CN111579671A - Method for determining content of three curcumins in turmeric - Google Patents

Method for determining content of three curcumins in turmeric Download PDF

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CN111579671A
CN111579671A CN202010457098.9A CN202010457098A CN111579671A CN 111579671 A CN111579671 A CN 111579671A CN 202010457098 A CN202010457098 A CN 202010457098A CN 111579671 A CN111579671 A CN 111579671A
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turmeric
curcumins
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宋科
薛永
秦秦
孙丽娟
孙雅菲
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Shanghai Academy of Agricultural Sciences
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Abstract

The invention provides a determination method of the content of three curcumins in turmeric, which comprises the steps of adding an extraction solvent into soaked turmeric, carrying out ultrasonic extraction and centrifugation, taking supernatant, filtering to obtain a sample solution, and carrying out qualitative and quantitative analysis on 3 curcumins in the sample solution by adopting a high performance liquid chromatography-electrochemical combined method. The method for determining the content of three curcumins in the turmeric integrates the good separation efficiency of HPLC and the high sensitivity of electrochemical detection, can simultaneously detect the curcumins, the demethoxycurcumin and the bisdemethoxycurcumin, improves the detection efficiency, shortens the detection time, is simple, convenient and quick, has higher sensitivity, shows a wide linear range and has good linearity and precision.

Description

Method for determining content of three curcumins in turmeric
Technical Field
The invention belongs to the technical field of agriculture, relates to a determination method of the content of three curcumins in turmeric, and particularly relates to a determination method of the content of curcumin (C), Demethoxycurcumin (DMC) and Bisdemethoxycurcumin (BDMC) in turmeric.
Background
Turmeric has been used since ancient times as a flavoring, preservative and coloring agent and as a folk medicine for the treatment of various diseases. Turmeric rhizome has a wide range of biological activities, but clinically the most important is its choleretic effect. In addition, they can act as potent antioxidants by inducing endogenous cellular antioxidant defense mechanisms, and are also useful in antibacterial, antiviral, chemopreventive and therapeutic applications due to their biological activities associated with anticancer agents.
Furthermore, turmeric may also exhibit anti-nutritional activity by reducing the bioavailability of iron. Nowadays, the use of turmeric as a dye is greatly increasing due to the trend towards natural compounds to replace synthetic additives. The yellow color of turmeric rhizome is due to the presence of a group of phenolic compounds named curcumin, also called curcuminoids, which mainly consist of curcumin (C), Demethoxycurcumin (DMC) and Bisdemethoxycurcumin (BDMC). Previous studies have reported various methods for quantifying curcuminoids, including ultraviolet spectrophotometry, HPLC, ultra high liquid chromatography (UHPLC), electrochemical detection (ECD), and the like. The ultraviolet spectrophotometry is a classical method, but the operation is complicated, and the sensitivity is not high; HPLC is known for its high efficiency and rapid separation, but since this analysis time frame does not include the time required to wash other compounds present in the column, much more time is actually spent in the sample-sampling interval after washing to return to the initial conditions and re-equilibrate the column/detector before the next run. In contrast, the analysis time can be greatly reduced with UHPLC systems. This method was used to rapidly quantify curcuminoids in turmeric, with a run time of only 2 minutes. The performance of the UHPLC method in terms of resolution and time is largely due to the use of short columns filled with small diameter particles, but these devices are too expensive to purchase and difficult to withstand in practical applications.
Electrochemical detection (ECD) is a promising assay because it is sensitive, economical and amenable to miniaturization. Has been widely applied in the fields of drug analysis and food quality control. However, no method for measuring the 3 curcuminoid component in turmeric by combining ECD and HPLC is available.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a method for determining the content of three curcumins in turmeric, which is used for solving the problem of the lack of a method for determining the content of 3 curcumins in turmeric by combining ECD and HPLC in the prior art.
In order to achieve the above purpose and other related purposes, the invention provides a method for measuring the content of three curcumins in turmeric, which comprises the steps of adding the soaked turmeric into an extraction solvent for ultrasonic extraction and centrifugation, then taking supernatant, filtering to obtain a sample solution, and then carrying out qualitative and quantitative analysis on 3 curcumins in the sample solution by adopting a high performance liquid chromatography-electrochemical combined method (HPLC-ECD).
