A quality evaluation method for determining the optimal processing technology of Mongolian medicine Herba Aconiti Kusnezoffii preparata

Abstract

The quality evaluation method for determining the best processing technology of Mongolian medicine terminalia chebula-processed aconiti kusnezoffii of the present invention belongs to the technical field of traditional Chinese medicine analysis and detection. The quality evaluation method of the present invention takes the content changes of terminalia chebula and alkaloids in terminalia chebula-processed aconiti kusnezoffii as evaluation indicators, establishes an orthogonal optimization test to determine the best processing method of terminalia chebula-processed aconiti kusnezoffii; establishes a method for comprehensively evaluating the quality of terminalia chebula-processed aconiti kusnezoffii, which is also applicable to raw aconiti kusnezoffii, and evaluates the quality of processed aconiti kusnezoffii by multiple detection means, including impurities, moisture, total ash, thin layer, content determination involving high performance liquid chromatography, and fingerprint. It is proved by test examples that the method is stable and reliable, and can evaluate the quality of terminalia chebula-processed aconiti kusnezoffii in a relatively comprehensive manner. At the same time, the Mongolian medicine terminalia chebula-processed aconiti kusnezoffii processing method optimized by the evaluation method is suitable for scientific research and production practice.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis and detection, and specifically relates to a quality evaluation method for determining the best processing technology of Mongolian medicine semen myristicae praeparata radix aconiti kusnezoffii. Background Art

[0002] Aconitum kusnezoffii Reichb. is the dried tuber of Aconitum kusnezoffii Reichb., an annual herbaceous plant of the Ranunculaceae family. It has a relatively hard texture and aliases such as "duck head", "medicinal sheep wormwood", "chicken head grass", "hundred-step grass", etc. When used as a medicinal material together with Radix Aconiti, they can be collectively referred to as "Aconitum". Aconitum kusnezoffii has a long history of application in the field of traditional Chinese medicine. It was first recorded in "Shennong Ben Cao Jing" and has the effects of dispelling wind and dampness, warming the meridians and relieving pain. Because its therapeutic effect coexists with its toxic effect, it must be detoxified before clinical use. Mongolian medicine is a characteristic ethnic medicine in China. It has rich experience and unique characteristics in processing Aconitum kusnezoffii. In the processing method of semen myristicae praeparata radix aconiti kusnezoffii, because of its significant detoxification effect and effective retention of the components in Aconitum kusnezoffii, it is widely used in various Mongolian medicine prescriptions. However, the specific processing methods of semen myristicae praeparata radix aconiti kusnezoffii are not the same in different medical books, and there is a lack of a complete quality evaluation system. Therefore, it is of great significance to standardize the processing and detoxification technology of semen myristicae praeparata radix aconiti kusnezoffii by means of modern detection means and construct a quality evaluation system. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a quality evaluation method for determining the best processing technology of semen myristicae praeparata radix aconiti kusnezoffii. The research content of this quality evaluation method is comprehensive and of great significance for constructing a quality evaluation system for Mongolian medicine processed Aconitum kusnezoffii.

[0004] To solve the above technical problems, the technical solution of the present invention is specifically as follows:

[0005] The present invention provides a quality evaluation method for determining the best processing technology of semen myristicae praeparata radix aconiti kusnezoffii, including the following steps:

[0006] Step 1: Establish an orthogonal optimization test for semen myristicae praeparata radix aconiti kusnezoffii to obtain semen myristicae praeparata radix aconiti kusnezoffii with different processing methods;

[0007] Step 2: Apply an Aobitrap LC-MS detector to compare the changes in alkaloids and tannin components in semen myristicae praeparata radix aconiti kusnezoffii with different processing methods;

[0008] Step 3: Refer to the identification methods of impurities, moisture, total ash, and thin-layer chromatography of processed Aconitum kusnezoffii in Part I of the Chinese Pharmacopoeia (2020 Edition) to conduct tests on semen myristicae praeparata radix aconiti kusnezoffii with different processing methods;

[0009] Step 4: Use high-performance liquid chromatography to determine the content of aconitum alkaloids in semen myristicae praeparata radix aconiti kusnezoffii;

[0010] Step 5: Establish the fingerprint of Terminalia chebula Retz. processed Kusnezoff Monkshood Root, evaluate the quality, and then determine the optimal processing technology of Mongolian medicine Terminalia chebula Retz. processed Kusnezoff Monkshood Root.

[0011] In the above technical solution, preferably: A specific implementation of the said Step 1 is:

[0012] After the crude Kusnezoff Monkshood Root is purified, taking the processing time, processing temperature, particle size of Terminalia chebula Retz., and dosage of decoction as factors, decoct the decoction of Terminalia chebula Retz. at a ratio of crude Kusnezoff Monkshood Root:Terminalia chebula Retz. = 10:1 every day. After changing the decoction of Terminalia chebula Retz. every day, wash it once with clear water, turn it over appropriately, then add the new decoction of Terminalia chebula Retz. for soaking, take it out and dry it; obtain Terminalia chebula Retz. processed Kusnezoff Monkshood Root with different processing methods.

