Aconitum quality control method based on quantitative nuclear magnetic resonance hydrogen spectrum technology

Abstract

The application discloses a quality control method of aconite medicinal herbs based on quantitative nuclear magnetic resonance hydrogen spectrum technology, which comprises the following steps: extracting aconite medicinal herbs by using ethyl acetate, dissolving the extracted liquid in deuterated acetone after drying, adjusting the pH to 1.0-2.0, measuring nuclear magnetic resonance hydrogen spectrum data, and calculating the content of monoester type alkaloids and diester type alkaloids through quantitative peaks. The method is simple, fast and does not need corresponding reference substances, and the quantitative limit can reach 0.2 mM. Moreover, more monoester type alkaloids and diester type alkaloids can be detected, the application range is wide, and the quality of aconite medicinal herbs can be more comprehensively evaluated and controlled.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of quality control of traditional Chinese medicinal materials, and particularly relates to a quality control method for Aconitum L. based on quantitative nuclear magnetic resonance hydrogen spectrum technology. BACKGROUND

[0002] Aconitum L. mainly includes dry mother roots and suberect roots of Aconitum carmichaelii Debx. plants and dry tubers of Aconitum kusnezoffii Reichb. plants, which are also called Chuanwu, Fuzi and Caowu respectively. Among them, Chuanwu is called as Chichuanwu after being processed. Fuzi is called as Yanzi, Heishunpian, Baifupian, Paofupian and Danfupian after being processed by different processing methods. Caowu is called as Dicauwu after being processed. Chichuanwu, Yanzi, Heishunpian, Baifupian, Paofupian, Danfupian and Dicauwu are a kind of Aconitum L. widely used in clinic, which has the effects of returning yang and rescuing reversal, warming kidney and assisting yang and dispelling cold and relieving pain, and is used for kidney yang deficiency, cold limbs, wind-cold-damp arthralgia and the like.

[0003] Aconitum L. mainly contains monoester and diester alkaloids, which are effective components of Aconitum L. but also main toxic components. During processing into decoction pieces, the monoester and diester alkaloids contained in Caowu, Chuanwu and Fuzi are largely degraded. However, it is found that even if the same processing technology is used, the monoester and diester alkaloid contents in these traditional Chinese medicinal material products will have great differences due to various factors such as medicinal material sources. Therefore, based on safety and product quality control, determination of total diester alkaloid and total monoester alkaloid contents in Aconitum L. products is of great significance for production and quality control of related products.

[0004] In the related art, the reported methods for determining the monoester and diester alkaloids in the aconite traditional Chinese medicines include capillary electrophoresis, high performance liquid chromatography-ultraviolet, high performance liquid chromatography-evaporative light scattering, high performance liquid chromatography-mass spectrometry, etc. However, the diester alkaloids determined by these methods are limited to aconitne, mesaconitne and hypaconitne, and the monoester alkaloids are limited to benzoylaconine, benzoylmesaconine and benzoylhypaconine. The determination of the monoester and diester alkaloids in the aconite traditional Chinese medicines by using a single method is not comprehensive. For example, deoxyactonitine and 10-OH-actonitine are also bioactive / toxic substances, but their contributions are not considered in the quality control of the aconite traditional Chinese medicines by using the above methods, thereby often leading to poisoning events. In addition, the analysis time of these methods is usually long. For example, the analysis time of the HPLC method for the quantitative analysis of the aconite traditional Chinese medicines in the Chinese Pharmacopoeia is as long as 65-70 minutes, which is not conducive to the rapid analysis of a large number of samples.

[0005] Therefore, it is of great significance to develop a method for rapidly and comprehensively determining the monoester and diester alkaloids in the aconite traditional Chinese medicines for the processing and production as well as the quality control of the aconite traditional Chinese medicines. SUMMARY

[0006] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the present application provides a method for determining the total monoester alkaloids and the total diester alkaloids in the aconite traditional Chinese medicines based on quantitative nuclear magnetic resonance hydrogen spectrum (qHMNR), thereby for the quality control of the aconite traditional Chinese medicines.

