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CN-121994574-A - Nuclear magnetic method for simultaneously quantifying 4 types of monophthalide components in ligusticum wallichii based on HSQC (high-speed quantitative) spectrum technology

CN121994574ACN 121994574 ACN121994574 ACN 121994574ACN-121994574-A

Abstract

The invention provides a nuclear magnetic method for simultaneously quantifying 4 types of monophthalide components in Ligusticum chuanxiong based on HSQC spectrogram technology, which comprises the following steps of 1) preparing internal standard solution; 2) preparation of a sample solution, 3) detection, 4) quantification, wherein the 4 types of monophthalide components are BT-I to BT-IV types of monophthalide, and the method has the advantages of high sampling efficiency, high accuracy and good repeatability, breaks through the limit of quantifying one type of component by one method, realizes the crossing type upgrading of quantifying four types of components simultaneously by one internal standard by one method, greatly improves the component quantification efficiency, and has better quantification accuracy and repeatability.

Inventors

  • XIONG LIANG
  • MENG CHUNWANG
  • Gou yan
  • FENG RUI
  • GENG ZHAO
  • PENG CHENG
  • ZHANG LILI
  • ZHANG KE

Assignees

  • 成都中医药大学
  • 四川省药品检验研究院(四川省医疗器械检测中心、四川养麝研究所)

Dates

Publication Date
20260508
Application Date
20260303

Claims (9)

  1. 1. A nuclear magnetic method for simultaneously quantifying 4 types of monophthalide components in Ligusticum chuanxiong based on HSQC spectrum technology is characterized by comprising the following steps: 1) Preparing an internal standard solution, namely dissolving magnolol in DMSO-d 6 , and adding a relaxation reagent for uniform mixing to obtain the internal standard solution; 2) Preparing test solution by extracting rhizoma Ligustici Chuanxiong with methanol, filtering and drying the extractive solution, and dissolving the residue with the internal standard solution obtained in step 1); 3) Detecting a sample solution by adopting a nuclear magnetic resonance instrument, wherein nuclear magnetic resonance parameters comprise relaxation delay time of 1.8 s; 4) The quantitative method comprises the steps of collecting an HSQC map, integrating a quantitative signal area, and calculating the content of various monophthalide components according to the integral value; The 4 types of monophthalide components are BT-I to BT-IV types of monophthalide: BT-I class of monophthalides are a class of compounds having the structure shown in formula I: Formula I, wherein R is independently selected from hydrogen, hydroxyl or C 1 –C 6 alkyl, m is 0-4, R 1 is C 1 –C 6 alkyl; BT-II class monophthalides are a class of compounds having the structure shown in formula II: Formula II, wherein R 5 is independently selected from hydrogen or hydroxy, m is 0-4, R 2 is C 1 –C 6 alkyl; BT-class III monophthalides are a class of compounds having the structure shown in formula III: Formula III, wherein R is each independently selected from hydrogen, hydroxy or C 1 –C 6 hydrocarbyl, m is 0-4, R 3 is C 1 –C 6 alkyl; BT-class IV monophthalides are a class of compounds having the structure shown in formula IV: Formula IV, wherein R 5 is independently selected from hydrogen or hydroxy, m is 0-4, and R 4 is C 1 –C 6 alkyl.
  2. 2. The nuclear magnetic resonance method as set forth in claim 1, wherein the concentration of magnolol in the internal standard solution in step 1) is 1.50 mM and the concentration of the relaxation agent is 3.00 mM.
  3. 3. The nuclear magnetic method according to claim 2, wherein the relaxation agent is chromium acetylacetonate.
  4. 4. The nuclear magnetic resonance method as set forth in claim 1, wherein the extraction in the step 2) is ultrasonic extraction, the temperature is not higher than 25 ℃, and the power is 300W.
  5. 5. The nuclear magnetic resonance method as set forth in claim 1, wherein the mass-to-volume ratio of the Ligusticum wallichii, methanol, filtrate and internal standard solution in the step 2) is 1~5 g:25ml:2 mL:400. Mu.L.
  6. 6. The nuclear magnetic resonance method as set forth in claim 1, wherein the nuclear magnetic resonance parameters in step 3) further include 25% NUS in sampling mode, HSQCETGPSISP.2 in pulse sequence, 298.25 in temperature, 16 in scanning times, 32 in idle scanning times, 156 Hz in coupling constant, 101 in gain, 16.0 ppm in spectral width, 200.0 ppm in spectral width, 2048 in sampling points, 128 in F1, 5.3 ppm in center frequency, 115.0 ppm in O2P.
  7. 7. The nuclear magnetic resonance method as set forth in claim 1, wherein the HSQC spectrum of step 4) is subjected to phase correction and baseline correction by Topspin.1.4 software.
  8. 8. The nuclear magnetic resonance method as set forth in claim 1, wherein in the step 4), the quantitative signal region of BT-I type monophthalide is δ H 5.3 ~ 5.6 ppm,δ C 109.2.2-114.0 ppm, the quantitative signal region of BT-II type monophthalide is δ H 5.9 ~ 6.0 ppm,δ C 107.8.8-110.8 ppm, the quantitative signal region of BT-III type monophthalide is δ H 5.0 ~ 5.2 ppm,δ C 79.3-83.7 ppm, the quantitative signal region of BT-IV type monophthalide is δ H 5.6 ~ 5.7 ppm,δ C 79.4.4-83.6 ppm, and the quantitative signal region of magnolol is δ H 5.8 ~ 5.9 ppm,δ C 136.9.9-140.2 ppm.
  9. 9. The nuclear magnetic resonance method as set forth in claim 1, wherein the content in the step 4) is calculated by the following formula: C S = N R /N S × C R × A S /A R Wherein C S is the molar concentration mM of a ligusticum wallichii sample to be detected, N R is the proton number of an internal standard quantitative signal, 2;N S is the proton number of a monophthalide quantitative signal, 1;C R is the molar concentration mM of an internal standard substance, A S is the integral peak volume of any one of BT-I-BT-IV quantitative signals in the ligusticum wallichii sample to be detected, and A R is the integral peak volume of an internal standard quantitative signal.