[0013] In the above technical solution, preferably: The detection method for the change of alkaloid components in the said Step 2 is as follows:

[0014] Take 1.0 g of the powder of Terminalia chebula Retz. processed Kusnezoff Monkshood Root with different processing methods, accurately weigh and place it in a stoppered conical flask, add 1 mL of ammonia test solution, accurately add 30 mL of ether, ultrasonically treat for 30 min, extract twice, combine the extracts, place the filtrate in an evaporating dish, naturally volatilize it, then ultrasonically redissolve it with 5 mL of 50% methanol solution, filter through a 0.22 μm sterile filter membrane, and use it as the test solution. Apply positive spectrum analysis with an Aobitrap LC-MS detector; the parameters during the analysis are as follows:

[0015] ESI source, positive ion scanning mode, capillary temperature 270 °C, capillary voltage 30 V, spray voltage 5 KV, sheath gas (N 2 ) flow rate 49 psi (1 psi ≈ 6.895 kPa), auxiliary gas (N 2 ) flow rate 10 psi; chromatographic column (C18 chromatographic column is Waters-C18, 4.6 mm * 100 mm, 1.7 μm;), methanol:acetonitrile = 50:50 as mobile phase A, 0.35 mol / L ammonium acetate ammonia water pH = 10.5 as mobile phase B, column temperature 30 °C, flow rate 0.3 mL / min, injection volume 10 μL, and perform elution;

[0016] The elution conditions are: 0 - 15 min, 10% - 45% A; 15 - 45 min, 45% - 60% A; 45 - 55 min, 60% - 85% A, 55 - 65 min, 85% - 90% A; 65 - 71 min, 90% - 10% A; 71 - 75 min, 10% A.

[0017] In the above technical solution, preferably: The detection method for the change of tannin components in the said Step 2 is as follows:

[0018] Take 0.5 g of Terminalia chebula Retz.-processed Kusnezoff Monkshood powder prepared by different processing methods, accurately weigh and place it in a stoppered conical flask. Accurately add 20 mL of a 50% methanol aqueous solution containing 0.2% formic acid, let it stand overnight, ultrasonically treat for 60 min, and filter through a 0.22 μm sterile filter membrane to obtain the test solution, and perform negative spectrum analysis using an Orbitrap LC-MS detector; the parameters during the analysis process are as follows:

[0019] ESI source, negative ion scanning mode, the mass spectrometry conditions and chromatographic column are the same as those in the positive spectrum conditions; acetonitrile is mobile phase A, 0.1% formic acid water is mobile phase B, the column temperature is 30 °C, the flow rate is 0.3 mL / min, and the injection volume is 5 μL for elution;

[0020] The elution conditions are as follows: 0 - 3 min, 5% A; 3 - 12 min, 5% - 13% A; 12 - 20 min, 13% - 25% A; 20 - 30 min, 25% - 30% A; 30 - 38 min, 30% - 60% A; 38 - 48 min, 60% - 95% A; 48 - 49 min, 95% - 5% A; 49 - 52 min, 5% A.

[0021] In the above technical solution, preferably: the method for determining the content of aconitum alkaloids in step four is as follows:

[0022] Take 1.0 g of raw Kusnezoff Monkshood and Terminalia chebula Retz.-processed Kusnezoff Monkshood powder obtained by different processing factors, accurately weigh and place it in a stoppered conical flask, add 1 mL of ammonia test solution, accurately add 50 mL of ether, weigh, ultrasonically treat for 30 min, extract twice, combine the extracts, place the filtrate in an evaporating dish, naturally evaporate to dryness, and then ultrasonically redissolve with 5 mL of a methanol solution containing 0.1% hydrochloric acid to obtain the test solution, and determine the content of aconitum alkaloids therein.

[0023] In the above technical solution, preferably: a specific implementation manner of step five is as follows:

[0024] Detect the test solution by high performance liquid chromatography, and the chromatographic parameters are: mobile phase A: 50 mmol·L -1 ammonium acetate solution; mobile phase B: acetonitrile; the C18 chromatographic column is Sunniest-C18 (4.6 mm * 250 mm, 5 μm); the detection wavelength is 235 nm; the flow rate is 1 mL·min -1 ; the column temperature is 30 °C; the injection volume is 20 μL; perform gradient elution;

[0025] Qualitatively analyze the active ingredients based on the peak emergence times of the obtained chromatograms; quantitatively analyze the active ingredients based on the covers of the obtained chromatograms; in the chromatogram obtained at a detection wavelength of 235 nm: benzoylmesaconine has a peak emergence time of 13.720 ± 0.2 min; benzoylaconine has a peak emergence time of 16.607 ± 0.2 min; benzoylhypaconine has a peak emergence time of 18.587 ± 0.2 min; mesaconitine has a peak emergence time of 35.127 ± 0.2 min; hypaconitine has a peak emergence time of 38.313 ± 0.2 min; aconitine has a peak emergence time of 39.340 ± 0.2 min.

