Determination of the contents of four alkaloids in Uncaria rhynchophylla

Abstract

The present invention belongs to the technical field of drug analysis, and discloses a method for determining the content of four alkaloids in Uncaria rhynchophylla. The determination method comprises the following steps: (1) preparing a test solution of total alkaloids of Uncaria rhynchophylla; (2) adding an internal standard to the test solution of total alkaloids of Uncaria rhynchophylla prepared in step (1), and then loading it into a nuclear magnetic resonance tube, performing a quantitative nuclear magnetic resonance hydrogen spectrum test, and integrating the selected signals of the internal standard and the four alkaloids respectively; (3) calculating using a formula, and respectively obtaining the content of the four alkaloids in the total alkaloids of Uncaria rhynchophylla. The present invention adopts a quantitative hydrogen nuclear magnetic resonance analysis technology to determine the content of the four alkaloids in Uncaria rhynchophylla, does not need to prepare a working curve with a standard reference substance, and can use a known common chemical substance as a reference, which can greatly save the test cost; and the sample test pretreatment is simple, the test time is short, and the measurement result has good linearity, sensitivity, precision and accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining the contents of four alkaloids in Uncaria rhynchophylla. Background Art

[0002] As a traditional Chinese medicine, Uncaria rhynchophylla has the effects of calming wind and relieving convulsions, clearing heat and calming the liver. It is used for internal liver wind movement, epilepsy convulsions, high fever convulsions, colds and convulsions, crying in children, eclampsia during pregnancy, and headache and dizziness. Uncaria rhynchophylla is the dried hooked stems and branches of the Rubiaceae plants Uncaria rhynchophylla (Miq.) Miq. ex Havil., Uncaria macrophylla Wall., Uncaria hirsuta Havil., Uncaria sinensis (Oliv.) Havil., or Uncaria sessilifructus Roxb., among which Uncaria rhynchophylla (Miq.) Miq. ex Havil. is the most commonly used. Total alkaloids of Uncaria rhynchophylla are important active ingredients of the traditional Chinese medicine Uncaria rhynchophylla, mainly composed of indole alkaloids represented by rhynchophylline and isorhynchophylline. Indole alkaloids represented by rhynchophylline and isorhynchophylline are indicator components for quality control of Uncaria rhynchophylla.

[0003] At present, the content of four alkaloids in the Chinese medicine Uncaria rhynchophylla - rhynchophylline, isorhynchophylline, conosine and isoconosine - is mainly determined by high performance liquid chromatography equipped with various detectors mainly based on DAD, or tandem mass spectrometry. Although the high performance liquid chromatography method has high accuracy, it needs to prepare the working curve with high purity and expensive standard products before determination, and the sample generally needs to be pre-treated before determination. The test process is also relatively complicated, including balancing the instrument, preparing the working curve, sample testing, etc. The overall test time is long and the reagent consumption is high.

[0004] Therefore, there is an urgent need to provide a method for determining the contents of the four alkaloids in Uncaria rhynchophylla that is simple, time-saving and accurate. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a method for determining the content of four alkaloids in Uncaria rhynchophylla, which is simple, time-saving and highly accurate.

[0006] The first aspect of the present invention provides a method for determining the contents of four alkaloids in Uncaria rhynchophylla.

[0007] The four alkaloids in the Uncaria rhynchophylla are rhynchophylline, isorhynchophylline, conosine and isoconosine, and their structural formulas are as follows:

[0008] The structural formula of rhynchophylline:

[0009]

[0010] The structural formula of isorhynchophylline:

[0011]

[0012] The structural formula of conosine:

[0013]

[0014] The structural formula of isoconoxine:

[0015]

[0016] Specifically, the method for determining the contents of four alkaloids in Uncaria rhynchophylla comprises the following steps:

[0017] (1) taking a sample of Uncaria rhynchophylla to prepare a test solution of total Uncaria rhynchophylla alkaloids;