[0007] In a first aspect, the present application provides a method for simultaneously detecting the monoester alkaloids and the diester alkaloids in the aconite traditional Chinese medicines, comprising the following steps:

[0008] The aconite traditional Chinese medicines are extracted by using ethyl acetate, the extracted solution is dried and then dissolved in deuterated acetone, the pH is adjusted to 1.0-2.0, the nuclear magnetic resonance hydrogen spectrum data is determined, and the contents of the monoester alkaloids and the diester alkaloids are calculated;

[0009] The quantitative peak (H-14) of the monoester alkaloids in the nuclear magnetic resonance hydrogen spectrum is 4.89-4.93 ppm or 4.95-4.99 ppm.

[0010] The quantitative peak (H-15) of the diester alkaloids in the nuclear magnetic resonance hydrogen spectrum is 4.72-4.77 ppm or 4.55-4.63 ppm.

[0011] NMR spectroscopy has unique advantages in the structural identification of natural products. Since the integral area of hydrogen in HNMR is proportional to the number of hydrogen, it can be used for quantitative analysis of specific components in complex matrix. The inventors found that quantitative analysis based on HNMR does not require corresponding standard products, and therefore is very suitable for simultaneous quantification of multiple diester alkaloids and monoester alkaloids in aconite traditional Chinese medicine.

[0012] In some embodiments of the present application, the aconite traditional Chinese medicine is obtained after pretreatment; the pretreatment comprises at least one of the following: crushing, sieving, acid treatment, ultrasonic treatment, centrifugation and alkalization.

[0013] In some embodiments of the present application, the pretreatment comprises crushing, sieving, acid treatment, ultrasonic treatment, centrifugation and alkalization in sequence.

[0014] In some embodiments of the present application, the crushing and sieving are crushing the aconite traditional Chinese medicine into a powder that can be sieved through a No. 2 or No. 3 sieve.

[0015] In the art, the No. 2 sieve has a mesh size of 850 ± 29 μm, and the No. 3 sieve has a mesh size of 355 ± 13 μm.

[0016] In some embodiments of the present application, the acid solution comprises hydrochloric acid.

[0017] In some embodiments of the present application, the hydrochloric acid is a 0.5-1.0% HCl (v / v) aqueous solution.

[0018] In some embodiments of the present application, the hydrochloric acid is a 1% HCl (v / v) aqueous solution.

[0019] In some embodiments of the present application, the ratio of the acid solution to the aconite traditional Chinese medicine is 5-20 mL: 1 g.

[0020] In some embodiments of the present application, the ratio of the acid solution to the aconite traditional Chinese medicine is 50 mL: 3 g.

[0021] In some embodiments of the present application, the acid treatment is mixing the aconite traditional Chinese medicine powder with the acid solution.

[0022] In the present application, the inventors consider that monoester alkaloids and diester alkaloids have weak alkalinity, and therefore can be extracted using an acidic aqueous solution.

[0023] In some embodiments of the present application, the ultrasonic treatment is ultrasonic treatment of the mixture of the aconite traditional Chinese medicine powder and the acid solution.

[0024] In some embodiments of the present application, the ultrasonic treatment is performed at a power of 250-350 W and a frequency of 35-45 kHz for 30 min at room temperature.

[0025] In the present application, the term "room temperature" also refers to "normal temperature", and unless otherwise specified, it specifically refers to a temperature condition of about 25°C. In the present application, the term "about" in relation to a numerical value means an error of ±5°C.

[0026] In some embodiments of the present application, the centrifugation is performed at 3000-5000 rpm for 5-15 min.

[0027] In some embodiments of the present application, the centrifugation is performed at 4000 rpm for 10 min.

[0028] In some embodiments of the present application, the alkalization is performed by adjusting the pH of the system to alkaline using an alkaline solution.

[0029] In some embodiments of the present application, the alkalization is performed by adjusting the pH of the system to 9-10 using an alkaline solution.

[0030] In some embodiments of the present application, the alkalization is performed by adjusting the pH of the system to 9.5-9.8 using an alkaline solution.