Preferably, the curcumin component comprises curcumin (C) (CAS No. 458-37-7), Demethoxycurcumin (DMC) (CAS No. 24939-17-1) and Bisdemethoxycurcumin (BDMC) (CAS No. 24939-16-0). The molecular structural formula of the curcumin component is as follows.
Figure BDA0002509625170000021
Preferably, the turmeric is ground into powder, and the particle size of the turmeric is 50-150 mesh, preferably 100 mesh.
Preferably, the ratio of the added mass g of the turmeric to the added volume mL of the soaking solution is 1: 40-60. More preferably, the ratio of the added mass g of the turmeric to the volume mL of the added soaking solution is 1: 50.
More preferably, the soaking solution is an acetic acid aqueous solution with the volume percentage (v/v) of 0.05-0.15%. Further preferably, the soaking solution is 0.1% by volume (v/v) of acetic acid aqueous solution.
Preferably, the soaking time of the turmeric is 20-40min, preferably 30 min.
Preferably, the ratio of the mass g of the turmeric to the volume mL of the extraction solvent is 1: 10-100. More preferably, the ratio of the mass g of turmeric added to the volume mL of extraction solvent added is 1: 50.
Preferably, the extraction solvent is methanol.
Preferably, the time of the ultrasonic extraction is 25-35 min. More preferably, the time of the ultrasonic extraction is 30 min.
Preferably, the frequency of the ultrasonic extraction is 30-50kHz, and the power of the ultrasonic extraction is 120-150W. More preferably, the frequency of the ultrasonic extraction is 40kHz and the power of the ultrasonic extraction is 135W.
Preferably, the rotation speed of the centrifugation is 8000-. More preferably, the rotation speed of the centrifugation is 10000 rpm.
Preferably, the time of centrifugation is 10-20 min. More preferably, the time of centrifugation is 15 min.
Preferably, the filtration is an organic phase membrane filtration. More preferably, the pore size of the organic phase filtration membrane is 0.22 μm.
Preferably, the high performance liquid chromatography-electrochemical combined method (HPLC-ECD) is used for qualitative and quantitative analysis of 3 curcumin components in the sample solution, and comprises the following steps:
1) preparing a standard solution: respectively taking standard samples of 3 curcumin components in the turmeric, adding a solvent for dilution and constant volume to prepare standard solutions;
2) and (3) qualitative and quantitative analysis: respectively measuring the sample solution and the standard solution prepared in the step 1) by a high performance liquid chromatography-electrochemical combined method, comparing retention time by the high performance liquid chromatography, performing qualitative determination according to a common characteristic peak, determining 3 curcumin components in the sample solution, and calculating the content of the 3 curcumin components in the sample solution by an electrochemical method by adopting a standard curve method.
More preferably, in the step 1), the standard solution is diluted by the stock solution to a constant volume.
Further preferably, the concentration of curcumin (C) in the stock solution is 0.24mg/mL, Demethoxycurcumin (DMC) is 0.21mg/mL, and Bisdemethoxycurcumin (BDMC) is 0.20 mg/mL. The stock solution was stored in a refrigerator.
More preferably, in the step 1), the concentration of curcumin (C) in the standard solution is 0.212-42.3 μmol/L, the concentration of Demethoxycurcumin (DMC) is 0.188-38.9 μmol/L, and the concentration of Bisdemethoxycurcumin (BDMC) is 0.232-117 μmol/L.
Further preferably, the concentration of curcumin (C) in the standard solution is 2.08. mu. mol/L, the concentration of Demethoxycurcumin (DMC) is 1.97. mu. mol/L, and the concentration of Bisdemethoxycurcumin (BDMC) is 2.27. mu. mol/L.
More preferably, in step 1), the solvent is acetonitrile.
More preferably, in the step 2), the measurement conditions of the high performance liquid chromatography in the electrochemical combination of high performance liquid chromatography are selected from any one of the following conditions:
a) a chromatographic column: c18A chromatographic column; column temperature: 35-55 ℃; flow rate: 0.5-2.5 mL/min; mobile phase A: acetonitrile; mobile phase B: 5-15mmol/L Na2HPO4-H3PO4An aqueous solution (pH 4.5-5.5); isocratic elution;
b) a chromatographic column: c18A chromatographic column; column temperature: 35-55 ℃; flow rate: 1.0-2.5 mL/min; mobile phase A: 0.05-0.15% (v/v) aqueous acetic acid; mobile phase B: 0.05-0.15% (v/v) acetonitrile or methanol in water; gradient elution.
