Two-dimensional nuclear magnetic quantitative method for rapidly quantifying C20-diterpenoid alkaloid component in radix aconiti carmichaeli

Abstract

The invention belongs to the field of medicine analysis, and particularly relates to a two-dimensional nuclear magnetic quantitative method for rapidly quantifying a C20-diterpenoid alkaloid component in radix aconiti carmichaeli. The method comprises the following steps: extracting and filtering coarse powder of traditional Chinese medicinal materials, taking filtrate, removing a solvent, and adding an internal standard solution containing magnolol to prepare a test solution; and detecting the test solution by adopting a two-dimensional nuclear magnetic resonance analyzer, then collecting an HSQC map, and calculating the content according to an integral value by taking 17-site exocyclic double bonds of the C20-diterpenoid alkaloid as key quantitative signals. The sampling efficiency is improved by applying a relaxation reagent and a non-uniform sampling (NUS) mode, and the added relaxation reagent is chromium acetylacetonate. The detection method provided by the invention has the advantages of high sampling efficiency, good repeatability and high stability, can accurately quantify the C20-diterpenoid alkaloid component in a radix aconiti carmichaeli sample in practical application, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a two-dimensional nuclear magnetic resonance quantitative method for rapidly quantifying C 20 -diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata. Background Art

[0002] C 20 -diterpenoid alkaloids are a class of alkaloid components with five-membered or six-membered rings, with relatively characteristic structures, and can be further subdivided into Atisine-type, Veatchine-type, and Denudatine-type, and are widely present in Aconitum plants. Aconiti Lateralis Radix Praeparata is a processed product of the lateral root of Aconitum carmichaelii Debx. of the Ranunculaceae family, which contains abundant C 20 -diterpenoid alkaloids. However, due to extremely weak ultraviolet absorption and large polarity, the research on C 20 -diterpenoid alkaloid components is extremely difficult, resulting in the lack of regulations on the content of C 20 -diterpenoid alkaloids in current standards at all levels of Aconiti Lateralis Radix Praeparata. However, modern research has found that C 20 -diterpenoid alkaloids have effects such as anti-arrhythmia, analgesia, anti-inflammatory, anti-tumor, and anti-anxiety, and are an important pharmacodynamic material basis indispensable in Aconiti Lateralis Radix Praeparata. Therefore, it is urgent to develop a method for determining the content of C 20 -type diterpenoid alkaloids in Aconiti Lateralis Radix Praeparata.

[0003] Due to the characteristics of extremely weak ultraviolet absorption, large polarity, and high structural specificity of C 20 -type diterpenoid alkaloids, quantitative nuclear magnetic resonance (qNMR) is more suitable than high-performance liquid chromatography and liquid chromatography-mass spectrometry, and can simultaneously detect the total amount of a class of components, which can greatly improve the detection efficiency. Therefore, it is particularly urgent to develop a quantitative nuclear magnetic resonance method for quantifying C 20 -type diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata.

[0004] Quantitative nuclear magnetic resonance is a general detection method, which has advantages such as no need for specific reference substances, no contact with samples, no need for separation, and high quantitative accuracy, and has developed rapidly in the past 20 years. It is particularly suitable for qualitative and quantitative analysis of a class of components in mixtures such as traditional Chinese medicine extracts and traditional Chinese medicine compound preparations. One-dimensional nuclear magnetic resonance quantification (1D qNMR) is usually based on the nuclear magnetic resonance signals of hydrogen nuclei and has the advantage of fast detection speed, and has been favored by many researchers. However, the hydrogen spectrum ( 1The spectral width range of 1D NMR is relatively narrow, and signal overlap is severe. Its signal discrimination and quantitative accuracy are inferior to those of two-dimensional nuclear magnetic resonance spectra. Compared with 1D qNMR, two-dimensional nuclear magnetic resonance quantification (2D qNMR) has advantages such as a wide spectral width range, no signal overlap, and high signal-structure correlation, and is more suitable for component quantification in traditional Chinese medicine mixtures.

[0005] In summary, based on the HSQC (heteronuclear single quantum correlation) spectral technology, a high-efficiency and high-accuracy two-dimensional nuclear magnetic resonance quantification technology is established for the rapid quantification of C 20 -type diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata, which is an urgent problem to be solved in this field. Summary of the Invention

[0006] Aiming at the defects of the prior art, the present invention provides a two-dimensional nuclear magnetic resonance quantification technology based on HSQC spectral technology, aiming to rapidly quantify and detect C 20 -type diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata.

[0007] The present invention provides a two-dimensional nuclear magnetic resonance quantification method for rapidly quantifying C 20 -diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata, which comprises the following steps:

[0008] Step 1: The crude powder of traditional Chinese medicine is extracted and filtered, and the filtrate is taken. After removing the solvent, it is dissolved with an internal standard solution to obtain a test solution, and the internal standard solution contains magnolol.

[0009] Step 2: Detect the test solution by a two-dimensional nuclear magnetic resonance analyzer.

[0010] Step 3: Collect the HSQC spectrum, and use the exocyclic double bond at the 17th position of C 20 -diterpenoid alkaloid as the key quantitative signal, and calculate the content according to the integral value.

[0011] Preferably, the internal standard solution further includes chromium acetylacetonate, and the concentration of chromium acetylacetonate is 1-4 mM.

[0012] Preferably, the concentration of chromium acetylacetonate in the internal standard solution is 3-4 mM.

[0013] Preferably, the parameters of the two-dimensional nuclear magnetic resonance analyzer in Step 2 are: the coupling constant is 157 or 158 Hz; and / or, the relaxation delay time is 1.5-10 s.