Description

Nuclear magnetic method for simultaneously quantifying 4 types of monophthalide components in ligusticum wallichii based on HSQC (high-speed quantitative) spectrum technology Technical Field The invention belongs to the field of medicine analysis, and in particular relates to a nuclear magnetic method for simultaneously quantifying 4 types of monophthalide components in ligusticum wallichii based on an HSQC (high-speed quantitative) spectrogram technology Background Ligusticum wallichii is a dried rhizome of Ligusticum chuanxiong Hort (Umbelliferae), which contains abundant phthalides. Wherein, the monophthalide is used as the most main phthalide type, forms the important active substance basis of the ligusticum wallichii, and the representative compounds comprise Z-ligustilide, 3-butenyl phthalide, senkyunolide A, butylphthalide and the like. Modern pharmacological researches prove that the phthalide component has wide bioactivity, can dilate blood vessels, improve microcirculation, has the effects of resisting inflammation, resisting oxidation, protecting nerves, inhibiting proliferation of tumor cells and the like, and has important value in preventing and treating cardiovascular and cerebrovascular diseases and nervous system diseases. The total content of phthalide components in the ligusticum wallichii is usually more than 1%, and more than 70 types of the components are reported at present. However, for a long time, the total control of the quality standard of ligusticum wallichii on phthalide components is weak, and the content measurement of ligustilide is introduced for the first time until the 2025 edition of Chinese pharmacopoeia, and the content of ligustilide is regulated to be not lower than 0.80%. The curative effect of the traditional Chinese medicine is derived from the synergistic effect of multiple components and multiple targets, and the whole quality of the medicinal materials is difficult to be comprehensively reflected by only measuring one or two monomer components. Therefore, establishing a component analysis method capable of rapidly quantifying phthalide components in ligusticum wallichii has become an urgent need for improving the quality control level and guaranteeing the clinical curative effect. Quantitative nuclear magnetic resonance (quantitative nuclear magnetic resonance, qNMR) is a general quantitative analysis method based on nuclear magnetic signals of organic molecular structures, and has been rapidly developed in recent 20 years. Compared with the traditional High Performance Liquid Chromatography (HPLC), liquid chromatography (LC-MS) and other technologies, qNMR not only can provide abundant structural information, but also has the advantages of no need of specific reference substances, nondestructivity, absolute quantification of one type of components and the like. The most widely applied qNMR methods at present mainly comprise 1 H NMR quantitative nuclear magnetism and 1H-13 C HSQC spectrum quantitative nuclear magnetism, and the latter has the characteristics of wide spectrum range and high signal resolution, and has remarkable advantages in quantitative analysis of traditional Chinese medicine mixtures. However, in practical application, especially analysis of complex systems of traditional Chinese medicines, a series of technical challenges still face, namely, on one hand, qNMR is influenced by multiple factors in quantitative accuracy. The traditional Chinese medicine components are complex, signals of different types of components can be overlapped, particularly, the overlapping of proton signals in a high field area is obvious, and even if a two-dimensional HSQC quantitative method is used, signal interference is difficult to avoid completely, and integration accuracy is affected. Meanwhile, a proper internal standard is also a key for influencing the accuracy of two-dimensional nuclear magnetism quantification. In view of the complexity of qNMR quantification, the monophthalide component in ligusticum wallichii has important pharmacological activity, is an important pharmacodynamic active component, and a specific and reliable qNMR quantification method for the component is not established at present. Disclosure of Invention In order to solve the problems, the invention provides a nuclear magnetic method for simultaneously quantifying 4 types of monophthalide components in ligusticum wallichii based on an HSQC spectrogram technology, which comprises the following steps: 1) Preparing an internal standard solution, namely dissolving magnolol in DMSO-d 6, and adding a relaxation reagent for uniform mixing to obtain the internal standard solution; 2) Preparing test solution by extracting rhizoma Ligustici Chuanxiong with methanol, filtering and drying the extractive solution, and dissolving the residue with the internal standard solution obtained in step 1); 3) Detecting a sample solution by adopting a nuclear magnetic resonance instrument, wherein nuclear mag