Method for improving safety of short-pedicel aconite root medicinal material or preparation

Abstract

The invention provides a method for improving the safety of a short-pedicel aconite root medicinal material or preparation, and belongs to the technical field of medicine quality control. The method established by the invention can accurately and reliably perform qualitative and quantitative analysis on the toxic components in the short-pedicel aconite root medicinal material and the preparation thereof, is simple and convenient to operate and good in repeatability, effectively overcomes the defects of the traditional quality control method, formulates a clear and feasible technical specification for the quality standard of the short-pedicel aconite root, and has a wide application prospect. Therefore, the clinical medication risk is managed and controlled from the source, the safety and effectiveness of the medicine are ensured, and the method has important practical significance for promoting reasonable development and safe application of the toxic medicinal material.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug quality control, and particularly relates to a method for improving the safety of Aconitum brachypodum medicinals or preparations. BACKGROUND

[0002] Aconitum brachypodum is an important folk medicine in southwest China, which is derived from various plants of the genus Aconitum in Ranunculaceae, among which Aconitum brachypodum is more common. The medicine is famous for its significant effects of promoting blood circulation to remove blood stasis and relieving swelling and pain, and is traditionally used to treat injuries, rheumatism and arthralgia. However, like the famous Aconitum medicines such as Aconitum carmichaelii and Aconitum kusnezoffii, Aconitum brachypodum also shows strong toxicity while playing a therapeutic role. If used improperly, it can easily lead to poisoning and even endanger life, and therefore is listed in the directory of toxic traditional Chinese medicines managed by the state.

[0003] The toxicity of Aconitum medicines mainly comes from the diester-type diterpene alkaloids contained therein. Such components have strong pharmacological activity but narrow safety window, and are the key substances leading to cardiotoxicity and neurotoxicity. In order to ensure the safety of clinical medication, traditional processing methods such as decoction and processing are often used to hydrolyze and transform the toxic components, reduce the content of diester-type alkaloids, and thus reduce toxicity while retaining efficacy. However, in the actual production and quality control process, due to the lack of precise detection methods for toxic components and clear quality control standards, production enterprises often face a dilemma: if the processing is insufficient, the product may exceed the standard of toxic components, posing a safety hazard; if the processing is excessive, although the safety is ensured, the loss of effective components may be too large, affecting the efficacy. This kind of "one-size-fits-all" extensive management not only restricts the rational application of the medicine, but also makes it difficult to ensure the consistency and reliability of the quality of different batches of products.

[0004] The quality control method of Aconitum brachypodum in the prior art is not sufficient, especially the specific and simple detection technology for the core toxic component, diester-type alkaloids, is lacking. Some methods may be tedious and costly, or cannot effectively exclude the interference of other coexisting components in the medicine, and are difficult to popularize in routine quality inspection. Therefore, it is urgent and significant to develop a quality control method that can specifically identify and accurately determine the main toxic alkaloids in Aconitum brachypodum, so as to scientifically evaluate the quality of the medicine, guide the reasonable processing, formulate scientific and reasonable quality standards, and ultimately ensure the safety of clinical medication. SUMMARY

[0005] The present application aims to provide a method for improving the safety of Aconitum brachypodum medicinals or preparations, and provides an effective means for the related field.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The application provides a method for improving the safety of Aconitum brachypodum medicine or preparation, which comprises identifying toxic alkaloids in Aconitum brachypodum by thin layer chromatography and determining the content of diester-type alkaloids in Aconitum brachypodum by ultraviolet spectrophotometry, so as to improve the safety by controlling the content of diester-type alkaloids.

[0007] Preferably, the thin layer chromatography comprises preparing a control solution and a sample solution, spotting on a thin layer plate, developing, and developing color.

[0008] Preferably, the control solution comprises at least one of aconitine, 3-deoxyaconitine, N-deethyl-3-deoxyaconitine, 12-epi-aconitine and neoline, and the concentration is 0.5 mg / mL to 2 mg / mL.

[0009] Preferably, the preparation of the sample solution comprises ultrasonic extraction of Aconitum brachypodum medicine powder with hydrochloric acid solution, filtration, extraction of the water layer with petroleum ether, extraction with chloroform, concentration of the chloroform extract, dissolution with ethanol, purification with basic aluminum oxide, elution, concentration and drying, and constant volume with ethanol.

[0010] Preferably, the mass concentration of the hydrochloric acid solution is 0.5% to 2%, the ultrasonic extraction is performed 2 to 4 times, and the ultrasonic extraction time is 15 to 25 minutes each time. The petroleum ether extraction is performed 3 to 5 times, and the petroleum ether is used in an amount of 40 to 60 mL each time; the chloroform extraction is performed 3 to 5 times, and the chloroform is used in an amount of 40 to 60 mL each time.

[0011] Preferably, the thin layer chromatography uses a silica gel G thin layer plate, the developing agent is a mixed solution of cyclohexane, ethyl acetate and methanol, the volume ratio is 6 to 7:3 to 4:0.8 to 1.2, the developing cylinder is saturated with ammonia vapor for 15 to 25 minutes, and the color developing agent is dilute bismuth potassium iodide test solution.

[0012] Preferably, the ultraviolet spectrophotometry comprises preparing a control solution and a sample solution, and determining the absorbance at a wavelength of 225 nm to 235 nm.

[0013] Preferably, the control solution is aconitine solution, and the concentration is 20 μg / mL to 30 μg / mL. The preparation of the sample solution comprises diluting the thin layer chromatography sample solution by 400 to 600 times. Further comprising drawing a standard curve, and the standard curve equation is Y equals aX plus b, wherein a is 20 to 22, b is negative 0.04 to negative 0.02, and the linear range is 0 μg / mL to 90 μg / mL.