[0014] Preferably, the coupling constant is 158 Hz; and / or, the relaxation delay time is 2.5 s.

[0015] Preferably, in Step 1, the internal standard solution is obtained by mixing magnolol with chromium acetylacetonate and a deuterated reagent; wherein, the concentration of magnolol is 1-10 mM; the deuterated reagent is selected from DMSO-d 6, Methanol-d 4 , Acetone-d 6 at least one of;

[0016] And / or, in the HSQC spectrum described in step 3, taking the terminal double bonds at the 9th and 9'-th positions in magnolol as the internal standard quantitative signals, the region of the quantitative signals of the internal standard is δ H 4.92 - 5.14 ppm, δ C 113.8 - 118.0 ppm;

[0017] And / or, the region of the key quantitative signals described in step 3 is δ H 4.56 - 5.16 ppm, δ C 105.0 - 115.0 ppm.

[0018] Preferably, the traditional Chinese medicine is aconite root;

[0019] And / or, in step 1, the extraction is carried out by mixing the crude powder of traditional Chinese medicine with ammonia test solution and isopropanol-ethyl acetate solution, and the mass-volume ratio of the aconite root, ammonia test solution and isopropanol-ethyl acetate solution is 2 g: 1 - 5 mL: 20 - 100 mL, and the volume ratio of isopropanol to ethyl acetate in the isopropanol-ethyl acetate solution is 1 - 5: 1;

[0020] And / or, in step 1, the extraction is ultrasonic extraction, the temperature ≤ 25°C, the power ≥ 300 W, and the time is 20 - 40 minutes; the removal of the solvent is carried out under reduced pressure at 30°C;

[0021] And / or, in step 1, the volume ratio of the filtrate to the internal standard solution is 10 - 50 mL: 400 μL.

[0022] Preferably, in step 2, the parameters of the nuclear magnetic resonance analyzer include:

[0023] The sampling method is non-uniform sampling NUS, and the sparsity of NUS is 20% to 50%; the pulse sequence is selected from any one of hsqcetgpsisp, hsqcetgpsisp.2, hsqcetgpsisp2, hsqcetgpsisp2.2, hsqcedetgpsisp, hsqcedetgpsisp.2, hsqcedetgpsisp2, hsqcedetgpsisp2.2, hsqcedetgpsisp2.3, hsqcedetgpsisp2.4; the temperature is 298K; the number of scans NS is 4 to 32; the number of dummy scans DS ≥ 16; the gain RG is 4 to 101; the frequency range scanned in the F2 dimension is 6 to 16 ppm, and the frequency range scanned in the F1 dimension is 80 to 200 ppm; the number of sampling points TD in the F2 dimension is ≥ 1024, and the number of sampling points TD in the F1 dimension is ≥ 128; 1 The central frequency of H is 3 to 8 ppm; 13 The central frequency of C is 50 to 100 ppm.

[0024] Preferably, the sparsity of NUS is 25%; the pulse sequence is hsqcetgpsisp2.2; the number of scans NS is 16; the number of dummy scans DS is 32; the gain RG is 101; the frequency range scanned in the F2 dimension is 16.0 ppm, and the frequency range scanned in the F1 dimension is 200.0 ppm; the number of sampling points TD in the F2 dimension is 2048, and the number of sampling points TD in the F1 dimension is 256; 1 The central frequency of H is 5.5 ppm; 13 The central frequency of C is 85.0 ppm.

[0025] Preferably, the HSQC spectrum described in step 3 is phase-corrected and baseline-corrected using Topspin 4.1.1 software;

[0026] And / or, the calculation formula for calculating the content according to the integral value described in step 3 is:

[0027] C S = N R / N S × C R × A S / A R

[0028] In the formula: C S is the molar concentration mM of C 20 -diterpenoid alkaloid in the sample; N R and N S respectively represent the number of protons of the internal standard quantitative signal and the key quantitative signal of C 20 -diterpenoid alkaloid; C Ris the molar concentration mM of the internal standard; A S is the integrated peak volume of the quantitative signal of the sample to be determined; A R is the integrated peak volume of the quantitative signal of magnolol.

[0029] Based on HSQC (heteronuclear single quantum correlation) spectral technology, the present invention has successfully established a two-dimensional NMR quantitative method for rapidly quantifying C 20 -diterpenoid alkaloid components in Aconiti Lateralis Radix Preparata. The conditions and steps in the determination method of the present invention are optimized. Using magnolol as the internal standard, the sampling efficiency is improved by applying relaxation reagents and / or non-uniform sampling (NUS) method. The added relaxation reagent is 3 mM chromium acetylacetonate, and the relaxation delay time is 2.5 s. Using the exocyclic double bond at the 17th position of C 20 -diterpenoid alkaloids as the key quantitative signal, and the terminal double bonds at the 9th and 9' positions in magnolol as the quantitative signals of the internal standard, and calculating the content according to the integral value. This method has the advantages of high sampling efficiency, good repeatability and high stability. The present invention is applicable to rapidly and accurately quantifying the content of C 20 -diterpenoid alkaloid components in raw Aconiti Lateralis Radix Preparata and its decoction pieces, and plants of the genus Aconitum in the Ranunculaceae family; It promotes the quality control of C 20 -diterpenoid alkaloids, the research on the pharmacodynamic mechanism and the development of related new drugs, and has good application prospects.

