Method for efficiently extracting and detecting tocopherol in hedyotis diffusa

Abstract

The invention discloses a method for determining tocopherol in hedyotis diffusa. According to the method, the content of the tocopherol in the hedyotis diffusa is measured by using liquid chromatography. According to the method, ethanol is used as an extracting solution, an ultrasonic method is adopted for extraction, and liquid chromatography is adopted for detection. Chromatographic conditions are as follows: a stationary phase is an Agilent C18 column, and a mobile phase is chromatographic grade methanol; the column temperature is 35 DEG C, and the flow velocity is 1ml / min; the detection wavelength is 295 nm; the sample size is 20 microliters. The method has the advantages of simple pretreatment, high efficiency, high analysis speed and high detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and particularly relates to a method for efficiently extracting and detecting three forms of tocopherol (α-tocopherol, δ-tocopherol, Υ-tocopherol) in Karelinia caspica. Background Art

[0002] Tocopherol, belonging to fat-soluble antioxidants, is an important component of vitamin E. As an effective non-enzymatic antioxidant, tocopherol can protect lipids from oxidative damage by scavenging lipid peroxyl radicals and singlet molecular oxygen. Tocopherol is an amphiphilic molecule, and its basic structure is a 6-hydroxy aromatic heterocycle and a saturated isoprenoid side chain with 16 carbon atoms. The number and position of methyl substituents on the aromatic ring determine the structures of four different isomers of tocopherol (α-tocopherol, β-tocopherol, δ-tocopherol, Υ-tocopherol). The total amount and composition of tocopherol in different plant tissues vary greatly. Among them, α-tocopherol is abundant in leaves, and Υ-tocopherol has a high content in seeds.

[0003] Karelinia caspica is a perennial herb of the genus Karelinia in the family Asteraceae. It is a halophyte that can grow normally in soil with a salt content of 2%. A large amount of antioxidant substances are contained in Karelinia caspica to resist lipid peroxidation damage caused by salt stress. Among them, tocopherol is an important antioxidant substance in Karelinia caspica. The content of tocopherol increases significantly under salt stress and plays an important role in maintaining the stability of reactive oxygen species in Karelinia caspica. Moreover, the high content of tocopherol in Karelinia caspica provides broad application prospects for extracting tocopherol from plants. The determination of the tocopherol content in Karelinia caspica plays an important role in the development and utilization of Karelinia caspica.

[0004] At present, the methods for determining the tocopherol content are mainly liquid chromatography and gas chromatography, and the most commonly used is liquid chromatography. The current national standards in China all adopt this method, but these standards all use the saponification method for extraction, which is time-consuming and has complex steps. And there is currently no efficient extraction and liquid-phase detection method for tocopherol in Karelinia caspica. The present invention provides a method for efficiently extracting and detecting tocopherol in Karelinia caspica. The sample to be tested only needs to be dissolved in ethanol, ultrasonically treated, centrifuged, and filtered. Compared with the saponification method, fewer reagents and instruments are used, reducing the consumable cost, and the operation process is simple and easy to master. Summary of the Invention

[0005] The present invention provides a method for detecting tocopherol in Karelinia caspica, comprising the following steps:

[0006] 1) Tocopherol extraction: collect leaves of Psoralea corylifolia, put them into paper bags, place them in an oven to dry at 60°C, grind the dried leaves in a plant grinder, weigh the ground sample, add 80% ethanol solution by volume, vortex mix, ultrasonicate for 30 minutes, centrifuge at 5000 rpm for 10 minutes, and filter the supernatant through a 0.45 μm filter membrane to obtain an extract, wherein the ratio of Psoralea corylifolia leaves to ethanol solution is 0.1 g: (5-10) ml;

[0007] 2) Preparation of standard substances: Weigh 0.1 g of tocopherol standard substances α-tocopherol, γ-tocopherol and δ-tocopherol respectively, add 1 ml of chromatographically pure anhydrous methanol, and dissolve to obtain a 100 mg / ml standard mixed stock solution;

[0008] 3) Preparation of standard curve solution: Pipette 25 μL of the tocopherol stock solution prepared in step 1) into a 25 mL volumetric flask, dilute to the mark with anhydrous methanol, and shake well to obtain a mixed standard stock solution of α-tocopherol, γ-tocopherol, and δ-tocopherol standards; Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of the tocopherol stock solution, respectively, and place them into 10 mL volumetric flasks, dilute to the mark with anhydrous methanol, and the concentrations are 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively, and store at 4°C in the dark for later use;

[0009] 4) Quantitative determination by external standard method: tocopherol mixed standard working solution is detected by liquid chromatograph; Chromatographic conditions are: C18 chromatographic column (4.6mm*250mm; particle size 5μm), diode array detector, detection wavelength 295nm, mobile phase: 100% methanol, flow rate 1ml / min, column temperature 35°C, injection volume 20μl. With peak area as ordinate, standard sample α-tocopherol, γ-tocopherol, δ-tocopherol concentration as abscissa, a standard curve of tocopherol is produced, and regression equation is calculated: α-tocopherol: f(x)=234384x+1356.81, δ-tocopherol: f(x)=557049x-4873.23, γ-tocopherol: f(x)=682361x-333.988; Calculate the content of different forms of tocopherol in the sample of Huahua Chai according to the peak area of the sample to be tested.

