Method for detecting phlorizin content in medicinal material
The content of root syringin in medicinal materials was detected by nuclear magnetic resonance hydrogen spectroscopy, and the mixed solvent of deuterated methanol and deuterated water was extracted and solid-liquid separation was performed. The problem of time-consuming and low sensitivity of the determination of root syringin content in the prior art was solved, and a fast and accurate detection effect was achieved.
Patent Information
- Application Number
- CN202411994922.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art has limitations such as time-consuming, low sensitivity, and high detection limit when determining the content of root senoids in medicinal materials, and there is no report on nuclear magnetic 1H NMR technology in this field.
The content of root sacin in medicinal materials was detected by nuclear magnetic resonance hydrogen spectrometry. The content of root sacin is calculated by extracting the internal standard substance and medicinal materials with a mixed solvent of deuterated methanol and deuterated water, solid-liquid separation and nuclear magnetic resonance hydrogen spectrometry were performed.
It realizes rapid and accurate detection of the content of root senin in medicinal materials, is easy to operate, has good data reproducibility, and is suitable for medicinal materials quality control and identification.
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Figure CN119715657A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting the content of phlorizin in medicinal materials. Background Art
[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.
[0003] Phlorizin, chemical name is 1-(2-(beta-D-pyranose glucoseoxy)-4,6-dihydroxyphenyl)-3-(4-hydroxyphenyl)-acetone, molecular formula is C 21 H 24 O 10 It is a rare dihydrochalcone glycoside substance in nature, which is mainly found in the leaves, stems and barks of apple plants in the Rosaceae family. It is rich in apple peels and branches, leaves of Malus hupehensis and its variant Polystachya. Phloridzin has biological activities of lowering blood sugar, regulating blood pressure, protecting the heart, balancing estrogen and anti-oxidation.
[0004] In order to evaluate the quality of medicinal materials, it is necessary to determine the content of phlorizin. At present, the methods used to quantify phlorizin in medicinal materials mainly include high performance liquid chromatography tandem mass spectrometry (HPLC-MS) and reverse phase high performance liquid chromatography (RP-HPLC). The HPLC method for determining the content of phlorizin has certain limitations, such as long time consumption, low sensitivity, high detection limit, etc. In addition, chalcone components have poor stability and are easily oxidized. Compared with the HPLC method, nuclear magnetic resonance spectroscopy (NMR) 1 H NMR technology can detect multiple substances at the same time; sample preparation is simple, using deuterated reagents to extract medicinal samples; good durability and repeatability; small sample volume is required, and the scanning time is short, which can be used as a good quantitative method. However, there are some questions about the use of 1 There is no report on the quantitative method of phlorizin in medicinal materials by H NMR. Summary of the invention
[0005] In view of the problems in the prior art, the present invention provides a method for determining the content of phlorizin, which uses nuclear magnetic resonance hydrogen spectrum for quantitative detection, has simple operation and accurate analysis data.
[0006] To achieve the above purpose, the present invention adopts the following technical solution.
[0007] A method for detecting the content of phlorizin in a medicinal material comprises the following steps: Extracting the internal standard substance and the medicinal material with a solvent, and performing solid-liquid separation to obtain an extract; The extract is subjected to nuclear magnetic resonance hydrogen spectrum detection to obtain the characteristic peak area of the internal standard substance and the characteristic peak area of phlorizin, and the phlorizin content is calculated according to the characteristic peak area of the internal standard substance, the characteristic peak area of phlorizin and the mass of the internal standard substance; The internal standard is 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS); The solvent is a mixture of deuterated methanol and deuterated water. Phlorizin is not easily soluble in water, but is easily soluble in organic solvents. Therefore, theoretically, the extraction of phlorizin in medicinal materials using organic solvents should be able to accurately detect phlorizin quantitatively using nuclear magnetic resonance hydrogen spectrum. However, the extraction of medicinal materials using deuterated methanol and then quantitative detection found that phlorizin could not be accurately detected quantitatively, so it is necessary to find a suitable deuterated organic solvent to extract phlorizin in medicinal materials. Phlorizin is not easily soluble in water. Theoretically, the use of a mixed solution of organic solvent and water should reduce the solubility of phlorizin. However, it was unexpectedly found through experiments that the extraction using a mixed solution of deuterated methanol and deuterated water can accurately detect phlorizin quantitatively through nuclear magnetic resonance hydrogen spectrum, thereby realizing the characteristics of simple operation and high work efficiency brought by the detection of phlorizin content using nuclear magnetic resonance hydrogen spectrum. Preferably, the volume ratio of the two is 1: (0.9-1.1) to detect the content of phlorizin more accurately. More preferably, the volume ratio between the two is 1:1.