Further preferably, the measurement conditions a) are: a chromatographic column: acclaimTMAmG C18HPLC column (4.6mm × 150mm, 3 μm), column temperature 40 deg.C, flow rate 1.0mL/min, mobile phase A of acetonitrile, and mobile phase B of 10mmol/L Na2HPO4-H3PO4Aqueous solution (pH 5.0); isocratic elution.
Further preferably, in the measurement condition a), the volume ratio of the mobile phase a to the mobile phase B in isocratic elution is 50: 50.
Further preferably, in the determination condition a), the mobile phase B is filtered through an organic phase filter before use.
Most preferably, the pore size of the organic phase filtration membrane is 0.22 μm.
Further preferably, the measurement conditions b) are: a chromatographic column: acclaimTMAmG C18HPLC column (4.6mm × 150mm, 3 μm), column temperature 55 deg.C, flow rate 2.5mL/min, mobile phase A0.1% (v/v) acetic acid aqueous solution, mobile phase B0.1% (v/v) acetonitrile or methanol aqueous solution, and gradient elution.
Further preferably, in the measurement conditions a) or b), the time required for equilibration before the measurement by high performance liquid chromatography is 1 to 5 minutes.
Further preferably, in the determination condition b), the specific procedure of the gradient elution is:
0-5min, phase A: the volume ratio of the phase B is 1:10-1: 5;
5-10min, phase A: the volume ratio of the phase B is 1:5-1: 100;
10-30min, phase A: the volume ratio of the phase B is 1:100-1: 10.
When the high performance liquid chromatography adopts a UV detector for detection, the detection wavelength is 240-600nm, preferably 425 nm; the sample amount is 1-10 μ L.
More preferably, in the step 2), in the electrochemical combination of high performance liquid chromatography and electrochemistry, the electrochemical measurement conditions are as follows: the measuring device is an electrochemical detection cell and comprises a glassy carbon electrode, an Ag/AgCl reference electrode and a counter electrode; the measuring temperature is 35-55 ℃; the flow rate is 1.0-2.5mL/min-1(ii) a The balance time is 1-5 minutes; the injection volume is 15-25L, preferably 20L; the detection potential is 0.6-1.2V, preferably 0.9V.
The electrochemical detection cell is a conventionally used electrochemical detection cell, and a glassy carbon electrode, an Ag/AgCl reference electrode and a counter electrode in the electrochemical detection cell are conventionally used electrodes.
The detection equipment adopted by the high performance liquid chromatography-electrochemical combined method comprises a high performance liquid chromatograph and an electrochemical detection cell.
More preferably, in step 2), the standard curve method is: a series of standard solutions with different concentrations of 3 curcumin components are respectively subjected to high performance liquid chromatography electrochemical combined method analysis, the 3 curcumin components in the standard solutions are determined through high performance liquid chromatography separation, the linear relation between the content of the 3 curcumin components in the standard solutions and the electrode potential is obtained through electrochemical measurement, corresponding standard working curves are drawn according to the corresponding content of each curcumin component electrode potential, and regression equations of the standard working curves are obtained through calculation. And then, analyzing the sample solution by a high performance liquid chromatography-electrochemical combined method, determining 3 curcumin components in the sample solution by high performance liquid chromatography separation, obtaining the electrode potentials of the 3 curcumin components in the sample solution by electrochemical determination, and respectively substituting the electrode potentials into the regression equation of each standard working curve to obtain the content of the 3 curcumin components in the sample solution.