[0031] In some embodiments of the present application, the alkaline solution used for the alkalization is an alkaline solution.

[0032] In some embodiments of the present application, the alkaline solution includes ammonia.

[0033] The inventors have found that when the solution system is adjusted to alkaline, the aconitum alkaloids are more easily extracted by ethyl acetate, thereby achieving their enrichment.

[0034] In some embodiments of the present application, the specific operation of the pretreatment is as follows: the aconite traditional Chinese medicine is crushed into powder, passed through a 2 or 3 mesh sieve, acidified with 0.5-1.5% hydrochloric acid according to a solid-liquid ratio of 5-20 mL: 1 g of the aconite traditional Chinese medicine, subjected to ultrasonic treatment, centrifuged, and the supernatant is adjusted to a pH of 9-10 by adding ammonia.

[0035] In some embodiments of the present application, the extraction liquid is an ethyl acetate layer solution.

[0036] In some embodiments of the present application, the volume ratio of the extraction liquid to the acid solution is 2-5:1.

[0037] In some embodiments of the present application, the aconite traditional Chinese medicine includes medicinal parts of Aconitum or aconite plants and / or products thereof.

[0038] In some embodiments of the present application, the nuclear magnetic resonance hydrogen spectrum data acquisition conditions comprise:

[0039] Scan times: 1-64 times; spectral width: 7-20.5 ppm; pulse angle: 30-90°.

[0040] In some embodiments of the present application, the nuclear magnetic resonance hydrogen spectrum data acquisition conditions are:

[0041] Irradiation frequency: 600 MHz; probe temperature: 298 K; close rotation; scan times: 1-64 times; spectral width: 7-20.5 ppm; open automatic filter; relaxation delay: 10 s; pulse angle: 30-90°; pulse width: 9.62 μs.

[0042] In some embodiments of the present application, the method specifically comprises:

[0043] The aconite traditional Chinese medicine (the supernatant after pH adjustment) after the above pretreatment is extracted with ethyl acetate, the ethyl acetate layer solution is taken, concentrated and dried, deuterated acetone is added, the pH is adjusted to 1.0-2.0 with deuterium chloride, and the nuclear magnetic resonance hydrogen spectrum data is determined after membrane filtration, so as to calculate the contents of the monoester type alkaloid and the diester type alkaloid.

[0044] In some embodiments of the present application, the volume of the deuterated acetone is 0.5-2 mL.

[0045] In the present application, the inventors finally determine that the H-14 or H-15 signal of the monoester type and diester type alkaloid in the acidic deuterated acetone is the characteristic signal thereof, which is not interfered by other signals, so as to realize the accurate quantification of the total monoester type alkaloid and the total diester type alkaloid. Compared with the current determination method, the present application is simple in operation, short in time consumption, and does not need the corresponding control product, thereby solving the problems of difficult acquisition and instability of the control product. In addition, the monoester type and diester type alkaloid determined by the quality control method is more comprehensive, so that the evaluation of the aconite traditional Chinese medicine is more accurate.

[0046] In a second aspect of the present application, the method of the first aspect of the present application is applied in any one of (1)-(3) as follows:

[0047] (1) Quality control of aconite traditional Chinese medicine;

[0048] (2) Identification of aconite traditional Chinese medicine;

[0049] (3) Analysis of components of aconite traditional Chinese medicine;

[0050] The components are monoester type alkaloid and / or diester type alkaloid.

[0051] The beneficial effects of the present application are:

[0052] 1. The present application provides a more accurate method for quality control of aconite traditional Chinese medicine, which is simple to operate, short in time consumption (for example, 3 min 48 s of sampling time for each sample when 16 times of conventional collection are calculated), does not require a corresponding control product (only one accurate concentration of hypaconitine is needed to accurately quantify other diester or monoester alkaloids), the LOQ (limit of quantification) is 0.2 mM when S / N is greater than 30 and the accuracy is between 90-110%, and is 0.07 mM when S / N is greater than 3 and the accuracy is between 85-115%, which has extremely high detection accuracy.