[0008] The mass volume ratio of the medicinal material and the solvent is 1: (10-50) g / mL.
[0009] The mass ratio of the internal standard to the medicinal material is 1:(1-40).
[0010] Preferably, the extraction is ultrasonic extraction; the extraction temperature is 30°C-40°C, and the extraction time is 30min-40min.
[0011] Preferably, the solid-liquid separation method is centrifugal separation, the centrifugal speed is 5000rpm-10000rpm, and the centrifugal time is 10min-15min.
[0012] The conditions of the nuclear magnetic resonance hydrogen spectrum are: pulse sequence zg, spectrum width of 15 ppm-25 ppm, radio frequency center of 6.17 ppm, scanning times of 8-32 times, relaxation time of 20 µs-30 µs, detection temperature of 295 K-300 K; time domain sampling number of 32 k-64 k.
[0013] More preferably, the conditions of nuclear magnetic resonance hydrogen spectrum are: pulse sequence zg, spectral width of 20 ppm, radio frequency center of 6.17 ppm, number of scans n of 8 times, relaxation time D1 of 25 µs, detection temperature T of 298.0 K, and time domain sampling number of 64 k; after optimization, it was found that the content of phlorizin was detected more accurately under this parameter.
[0014] Under optimal conditions, d H The signal peak at 6.207 (H-6) ppm is the characteristic peak of phlorizin. d H 6.207(H-6) has good separation degree from other signal peaks in medicinal materials, so the signal peak of this chemical shift is used as the characteristic peak for phlorizin. 1 H NMR quantification.
[0015] Preferably, the method for obtaining the characteristic peak area is: determine the chemical shift standard with the internal standard signal as a reference; adjust the phase and baseline to make the peak symmetrical on the left and right, and adjust the baseline; perform manual integration, set the characteristic peak area interception range, and the peak area interception method is to intercept the same distance from the center of the characteristic peak to both sides, integrate the peak area between the two interception lines, repeat the integration multiple times, and calculate the average value, which is the characteristic peak area.
[0016] Specifically, the formula for calculating the phlorizin content is: Ws = Wr × (As / Ar) × (Es / Er) Wherein, Ws is the mass of phlorizin, Wr is the mass of the internal standard, As and Ar are the peak areas of the characteristic peak of phlorizin and the characteristic peak of the internal standard, respectively, Es and Er are the proton equivalent masses of phlorizin and the internal standard, respectively, and the proton equivalent mass is calculated by dividing the molecular weight by the number of protons of the characteristic peak.
[0017] The present invention has the following advantages: The present invention adopts a method to determine the hydrogen resonance frequency of the internal standard and the medicinal material, and can quickly determine the content of phlorizin in the medicinal material. The present invention has the advantages of no need for a reference substance, simple operation, and combined use of an automatic sampling system to achieve rapid batch determination. The experimental data has good reproducibility, is intuitive and reliable, and can be applied to the quality control and identification of medicinal materials. At the same time, the present invention adopts a non-destructive analysis method, and precious samples are convenient to recover after detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 is the phlorizin under optimized conditions 1 H NMR spectrum. DETAILED DESCRIPTION
[0019] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.