[0030] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0031] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 are the chemical structural formulas of the C 20 -diterpenoid alkaloid skeleton, songorine and magnolol;

[0033] Figure 2 are the 1 H- 13 C HSQC spectra (DMSO-d 6 ) of the songorine reference substance and magnolol (internal standard) superimposed full map;

[0034] Figure 3 are the 1 H- 13 C HSQC spectra (DMSO-d6 )Key quantitative signal superposition diagram;

[0035] Figure 4 For the 1 H- 13 C HSQC spectrum (DMSO-d 6 ) Superposition full diagram;

[0036] Figure 5 For the 1 H- 13 C HSQC spectrum (DMSO-d 6 ) Quantitative signal region superposition diagram;

[0037] Figure 6 For ginsenoside Re's 1 H- 13 C HSQC spectrum diagram;

[0038] Figure 7 For ginsenoside Rb1's 1 H- 13 C HSQC spectrum diagram;

[0039] Figure 8 For magnolol's 1 H- 13 C HSQC spectrum diagram;

[0040] Figure 9 For baicalin's 1 H- 13 C HSQC spectrum diagram;

[0041] Figure 10 For arctiin's 1 H- 13 C HSQC spectrum diagram;

[0042] Figure 11 For magnolol (internal standard) and aconite sample (FZ-1)'s 1 H- 13 C HSQC spectrum (DMSO-d 6 ) Superposition full diagram;

[0043] Figure 12 For magnolol (internal standard) and aconite sample (FZ-1)'s 1 H- 13 C HSQC spectrum (DMSO-d 6 ) Quantitative signal region superposition diagram;

[0044] Figure 13 For the key quantitative signal peaks of magnolol (internal standard) at different chromium acetylacetonate concentrations 1Partial 1H NMR spectrum; where A is the superimposed display mode and B is the list display mode;

[0045] Figure 14 The key quantitative signal peaks of songorine reference substance under different chromium acetylacetonate concentrations 1 Partial 1H NMR spectrum; where A is the superimposed display mode and B is the list display mode. Detailed implementation mode

[0046] In the following examples and experimental examples, reagents and materials not specifically stated are commercially available products.

[0047] Example 1 A two-dimensional NMR quantitative method for rapidly quantifying C 20 -diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata

[0048] This example provides a two-dimensional NMR method for quantitatively detecting C 20 -diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata. I. Preparation of solutions

[0049] 1. Chromium acetylacetonate stock solution

[0050] Precisely weigh an appropriate amount of chromium acetylacetonate, dissolve it in 1.0 mL of DMSO-d 6 , shake well and dissolve by ultrasonic treatment to prepare a 0.1 M stock solution.

[0051] 2. Internal standard solution

[0052] Take the chromium acetylacetonate stock solution and dissolve magnolol, the internal standard substance, in DMSO-d 6 to make the final concentrations of magnolol and chromium acetylacetonate about 2.15 mM and 3.00 mM respectively after mixing.

[0053] 3. Test solution

[0054] Precisely weigh about 2.0 g of Aconiti Lateralis Radix Praeparata powder (No. 3 sieve), transfer it to a stoppered Erlenmeyer flask, add 3 mL of ammonia test solution, precisely add 50 mL of isopropanol-ethyl acetate mixed solution (the volume ratio of isopropanol to ethyl acetate is 1:1), weigh it, ultrasonically treat it at a power of 300 W and a water temperature not higher than 25 °C for 30 min, cool it to 20 °C, weigh it again, make up the lost weight with isopropanol-ethyl acetate (1:1) mixed solution, shake well, filter, precisely absorb 25 mL of the filtrate, recover the solvent under reduced pressure to dryness at 30 °C, precisely add 400 μL of the internal standard solution to dissolve the residue, and transfer it to a 5 mm NMR tube to obtain the test solution.

[0055] II. Detection

[0056] The test solution was detected using a two-dimensional nuclear magnetic resonance analyzer; the HSQC nuclear magnetic resonance quantitative analysis parameters were as follows: 25% NUS sampling method was adopted; pulse sequence: hsqcetgpsisp2.2; temperature: 298K; number of scans (NS): 16; number of dummy scans (DS): 32; relaxation delay time (D 1 ): 2.5 s; coupling constant (CNST2): 158 Hz; gain (RG): 101; spectral width (SW): 16.0 ppm (F2), 200.0 ppm (F1); number of data points (TD): 2048 (F2), 256 (F1); center frequency: 5.5 ppm (O1P), 85.0 ppm (O2P).

[0057] III. Calculation of Content

[0058] Using Topspin 4.1.1 software, the measured HSQC spectrum was phase-corrected and baseline-corrected. The quantitative signal regions of the analyte and the internal standard were selected for integration, and then the content was calculated.

[0059] Among them, for the exocyclic double bond at the 17th position of C 20 -diterpenoid alkaloid, the quantitative signal region was δ H 4.56 - 5.16 ppm, δ C 105.0 - 115.0 ppm;

[0060] For the terminal double bonds at the 9th and 9' positions of magnolol, the quantitative signal region was δ H 4.92 - 5.14 ppm, δ C 113.8 - 118.0 ppm;

[0061] The calculation formula was: C S = N R / N S × C R × A S / A R

[0062] In the formula: C S was the molar concentration mM of C 20 -diterpenoid alkaloid in the sample; N R and N S represented the number of protons of the internal standard quantitative signal and the key quantitative signal of C 20 -diterpenoid alkaloid respectively; C R was the molar concentration mM of the internal standard; A S was the integrated peak volume of the quantitative signal of the sample to be determined; A R was the integrated peak volume of the quantitative signal of the internal standard.