[0026] The quality evaluation method for determining the optimal processing technology of Mongolian medicine Terminalia chebula processed kusnezoff monkshood provided by the present invention is also applicable to raw kusnezoff monkshood.

[0027] Using the above content determination chromatographic conditions, obtain the superposition map of the fingerprint chromatograms of nine batches of different processed products, and analyze the stability and quality consistency of the alkaloid components.

[0028] The beneficial effects of the present invention are:

[0029] The quality evaluation method for determining the optimal processing technology of Mongolian medicine Terminalia chebula processed kusnezoff monkshood of the present invention takes the content changes of Terminalia chebula and alkaloids in Terminalia chebula processed kusnezoff monkshood as evaluation indicators, establishes an orthogonal optimization experiment to determine the optimal processing method of Terminalia chebula processed kusnezoff monkshood; establishes a method for multi-faceted evaluation of the quality of Terminalia chebula processed kusnezoff monkshood, and this method is also applicable to raw kusnezoff monkshood at the same time. Evaluate the quality of processed kusnezoff monkshood through a variety of detection means, including impurities, moisture, total ash, thin layer, content determination involving high performance liquid chromatography, and fingerprint chromatogram. It is proved by experimental examples that this method is stable and reliable, can comprehensively evaluate the quality of Terminalia chebula processed kusnezoff monkshood, and at the same time, the processing method of Mongolian medicine Terminalia chebula processed kusnezoff monkshood optimized by this evaluation method is applicable to scientific research and production practice.

[0030] The quality evaluation method for determining the optimal processing technology of Mongolian medicine Terminalia chebula processed kusnezoff monkshood of the present invention is a relatively comprehensive quality evaluation method for Terminalia chebula processed kusnezoff monkshood. On the basis of the first part of the Chinese Pharmacopoeia 2020 edition, identify impurities, moisture, total ash and thin layer chromatography, and establish a new HPLC content determination method for aconite alkaloids. Use an Orbitrap LC-MS detector to analyze the alkaloids and tannin components in the processed products, and analyze the influence of different processing factors on their content changes. Compared with the current Pharmacopoeia evaluation method, the content determination method of the present invention can simultaneously measure six alkaloids, namely aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylhypaconine, and benzoylmesaconine. Moreover, this method has better resolution, no false positives and interference peaks, and has a matrix for liquid mass spectrometry. Based on the above results, determine the optimal processing technology of Terminalia chebula processed kusnezoff monkshood. It has far-reaching significance for the use of Terminalia chebula processed kusnezoff monkshood. Description of the Drawings

[0031] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0032] Figure 1 It is a thin-layer identification diagram of Kusnezoff Monkshood processed with Terminalia chebula by different processing methods.

[0033] Figure 2 It is a diagram showing the influence of different factors on the total alkaloids of Kusnezoff Monkshood processed with Terminalia chebula.

[0034] Figure 3 It is a diagram showing the influence of different factors on the total tannin components of Kusnezoff Monkshood processed with Terminalia chebula.

[0035] Figure 4 It is an overlay diagram of different processing methods for Kusnezoff Monkshood from different origins.

[0036] Figure 5 It is a control spectrum of Kusnezoff Monkshood from different origins.

[0037] Figure 6 It is an overlay diagram of different processing methods.

[0038] Figure 7 It is a control spectrum of Kusnezoff Monkshood processed with Terminalia chebula by different processing methods. Specific Embodiments

[0039] Example 1

[0040] The present invention provides a quality evaluation method for determining the optimal processing technology of Mongolian medicine Kusnezoff Monkshood processed with Terminalia chebula, including the following steps:

[0041] Step 1: Take raw Kusnezoff Monkshood and establish an orthogonal optimization experiment with four factors: processing time, processing temperature, particle size of Terminalia chebula, and dosage of decoction to obtain Kusnezoff Monkshood processed with Terminalia chebula by different processing methods; the four different factors in Step 1 at three levels are compared to study the influence of different factors on the components in Kusnezoff Monkshood processed with Terminalia chebula.

[0042] Step 2: Apply an Aobitrap LC-MS detector to compare the changes in alkaloids in Kusnezoff Monkshood processed with Terminalia chebula by different processing methods and the tannin components in Terminalia chebula; the types of alkaloids that can be compared in Step 2 are more than those of the HPLC method, such as alkaloids without ultraviolet absorption like neoline; the changes in tannin components in Kusnezoff Monkshood processed with Terminalia chebula by different processing methods with respect to processing factors can be compared.

[0043] Step 3: Identify the Kusnezoff Monkshood processed with Terminalia chebula obtained in Step 1 with reference to the requirements for impurities, ash, moisture, and thin-layer identification of processed Kusnezoff Monkshood in Part I of the Chinese Pharmacopoeia 2020 Edition.