[0020] Example 1 Establishment and verification of detection method 1. Method development Phlorizin standard was used as the detection object, 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as the internal standard compound, and the measurement was carried out on a Bruker DRX-600 nuclear magnetic resonance spectrometer with a pulse sequence of zg and a radio frequency center of 6.17 ppm. The measurement was repeated 6 times by changing one of the parameters including the number of scans (n), spectrum width (SW), relaxation time (D1), time domain sampling number (TD) and detection temperature (T) each time, and the sample recovery rate was calculated to determine the optimal parameters.
[0021] Table 1 Effect of various parameters on sample recovery It can be seen from Table 1 that when the scanning number n=8, the relative error of the recovery rate is the smallest; when the spectrum width SW=20 ppm, the relative error of the recovery rate is the smallest; when the relaxation time D1=25 µs, the relative error of the recovery rate is the smallest; when the time domain sampling number TD is 64 k, the relative error of the recovery rate is the smallest; when the detection temperature T is 298 K, the relative error of the recovery rate is the smallest.
[0022] Under the above optimized conditions, the 1 H NMR spectrum Figure 1 shown.
[0023] 2. Linear correlation (or detection limit) Accurately weigh 0.1643 mg, 0.3286 mg, 0.6572 mg, 1.31475 mg, 2.62875 mg, 5.2575 mg, and 10.5150 mg of the standard and add them to 1 mL of a solvent with a volume ratio of 1:1 of heavy water and deuterated methanol. Place them in an NMR tube and perform NMR analysis under the following conditions: The measurements were performed on a Bruker DRX-600 NMR spectrometer with a pulse sequence of zg, scan number n=8, spectral width SW=20 ppm, RF center 6.17 ppm, relaxation time D1=25 µs, time domain sampling number TD of 64 k, and temperature T of 298 K.
[0024] Obtain the peak area, and draw a standard curve with the injection mass as the horizontal axis and the peak area as the vertical axis. The regression line equation is: Y=0.838X-0.083, R 2 =0.997.
[0025] 3. Precision The same test solution was measured 5 times continuously. 6’ The RSD of the ratio of the double signal peak of -H to the quantitative peak of internal standard DSS was 0.08%, indicating that this method has good precision.
[0026] 4. Stability The instrument's built-in parameters 1 The H NMR experiment was repeated five times by the same experimenter on the same day, and 18.33 mg, 24.47 mg, 19.09 mg, 21.73 mg, and 20.79 mg were weighed for testing, respectively. The stability of the instrument was tested, the peak area was integrated, and the recovery rate was calculated. The results were between 98.2% and 102.1%, which met the experimental requirements.
[0027] Example 2 Detection of Phlorizin Content in Malus hupehensis Leaves Accurately weigh 20 mg of Hubei Begonia medicinal material powder and about 2.84 mg of internal standard (DSS), and transfer them to a centrifuge tube. Add 1000 µL of heavy water and 1000 µL of deuterated methanol and vortex at room temperature for 2 min. Ultrasonicate at room temperature for 15 min, centrifuge for 15 min, take the supernatant, and transfer it to a 5 mm NMR tube as the sample solution for determination.
[0028] The Bruker DRX-600 NMR spectrometer was set with the following parameters for measurement: temperature of 25 °C, shim-field three times, pulse sequence zg, spectral width SW = 20 ppm, RF center 6.17 ppm; time domain sampling number TD = 64 k, scanning number n = 8 times, relaxation time D1 = 25 µs, time domain sampling number TD = 64 k, temperature T = 298.0 K, and other values were set by the instrument.
[0029] Accurately locate the characteristic peak, adjust the baseline, intercept the peak area of the characteristic peak of phlorizin, intercept the peak area of the internal standard DSS, and use it to calculate the content of phlorizin in the medicinal powder. The method for obtaining the characteristic peak area is: determine the chemical shift standard with the internal standard signal as a reference; adjust the phase and baseline to make the peak symmetrical on the left and right, and adjust the baseline; perform manual integration, set the characteristic peak area interception range, and the peak area interception method is to intercept the same distance from the center of the characteristic peak to both sides, integrate the peak area between the two interception lines, repeat the integration multiple times, and calculate the average value as the characteristic peak area; The phlorizin content in the sample was calculated according to the following formula: Ws = Wr × (As / Ar) × (Es / Er) Wherein, Ws is the mass of phlorizin, Wr is the mass of the internal standard, As and Ar are the peak areas of the characteristic peak of phlorizin and the characteristic peak of the internal standard, respectively, Es and Er are the proton equivalent masses of phlorizin and the internal standard, respectively, and the proton equivalent mass is calculated by dividing the molecular weight by the number of protons of the characteristic peak.
