingScientificInstrument

[0038] Example 1

[0039] 1. Draw a standard curve

[0040] Accurately weigh 20 mg of tannic acid, gallic acid, and catechin standard products, dissolve in 10 mL of methanol solution to prepare a 2000 μg / mL standard stock solution. Then dilute the stock solution with methanol to prepare a series of standard solutions with concentrations of 0.0 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL.

[0041] The standard working solution was injected and analyzed by high performance liquid chromatography according to the chromatographic conditions provided below. The concentration and peak area of ​​the standard solution are shown in Table 2. The concentration of the reference substance was used as the horizontal coordinate and the chromatographic peak area was used as the vertical coordinate. The standard curves of tannic acid, gallic acid and catechin were Y=0.7343X+0.0000 (r=0.9997), Y=0.1626X+0.0000 (r=0.9999), and Y=0.0655X+0.0000 (r=1.0000), respectively, with good linear relationships. The chromatographic conditions were: the wavelength of the liquid chromatography detection was 280nm; the mobile phase A of the liquid chromatography detection was acetonitrile, and the mobile phase B was 0.1wt% phosphoric acid solution; the flow rate of the mobile phase was 1mL / min; the chromatographic column was NovaAtom C18; the column temperature of the chromatographic detection was 30°C; the injection volume was 10μL, and the elution gradient was:

[0042]

[0043]

[0044] Table 2 Peak area of ​​standard products

[0045]

[0046] Linear evaluation: Correlation coefficient R 2 >0.99, in line with the requirements of GB / T27404 "Laboratory Quality Control Standards for Food Physical and Chemical Testing" (GB / T 27404 requires the correlation coefficient R 2 >0.99), so it is feasible to use this method to determine the contents of three tannins.

[0047] 2. Limit of detection and limit of quantification

[0048] The signal-to-noise ratio S / N ≥ 3 was defined as the limit of detection (LOD) (Table 3), and S / N ≥ 10 was defined as the limit of quantification (LOQ) (Table 4).

[0049] Table 3

[0050]

[0051]

[0052] Table 4

[0053]

[0054] 3. Sample determination

[0055] Optimization of sample pretreatment

[0056] Accurately weigh 100 mg of rapeseed sample and place it in a 15 mL centrifuge tube. Add 10 ml of mixed solvent to the centrifuge tube and soak for 24 hours. Then, ultrasonicate at 40 kHz for 60 minutes at room temperature, and then centrifuge at 2500 r / min for 30 minutes at 4°C to collect the supernatant. Filter the obtained supernatant with a 0.45 μm filter membrane, and collect the filtrate as the sample to be tested for HPLC analysis.

[0057] Select two mixed solvent systems V 乙醇 :V (0.1％乙酸水) (3:2) and V 甲醇 :V (0.1％磷酸水) (1:1) Then, tannic acid, gallic acid and catechins in the samples were extracted according to the solid-liquid ratio (1:70, g / mL) and (1:100, g / mL), and the differences in extraction rates were compared. The results are shown in Table 5.

[0058] Table 5

[0059]

[0060] It can be seen from Table 5 that the solvent system methanol:0.1% phosphoric acid aqueous solution (1:1, V / V) has the highest extraction rate according to the solid-liquid ratio (1:100).

[0061] 3.2 Optimization of extraction method

[0062] According to the best extraction solvent system obtained in experiment 3.1, methanol: 0.1% phosphoric acid aqueous solution (1:1, V / V), solid-liquid ratio (1:100), two extraction methods were selected: oscillation extraction (180rmin') for 60min and ultrasonic extraction for 60min to extract tannic acid, gallic acid and catechins in the sample, and the difference in the two extraction rates was compared. The results are shown in Table 6.

[0063] Table 6

[0064]

[0065] It can be seen from Table 6 that the extraction rates of tannic acid vary greatly with different extraction methods. The ultrasonic extraction rate is the highest when using a solvent system of methanol: water: phosphoric acid (50:49:1) with a solid-liquid ratio of (1:100).

[0066] 3.3 Optimization of extraction time

[0067] According to the optimal extraction solvent system and extraction method obtained in 3.1 and 3.2, three extraction times were set up to compare the effects of different extraction times on the recovery rates of tannic acid, gallic acid and catechins in the samples. The results are shown in Table 7.

[0068] Table 7

[0069]

[0070] It can be seen from Table 7 that with the extension of extraction time, the extraction rate of the three tannins gradually increased, but when the ultrasonic time was too long (more than 60 min), the extraction rate of the tannins would be reduced.

[0071] 4. Repeatability, stability and precision examination

[0072] Weigh 0.1008g of sample and prepare the test solution according to the optimized pretreatment method. Inject the sample at 0, 2, 4, 6, 12, and 24 hours respectively, calculate the RSD value of each test result, and examine the stability. The results are shown in Table 7.

[0073] Table 7

[0074]

[0075]

[0076] Weigh 0.0985g of sample and prepare the test solution according to the optimized pretreatment method. Inject the sample 6 times continuously and calculate the RSD value of each test result to examine the precision. The results are shown in Table 8.

[0077] Table 8

[0078]

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously determining the contents of three tannin substances in rapeseed, characterized in that: The following steps are involved: The rapeseed is soaked in a mixed solvent, then subjected to ultrasound and centrifugation, and the supernatant obtained by centrifugation is filtered to obtain a test solution; Detecting the liquid to be tested by HPLC; The mixed solvent is a mixed solution of methanol and phosphoric acid aqueous solution in a volume ratio of 1:1; the mass concentration of the phosphoric acid aqueous solution is 0.1%.

2. The method for simultaneously determining the contents of three tannin substances in rapeseed according to claim 1, characterized in that: The tannin substances are gallic acid, catechin and tannic acid.

3. The method for simultaneously determining the contents of three tannin substances in rapeseed according to claim 1, characterized in that: The mass volume ratio of the rapeseed to the mixed solvent is 100 mg:10 mL.

4. The method for simultaneously determining the contents of three tannin substances in rapeseed according to claim 1, characterized in that: The soaking time is 20-24h.

5. The method for simultaneously determining the contents of three tannins in rapeseed according to claim 1, characterized in that: The frequency of the ultrasound is 40 KHz and the duration is 60 min.

6. The method for simultaneously determining the contents of three tannins in rapeseed according to claim 1, characterized in that: The centrifugal conditions are: centrifugation at 2500 r / min at 4°C for 30 min.

7. The method for simultaneously determining the contents of three tannins in rapeseed according to claim 1, characterized in that: The filtration is carried out using a 0.45 μm filter membrane.

8. The method for simultaneously determining the contents of three tannins in rapeseed according to claim 1, characterized in that: The parameters of the HPLC are as follows: mobile phase A is acetonitrile, mobile phase B is 0.1 wt% phosphoric acid solution, the flow rate of the mobile phase is 1 mL / min, the elution method is gradient elution, the chromatographic column is NovaAtom C18, the column temperature of the chromatographic column is 30° C., and the injection volume is 10 μL.

9. The method for simultaneously determining the contents of three tannins in rapeseed according to claim 8, characterized in that: The gradient elution is as follows: from 0 to 10 minutes, the volume of mobile phase B is maintained at 87.5%; from 10 to 14 minutes, the volume of mobile phase B is reduced from 87.5% to 80.0%; from 14.0 to 14.5 minutes, the volume of mobile phase B is maintained at 80.0%; from 14.5 to 16 minutes, the volume of mobile phase B is increased from 80.0% to 95%.

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