As described above, the determination method for the content of three curcumins in the turmeric provided by the invention researches technical parameters influencing separation and chemical detection, combines a high performance liquid chromatography method adopting optimized conditions with an electrochemical method, integrates good separation efficiency of HPLC and high sensitivity of electrochemical detection, is successfully applied to analysis of 3 curcumins in the turmeric, can simultaneously detect the curcumins, demethoxycurcumin and bisdemethoxycurcumin, improves the detection efficiency, enables the 3 curcumins to be well separated and detected, and shortens the detection time. The invention overcomes the defects of low content of pharmacological active ingredients in the herbal medicine and complex matrix, improves the defects of low sensitivity and limited selectivity of an ultraviolet spectrophotometry method and a capillary electrophoresis method, avoids the defects of atomization and evaporation steps of an HPLC-MS method, ionization of a sample and expensive instruments, develops a new method for detecting and analyzing the curcuminoid by combining HPLC with electrochemistry, is simple, convenient and quick, has higher sensitivity, shows a wide linear range and good linearity and precision, and can be practically applied to the detection of the curcumin sample. Compared with the common HPLC-UV method, the method has good analytical performance in curcumin determination, and can be used for qualitative analysis of real herbal medicines and medical samples.
Drawings
FIG. 1 shows a liquid chromatogram of three curcumins in turmeric of the present invention, wherein a is bisdemethoxycurcumin; b is demethoxycurcumin; c is curcumin.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and equipment used in the following examples are as follows:
1. reagent
Turmeric (produced in Shanghai Chongming district, Sanxingzhen town); curcumin (C), Demethoxycurcumin (DMC) and Bisdemethoxycurcumin (BDMC) standards (purity > 95%, chengdu traditional Chinese medicine purification limited); acetonitrile, methanol, acetic acid, ethanol (HPLC pure, pharmaceutical chemicals limited); sodium dihydrogen phosphate, disodium hydrogen phosphate, phosphoric acid (analytically pure, national chemical reagents ltd.); ultrapure water (national chemical agents, ltd.).
2. Instrument for measuring the position of a moving object
LC-20AD high performance liquid chromatograph (Shimadzu corporation); CHI842B electrochemical workstation (Beijing Huakepu Tian science and technology, Inc.); model AD3-543 ultrasonograph (Japan meal Mozier Co.).
The specific detection process of the determination method for the content of three curcumins in the turmeric in the invention is as follows.
1. Sample pretreatment
Grinding Curcuma rhizome sample into powder with particle size of 50-150 meshes, weighing Curcuma rhizome sample with a certain weight, and soaking in 0.05-0.15 v/v% acetic acid water solution for 20-40 min. The ratio of the weight g of the turmeric to the volume mL of the soaking solution is 1: 40-60. Then ultrasonic extracting with extraction solvent in ultrasonic device for 25-35 min. The extraction solvent is methanol. The ratio of the mass g of the turmeric to the volume mL of the extraction solvent is 1: 10-100. The frequency of ultrasonic extraction is 30-50kHz, and the power of ultrasonic extraction is 120-150W. Centrifuging at 8000-12000rpm for 10-20min, collecting supernatant, and filtering with 0.22 μm organic phase filter membrane to obtain sample solution.
2. Qualitative and quantitative analysis
Taking standard samples of 3 curcumin components in the turmeric, adding a solvent for dilution and constant volume to prepare a stock solution, wherein the concentration of curcumin in the stock solution is 0.24mg/mL, the concentration of demethoxycurcumin is 0.21mg/mL, and the concentration of bisdemethoxycurcumin is 0.20 mg/mL. The stock solution was stored in a refrigerator until use.
And adding the stock solution into a solvent for dilution and volume fixing to prepare a standard solution with single concentration. The standard solution contains curcumin 2.08 μmol/L, demethoxycurcumin 1.97 μmol/L, and bisdemethoxycurcumin 2.27 μmol/L. It can also be prepared into a series of standard solutions with different concentrations. The standard solution contains curcumin 0.212-42.3 μmol/L, demethoxycurcumin 0.188-38.9 μmol/L, and bisdemethoxycurcumin 0.232-117 μmol/L.
The solvent is acetonitrile.
And then, the sample solution and the standard solution are subjected to high performance liquid chromatography-electrochemical combined method determination, retention time is compared through the high performance liquid chromatography, qualitative determination is carried out according to a common characteristic peak, 3 curcumin components in the sample solution are determined, and the content of the 3 curcumin components in the sample solution is calculated through electrochemistry by adopting a standard curve method.