[0044] Step 4: Establish a high performance liquid chromatography method to determine the content of aconitum alkaloids in the processed kusnezoff monkshood root; the content determination method in Step 4 can meet the simultaneous determination of diester alkaloids and monoester alkaloids in the processed kusnezoff monkshood root with Terminalia chebula, and has better resolution and a matrix for liquid chromatography-mass spectrometry; the extraction method of the test sample in Step 4 uses ultrasonic extraction with ether.

[0045] Step 5: Establish a fingerprint of the processed kusnezoff monkshood root to evaluate the quality, and thus determine the optimal processing technology of the Mongolian medicine processed kusnezoff monkshood root with Terminalia chebula; the fingerprint established in Step 5 can provide comprehensive and reliable information and objectively reflect the consistency and authenticity of the processed kusnezoff monkshood root with Terminalia chebula.

[0046] Each step of the above quality evaluation method specifically includes the following steps:

[0047] According to the present invention, after the raw kusnezoff monkshood root is purified, taking the processing time, processing temperature, particle size of Terminalia chebula, and amount of decoction as factors, decoct the Terminalia chebula decoction at a ratio of raw kusnezoff monkshood root: Terminalia chebula = 10:1 every day. After changing the Terminalia chebula decoction every day, wash it once with clear water, turn it over appropriately, then add new Terminalia chebula decoction for soaking, take it out and dry it to obtain the processed kusnezoff monkshood root with Terminalia chebula, as shown in Table 1 below.

[0048] Table 1 Different processing methods

[0049]

[0050] According to the present invention, referring to the Pharmacopoeia of the People's Republic of China (2020 Edition), Volume I, the impurities in the processed kusnezoff monkshood root shall not exceed 1% (General Rule 2301). The moisture content shall not exceed 12.0% (Second Method of General Rule 0832). The total ash content shall not exceed 6.0% (General Rule 2302) and thin layer identification. Identify the processed kusnezoff monkshood root. The inspection results of moisture and total ash content are shown in Table 2 below, and the thin layer identification is shown in Figure 1 .

[0051] Table 2 Inspection results of moisture and total ash content

[0052]

[0053] Figure 1 Among them: from top to bottom, s3 and s4 are mixed reference substances of benzoylmesaconine, benzoylaconine, and benzoylhypaconine, 1 is the first processed product; 2 is the second processed product; 3 is the third processed product; 4 is the fourth processed product; 5 is the fifth processed product; 6 is the sixth processed product; 7 is the seventh processed product; 8 is the eighth processed product; 9 is the ninth processed product; at the position corresponding to the reference substance chromatogram, spots of the same color appear.

[0054] According to the present invention, take 1.0 g of the powder of Terminalia chebula processed Kusnezoff Monkshood Root with different processing methods, accurately weigh and place it in a stoppered conical flask, add 1 mL of ammonia test solution, accurately add 30 mL of ether, ultrasonically treat for 30 min, extract twice, combine the extracts, place the filtrate in an evaporating dish, naturally volatilize to dryness, then ultrasonically redissolve with 5 mL of a methanol solution containing 50%, and filter through a 0.22 μm sterile filter membrane to obtain the test solution, and perform positive spectrum analysis using an Aobitrap LC-MS detector; ESI source, positive ion scanning mode, capillary temperature 270 °C, capillary voltage 30 V, spray voltage 5 KV, sheath gas (N 2 ) flow rate 49 psi (1 psi ≈ 6.895 kPa), auxiliary gas (N 2 ) flow rate 10 psi; the chromatographic column is a C18 chromatographic column, Waters-ACQUITY-UPLC-C18, (2.1mm * 100mm, 1.7μm;), methanol:acetonitrile = 50:50 as mobile phase A, 0.35mol / L ammonium acetate ammonia water pH = 10.5 as mobile phase B, column temperature 30°C, flow rate 0.3mL / min, injection volume 10 μL, and elute under the following conditions. 0 - 15 min, 10% - 45%A; 15 - 45 min, 45% - 60% A; 45 - 55 min, 60% - 85% A, 55 - 65 min, 85% - 90% A; 65 - 71 min, 90% - 10% A; 71 - 75 min, 10% A. Calculate the influence of different processing methods on the change of alkaloid content according to the change of the peak area ratio with raw Kusnezoff Monkshood Root, and the results are shown in Table 3 below.