[0053] 2. The method of the present application can detect more monoester and diester alkaloids than the traditional method, so as to achieve the purpose of more accurate and comprehensive control of the quality of aconite traditional Chinese medicine. Moreover, the method has wide application range and can be used for quality control of various aconite traditional Chinese medicines. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 The total monoester alkaloids (MDAs) and total diester alkaloids (DDAs) in the white aconite tablet of the present application are 1 The quantitative peaks of total monoester alkaloids (MDAs) and total diester alkaloids (DDAs) in the H-NMR spectrum. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical scheme and technical effects of the present application clearer, the present application will be further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described in the present specification are only for the purpose of explaining the present application, and are not intended to limit the present application.

[0056] The experimental materials and reagents used are conventional consumables and reagents that can be obtained from commercial channels, unless otherwise specified.

[0057] Example 1

[0058] In this embodiment, salt aconite is selected as the aconite traditional Chinese medicine detection sample for detection, and the specific steps are as follows:

[0059] The salt aconite was crushed into powder and passed through a No. 2 sieve (pore size 850 ± 29 μm, 24 mesh). About 3 g of the salt aconite powder was taken for testing. The salt aconite powder was placed in a 50 mL conical flask with a plug, 50.0 mL of an aqueous hydrochloric acid solution containing 1% HC1 (v / v) was added, and ultrasonic extraction was performed at a power of 300 W, a frequency of 40 kHz, and room temperature for 30 min. Centrifugation was performed at 4000 rpm for 10 min. 25 mL of the supernatant was taken, and concentrated aqueous ammonia solution was added to adjust the pH to 9.5. The supernatant after pH adjustment was transferred to a 250 mL separation funnel, and extraction was performed three times with 70 mL of ethyl acetate. The ethyl acetate layers were combined, concentrated and dried, and then dissolved in 2 mL of deuterated acetone. The pH was adjusted to 2.0 with deuterium chloride solution (35% deuterium chloride diluted 10 times with deuterated methanol), and the sample was filtered through a 0.22 μm membrane. The 1H-NMR data of the sample were collected using a Bruker AVANCE 600 nuclear magnetic resonance spectrometer.

[0060] The 1H-NMR data collection conditions were as follows: irradiation frequency 600 MHz; probe temperature 298 K; off spin; number of scans 16; spectral width 20.5 ppm; on (eight times) automatic filter; acquisition time 3 min 48 s; relaxation delay 10 s; pulse angle 30°; pulse width 9.62 μs.

[0061] The peak settings were as follows: total monoester alkaloid quantitative peak H-14 (4.89-4.93 ppm), total diester alkaloid quantitative peak H-14 (4.95-4.99 ppm).

[0062] The total monoester alkaloid and total diester alkaloid contents were calculated according to the collected data in combination with the ERETIC 2 function.

[0063] It was found that in the salt aconite of the present example, the content of total diester alkaloids was 0.0069%, and the content of total monoester alkaloids was 0.0238%, which met the requirements of the Chinese Pharmacopoeia, indicating that the salt aconite product was in compliance, and there were no quality and safety problems.

[0064] Example 2

[0065] In the present example, white smooth tablets were selected as the aconite medicinal sample for testing, and the specific steps were as follows:

[0066] The Bai Sunpian was crushed into powder and passed through a No. 2 sieve (pore size 850 ± 29 μm, 24 mesh). About 3 g of the Bai Sunpian powder was taken for testing. The Bai Sunpian powder was placed in a 50 mL conical flask with a stopper, and 50.0 mL of an aqueous hydrochloric acid solution containing 1% HC1 (v / v) was added. Ultrasonic extraction was performed at a power of 300 W and a frequency of 40 kHz at room temperature for 30 min. Centrifugation was performed at 4000 rpm for 10 min. 25 mL of the supernatant was taken, and concentrated aqueous ammonia solution was added to adjust the pH to 9.8. The supernatant after pH adjustment was transferred to a 250 mL separation funnel, and extracted with 75 mL of ethyl acetate three times. The ethyl acetate layers were combined, concentrated and dried, and then dissolved in 2 mL of deuterated acetone. The pH was adjusted to 1.0 with deuterium chloride solution (35% deuterium chloride diluted 10 times with deuterated methanol), and the sample was filtered through a 0.22 μm membrane and collected using a Bruker AVANCE 600 nuclear magnetic resonance spectrometer 1 H-NMR data.