[0063] The technical solution of the present invention will be further described through experiments below.

[0064] Experimental Example 1 Aconite C 20 -Development and Application Research on a New Method for Rapid Two-Dimensional NMR Quantification of Diterpenoid Alkaloid Components

[0065] I. Instruments and Reagents

[0066] Bruker Avance NEO 600MHz nuclear magnetic resonance spectrometer (Bruker), BP221S electronic analytical balance (Sartorius, Germany).

[0067] The information of aconite samples is shown in Table 1. Songguoling (PS001150, purity: 99.27%) was purchased from Chengdu Purigen Biotech Co., Ltd.; Magnolol (110729-202015, purity: 99.00%) was purchased from the National Institutes for Food and Drug Control. Chromium acetylacetonate (Sigma, batch number: MKCN5034, purity: 97.00%), deuterated DMSO-d 6 (Shanghai Titan Scientific Co., Ltd., batch number: P1867731); Isopropanol, Ethyl acetate, Ammonia water (analytical pure, Chengdu Kelong Chemical Co., Ltd.).

[0068] Table 1. Source and Origin Information Table of Aconite Samples

[0069]

[0070] II. Experimental Test Solutions

[0071] 1. Chromium Acetylacetonate Stock Solution

[0072] Precisely weigh 35.08 mg of chromium acetylacetonate reference substance, dissolve it in 1.0 mL of DMSO-d 6 , shake well and dissolve it ultrasonically to prepare a 0.1 M stock solution.

[0073] 2. Candidate Internal Standard Solution

[0074] Precisely weigh appropriate amounts of reference substances of Ginsenoside Re, Ginsenoside Rb1, Magnolol, Arctiin, and Baicalin, add them to DMSO-d 6 to dissolve, and prepare a candidate internal standard solution with a concentration of 4 mg / ml for each.

[0075] 3. Internal Standard Solution

[0076] Take an appropriate amount of chromium acetylacetonate stock solution, dissolve the internal standard substance magnolol in DMSO-d 6 so that the final concentrations of magnolol and chromium acetylacetonate after mixing are approximately 2.15 mM and 3.00 mM respectively.

[0077] 4. Standard solution

[0078] (1) Magnolol standard relaxation solution and sungucoline standard relaxation solution

[0079] Take appropriate amounts of chromium acetylacetonate stock solution, magnolol and sungucoline, and prepare magnolol standard relaxation solutions containing 0, 1, 1.5, 2.5, 3, 4, 5, 50 mM chromium acetylacetonate and sungucoline standard relaxation solutions containing 0, 1, 1.5, 2.5, 3,

[0080] 4, 5, 50 mM chromium acetylacetonate, and investigate the effect of different doses of chromium acetylacetonate on the T of the quantitative signal 1 .

[0081] (2) Sungucoline standard sample solution and magnolol standard sample solution

[0082] Take appropriate amounts of internal standard solution and sungucoline standard internal standard solution and prepare sungucoline standard sample solution 1 (SGL1) containing 3.00 mM chromium acetylacetonate, 2.15 mM magnolol and 2.80 mM sungucoline;

[0083] Accurately weigh the sungucoline reference substance and dissolve it in DMSO-d 6 to make the concentration of sungucoline 24.06 mM, that is, obtain sungucoline standard sample solution 2 (SGL2);

[0084] Accurately weigh the magnolol reference substance and dissolve it in DMSO-d 6 to make the concentration of magnolol 29.74 mM, that is, obtain magnolol standard sample solution. The above standard sample solutions are used to screen the characteristic NMR quantitative signals of C 20 -diterpenoid alkaloid components, internal standard quantitative NMR signals, optimize the coupling constant 1 J C-H , measure T 1 , etc. experiments

[0085] 5. Test solution of Aconiti Lateralis Radix Praeparata

[0086] Accurately weigh about 2.0 g of Aconiti Lateralis Radix Praeparata powder (No. 3 sieve), transfer it to a stoppered Erlenmeyer flask, add 3 mL of ammonia test solution, fully moisten it, accurately add 50 mL of isopropanol-ethyl acetate mixed solution (volume ratio of isopropanol to ethyl acetate is 1:1), weigh it, sonicate at a power of 300 W and a water temperature not higher than 25 °C for 30 min, let it cool, weigh it, make up the lost weight with isopropanol-ethyl acetate (1:1) mixed solution, shake well, filter, accurately absorb 25 mL of the filtrate, recover the solvent under reduced pressure to dryness at 40 °C or below. The residue is accurately added with 400 μL of internal standard solution to dissolve, and transferred to a 5 mm NMR tube, that is, obtained.

[0087] III. Experimental method

[0088] (1) Determination of characteristic NMR quantitative signals and internal standard quantitative NMR signals of C 20 -diterpenoid alkaloid components in Aconitum carmichaelii Debx

[0089] Take 400 μL of the test solution of Aconitum carmichaelii Debx, standard sample solution 2 of songorine (SGL2), and standard sample solution of magnolol, and transfer them into 5 mm NMR tubes respectively for 1 H- 13 C HSQC two-dimensional NMR detection.

[0090] The detection method is as follows: 25% NUS sampling mode; pulse sequence: hsqcetgpsisp2.2; temperature: 298 K; number of scans (NS): 16; number of dummy scans (DS): 32; relaxation delay time (D 1 ): 2.5 s; coupling constant (CNST2): 158 Hz; gain (RG): 101; spectral width (SW): 16.0 ppm (F2), 200.0 ppm (F1); number of sampling points (TD): 2048 (F2), 256 (F1); center frequency: 5.5 ppm (O1P), 85.0 ppm (O2P).