[0030] The signal peaks of the aromatic region of phloridzin in 5 batches of Hubei crabapple leaves were analyzed. d H 6.207 (H-6) ppm, signal peak of three methyl groups on internal standard DSS silicon d H 0.0ppm, with good separation from other signals in medicinal materials, and can be used for phlorizin 1 HNMR quantification, the intercepted sample peak area and the internal standard peak area integral value were substituted into the formula to calculate the mass fraction of phloridzin in the sample. The content of phloridzin in the leaves of Malus Hubeiensis was 32.5 ± 0.4 mg / g.
[0031] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims
1. A method for detecting the content of phlorizin in medicinal materials, characterized in that: The following steps are involved: Extracting the internal standard substance and the medicinal material with a solvent, and performing solid-liquid separation to obtain an extract; The extract is subjected to nuclear magnetic resonance hydrogen spectrum detection to obtain the characteristic peak area of the internal standard substance and the characteristic peak area of phlorizin, and the phlorizin content is calculated according to the characteristic peak area of the internal standard substance, the characteristic peak area of phlorizin and the mass of the internal standard substance; The internal standard is 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS); The solvent is a mixture of deuterated methanol and deuterated water.
2. The method according to claim 1, characterized in that The volume ratio of deuterated methanol to deuterated water is 1:(0.9-1.1); The mass volume ratio of the medicinal material and the solvent is 1: (10-50) g / mL; The mass ratio of the internal standard to the medicinal material is 1:(1-40); The conditions of the nuclear magnetic resonance hydrogen spectrum are: pulse sequence zg, spectrum width of 15 ppm-25 ppm, radio frequency center of 6.17 ppm, scanning times of 8-32 times, relaxation time of 20 µs-30 µs, detection temperature of 295 K-300 K; time domain sampling number of 32k-64 k.
3. The method according to claim 1, characterized in that The conditions of H NMR spectrum were as follows: pulse sequence zg, spectral width of 20 ppm, RF center of 6.17 ppm, number of scans n of 8 times, relaxation time D1 of 25 µs, detection temperature T of 298.0 K, and time domain sampling number of 64 k.
4. The method according to claim 1, characterized in that: The volume ratio of deuterated methanol to deuterated water is 1:1; The extraction is ultrasonic extraction; the extraction temperature is 30°C-40°C, and the extraction time is 30min-40min; The solid-liquid separation method is centrifugal separation; the centrifugal speed is 5000rpm-10000rpm; the centrifugal time is 10min-15min.
5. The method according to claim 1, characterized in that The characteristic peaks of phlorizin are δ H 6.207ppm.
6. The method according to claim 1, characterized in that The method for obtaining the characteristic peak area is as follows: determine the chemical shift standard using the internal standard signal as a reference; adjust the phase and baseline to make the peak symmetrical on the left and right, and adjust the baseline; perform manual integration, set the characteristic peak area interception range, and the peak area interception method is to intercept the same distance from the center of the characteristic peak to both sides, integrate the peak area between the two interception lines, repeat the integration multiple times, and calculate the average value, which is the characteristic peak area.
7. The method according to claim 1, characterized in that The formula for phlorizin content is: Ws = Wr × (As / Ar) × (Es / Er) Wherein, Ws is the mass of phlorizin, Wr is the mass of the internal standard, As and Ar are the peak areas of the characteristic peak of phlorizin and the characteristic peak of the internal standard, respectively, Es and Er are the proton equivalent masses of phlorizin and the internal standard, respectively, and the proton equivalent mass is calculated by dividing the molecular weight by the number of protons of the characteristic peak.