Wherein the measuring conditions of the high performance liquid chromatography are selected from any one of the following conditions:
a) a chromatographic column: a C18 chromatography column; column temperature: 35-55 ℃; flow rate: 0.5-2.5 mL/min; mobile phase A: acetonitrile; mobile phase B: 5-15mmol/L Na2HPO4-H3PO4An aqueous solution (pH 4.5-5.5); isocratic elution;
b) a chromatographic column: a C18 chromatography column; column temperature: 35-55 ℃; flow rate: 1.0-2.5 mL/min; mobile phase A: 0.05-0.15% (v/v) aqueous acetic acid; mobile phase B: 0.05-0.15% (v/v) acetonitrile or methanol in water; gradient elution.
Under the measurement condition a), the volume ratio of the mobile phase A to the mobile phase B in isocratic elution was 50: 50. Mobile phase B was filtered through a 0.22 μm organic phase filter before use.
In the measurement conditions a) or b), the time required for equilibration before the measurement by high performance liquid chromatography is 1 to 5 minutes.
In the determination condition b), the specific procedure of the gradient elution is as follows:
0-5min, phase A: the volume ratio of the phase B is 1:10-1: 5;
5-10min, phase A: the volume ratio of the phase B is 1:5-1: 100;
10-30min, phase A: the volume ratio of the phase B is 1:100-1: 10.
The electrochemical measurement conditions were: the measuring device is an electrochemical detection cell and comprises a glassy carbon electrode, an Ag/AgCl reference electrode and a counter electrode; the measuring temperature is 35-55 ℃; the flow rate is 1.0-2.5mL/min-1(ii) a The balance time is 1-5 minutes; the injection volume was 20L; the detection potential was 0.9V.
Example 1
1. Sample pretreatment
Grinding the turmeric sample into powder with particle size of 100 mesh, weighing 0.1g of turmeric sample into a 10mL centrifuge tube, and soaking in 5mL of 0.1 v/v% acetic acid aqueous solution for 30 min. Then ultrasonically extracted with 5mL of methanol in an ultrasonic device for 30 min. The frequency of ultrasonic extraction was 40kHz and the power of ultrasonic extraction was 135W. Centrifuging at 10000rpm for 15min, collecting supernatant, and filtering with 0.22 μm organic phase filter membrane to obtain sample solution 1 #.
2. Qualitative and quantitative analysis
Taking standard samples of 3 curcumin components in the turmeric, adding a solvent for dilution and constant volume to prepare a stock solution, wherein the concentration of curcumin in the stock solution is 0.24mg/mL, the concentration of demethoxycurcumin is 0.21mg/mL, and the concentration of bisdemethoxycurcumin is 0.20 mg/mL. The stock solution was stored in a refrigerator until use.
And adding the stock solution into a solvent for dilution and volume fixing to prepare a standard solution with single concentration. The standard solution contains curcumin 2.08 μmol/L, demethoxycurcumin 1.97 μmol/L, and bisdemethoxycurcumin 2.27 μmol/L. It can also be prepared into a series of standard solutions with different concentrations. The standard solution contains curcumin 0.212-42.3 μmol/L, demethoxycurcumin 0.188-38.9 μmol/L, and bisdemethoxycurcumin 0.232-117 μmol/L.
The solvent is acetonitrile.
And then, the sample solution and the standard solution are subjected to high performance liquid chromatography-electrochemical combined method for determination, as shown in figure 1, the retention time is compared through the high performance liquid chromatography, the qualitative determination is carried out according to the shared characteristic peak, 3 curcumin components in the sample solution are determined, and the content of the 3 curcumin components in the sample solution is calculated through electrochemistry by adopting a standard curve method.
Wherein the measuring conditions of the high performance liquid chromatography are selected from any one of the following conditions:
a) a chromatographic column: acclaimTMAmG C18HPLC column (4.6mm × 150mm, 3 μm), column temperature 40 deg.C, flow rate 1.0mL/min, mobile phase A of acetonitrile, and mobile phase B of 10mmol/L Na2HPO4-H3PO4Aqueous solution (pH 5.0); isocratic elution;
b) a chromatographic column: acclaimTMAmG C18HPLC column (4.6mm × 150mm, 3 μm), column temperature 55 deg.C, flow rate 2.5mL/min, mobile phase A0.1% (v/v) acetic acid aqueous solution, mobile phase B0.1% (v/v) acetonitrile or methanol aqueous solution, and gradient elution.
Under the measurement condition a), the volume ratio of the mobile phase A to the mobile phase B in isocratic elution was 50: 50. Mobile phase B was filtered through a 0.22 μm organic phase filter before use.