[0091] (2) Screening experiment of candidate internal standards

[0092] Take 400 μL of the candidate internal standard solution, transfer them into 5 mm NMR tubes respectively for 1 H- 13 C HSQC two-dimensional NMR detection.

[0093] The detection method is as follows: 25% NUS sampling mode; pulse sequence: hsqcetgpsisp2.3; temperature: 298 K; number of scans (NS): 16; number of dummy scans (DS): 16; relaxation delay time (D 1 ): 1.8 s; coupling constant (CNST2): 145 Hz; gain (RG): 101; spectral width (SW): 16.0 ppm (F2), 180.0 ppm (F1); number of sampling points (TD): 4096 (F2), 192 (F1); center frequency: 5.5 ppm (O1P), 85.0 ppm (O2P).

[0094] (3) Optimization experiment of coupling constant CNST2

[0095] Take 400 μL of standard sample solution 2 of songorine (SGL2) and standard sample solution of magnolol, transfer them into 5 mm NMR tubes respectively for HETLOC NMR experiment detection.

[0096] Determine the quantitative signals by HETLOC experiment1 J C-H Experimental method: Pulse sequence: dipsi2etgpjcsix1; 25% NUS was used; Temperature: 298K; Number of scans (NS): 16; Number of dummy scans (DS): 16; Relaxation delay time (D 1 ): 2.0 s; Gain (RG): 101; Spectral width (SW): 16.0 ppm (F2), 16.0 ppm (F1); Number of data points (TD): 4096 (F2), 512 (F1); Center frequency: 5.5 ppm (O1P), 5.5 ppm (O2P). After sampling, the coupling constant value was directly read in Topspin 4.1.4 software.

[0097] Take 400 μL of songorine standard sample solution (SGL1), transfer it to a 5 mm NMR tube, and perform 1 H- 13 C HSQC two-dimensional NMR detection to verify the accuracy of quantification using different CNST2 values. The detection parameters are as follows: 25% NUS sampling mode; Pulse sequence: hsqcetgpsisp2.2; Temperature: 298K; Number of scans (NS): 16; Number of dummy scans (DS): 32; Relaxation delay time (D 1 ): 2.5 s; Coupling constant (CNST2): 157 or 158 Hz; Gain (RG): 101; Spectral width (SW): 16.0 ppm (F2), 200.0 ppm (F1); Number of data points (TD): 2048 (F2), 256 (F1); Center frequency: 5.5 ppm (O1P), 85.0 ppm (O2P).

[0098] (4)C 20 -diterpenoid alkaloid component and internal standard quantitative signal T 1 Measurement

[0099] Take 400 μL of songorine standard sample solution 2 (SGL2), magnolol standard sample solution, songorine standard relaxation solution, and magnolol standard relaxation solution respectively, transfer them to 5 mm NMR tubes respectively, and perform T 1 Test or two-dimensional NMR detection.

[0100] Method for measuring longitudinal relaxation time T 1 : Pulse sequence: t1ir; Uniform sampling mode (traditional) was used; Temperature: 298K; Number of scans (NS): 16; Number of dummy scans (DS): 4; Relaxation delay time (D 1): 20 s; Gain: 33; Spectral width (SW): 16.0 ppm (F2), 2.0 ppm (F1); Number of sampling points (TD): 16384 (F2), 10 (F1); Central frequency (O1P): 5.5 ppm (F2), 5.5 ppm (F1). After sampling, the T1T2 process in the Dynamics function of Topspin 4.1.4 software was used to obtain T 1 。

[0101] 1 H- 13 The traditional sampling method for 1H-13C HSQC is as follows: Uniform sampling mode (traditional); Pulse sequence: hsqcetgpsisp2.2; Temperature: 298 K; Number of scans (NS): 16; Number of dummy scans (DS): 32; Relaxation delay time (D 1 ): 2.5 s; Coupling constant (CNST2): 158 Hz; Gain (RG): 101; Spectral width (SW): 16.0 ppm (F2), 200.0 ppm (F1); Number of sampling points (TD): 2048 (F2), 256 (F1); Central frequency: 5.5 ppm (O1P), 85.0 ppm (O2P).

[0102] IV. Experimental Results

[0103] (1) Determination of the characteristic NMR quantitative signals of C- diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata 20 - The characteristic NMR quantitative signals of C- diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata

[0104] The C- diterpenoid alkaloids in Aconiti Lateralis Radix Praeparata are mainly of hetisine type and napelline type, both of which have an exocyclic terminal double bond at the 17th position. Songorine is a representative component of C- diterpenoid alkaloids with a relatively high content in Aconiti Lateralis Radix Praeparata. The structure is as 20 shown. The double bond at the 17th position of songorine was selected as the key quantitative signal, with its δ 20 in the range of 4.56 - 5.16 ppm and δ Figure 1 in the range of 105.0 - 115.0 ppm (see H and C ). In the 1H-13C HSQC spectrum of the Aconiti Lateralis Radix Praeparata sample, it can be found that the key quantitative signal of this C- diterpenoid alkaloid is not interfered by other signals (see Figure 2 and 3 ), and can be used for the NMR quantification of C- diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata. 1 H- 13 The C- diterpenoid alkaloid in the 1H-13C HSQC spectrum of the Aconiti Lateralis Radix Praeparata sample, it can be found that the key quantitative signal of this C- diterpenoid alkaloid is not interfered by other signals (see 20 and Figure 4 and 5 ), and can be used for the NMR quantification of C- diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata. 20 - The NMR quantification of C- diterpenoid alkaloid components in Aconiti Lateralis Radix Praeparata.