[0018] (2) adding an internal standard to the total alkaloids of Uncaria rhynchophylla test solution prepared in step (1), then loading it into a nuclear magnetic resonance tube, performing a quantitative nuclear magnetic resonance hydrogen spectrum test, and integrating the selected signals of the internal standard and the four alkaloids respectively;

[0019] (3) Using formula C X =100%×(S X ×m I.S ×M X ) / (S I.S ×M I.S ×m TA ) to calculate the content of the alkaloids to be tested in the total alkaloids of Uncaria rhynchophylla;

[0020] Where: C X is the content of the alkaloid to be tested in the total alkaloids of Uncaria rhynchophylla,

[0021] S X and S I.S are the integrated areas of the quantitative peaks of the alkaloid to be tested and the internal standard in the Uncaria rhynchophylla sample,

[0022] M X and M I.S are the molar molecular weights (g / mol) of the alkaloid to be tested and the internal standard quantitative peaks in the Uncaria rhynchophylla sample,

[0023] m TA and m I.S are respectively the masses (μg) of the alkaloids to be tested, the total alkaloids of Uncaria rhynchophylla and the internal standard in the Uncaria rhynchophylla sample.

[0024] Preferably, in step (1), the process of preparing the total alkaloids of Uncaria rhynchophylla test solution is: crushing the Uncaria rhynchophylla sample, extracting it with an acid solution, then adjusting the pH to be alkaline, then extracting it with an organic solvent, drying it to obtain the total alkaloids of Uncaria rhynchophylla, and finally dissolving the total alkaloids of Uncaria rhynchophylla in a deuterated solvent to obtain the total alkaloids of Uncaria rhynchophylla test solution.

[0025] Preferably, the Uncaria rhynchophylla sample is a sample containing rhynchophylline, isorhynchophylline, conosine and isorhynchophylline, including but not limited to the traditional Chinese medicine Uncaria rhynchophylla.

[0026] Preferably, the acid solution is hydrochloric acid with a mass fraction of 0.5%-2%; further preferably, the acid solution is hydrochloric acid with a mass fraction of 0.8%-1.5%.

[0027] Preferably, the alkalinity has a pH value of 8-11; further preferably, the alkalinity has a pH value of 9-10.

[0028] Preferably, the organic solvent is selected from at least one of organic acids, esters, acid esters, hydrocarbon derivatives or ethers; further preferably, the organic solvent is at least one of ethyl acetate, dichloromethane or chloroform.

[0029] Preferably, the deuterated solvent is selected from one of deuterated benzene, deuterated acetonitrile, deuterated acetone, deuterated dimethyl sulfoxide (DMSO-d6) or deuterated pyridine; further preferably, the deuterated solvent is deuterated dimethyl sulfoxide (DMSO-d6). By selecting a solvent for dissolving the total alkaloids of Uncaria rhynchophylla, the peak effect can be optimized, so that the internal standard and the sample to be tested can be well separated.

[0030] Preferably, in step (2), the internal standard is 3,4,5-trimethoxybenzaldehyde. 3,4,5-trimethoxybenzaldehyde is used as the internal standard, and its signal is close to the quantitative signal of the analyte without overlapping, which solves the problem of separation between the internal standard and the analyte signal, and avoids the accuracy problem caused by inconsistent baselines.

[0031] Preferably, the selected signal in step (2) is an NH signal, and the NH signal is used as the quantitative peak. Using the low-field NH signal as the quantitative peak can effectively solve the problem of overlapping signals of rhynchophylline components.

[0032] Preferably, in step (2), the NH signal peaks of rhynchophylline, isorhynchophylline, konocine and isorhynchophylline are located at δ H 10.11s, δ H 10.32s, δ H 10.13s, and δ H 10.33s.

[0033] Preferably, in step (2), the NH signal peak of the internal standard is located at δH 9.88s.