In the measurement conditions a) or b), the time required for equilibration before measurement by high performance liquid chromatography is 1 to 5 minutes.
In the determination condition b), the specific procedure of the gradient elution is as follows:
0-5min, phase A: the volume ratio of the phase B is 1:10-1: 5;
5-10min, phase A: the volume ratio of the phase B is 1:5-1: 100;
10-30min, phase A: the volume ratio of the phase B is 1:100-1: 10.
The electrochemical measurement conditions were: the measuring device is an electrochemical detection cell and comprises a glassy carbon electrode, an Ag/AgCl reference electrode and a counter electrode; the measuring temperature is 35-55 ℃; the flow rate is 1.0-2.5mL/min-1(ii) a The balance time is 1-5 minutes; the injection volume was 20L; the detection potential was 0.9V.
Example 2
The 3 curcumin components in the standard solution were determined based on retention time, kprime, selectivity, symmetry factor, baseline width and resolution of the three peaks of the curcuminoids: chromatographic profiles of curcumin (C), Demethoxycurcumin (DMC) and Bisdemethoxycurcumin (BDMC), all of which can be calculated by Empower 3 software using US Pharmacopeia (USP) selection. The specific data are shown in Table 1.
TABLE 1 chromatographic characteristics
Curcumin (curcumin) Retention time (min) K prime Selectivity is Symmetry factor Base line width Linear range
C 1.25 2.31 1.15 1.23 0.18 8.62
DMC 1.13 1.87 1.14 1.32 0.1 6.21
BDMC 1.06 1.56 0 1.45 0.12 6.87
A series of different concentrations (0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 75 and 100mg L) within the above range were precisely weighed-1) And measured under the conditions of High Performance Liquid Chromatography (HPLC) and Electrochemistry (ECD) described above in example 1, using the sps software to calculate the regression equation, correlation coefficient (r)2) And fitting tests. The standard regression equation, correlation coefficient and linear range of the 3 curcumin components are obtained by calculation, and the specific data are shown in table 2. Then, the detection limit was determined by dividing 3 times the deviation of the background noise obtained for the blank sample (N ═ 10) by the slope of the calibration curve, i.e., S/N ═ 3. The quantitation limit was determined by dividing 10 times the deviation of the background noise obtained for the blank sample (N-10) by the slope of the calibration curve, i.e., S/N-10, and the data is shown in table 2.
TABLE 2
Figure BDA0002509625170000081
As can be seen from Table 2, the standard regression equation takes the chromatographic peak area as the ordinate (y) and the compound concentration as the abscissa (x), and meanwhile, the 3 components all have good linear relations in a certain linear range, and the correlation coefficient r of the standard regression equation is2Are all greater than 0.996. In addition, the detection limit of the detection method in the invention reaches 10-8And M is lower than the detection limit of the conventional high performance liquid chromatography by adopting a UV detector.
Example 3
According to the pretreatment and qualitative and quantitative analysis steps in example 1, 10 replicates of turmeric samples were selected within 1 day, and the accuracy of the day-to-day reproducibility of the method was examined. Turmeric samples were also selected and assayed for three consecutive days, 10 replicates per day, and the accuracy of the day-to-day reproducibility of the method was examined. The intra-day and inter-day precision was evaluated according to the F-test. The Relative Standard Deviation (RSD) for the intra-day accuracy was 1.15% to 1.94%, respectively, and the inter-day accuracy was 4.27% to 5.85%, respectively, as shown in table 3. As can be seen from table 3, the Relative Standard Deviation (RSD) of the 3 curcumin components was below 6%, indicating that the reproducibility of the method was good.
TABLE 3 accuracy relative standard deviation table
Name of Chinese Day inner RSD (%) Day time RSD (%)
Curcumin (curcumin) 1.15 4.27
Demethoxycurcumin 1.26 5.24
Bisdemethoxycurcumin 1.94 5.85
Example 4
The pretreatment and qualitative and quantitative analysis steps in example 1 were followed to select three standard addition levels, low, medium and high, of the turmeric sample, and the standard addition recovery rates of the sample were determined, the standard addition levels were 50, 100 and 150. mu.g/mL, respectively, and the corresponding recovery rates are shown in Table 4. And calculating the recovery rate according to the difference between the concentration of the known sample and the standard sample relative to the amount of the standard sample, wherein the recovery rate is 85.8-108%, and the recovery rate of each component to be detected is good.