[0105] (2) Optimization of the coupling constant CNST2 and selection of the internal standard

[0106] (2.1) Selection of internal standard

[0107] For HSQC qNMR, when selecting an internal standard, in addition to having a stable structure, not reacting with the solvent and sample, and the quantitative signal not overlapping with the quantitative signals of the components to be measured and other signal peaks, the most important point is that the hybridization types of the quantitative signals of the internal standard and the components to be measured should be the same. By consulting a large amount of literature and considering factors such as chemical structure similarity, sample stability, chemical shift, and economic cost, ginsenoside Re, ginsenoside Rb1, magnolol, arctiin, and baicalin were selected as candidate internal standards. By measuring the HSQC spectra, as shown in Figures 6 - 10 . After comprehensively evaluating the hybridization type of the quantitative signal, chemical shift, etc., magnolol was selected as the optimal internal standard, and its key quantitative signals δ H are in the range of 4.56 - 5.16 ppm, and δ C are in the key quantitative signal region of 105.0 - 115.0 ppm. The HSQC spectra signals of magnolol and songorine or aconite samples were superimposed. As shown in Figure 2 and 11 , the signal ranges of the terminal double bonds at positions 9 and 9' in the structure of magnolol in the HSQC spectrum are δ H 4.92 - 5.14 ppm, δ C 113.8 - 118.0 ppm, and this region does not overlap with the other signals of songorine (as shown in Figure 3 ) and aconite samples (as shown in Figure 12 ); the HETLOC experiment results found that the 1 J C-H value of the key quantitative signal of songorine is 158 Hz, and the 1 J C-H value of the terminal double bonds at positions 9 and 9' in the structure of magnolol is 157 Hz, which is close to the 20 J 1 value of the C C-H -diterpenoid alkaloid components. Therefore, it was comprehensively determined that magnolol is an ideal choice for the internal standard of HSQC qNMR of aconite C 20 -diterpenoid alkaloid components.

[0108] (2.2) Optimization of coupling constant CNST2

[0109] Using songorine as the representative component of C 20 -diterpenoid alkaloids, the quantitative accuracy of HSQC experiments with different CNST2 values was evaluated. Taking the SGL1 standard sample solution as the research object, the hsqcetgpsisp2.2 pulse sequence was used, and the measurements were carried out with CNST2 being 157 and 158 Hz respectively. The results are shown in Table 2.

[0110] Table 2. Influence of Different CNST2 on the Accuracy of Songguolin Content in SGL1 Sample Solution

[0111]

[0112] The experimental results show that when CNST2 is 158 Hz, the experimental value of the songguolin content in the SGL1 sample is closest to the true value. Therefore, the CNST2 parameter is determined to be 158 Hz.

[0113] (3) Determination of the Relaxation Delay Time (D 1 ) of the HSQC qNMR Method

[0114] (3.1) Measurement of the T 20 of the Quantitative Signals of C 1 -Diterpenoid Alkaloid Components and Internal Standard

[0115] The experimental results show that the T 1 values of the quantitative signals of songguolin and magnolol are 1.15 and 2.61 s, respectively. Since the T 1 value of the quantitative signal of magnolol is the longest, which is 2.61 s, according to D 1 ≥5×T 1 to ensure the accuracy of the NMR quantitative results, D 1 should be set to 13.0 s. In the traditional sampling mode, the sampling time is 15 h 3 min (as shown in Table 3). This method takes too long and is not conducive to realizing high-throughput NMR quantitative analysis. At the same time, problems such as poor stability of the instrument field strength or 13 C offset effect will occur during the long sampling time, which will inevitably affect the accuracy of the quantitative results. Therefore, the method needs to be further optimized to shorten the sampling time as much as possible.

[0116] Table 3. Sampling Times of Different Sampling Modes

[0117]

[0118] (3.2) Determination of the Relaxation Delay Time D 1

[0119] To shorten the sampling time of HSQC qNMR as much as possible, a relaxation reagent (chromium acetylacetonate) is added to optimize the longitudinal relaxation time (T 1 ) of the quantitative signal. The T 1 values of the quantitative signals of a series of magnolol standard relaxation solutions containing 0, 1, 1.5, 2.5, 3, 4, 5, and 50 mM chromium acetylacetonate are measured. The results are shown in Table 4 and Figure 13 .

[0120] Table 4. Influence of Different Concentrations of Chromium Acetylacetonate on the T 1 Value of the Quantitative Signal of the Internal Standard​

[0121]

[0122] The experimental results show that as the concentration of the relaxation reagent increases, the T of the internal standard quantitative signal 1 gradually shortens ( Figure 13 ), and its T 1 can be shortened to 300.13 ms at 5 mM, which means that D 1 can be shortened to about 1.5 s. However, the peak shape of this quantitative signal deteriorates significantly, which will affect the accuracy of the quantitative signal integration and thus the accuracy of the quantitative result; when the concentration of the relaxation reagent is 4 mM and 3 mM, the peak width, resolution and T 1 of the quantitative signal are relatively similar, and the latter is slightly better; due to the diverse types and large numbers of components in the actual sample, 3 mM is comprehensively considered as the addition concentration of chromium acetylacetonate, and D 1 is set to 2.5 s. Applying this method will save about 82% of the sampling time.