[0067] The qH NMR data acquisition conditions were: irradiation frequency 600 MHz; probe temperature 298 K; off spin; number of scans 16; spectral width 20.5 ppm; on (eight times) automatic filter; acquisition time 3 min 48 s; relaxation delay 10 s; pulse angle 30°; pulse width 9.62 μs.

[0068] The peak settings were: total monoester-type alkaloid quantitative peak H-14 (4.89-4.93 ppm), total diester-type alkaloid quantitative peak H-14 (4.95-4.99 ppm).

[0069] The total monoester-type alkaloid and total diester-type alkaloid contents were calculated according to the data collected in combination with the ERETIC 2 function.

[0070] It was found that the content of total diester-type alkaloids in the Bai Sunpian in this example was 0.0049%, and the content of total monoester-type alkaloids was 0.0146%, which met the requirements of the Chinese Pharmacopoeia, indicating that the Bai Sunpian product was in compliance and had no quality and safety problems.

[0071] Example 3

[0072] In this example, the Radix Aconiti was selected as the aconite medicinal sample for testing, and the specific steps were as follows:

[0073] The prepared radix aconiti powder was crushed into powder and passed through a No. 2 sieve (pore size 850 ± 29 μm, 24 mesh). About 3 g of the prepared radix aconiti powder was taken for testing. The prepared radix aconiti powder was placed in a 50 mL conical flask with a plug, 50.0 mL of an aqueous hydrochloric acid solution containing 1% HC1 (v / v) was added, and ultrasonic extraction was performed at a power of 300 W, a frequency of 40 kHz, and room temperature for 30 min. Centrifugation was performed at 4000 rpm for 10 min. 25 mL of supernatant was taken, and concentrated aqueous ammonia solution was added to adjust the pH to 9.8. The supernatant after pH adjustment was transferred to a 250 mL separation funnel, and extraction was performed three times with 65 mL of ethyl acetate. The ethyl acetate layers were combined, concentrated and dried, and then dissolved in 2 mL of deuterated acetone. The pH was adjusted to 1.0 with deuterium chloride solution (35% deuterium chloride diluted 10 times with deuterated methanol), and the sample was filtered through a 0.22 μm membrane. The sample was subjected to 1H-NMR data acquisition using a Bruker AVANCE 600 nuclear magnetic resonance spectrometer.

[0074] The 1H-NMR data acquisition conditions were as follows: irradiation frequency 600 MHz; probe temperature 298 K; off spin; number of scans 16; spectral width 20.5 ppm; on (eight times) automatic filter; acquisition time 3 min 48 s; relaxation delay 10 s; pulse angle 30°; pulse width 9.62 μs.

[0075] The peak settings were as follows: total monoester alkaloid quantitative peak H-14 (4.89-4.93 ppm), total diester alkaloid quantitative peak H-14 (4.95-4.99 ppm).

[0076] The total monoester alkaloid and total diester alkaloid contents were calculated according to the acquired data in combination with the ERETIC 2 function.

[0077] It was found that the content of total diester alkaloids in the white smooth tablet in this example was 0.0091%, and the content of total monoester alkaloids was 0.018%, which met the requirements of the Chinese Pharmacopoeia, indicating that the white smooth tablet product was in compliance, and there were no quality and safety problems.

[0078] Example 4

[0079] In order to further test the detection accuracy of the qHNMR method in the present application, the inventors prepared standard solutions of different concentrations of hypaconitine and benzoyl mesaconitine standard (the concentration of hypaconitine was 5.5546, 1.8515, 0.6172, 0.2057, 0.0686 mM, respectively; the concentration of benzoyl mesaconitine was 5.6113, 1.8704, 0.6235, 0.2078, 0.0693 mM, respectively), and the qHNMR data acquisition conditions in the above examples were used for testing. The test results are shown in Table 1.