[0034] Preferably, in step (2), when the deuterated solvent is deuterated dimethyl sulfoxide, the NH signal peaks of rhynchophylline, isorhynchophylline, konosine and isorhynchophylline are located at δ H 10.11s, δ H 10.32s, δ H 10.13s, and δ H 10.33s.

[0035] Preferably, in step (2), the longitudinal relaxation time (T1 value) of each quantitative peak is determined by t1ir pulse, and the longitudinal relaxation time of rhynchophylline, isorhynchophylline, conosine and isoconosine is 0.99s, 0.99s, 1.40s, 1.11s, respectively, and the longitudinal relaxation time of the internal standard is 2.33s. By the longitudinal relaxation time of the quantitative target proton obtained, parameters such as relaxation delay time (d1) are set on this basis to ensure the accuracy of the established quantitative method.

[0036] Preferably, the relaxation delay time is 4-6 times the longest T1 of the protons to be measured.

[0037] Preferably, in step (2), the parameters also include a relaxation delay time (d1) of 8-13 s. Setting a suitable d1 can ensure that all quantitative protons have sufficient time to reach magnetization equilibrium (recover to a low energy state) after being excited, thereby avoiding insufficient measured proton integration due to insufficient relaxation time.

[0038] Preferably, in step (2), the parameters also include setting the number of scans to 32-128 times; further preferably, the number of scans is 64 times. Increasing the number of scans can improve 1 The signal-to-noise ratio of H NMR spectra can improve the precision and sensitivity of analytical methods.

[0039] Preferably, in step (2), the parameters of the nuclear magnetic resonance spectrum test are: flip angle (flip angle) = 90°, relaxation delay time (relaxation delay, d1) = 10 s, time domain data length (size of fid, TD)) = 65536, number of scans (NS) = 64, spectral width (spectral width, SW) = 16.0221 ppm, sampling time (acquisition time, AQ) = 3.408 s, probe temperature (requested probe temperature, TE) = 300 K.

[0040] The second aspect of the present invention provides the application of the above determination method in the preparation of Uncaria rhynchophylla medicine.

[0041] The Uncaria rhynchophylla medicine is a medicine containing Uncaria rhynchophylla, rhynchophylline, isorhynchophylline, conosine and isoconosine.

[0042] The preparation of Uncaria rhynchophylla medicines includes, but is not limited to, the control of the quality of the raw materials, the analysis of the activity of the medicine during the preparation process, and the monitoring of the quality of the finished medicine.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] (1) The present invention uses quantitative hydrogen nuclear magnetic analysis technology to measure the content of four alkaloids in Uncaria rhynchophylla, and achieves quantification based on the response of protons. The resonance response of proton signals is non-selective and does not rely on high-purity standard reference substances. This method does not require the preparation of working curves using standard reference substances of the four alkaloids in Uncaria rhynchophylla, and can use known common chemical substances as references. This method can greatly save testing costs.

[0045] (2) Compared with quantitative techniques such as high performance liquid chromatography and liquid chromatography-mass spectrometry, the determination method provided by the present invention is efficient and rapid, the sample pretreatment is simple, and no balancing instrument is required. The measurement time of a single sample is generally within a few minutes to more than ten minutes, and the test time is short.

[0046] (3) The determination method provided by the present invention is used to determine the contents of the four alkaloids in Uncaria rhynchophylla, and the determination results have good linearity, sensitivity, precision and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 For the rhynchophylline in Example 1 1 H-NMR spectrum;

[0048] Figure 2 is the isorhynchophylline in Example 1 1 H-NMR spectrum;

[0049] Figure 3 For the conosine alkali in embodiment 1 1 H-NMR spectrum;

[0050] Figure 4 is the isoconoxine in Example 1 1 H-NMR spectrum;

[0051] Figure 5 is the total alkaloids of Uncaria rhynchophylla in Example 1 1 H-NMR spectrum;

[0052] Figure 6 This is the HMBC spectrum of the total alkaloids of Uncaria rhynchophylla in Example 1. DETAILED DESCRIPTION