[0010] The method also includes subjecting the schizonepeta to salt stress treatment with 100 mM NaCl for 14 days before collecting the leaves of the schizonepeta.

[0011] Specifically, the cultivation method of the above-mentioned Karelinia caspica can be as follows: Select plump and consistent Karelinia caspica seeds, disinfect them with 75% ethanol for 30 s and 5% sodium hypochlorite for 5 min, and then sow them on 1 / 2 MS medium. After the seedlings germinate and grow for 6 weeks, transfer the seedlings into a hydroponic box containing Hoagland nutrient solution for acclimatization for 1 week, and then treat them with 100 mM NaCl for salt stress for 14 days.

[0012] During the acclimatization and treatment of the seedlings, the conditions in the artificial climate chamber are as follows: during the day, the light intensity is 450 μmol·m -2 ·s -1 , the temperature is 25 °C, and the duration is 16 h; at night, it is dark, the temperature is 20 °C, the duration is 8 h, and the relative humidity is 50%.

[0013] Advantages of the present invention:

[0014] The present invention has established a rapid, effective, simple and reliable detection and analysis method for three forms (α-tocopherol, γ-tocopherol and δ-tocopherol) of tocopherol in the halophyte Karelinia caspica. Compared with the traditional method, it is simple and efficient and can be used for batch extraction. And it is further confirmed that under 100 mM salt treatment, the three forms of tocopherol can be increased by 59.7%, 37.6% and 17.3% compared with the control, which is of great significance for improving the yield of tocopherol using plant reactors and enhancing the application prospect of Karelinia caspica in the development of tocopherol products. Description of the drawings

[0015] Figure 1 It is the liquid chromatogram of the tocopherol standard solution; Note: In the figure, tocopherol 1 is a-tocopherol, tocopherol 2 is δ-tocopherol, and tocopherol 3 is γ-tocopherol.

[0016] Figure 2 It is the standard curve of tocopherol;

[0017] Figure 3 It is the tocopherol content under different concentrations of ethanol extracts;

[0018] Figure 4 It is the tocopherol content under different material-liquid ratios. Specific embodiments

[0019] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0020] The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0021] Example 1. A method for efficient extraction and detection of tocopherols in Karelinia caspica

[0022] 1) Preparation of standard products: Weigh 0.1 g of tocopherol standard products (α-tocopherol, Υ-tocopherol, δ-tocopherol) respectively, add 1 ml of anhydrous methanol of chromatographic purity, and dissolve to obtain a standard mixed stock solution of 100 mg / ml.

[0023] 2) Preparation of solutions for standard curve: Pipette 25 μl of tocopherol stock solution (α-tocopherol, γ-tocopherol, δ-tocopherol) into a 25-ml volumetric flask, make up the volume to the mark with anhydrous methanol (chromatographic purity), shake well, and obtain a mixed standard stock solution of tocopherol (α-tocopherol, γ-tocopherol, δ-tocopherol). Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1 mL of tocopherol stock solution into 10-ml volumetric flasks respectively, make up the volume with anhydrous methanol (chromatographic purity), and obtain solutions with concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL respectively. Store in the dark at 4°C for later use.

[0024] 3) External standard quantification: Detect the mixed standard working solution of tocopherols by a liquid chromatograph, and the obtained peak diagram is shown in Figure 1 . Take the peak area as the ordinate and the concentrations of standard samples α-tocopherol, γ-tocopherol, δ-tocopherol as the abscissa to make a standard curve of tocopherols, and calculate the regression equations: α-tocopherol: f(x)=234384x + 1356.81, δ-tocopherol: f(x)=557049x - 4873.23, γ-tocopherol: f(x)=682361x - 333.988. Calculate the contents of different forms of tocopherols in Karelinia caspica samples according to the peak areas of the samples to be detected. Figure 2 It is the standard curve diagram of tocopherols.

[0025] 4) Preparation of samples to be detected:

[0026] (1) Influence of ethanol extraction solutions with different concentrations on the extraction of tocopherols: Weigh 0.1 g of Karelinia caspica samples, add 5 ml of 80%, 90% and 100% ethanol respectively, vortex and mix well, then ultrasonicate for 30 min, centrifuge at 5000 rpm for 10 min, pipette the supernatant and filter through a 0.45-μm filter membrane for injection. The results are shown in Figure 3 . The results show that the concentration of 80% ethanol extraction solution has the highest content of tocopherols in Karelinia caspica (α-tocopherol is 23.80 ± 0.58, γ-tocopherol is 0.14 ± 0.01, δ-tocopherol is 1.52 ± 0.07), which is significantly higher than the extraction amounts of 90% and 100% ethanol.