[0080] The scanning times are set to 16 and 32 respectively, and the theoretical value is the concentration of hypaconitine and benzoyl neoaconitine in the prepared hypaconitine and benzoyl neoaconitine standard solution.

[0081] Table 1 Accuracy of qHNMR method for hypaconitine and benzoyl neoaconitine

[0082]

[0083]

[0084] It can be found that the qHNMR method in the present application has excellent detection effect for both hypaconitine and benzoyl neoaconitine, and even at an extremely low concentration of 0.07 mM, the detection accuracy can still reach more than 85%, which has a better detection rate than the existing detection technology.

[0085] Example 5

[0086] In this embodiment, Bai Fu Pian containing hypaconitine (adding high (0.2300 mg), medium (0.1150 mg), and low (0.0575 mg) quality of hypaconitine standard to Bai Fu Pian samples) is used as a detection sample for detection, and the method in the present application is tested for sample recovery rate by referring to the method in Chinese Pharmacopoeia.

[0087] At the same time, Bai Fu Pian samples without adding hypaconitine are used as blank controls, and the total double glycoside alkaloid content is determined by the same method. The recovery amount is defined as the difference between the total double glycoside alkaloid determined by adding the standard and the total double glycoside alkaloid determined by not adding the standard. The ratio of the recovery amount to the added standard amount is the recovery rate.

[0088] The results are shown in Table 2 and Figure 1 .

[0089] Table 2 Recovery rate of hypaconitine

[0090]

[0091]

[0092] Figure 1 The detection spectrum of Bai Fu Pian samples without adding hypaconitine can be found that the method in the present application can accurately detect specific target peaks. From the recovery rate of hypaconitine standard in Bai Fu Pian, it can be seen that the method in the present application can effectively recover the target, and even at an extremely low amount, the recovery rate can still remain more than 90%, which has excellent recovery effect.

[0093] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for simultaneously detecting monoester alkaloids and diester alkaloids in aconite traditional Chinese medicine, comprising the following steps: extracting aconite traditional Chinese medicine with ethyl acetate, taking ethyl acetate layer solution, adding deuterated acetone after drying by concentration, adjusting pH to 1.0-2.0 with deuterium chloride, filtering with filter membrane, and then determining nuclear magnetic resonance hydrogen spectrum data to calculate monoester alkaloid and diester alkaloid contents; wherein quantitative peaks of monoester alkaloids in nuclear magnetic resonance hydrogen spectrum are 4.89-4.93 ppm or 4.95-4.99 ppm; quantitative peaks of diester alkaloids in nuclear magnetic resonance hydrogen spectrum are 4.72-4.77 ppm or 4.55-4.63 ppm; the nuclear magnetic resonance hydrogen spectrum data acquisition conditions include: scan times: 1-64 times; spectral width: 7-20.5 ppm; pulse angle: 30-90°; wherein the aconite traditional Chinese medicine is obtained after pretreatment; the pretreatment includes crushing, sieving, acid treatment, ultrasonic treatment, centrifugation and alkalization; the alkalization is adjusting system pH to 9-10 with alkali.

2. The method of claim 1, wherein, the acid includes hydrochloric acid.

3. The method of claim 1, wherein, the acid to aconite traditional Chinese medicine ratio is 5-20 mL: 1 g.

4. The method of claim 1, wherein, the volume ratio of ethyl acetate layer solution to acid is 2-5:

1.

5. The method of claim 1, wherein, the aconite traditional Chinese medicine includes aconite or medicinal parts of aconite plants and / or products thereof. 6.Use of the method of any one of claims 1-5 in any one of (1)-(3): (1) aconite traditional Chinese medicine quality control; (2) aconite traditional Chinese medicine identification; (3) aconite traditional Chinese medicine component analysis; wherein the components are monoester alkaloids and / or diester alkaloids.

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