[0123] To further determine the feasibility of using 3 mM chromium acetylacetonate for quantifying the C 20 -diterpenoid alkaloid components, the effects of different concentrations of chromium acetylacetonate on the T 1 value of songorine were measured. The results show that 3 mM chromium acetylacetonate is also applicable to songorine, and can shorten the T 1 of its quantitative signal from 1153 ms to 288.24 ms, as shown in Table 5 and Figure 14 . Therefore, the concentration of chromium acetylacetonate is determined to be 3 mM, and D 1 is determined to be 2.5 s.

[0124] Table 5. Effects of different concentrations of chromium acetylacetonate on the T 1 value of songorine quantitative signal

[0125]

[0126] The above results show that the key parameters of the NMR quantitative method for C 20 -type diterpenoid alkaloids are determined as follows: the quantitative method is the internal standard method, the internal standard is magnolol, the relaxation reagent is chromium acetylacetonate (CA, added at 3 mM), the relaxation delay time (D1 = 2.5 s), and the coupling constant (CNST2 = 158).

[0127] Experimental Example 2 Methodology Verification

[0128] I. Linear Relationship

[0129] 1. Songorine standard internal standard solution

[0130] Accurately weigh the stigmasterol reference substance, dissolve it in the internal standard solution to prepare a stock solution containing 11.36 μM of stigmasterol per 1 mL, and then dilute and prepare stigmasterol standard internal standard solutions with concentrations of 0.1136, 0.5678, 1.1357, 2.2714, 4.5428, 6.8142, and 9.0856 mM for investigating the linear relationship, detection limit, quantification limit, and precision.

[0131] 2. NMR detection

[0132] Take 400 μL of each of the 8 different concentrations of stigmasterol standard internal standard solutions in this experimental example, transfer them to 5 mm NMR tubes respectively, and measure the HSQC spectra of each standard solution according to the detection method described in Example 1. Using the 2n 宋果灵 / n 厚朴酚 (n is the amount of substance of the compound) ratio as the ordinate y and the stigmasterol quantitative signal volume / honokiol quantitative signal volume as the abscissa x, plot a linear relationship graph, and obtain the regression equation y = 0.5038x + 0.0477, r 2 = 0.9996. The results show a good linear relationship, and the results are shown in Table 6.

[0133] Table 6. Results of linear investigation

[0134]

[0135] II. Detection limit and quantification limit

[0136] Take the experimental results of Sample No. 1 in Table 6 for calculation. When S / N = 10, the quantification limit is 211.32 μM, and when S / N = 3, the detection limit is 63.39 μM.

[0137] III. Repeatability

[0138] Take the FZ-1 sample, prepare 6 parallel sample solutions according to the preparation method of the test solution described in Experimental Example 1, and calculate the RSD by the integral peak volume ratio of the C 20 -diterpenoid alkaloid component to the honokiol quantitative signal in the sample to judge the repeatability of this method. The results show that the RSD is 2.10% (see Table 7), and the repeatability is good.

[0139] Table 7 Repeatability experiment

[0140]

[0141] IV. Stability test

[0142] Take the FZ-1 sample, prepare 1 test solution according to the preparation method of the test solution described in Experimental Example 1, and measure it at 0, 2, 4, 8, 12, 24, and 48 h respectively, and record the C in the sample 20-The integral peak volume ratio of the key quantitative signal of the diterpenoid alkaloid component to the quantitative signal of magnolol was calculated, and the RSD was <2.0%, indicating that the sample solution remained relatively stable after standing at room temperature for 48 h (Table 8).

[0143] Table 8 Stability experiment

[0144]

[0145] V. Quantitative results

[0146] The test solution of 12 batches of raw Aconiti Lateralis Radix Preparata samples (FZ-1 to FZ-12 in Table 1) was prepared according to the preparation method of the test solution described in Experimental Example 1, and quantitative detection was carried out according to the detection method of Example 1. The results are shown in Table 9: The content of C 20 -diterpenoid alkaloid components in raw Aconiti Lateralis Radix Preparata was 0.2989 - 1.4002 μM / g, and the percentage content was 0.0056 - 0.0668%, indicating that the C 20 -diterpenoid alkaloid components and C 19 -ester-type alkaloid contents were in the same order of magnitude and were also important components that needed to be quality controlled.

[0147] Table 9 Content of C 20 -diterpenoid alkaloid components in raw Aconiti Lateralis Radix Preparata

[0148]

[0149] The above results indicate that the detection method established in Example 1 of the present invention has a good linear relationship, the quantitative limit is 211.32 μM, the detection limit is 63.39 μM, has good repeatability and stability, and can be used for quantitative detection of C 20 -diterpenoid alkaloid components in raw Aconiti Lateralis Radix Preparata in practical applications.

[0150] As can be seen from the above examples and experimental examples, the present invention has established a two-dimensional NMR quantitative method for rapidly quantifying C 20 -diterpenoid alkaloid components in Aconiti Lateralis Radix Preparata based on HSQC NMR technology. The present invention uses magnolol as an internal standard, applies a relaxation reagent and / or non-uniform sampling (NUS) method to improve the sampling efficiency. The added relaxation reagent is 3 mM chromium acetylacetonate, and the relaxation delay time is 2.5 s. Using the exocyclic double bond at the 17th position of C 20 -diterpenoid alkaloids as the key quantitative signal and the terminal double bonds at the 9th and 9' positions in magnolol as the quantitative signal of the internal standard, the content is calculated according to the integral value. This method has the advantages of high sampling efficiency, good repeatability, and high stability. The present invention promotes C 20- The quality control, research on pharmacodynamic mechanisms, and development of related new drugs of diterpenoid alkaloids have good application prospects. This method has the advantages of high sampling efficiency and good repeatability. The present invention is applicable to rapidly and accurately quantifying the content of C 20 - diterpenoid alkaloid components in processed Aconiti Lateralis Radix Praeparata and its cut pieces, and plants of the genus Aconitum of the Ranunculaceae family.