[0055] Table 3 Changes in alkaloid content of nine different processing methods

[0056]

[0057] Take 0.5 g of Terminalia chebula - processed Kusnezoff Monkshood and Terminalia chebula powder prepared by different processing methods, accurately weigh and place in a stoppered conical flask. Accurately add 20 mL of 50% methanol aqueous solution containing 0.2% formic acid, let stand overnight, ultrasonically treat for 60 min, filter through a 0.22 μm sterile filter membrane to obtain the test solution, and perform negative - spectrum analysis using an Orbitrap LC - MS detector; ESI source, negative - ion scanning mode, the mass spectrometry conditions and chromatographic column are the same as those in the positive - spectrum conditions; acetonitrile is mobile phase A, 0.1% formic acid water is mobile phase B, column temperature is 30 °C, flow rate is 0.3 mL / min, injection volume is 5 μL, and elute under the following conditions: 0 - 3 min, 5% A; 3 - 12 min, 5% - 13% A; 12 - 20 min, 13% - 25% A; 20 - 30 min, 25% - 30% A; 30 - 38 min, 30% - 60% A; 38 - 48 min, 60% - 95% A; 48 - 49 min, 95% - 5% A; 49 - 52 min, 5% A. Calculate the influence of different processing methods on the changes of tannin - like components in Terminalia chebula according to the change in the ratio of the peak area of Terminalia chebula, and the results are shown in Table 4 below.

[0058] Table 4 Changes in the content of tannin components in Terminalia chebula prepared by nine different processing methods

[0059]

[0060] According to the above - mentioned total component changes, combined with the orthogonal experiment, make an intuitive diagram of the influence of different processing factors on the components. The influence of different factors on the total alkaloids in Terminalia chebula - processed Kusnezoff Monkshood is shown in Figure 2 and the diagram of the influence of different factors on the total tannin - like components in Terminalia chebula - processed Kusnezoff Monkshood is shown in Figure 3, it can be seen that the processing time has the greatest impact on the total alkaloids. The total alkaloids will significantly decrease with the extension of the processing time. Secondly, the processing temperature has a greater impact on the total alkaloids. The increase in temperature will also affect the content of the total alkaloids. The size of Terminalia chebula fruits and the amount of decoction have a relatively small impact on the total alkaloids. Regarding the tannin components in processed Kusnezoff Monkshood Root with Terminalia chebula fruits, the size of Terminalia chebula fruits has the greatest impact on the total tannin components. The smaller the particle size, the more tannin components enter the Kusnezoff Monkshood Root. Secondly, it is the processing time. The tannin components change greatly from 2 days to 4 days of processing, and the change is relatively small from 4 days to 6 days of processing. The components of Terminalia chebula fruits at 50 °C are more than those at 4 °C, and the least at 25 °C. The amount of decoction has a relatively small impact on the change of tannin components in Terminalia chebula fruits, and shows a negative correlation, while it shows a positive correlation with the change of the total alkaloid content. According to the "Drug Standards of the Ministry of Health of the People's Republic of China (Mongolian Medicine Volume)", the method of soaking Kusnezoff Monkshood Root in Terminalia chebula decoction is as follows: Take Kusnezoff Monkshood Root and put it into Terminalia chebula decoction, soak it at room temperature for 3 - 5 days, turn it over 3 - 5 times a day, take it out when it tastes slightly numb on the tongue, and dry it at low temperature. The processing method of processed Kusnezoff Monkshood Root with Terminalia chebula fruits mentioned in the "Inner Mongolia Mongolian Medicine Processing Specification" is: For every 100 kg of Kusnezoff Monkshood Root, use 300 kg of Terminalia chebula decoction (30 kg of Terminalia chebula fruits plus 300 kg of water to make decoction). The process of processed Kusnezoff Monkshood Root with Terminalia chebula fruits recorded in "Mongolian Medicine Processing Science" is: Remove the impurities from Kusnezoff Monkshood Root, soak it in Terminalia chebula decoction for 1 - 3 days (10 kg of Kusnezoff Monkshood Root, 3 kg of Terminalia chebula fruits to make 30 L of decoction). And according to the fact that Mongolian medicine processed Kusnezoff Monkshood Root is generally processed in spring and autumn, the processing temperature should not be too high. Therefore, combining the experimental results and literature reports, finally, the optimal processing method is selected as follows: process for 4 days, processing temperature of 4 °C, select the fragments of Terminalia chebula fruits with a particle size of 0.3 cm after being broken to make Terminalia chebula decoction, and soak it according to the ratio of Kusnezoff Monkshood Root:Terminalia chebula decoction = 1:3 (Kusnezoff Monkshood Root:Terminalia chebula fruits = 10:3 to make decoction) every day.

[0061] According to the present invention, take 1.0 g of processed Kusnezoff Monkshood Root and raw Kusnezoff Monkshood Root powders of different batches, accurately weigh and place them in a stoppered conical flask, add 1 mL of ammonia water, accurately add 50 mL of ether, weigh, ultrasonically treat for 30 min, extract twice, combine the extracts, place the filtrate in an evaporating dish, naturally volatilize, and then ultrasonically redissolve with 5 mL of methanol solution containing 0.1% hydrochloric acid as the test solution. Use high performance liquid chromatography to detect the test solution. After exploring various chromatographic conditions, the optimal chromatographic conditions are finally determined according to the separation of chromatographic peaks and the precision, stability, repeatability, accuracy and linearity of detection as follows: The chromatographic parameters are: C18 chromatographic column; Mobile phase A: 50 mmol·L -1 ammonium acetate solution; Mobile phase B: acetonitrile; The gradient elution program is as described in Table 5 below; The C18 chromatographic column is Sunniest-C18, size 4.6mm * 250mm, 5μm; The detection wavelength is 235 nm; The flow rate is 1 mL·min -1 ; The column temperature is 30 °C; The injection volume is 20 μL.