[0053] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0054] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0055] Example 1: Determination of four index components in commercially available Chinese medicine Uncaria rhynchophylla

[0056] The Chinese herbal medicine Uncaria rhynchophylla was crushed, 10.0 g was weighed, and ultrasonic extraction was performed with 100 mL HCl (1%) for 30 minutes, and the extraction was performed three times. The extracts were combined, and the pH was adjusted to 9-10 with ammonia water, and extracted with ethyl acetate three times. After the extracts were combined, the solvent was evaporated under reduced pressure in a rotary evaporator to obtain a total alkali solid, and the total alkali was dried overnight in a nitrogen blower.

[0057] Accurately weigh 30.0 mg of total alkali, quantitatively add 50 μg of 3,4,5-trimethoxybenzaldehyde, add 0.5 mL of DMSO-d6 to fully dissolve, shake well and then take samples.

[0058] The test was conducted using a Bruker AVANCE III 600MHz NMR instrument equipped with an ultra-low temperature probe, and a NMR tube of NORELL5mm ST-500-7NMR. The Bruker standard pulse zg30 was used, and the parameters were set as follows: flip angle = 90°, relaxation delay (d1) = 10s, size of fid (TD) = 65536, number of scans (NS) = 64, spectral width (SW) = 16.0221ppm, acquisition time (AQ) = 3.408s, requested probe temperature (TE) = 300K. The test time for a single sample was 14 minutes. The obtained spectrum was processed by TopSpin 4.1.0 provided by Bruker Instruments, and the selected quantitative peaks were integrated, and the content of the analyte was calculated by the following formula.

[0059] C X =100%×(S X ×m I.S ×M X ) / (S I.S ×M I.S ×m TA ) to calculate the content of the alkaloids to be tested in the total alkaloids of Uncaria rhynchophylla;

[0060] Where: C Xis the content of the alkaloid to be tested in the total alkaloids of Uncaria rhynchophylla,

[0061] S X and S I.S are the integrated areas of the quantitative peaks of the alkaloid to be tested and the internal standard in the Uncaria rhynchophylla sample,

[0062] M X and M I.S are the molar molecular weights (g / mol) of the alkaloid to be tested and the internal standard quantitative peaks in the Uncaria rhynchophylla sample,

[0063] m TA and m I.S are respectively the masses (μg) of the alkaloids to be tested, the total alkaloids of Uncaria rhynchophylla and the internal standard in the Uncaria rhynchophylla sample.

[0064] Rhynchophylline, isorhynchophylline, conosine and isorhynchophylline 1 H-NMR images Figures 1 to 4 In DMSO-d6, the NH signal peaks of rhynchophylline, isorhynchophylline, konosine and isorhynchophylline are located at δ H 10.11s, δ H 10.32s, δ H 10.13s, and δ H 10.33s, and they do not overlap with each other or other signals in the total base. Figure 5 Total alkaloids from Uncaria rhynchophylla 1 H-NMR spectrum, Figure 5 In the figure, the number 1 represents rhynchophylline, the number 2 represents isorhynchophylline, the number 3 represents conosine, and the number 4 represents isoconosine. In addition, the two-dimensional HMBC spectrum further shows that the above NH has specificity and can be used as a quantitative peak. To improve the accuracy of the method, the present embodiment tests the T1 value of each quantitative peak determined by t1ir pulse, and the T1 values ​​of rhynchophylline, isorhynchophylline, conosine and isoconosine are 0.99s, 0.99s, 1.40s, 1.11s, respectively, and the T1 value of the internal standard is 2.33s.