[0027] (2) Effect of different solid-liquid ratios on the extraction of tocopherols: Weigh 0.1 g of Karelinia caspica powder, add 3 ml, 5 ml, and 10 ml of 80% ethanol for dissolution respectively. After vortex mixing, ultrasonic for 30 min, centrifuge at 5000 rpm for 10 min, absorb the supernatant and filter through a 0.45 μm filter membrane, and then perform machine analysis. The results are as Figure 4 shown. The results indicate that the solid-liquid ratios of 0.1 g / 10 ml and 0.1 g / 5 ml are both significantly higher than that of 0.1 g / 3 ml in terms of the extraction amount of tocopherols from Karelinia caspica. However, due to the high dilution factor, γ-tocopherol with a very low content cannot be detected at the solid-liquid ratio of 0.1 g / 10 ml. Therefore, the solid-liquid ratio of 0.1 g / 5 ml is the optimal solid-liquid ratio.

[0028] Select plump and consistent Karelinia caspica seeds, disinfect them with 75% ethanol for 30 s and 5% sodium hypochlorite for 5 min, and then sow them on 1 / 2 MS medium. After the seedlings germinate and grow for 6 weeks, transfer the seedlings into a hydroponic box containing Hoagland nutrient solution for acclimation for 1 week, and then select seedlings with consistent growth for salt stress experiments. This experiment sets one concentration gradient of 100 mM NaCl, and uses 0 mM NaCl treatment as the control (CK). Each treatment has 3 replicates, and each replicate has 2 seedlings. The conditions in the artificial climate chamber during the acclimation and treatment of the seedlings are as follows: daytime light intensity is 450 μmol·m -2 ·s -1 , temperature is 25 °C, and the duration is 16 h; at night, it is dark, the temperature is 20 °C, the duration is 8 h, and the relative humidity is 50%. After 14 days of stress treatment, the leaves of Karelinia caspica under normal growth and 100 mM NaCl treatment were collected for the determination of tocopherol content. First, dry the leaves and grind them into powder. Weigh 0.1 g of the sample respectively, add 5 ml of 80% ethanol, vortex mix and then ultrasonic for 30 min, centrifuge at 5000 rpm for 10 min, absorb the supernatant, filter through a 0.45 μm filter membrane, and then perform machine analysis. The measurement results show that the tocopherol content under the salt treatment concentration has a significant increase. The results are shown in Table 1. The results indicate that the content of α-tocopherol is the highest at 7.62 ± 0.20 a under 100 mM NaCl treatment. The content of δ-tocopherol is 4.61 ± 0.07 a, and the content of γ-tocopherol is 7.13 ± 0.18 a, which are increased by 59.7%, 37.6% and 17.3% respectively compared with the control.

[0029] Table 1. Tocopherol content of Karelinia caspica under different salt treatment concentrations

[0030]

[0031]

[0032] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made thereto based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A method for detecting tocopherols in Karelinia caspica, comprising the following steps: 1) Tocopherol extraction: Collect the leaves of Psoralea corylifolia, put them into a paper bag, dry them in an oven at 60°C, grind the dried leaves in a plant grinder, weigh the ground sample, add 80% ethanol solution by volume, vortex mix, ultrasonicate for 30 minutes, centrifuge at 5000 rpm for 10 minutes, and filter the supernatant through a 0.45 μm filter membrane to obtain an extract, wherein: The ratio of Karelinia caspica leaves to ethanol solution is 0.1 g : (5 - 10) ml; 2) Preparation of standard products: Weigh 0.1 g of tocopherol standard products α-tocopherol, Υ-tocopherol, and δ-tocopherol respectively, add 1 ml of chromatographically pure anhydrous methanol, and dissolve to obtain a standard mixed stock solution of 100 mg / ml; 3) Preparation of solutions for standard curves: Pipette 25 μl of the tocopherol stock solution prepared in step 1) into a 25 ml volumetric flask, make up to the mark with anhydrous methanol, and shake well to obtain a mixed standard stock solution of α-tocopherol, Υ-tocopherol, and δ-tocopherol standard products; Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of the tocopherol stock solution respectively into 10 mL volumetric flasks, make up with anhydrous methanol, and store in the dark at 4°C for later use. The concentrations of the solutions are 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL respectively; 4) External standard quantification: Detect the mixed tocopherol standard working solution by a liquid chromatograph; Chromatographic conditions are as follows: the stationary phase is an Agilent C18 column, the mobile phase is chromatographic grade methanol; the column temperature is 35°C, the flow rate is 1 ml / min; the detection wavelength is 295 nm; the injection volume is 20 μl; Use the peak area as the ordinate and the concentrations of the standard products α-tocopherol, Υ-tocopherol, and δ-tocopherol as the abscissa to prepare a standard curve of tocopherols, and calculate the regression equations: α-tocopherol: f(x) = 234384x + 1356.81, δ-tocopherol: f(x) = 557049x - 4873.23, γ-tocopherol: f(x) = 682361x - 333.988; Calculate the contents of different forms of tocopherols in the Karelinia caspica samples according to the peak areas of the samples to be measured.

2. The method according to claim 1, characterized in that, In the above method, before collecting Karelinia caspica leaves, the Karelinia caspica is treated with 100 mM NaCl for salt stress for 14 days.

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