TABLE 4 Bidding recovery Rate Table
Figure BDA0002509625170000091
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims (10)

1. A method for determining the content of three curcumins in Curcuma rhizome comprises adding soaked Curcuma rhizome into extraction solvent, ultrasonic extracting, centrifuging, collecting supernatant, filtering to obtain sample solution, and performing qualitative and quantitative analysis on 3 curcumins in the sample solution by high performance liquid chromatography electrochemical combination method.
2. The method for determining the content of three curcumins in turmeric according to claim 1, wherein the ratio of the added mass g of turmeric to the added volume mL of the soaking solution is 1: 40-60; the soaking solution is 0.05-0.15% of acetic acid aqueous solution by volume percentage; the soaking time of the turmeric is 20-40 min.
3. The method for determining the content of three curcumins in turmeric according to claim 1, wherein the ratio of the added mass g of turmeric to the added volume mL of extraction solvent is 1: 10-100; the extraction solvent is methanol.
4. The method for determining the content of three curcumins in turmeric according to claim 1, wherein the ultrasonic extraction time is 25-35 min; the frequency of the ultrasonic extraction is 30-50kHz, and the power of the ultrasonic extraction is 120-150W.
5. The method as claimed in claim 1, wherein the rotation speed of the centrifugation is 8000-12000 rpm; the centrifugation time is 10-20 min.
6. The method for determining the content of three curcumins in turmeric according to claim 1, wherein the combined high performance liquid chromatography and electrochemical method is used for qualitative and quantitative analysis of 3 curcumins in sample solution, and comprises the following steps:
1) preparing a standard solution: respectively taking standard samples of 3 curcumin components in the turmeric, adding a solvent for dilution and constant volume to prepare standard solutions;
2) and (3) qualitative and quantitative analysis: respectively measuring the sample solution and the standard solution prepared in the step 1) by a high performance liquid chromatography-electrochemical combined method, comparing retention time by the high performance liquid chromatography, performing qualitative determination according to a common characteristic peak, determining 3 curcumin components in the sample solution, and calculating the content of the 3 curcumin components in the sample solution by an electrochemical method by adopting a standard curve method.
7. The method for determining the content of three curcumins in turmeric according to claim 6, wherein in the step 2), the determination condition of the high performance liquid chromatography is selected from any one of the following conditions:
a) a chromatographic column: c18A chromatographic column; column temperature: 35-55 ℃; flow rate: 0.5-2.5 mL/min; mobile phase A: acetonitrile; mobile phase B: 5-15mmol/L Na2HPO4-H3PO4An aqueous solution, pH 4.5-5.5; isocratic elution;
b) a chromatographic column: c18A chromatographic column; column temperature: 35-55 ℃; flow rate: 1.0-2.5 mL/min; mobile phase A: 0.05-0.15 v/v% aqueous acetic acid; mobile phase B: 0.05-0.15 v/v% acetonitrile or methanol in water; gradient elution.
8. The method for measuring the content of three curcumins in turmeric according to claim 7, wherein in the measurement condition a), the volume ratio of the mobile phase A to the mobile phase B in isocratic elution is 50: 50.
9. The method for determining the content of three curcumins in turmeric according to claim 7, wherein in the determination condition b), the specific procedure of gradient elution is as follows: 0-5min, phase A: the volume ratio of the phase B is 1:10-1: 5; 5-10min, phase A: the volume ratio of the phase B is 1:5-1: 100; 10-30min, phase A: the volume ratio of the phase B is 1:100-1: 10.
10. The method for measuring the content of three curcumins in turmeric according to claim 6, wherein in the step 2), the electrochemical measurement conditions are as follows: the measuring device is an electrochemical detection cell and comprises a glassy carbon electrode, an Ag/AgCl reference electrode and a counter electrode; the measuring temperature is 35-55 ℃; the flow rate is 1.0-2.5mL/min-1(ii) a The balance time is 1-5 minutes; injection bodyThe product is 15-25L; the detection potential is 0.6-1.2V.
CN202010457098.9A 2020-05-26 2020-05-26 Method for determining content of three curcumins in turmeric Pending CN111579671A (en)

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