[0065] Figure 6 is the HMBC spectrum of total alkaloids of Uncaria rhynchophylla ( 1 H detected heteronuclear multiple bondcorrelation, 1 H heteronuclear multiple bond correlation). Figure 6 The number 1 represents rhynchophylline, the number 2 represents isorhynchophylline, the number 3 represents conosine, and the number 4 represents isoconosine. Through the attribution of the relevant cross peaks shown in the HMBC spectrum, it was further determined that each quantitative peak did not overlap with other signals, and the selected quantitative peak was specific. For example, the NH signal δ HOnly four HMBC correlations are shown at 10.11s, which can be attributed to NH and C-7 (δ C 55.2s), C-8(δ C 133.8s), C-13(δ C 141.9s), and C-2 (δ C 179.6s), no correlation peaks of other impurities were observed, which further confirmed that the NH signal of rhynchophylline did not overlap with other signals.

[0066] LC-MS was used to verify the results of H NMR quantification. The verification results are shown in Table 1.

[0067] Table 1

[0068]

[0069] The results show that the contents of the four rhynchophylline detected by the detection method provided by the present invention are basically consistent with the LC-MS method, and the detection accuracy is high. However, compared with the traditional high-performance liquid phase detection method, the detection method provided by the present invention has simple pre-treatment, does not require expensive standard reference substances, and has lower detection costs.

[0070] In this embodiment, deuterated dimethyl sulfoxide is used as the solvent for dissolving total alkaloids of Uncaria rhynchophylla. If it is replaced with deuterated benzene, deuterated acetonitrile, deuterated acetone or deuterated pyridine, similar effects can be achieved. If other solvents are used, the ideal separation effect cannot be achieved, and the accuracy of content determination is low, such as deuterated chloroform and deuterated methanol.

[0071] Example 2: Method Validation

[0072] (1) Linear range

[0073] Six different concentrations (25-400 μg / mL) of rhynchophylline, isorhynchophylline, conosine and isoconosine reference substances were prepared in triplicate, 0.5 mL was transferred to NMR tubes, 50 μg of internal standard was quantitatively added, and the optimized experimental parameters and conditions were used to measure the concentrations of rhynchophylline, isorhynchophylline and conosine. 1 H NMR spectrum. The obtained spectrum is processed to obtain the integral results of each quantitative peak, and then the measured value of each substance content can be obtained according to the formula. x ), the ratio of the peak area of ​​the analyte to the internal standard quantitative peak is the ordinate (S x / S I.S ), draw the standard curve. 2 The linearity of the analytes was evaluated. The results are shown in Table 2. The correlation coefficients of the four compounds were all greater than 0.999, showing good linearity.

[0074] (2) Sensitivity

[0075] The detection line (LOD) was calculated based on the signal-to-noise ratio S / N=3. The LODs of the analytes rhynchophylline, isorhynchophylline, conosine and isoconosine were 10.2μg / mL, 4.2μg / mL, 5.1μg / mL and 5.4μg / mL, respectively, obtained by diluting the standard solution. The limit of quantification (LOQ) was determined based on the lower limit of the working curve, and the LOQs of the analytes were all 25.0μg / mL. The experimental results are shown in Table 2. The above data show that the method has good sensitivity.

[0076] (3) Intra-day precision and inter-day precision

[0077] Three different concentration levels (50 μg / mL, 100 μg / mL and 300 μg / mL) were prepared in parallel, and 0.5 mL was transferred to the NMR tube for measurement. 1 H NMR spectrum, the data measured once were used to test the intra-day precision, and the data measured for three consecutive days were used to test the inter-day precision. The experimental results are shown in Table 2. The RSD value range of intra-day precision was 0.40%-2.51%, and the RSD range of inter-day precision was 0.41%-1.73%, indicating that the quantitative NMR method has good precision.

[0078] (4) Accuracy

[0079] In this experiment, three concentration levels of 80%, 100% and 120% of the reference substances were added to a certain amount of total alkaloids of Rhynchophylla, and the recovery results were used to test the accuracy of the quantitative NMR method. Three groups of solutions at each concentration level were prepared in parallel and then transferred to 0.5 mL NMR tubes for measurement. 1 H NMR spectrum. The experimental results are shown in Table 2. From the data, it can be seen that the recovery rates of the four quantified substances are between 97.1% and 104.4%, respectively. This shows that the experimental analysis method has good accuracy.