[0062] Table 5 Gradient Elution Program

[0063]

[0064] Example 2 Methodology Investigation

[0065] 1. Investigation of Linear Relationship

[0066] Prepare mixed standard solution with different concentrations. Draw a standard curve with the peak area of each standard as the Y-axis and the concentration as the X-axis. The results are shown in Table 6 below. The results indicate that: using the method provided by the present invention, the alkaloid components in Aconitum kusnezoffii Reichb. show good linearity within the corresponding concentration ranges.

[0067] Table 6 Linear Relationship of Six Aconitum Alkaloids

[0068]

[0069] 2. Precision Experiment

[0070] Take the reference solution and inject it continuously for 6 times under the above chromatographic conditions. Calculate the RSD of the peak area of each component to investigate the precision of the instrument. The RSD(%) of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine are 2.876, 2.195, 2.506, 1.331, 2.387, and 1.476 respectively. It shows that the precision of the instrument is good.

[0071] 3. Stability Test

[0072] Precisely measure the solution of the same test sample, seal it and place it at room temperature. Determine it under the chromatographic conditions at 0, 2, 4, 8, 12, 16, 20, and 24 h respectively. The results show that the RSD(%) of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine are 1.449, 0.538, 2.284, 0.863, 0.522, and 2.36 respectively. It shows that the test sample solution is basically stable after being placed for 24 h.

[0073] 4. Repeatability Experiment

[0074] Precisely weigh the same batch of test samples, prepare the test sample solution, inject it continuously for 6 times, and detect it under the chromatographic conditions. The results show that the RSD(%) of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine are 0.893, 0.38, 1.301, 1.29, 1.381, and 1.282 respectively. It shows that the method has good repeatability.

[0075] 5. Reproducibility Experiment

[0076] Accurately weigh the same batch of test samples, prepare 6 portions of test sample solutions and perform detection according to the chromatographic conditions. The results show that the RSD(%) of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine are 1.442, 1.502, 2.318, 1.586, 1.459, and 2.175 respectively, indicating that the method has good reproducibility.

[0077] 6. Spike recovery experiment

[0078] Accurately measure 6 portions of test samples with known content, accurately add a certain amount of mixed reference substances to each component according to a ratio of 1:1, perform detection under the chromatographic conditions. The calculation method for spike recovery is (the content of each compound in the sample after adding the reference substance - the content of each compound in the known test sample) / the content of each component reference substance added × 100% = spike recovery rate. The results show that the average recovery rates(%) of aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, and benzoylhypaconine are 97.41%, 91.79%, 98.50%, 106.61%, 99.53%, and 93.93% respectively, indicating that the recovery rates of each component are good.

[0079] 7. Determination of sample content

[0080] According to the preparation method, each sample is extracted twice to obtain the test sample solution, each portion of the test sample solution is injected twice, and the determination is carried out according to the content determination method. The component contents in each batch of samples are calculated, and the results are shown in Table 7 below.

[0081] Table 7 Determination of aconitum alkaloids in raw and processed kusnezoff monkshood

[0082]

[0083] According to the preparation method, establish the fingerprint of semen myristicae processed kusnezoff monkshood. Since there are significant differences in the types of alkaloids in kusnezoff monkshood from different origins, and the difference in the types of alkaloids before and after processing is relatively small, it is not representative to establish the fingerprint of semen myristicae processed kusnezoff monkshood using raw kusnezoff monkshood from a single origin. Therefore, in this experiment, the above-mentioned liquid phase method for content determination is used to establish the fingerprint methodology through common peaks for 12 batches of raw kusnezoff monkshood from 3 origins in Inner Mongolia, Xinjiang, and Shaanxi. This method can more intuitively show the changes in the alkaloid content of semen myristicae processed kusnezoff monkshood before and after different processing methods. The superimposed maps of kusnezoff monkshood from different origins and different batches and the superimposed maps of different processing methods are shown in Figure 4 , and the reference maps of kusnezoff monkshood from different origins are shown in Figure 5 , Figure 5 A total of 9 common peaks are detected in the 12 batches of kusnezoff monkshood, and the 9 common peaks are numbered 1 - 9 in sequence according to the retention time.