Claims

1. A rapid quantitative method for the determination of C in Radix Aconiti Lateralis 20 - A two-dimensional nuclear magnetic resonance quantification method for diterpene alkaloid components, characterized in that: It includes the following steps: Step 1, extracting and filtering the crude powder of Chinese medicinal materials, taking the filtrate, removing the solvent and adding the internal standard solution to dissolve it to prepare a test solution, wherein the internal standard solution contains magnolol; Step 2, using a two-dimensional nuclear magnetic resonance analyzer to detect the test solution; Step 3: Collect HSQC spectra and use C 20 The 17-position exocyclic double bond of diterpene alkaloids was used as the key quantitative signal, and the content was calculated based on the integral value.

2. The quantitative method according to claim 1, characterized in that: The internal standard solution also includes chromium acetylacetonate, wherein the concentration of chromium acetylacetonate is 1-4 mM.

3. The quantitative method according to claim 2, characterized in that: The concentration of chromium acetylacetonate in the internal standard solution is 3-4 mM.

4. The quantitative method according to claim 1 or 2, characterized in that: The parameters of the two-dimensional nuclear magnetic resonance analyzer in step 2 are: a coupling constant of 157 or 158 Hz; and / or a relaxation delay time of 1.5-10 s.

5. The quantitative method according to claim 4, characterized in that: The coupling constant is 158 Hz; and / or the relaxation delay time is 2.5 s.

6. The quantitative method according to claim 1, characterized in that: In step 1, the internal standard solution is obtained by mixing magnolol with chromium acetylacetonate and a deuterated reagent; wherein the concentration of magnolol is 1-10 mM; and the deuterated reagent is selected from at least one of DMSO-d6, methanol-d4, and acetone-d6; And / or, in the HSQC spectrum described in step 3, the terminal double bonds at positions 9 and 9' in magnolol are used as the quantitative signals of the internal standard, and the region of the quantitative signals of the internal standard is δ H 4.92~5.14ppm,δ C 113.8~118.0ppm; And / or, the region of the key quantitative signal in step 3 is δ H 4.56~5.16ppm,δ C 105.0~115.0ppm.

7. The quantitative method according to claim 1, characterized in that: The Chinese herbal medicine is aconite root; And / or, in step 1, the extraction is a mixture of crude Chinese medicinal material powder, ammonia test solution and isopropanol-ethyl acetate solution, the mass volume ratio of the aconite root, ammonia test solution and isopropanol-ethyl acetate solution is 2g:1-5mL:20-100mL, and the volume ratio of isopropanol to ethyl acetate in the isopropanol-ethyl acetate solution is 1-5:1; And / or, in step 1, the extraction is ultrasonic extraction, the temperature is ≤25°C, the power is ≥ 300W, time is 20 to 40 minutes; the solvent removal is carried out under reduced pressure at 30°C; And / or, in step 1, the volume ratio of the filtrate to the internal standard solution is 10-50 mL:400 μL.

8. The quantitative method according to claim 1, characterized in that: In step 2, the parameters of the nuclear magnetic resonance analyzer include: The sampling method is non-uniform sampling NUS, and the sparsity of NUS is 20% to 50%; the pulse sequence is selected from any one of hsqcetgpsisp, hsqcetgpsisp.2, hsqcetgpsisp2, hsqcetgpsisp2.2, hsqcedetgpsisp, hsqcedetgpsisp.2, hsqcedetgpsisp2, hsqcedetgpsisp2.2, hsqcedetgpsisp2.3, and hsqcedetgpsisp2.4; the temperature is 298K; the number of scans NS is 4 to 32; the number of empty scans DS is ≥16; the gain RG is 4 to 101; the frequency range of the scan in the F2 dimension is 6 to 16ppm, and the frequency range of the scan in the F1 dimension is 80 to 200ppm; the number of sampling points TD in the F2 dimension is ≥1024, and the number of sampling points TD in the F1 dimension is ≥128; 1 The center frequency of H is 3 to 8 ppm; 13 The center frequency of C is 50~100ppm.

9. The quantitative method according to claim 8, characterized in that: The sparsity of NUS is 25%; the pulse sequence is hsqcetgpsisp2.2; the number of scans NS is 16; the number of empty scans DS is 32; the gain RG is 101; the frequency range scanned in the F2 dimension is 16.0ppm, and the frequency range scanned in the F1 dimension is 200.0ppm; the number of sampling points TD in the F2 dimension is 2048, and the number of sampling points TD in the F1 dimension is 256; 1 The center frequency of H is 5.5ppm; 13 The center frequency of C is 85.0ppm.

10. The quantitative method according to claim 1, characterized in that: The HSQC spectrum described in step 3 was phase corrected and baseline corrected using Topspin4.1.1 software; And / or, the calculation formula for calculating the content according to the integral value in step 3 is: C S =N R / N S ×C R ×A S / A R Where: C S C in the sample 20 -Molar concentration of diterpene alkaloids mM; N R and N S represent the internal standard quantitative signal and C 20 -The number of protons of the key quantitative signal of diterpene alkaloids; C R is the molar concentration of the internal standard in mM; A S A is the integrated peak volume of the quantitative signal of the measured sample; R is the integrated peak volume of magnolol quantitative signal.

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