[0080] Table 2

[0081] 1 2 3 4 Linearity (μg / mL) 25-400 25-400 25-400 25-400 <![CDATA[Correlation coefficient (R 2 )]]> 0.9995 0.9996 0.9994 0.9995 LOD (μg / mL) 10.2 4.2 5.1 5.4 LOQ (μg / mL) 25.0 25.0 25.0 25.0 Intra-day precision (RSD%) 0.40-1.38 0.85-2.51 0.19-0.86 1.22-2.19 Inter-day precision 0.85-1.73 1.01-1.63 0.41-1.53 0.99-1.49 Collection rate (%) 98.3-103.6 99.5-104.4 97.9-103.5 97.1-104.0

Claims

1. A method for determining the contents of four alkaloids in Rhynchophylla, wherein the four alkaloids are rhynchophylline, isorhynchophylline, conosine and isoconosine, characterized in that: The following steps are involved: (1) taking a sample of Uncaria rhynchophylla to prepare a test solution of total Uncaria rhynchophylla alkaloids; (2) adding an internal standard to the total alkaloids of Uncaria rhynchophylla test solution prepared in step (1), performing a quantitative nuclear magnetic resonance hydrogen spectrum test, and integrating the selected signals of the internal standard and the four alkaloids respectively; (3) Using formula C X =100%×(S X ×m I.S ×M X ) / (S I.S ×M I.S ×m TA ) to calculate the content of the alkaloids to be tested in the total alkaloids of Uncaria rhynchophylla; Where: C X is the content of the alkaloid to be tested in the total alkaloids of Uncaria rhynchophylla, S X and S I.S are the integrated areas of the quantitative peaks of the alkaloid to be tested and the internal standard in the Uncaria rhynchophylla sample, M X and M I.S are the molar molecular weights of the alkaloid to be tested and the internal standard quantitative peaks in the Uncaria rhynchophylla sample, m TA and m I.S are the masses of total alkaloids of Uncaria rhynchophylla and internal standard in the Uncaria rhynchophylla sample, respectively; In step (1), the process of preparing the total alkaloids of Uncaria rhynchophylla test solution is as follows: crushing the Uncaria rhynchophylla sample, extracting it with an acid solution, adjusting the pH to be alkaline, extracting it with an organic solvent, drying it, and finally dissolving the total alkaloids of Uncaria rhynchophylla in a deuterated solvent to obtain the total alkaloids of Uncaria rhynchophylla test solution; In step (2), the longitudinal relaxation time of each quantitative peak is determined by using a t1ir pulse, and the longitudinal relaxation times of rhynchophylline, isorhynchophylline, conosine and isoconosine are 0.99s, 0.99s, 1.40s, and 1.11s, respectively, and the longitudinal relaxation time of the internal standard is 2.33s; The selected signal in step (2) is the NH signal, and the NH signal is used as the quantitative peak. The two-dimensional HMBC spectrum further shows that the above NH has specificity as the quantitative peak; in step (2), in DMSO-d6, the NH signal peaks of rhynchophylline, isorhynchophylline, conosine and isoconosine are respectively located at δ H 10.11s, δ H 10.32s, δ H 10.13s and δ H 10.33s; In step (2), the parameters of the nuclear magnetic resonance spectrum test are: deflection angle = 90°, relaxation delay time = 10 s, time domain data length = 65536, number of scans = 64, spectrum width = 16.0221 ppm, sampling time = 3.408 s, and probe temperature = 300 K.

2. The measuring method according to claim 1, characterized in that The alkalinity is a pH value of 8-11.

3. The measuring method according to claim 1, characterized in that In step (2), the internal standard is 3,4,5-trimethoxybenzaldehyde.

4. Use of the determination method according to any one of claims 1 to 3 in the preparation of Uncaria rhynchophylla medicine.

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