[0084] 1. Investigation of fingerprint precision

[0085] Taking Peak 1 as the reference peak for Peaks 2, 3, 4, and 5, and Peak 6 as the reference peak for Peaks 7, 8, and 9, the relative retention time and the RSD of the relative peak area of the common peaks were calculated based on the retention time and peak area 1 of the reference peak. Six consecutive injections were performed, and the results showed that the RSDs (%) of the relative retention times of the 9 peaks were 0.00, 0.20, 0.20, 0.29, 0.53, 0.00, 0.12, 0.08, and 0.10, respectively, and the RSDs (%) of the relative peak areas of the 9 peaks were 0.00, 1.23, 1.86, 1.26, 2.91, 0.00, 1.62, 2.86, and 2.25, respectively. The similarity was greater than 0.9, indicating that the instrument precision was good and met the requirements for fingerprint determination.

[0086] 2. Investigation on the stability of the fingerprint

[0087] Taking the solution of the same test sample, it was sealed and placed at room temperature. Determinations were carried out according to the chromatographic conditions at 0, 2, 4, 8, 12, 16, 20, and 24 h. The RSDs (%) of the relative retention times of the 9 peaks were 0.00, 0.11, 0.11, 0.21, 0.53, 0.00, 0.15, 0.12, and 0.16, respectively, and the RSDs (%) of the relative peak areas of the 9 peaks were 0.00, 2.00, 1.67, 1.99, 1.76, 0.00, 0.54, 1.62, and 2.87, respectively. The similarity was greater than 0.9, indicating that the test sample solution was basically stable after being placed for 24 h.

[0088] 3. Investigation on the reproducibility of the fingerprint

[0089] For the repeatability experiment, the RSDs (%) of the relative retention times of the 9 peaks were 0.00, 0.11, 0.13, 0.15, 0.26, 0.00, 1.03, 1.74, and 0.24, respectively, and the RSDs (%) of the relative peak areas were 0.00, 0.27, 1.38, 0.85, 1.51, 0.00, 0.51, 1.63, and 1.73, respectively. The similarity was greater than 0.9, indicating that the method had good repeatability.

[0090] Overlaying the chromatograms obtained by different processing methods, the overlay map of the fingerprints of Radix Aconiti Kusnezoffii Praeparata cum Fructu Chebulae can be seen in Figure 6 and the reference map of Radix Aconiti Kusnezoffii Praeparata cum Fructu Chebulae with different processing methods can be seen in Figure 7 . Combining the common peaks of different batches of Radix Aconiti Kusnezoffii and the common peaks of different processing methods, Figure 7 such common peaks were used as the characteristic peaks for evaluating Radix Aconiti Kusnezoffii Praeparata cum Fructu Chebulae.

[0091] In summary, for the quality evaluation method of Radix Aconiti Kusnezoffii Preparata, the identification methods of ash, moisture, and thin layer in Part I of the Chinese Pharmacopoeia 2020 Edition can be referred to for identification. The content determination and fingerprint evaluation methods can be referred to the methods established in the present invention. This evaluation method can lay a foundation for the in-depth study of the chemical components and quality evaluation of Radix Aconiti Kusnezoffii Preparata in the future.

[0092] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. The obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for determining the optimal processing technology of Mongolian medicine Terminalia chebula processed Aconitum kusnezoffii Reichb., characterized in that, it comprises the following steps: Step 1: Establish an orthogonal optimization test for Terminalia chebula processed Aconitum kusnezoffii Reichb. to obtain Terminalia chebula processed Aconitum kusnezoffii Reichb. with different processing methods; Step 2: Apply an Orbitrap LC-MS detector to compare the changes in alkaloids and tannin components in Terminalia chebula processed Aconitum kusnezoffii Reichb. with different processing methods; The detection method for the change in alkaloid components in Step 2 is as follows: Take 1.0 g of the powder of Terminalia chebula processed Aconitum kusnezoffii Reichb. with different processing methods, accurately weigh and place it in a stoppered conical flask, add 1 mL of ammonia test solution, accurately add 30 mL of ether, ultrasonically treat for 30 min, extract twice, combine the extracts, place the filtrate in an evaporating dish, naturally volatilize to dryness, ultrasonically redissolve with 5 mL of 50% methanol solution, and filter through a 0.22 μm sterile filter membrane to obtain a test solution, and perform positive spectrum analysis using an Aobitrap LC-MS detector; the analysis process parameters are as follows: ESI source, positive ion scan mode, capillary temperature 270 °C, capillary voltage 30 V, spray voltage 5 kV, sheath gas is N 2 , its flow rate is 49 psi, auxiliary gas is N 2 , its flow rate is 10 psi; the chromatographic column is a C18 chromatographic column, its size is 4.6 mm * 100 mm, 1.7 μm; Methanol: acetonitrile = 50:50 as mobile phase A, 0.35 mol / L ammonium acetate ammonia water pH = 10.5 as mobile phase B, column temperature 30 °C, flow rate 0.3 mL / min, injection volume 10 μL, and perform elution; The elution conditions are: 0 - 15 min, 10% - 45% A; 15 - 45 min, 45% - 60% A; 45 - 55 min, 60% - 85% A, 55 - 65 min, 85% - 90% A; 65 - 71 min, 90% - 10% A; 71 - 75 min, 10% A; The detection method for the change in tannin components in Step 2 is as follows: Take 0.5 g of the powder of Terminalia chebula processed Aconitum kusnezoffii Reichb. with different processing methods, accurately weigh and place it in a stoppered conical flask, accurately add 20 mL of 50% methanol aqueous solution containing 0.2% formic acid, let stand overnight, ultrasonically treat for 60 min, filter through a 0.22 μm sterile filter membrane to obtain a test solution, and perform negative spectrum analysis using an Orbitrap LC-MS detector; the analysis process parameters are as follows: ESI source, negative ion scanning mode, capillary temperature 270 °C, capillary voltage 30 V, spray voltage 5 kV, sheath gas is N 2 , its flow rate is 49 psi, auxiliary gas is N 2 , its flow rate is 10 psi; the chromatographic column is a C18 chromatographic column, with dimensions of 4.6 mm * 100 mm, 1.7 μm; acetonitrile is mobile phase A, 0.1% formic acid water is mobile phase B, column temperature 30 °C, flow rate 0.3 mL / min, injection volume 5 μL, for elution; The elution conditions are: 0 - 3 min, 5% A; 3 - 12 min, 5% - 13% A; 12 - 20 min, 13% - 25% A; 20 - 30 min, 25% - 30% A; 30 - 38 min, 30% - 60% A; 38 - 48 min, 60% - 95% A; 48 - 49 min, 95% - 5% A; 49 - 52 min, 5% A; Step 3: Identify the impurities, moisture, total ash, and thin layer in Terminalia chebula processed Aconitum kusnezoffii Reichb. with different processing methods; Step 4: Determine the content of aconite alkaloids contained in Terminalia chebula processed Aconitum kusnezoffii Reichb. by high performance liquid chromatography; The method for determining the content of aconite alkaloids in Step 4 is as follows: Take 1.0 g of the powder of raw Aconitum kusnezoffii Reichb. and Terminalia chebula processed Aconitum kusnezoffii Reichb. obtained under different processing factors, accurately weigh and place it in a stoppered conical flask, add 1 mL of ammonia test solution, accurately add 50 mL of ether, weigh, ultrasonically treat for 30 min, extract twice, combine the extracts, place the filtrate in an evaporating dish, naturally volatilize to dryness, ultrasonically redissolve with 5 mL of methanol solution containing 0.1% hydrochloric acid to obtain a test solution, and determine the content of aconite alkaloids therein; Step 5: Detect the test solution by high performance liquid chromatography, establish the fingerprint of processed Kusnezoff Monkshood Root with Terminalia chebula, evaluate the quality, and then the best processing technology of Mongolian medicine processed Kusnezoff Monkshood Root can be determined; The chromatographic parameters in Steps 4 and 5 are as follows: the chromatographic column is a C18 chromatographic column with dimensions of 4.6 mm * 250 mm and 5 μm; mobile phase A: 50 mmol·L -1 ammonium acetate solution; mobile phase B: acetonitrile; detection wavelength is 235 nm; flow rate is 1 mL·min -1 ; column temperature is 30 °C; injection volume is 20 μL; gradient elution is performed; the gradient elution program is as follows:

2. The method for determining the best processing technology of Mongolian medicine processed Kusnezoff Monkshood Root according to claim 1, characterized in that, the said Step 1 is: After taking the crude Kusnezoff Monkshood Root and cleaning it, taking the processing time, processing temperature, particle size of Terminalia chebula, and dosage of decoction as factors, decoct the Terminalia chebula decoction at a ratio of crude Kusnezoff Monkshood Root: Terminalia chebula = 10:1 every day. After changing the Terminalia chebula decoction every day, wash it once with clear water, turn it over, then add new Terminalia chebula decoction for soaking, take it out and dry it; obtain processed Kusnezoff Monkshood Root with different processing methods.

3. The method for determining the best processing technology of Mongolian medicine processed Kusnezoff Monkshood Root according to claim 1, characterized in that, in the said Step 5: After detecting the test solution by high performance liquid chromatography, qualitatively analyze the active ingredients according to the peak emergence time of the obtained chromatogram; quantitatively analyze the active ingredients according to the cover of the obtained chromatogram; in the chromatogram obtained at a detection wavelength of 235 nm: the peak emergence time of 13.720 ± 0.2 min is benzoylaconine; the peak emergence time of 16.607 ± 0.2 min is benzoylaconitine; the peak emergence time of 18.587 ± 0.2 min is benzoylhypaconitine; the peak emergence time of 35.127 ± 0.2 min is aconine; the peak emergence time of 38.313 ± 0.2 min is hypaconitine; the peak emergence time of 39.340 ± 0.2 min is aconitine.

4. The method for determining the best processing technology of Mongolian medicine processed Kusnezoff Monkshood Root according to claim 1, characterized in that, it is also applicable to crude Kusnezoff Monkshood Root.

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