[0014] Preferably, the method further comprises a repeatability experiment, and the content of the diester alkaloid is determined by taking multiple samples, and the relative standard deviation is less than 5%. The method further comprises a sample addition recovery experiment, and the recovery rate is 95% to 105%, and the relative standard deviation is less than 5%.

[0015] Preferably, the basic aluminum oxide purification comprises elution with ethanol to an alkaloid-free reaction, and the reduced pressure drying temperature is 70 to 90 degrees Celsius, and the drying time is 3 to 5 hours.

[0016] Technical effects and advantages of the present application: The method established in the present application can accurately and reliably qualitatively and quantitatively analyze the toxic components in the Herba Potentillae Chinensis and its preparations, and the method is simple in operation and good in repeatability, effectively overcomes the shortcomings of the traditional quality control method, and formulates clear and feasible technical specifications for the quality standard of Herba Potentillae Chinensis, thereby controlling the clinical drug risk from the source and ensuring the safety and effectiveness of the drug, which has important practical significance for promoting the reasonable development and safe application of the toxic medicinal material. DETAILED DESCRIPTION

[0017] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0018] Examples 1. Instruments and reagents 1.1 Instruments UV-265-FW ultraviolet visible spectrophotometer (Japan Shimadzu), AUW-220D electronic analytical balance (Japan Shimadzu), ZF-7 ultraviolet analyzer (Shanghai Jiapeng).

[0019] 1.2 Reagents Aconitine, 3-deoxyaconitine, N-deethyl-3-deoxyaconitine, 12-epi-aconitine, and neoline control samples (self-made); modified bismuth potassium iodide reagent (self-made); other reagents are analytical pure: chloroform (Yunnan Yanglin Industrial Development Zone Shandian Pharmaceutical Co., Ltd., batch number: 20180604), petroleum ether (Tianjin Zhiyuan Chemical Reagent Co., Ltd., batch number: 202102014), hydrochloric acid (Yunnan Yanglin Industrial Development Zone Shandian Pharmaceutical Co., Ltd., batch number: 20120818), sodium hydroxide (Tianjin Zhiyuan Chemical Reagent Co., Ltd., batch number: 20200220), methanol (Tianjin Fenshen Chemical Reagent Technology Co., Ltd., batch number: 2021091601), cyclohexane (Tianjin Fenshen Chemical Reagent Technology Co., Ltd., batch number: 2021083001), ethyl acetate (Tianjin Fenshen Chemical Reagent Technology Co., Ltd., batch number: 2021091701).

[0020] 1.3 Drug material The experimental Aconitum brachypodum drug material was collected from the Aconitum brachypodum planting base of Kunming Yusi Pharmaceutical Co., Ltd. in Tongdan Town, Dongchuan District, Kunming City in November 2020 (batch number: 201110-02, 201110-03, 210510-01, 210510-02, 210510-03). The plant specimen was identified by Mr. Yang Wenguang of the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences as Aconitum brachypodum of Ranunculaceae Aconitum brachypodum Diels., and the specimen (specimen number: 2021081701-1) was stored in the College of Chinese Medicine, Yunnan University of Chinese Medicine.

[0021] 2. Methods and results 2.1 Thin layer chromatography identification 2.1.1 Preparation of control solution Precisely weigh aconitine, 3-deoxyaconitine, N-deethyl-3-deoxyaconitine, 12-epi-aconine, neoline, etc. control samples, dissolve in methanol and prepare 1 mg / ml solution as control solution.

[0022] 2.1.2 Preparation of test solution Take Aconitum brachypodum drug material, crush, pass through a No. 3 sieve, and precisely weigh 5 g. Add 1% hydrochloric acid 50 ml and ultrasonic extraction three times (20 minutes each time). Filter and combine the extract. The water layer is extracted with petroleum ether four times, 50 ml each time, and the residue is extracted with chloroform four times, 50 ml each time. Combine the chloroform extract, concentrate to dryness, dissolve in ethanol, add 10 g of basic alumina, and elute with ethanol until there is no alkaloid reaction. Concentrate, reduce pressure and dry at 80°C for 4 hours (weigh). Dissolve the residue in ethanol and dilute to 25 ml in a volumetric flask, shake well, filter, and take the filtrate as the diester type alkaloid test solution.

[0023] 2.1.3 Thin layer chromatography According to the test of thin layer chromatography (general rule 0502), take 5 μl of the above test solution (0.2 g / ml), 5 μl of diester type alkaloid solution (diluted to 1 mg / ml), and 2 μl of control solution (1 mg / ml) respectively, and point them on the same silica gel G thin layer plate. Use cyclohexane-ethyl acetate-methanol (6.4:3.6:1) as the developing agent in an ammonia vapor saturated developing jar for 20 minutes, develop, take out, air dry, and spray with dilute bismuth potassium iodide solution. In the test sample chromatogram, the same colored spots appear at the corresponding positions of the control sample chromatogram. The thin layer chromatography results show that the spots of each sample are clear and well separated, which can be used for the identification of toxic alkaloids.

[0024] 2.2 Determination of content 2.2.1 Preparation of control solution Accurately weigh the aconitine reference substance, dissolve in methanol and prepare a solution containing 25 μg / ml as the reference solution.

[0025] 2.2.2 Preparation of test solution Accurately take 1 ml of the test solution under 2.1.2, place it in a 500 ml volumetric flask, add ethanol to the mark, shake well, and you get it.

[0026] 2.2.3 Selection of determination wavelength Take methanol as the blank solvent, respectively, scan the absorption spectrum of the reference solution and the test solution. The maximum absorption wavelength of the reference solution is 230 nm, and the maximum absorption wavelength of the test solution is 226 nm, so 230 nm is selected as the determination wavelength. The wavelength scanning spectrum shows that there is a characteristic absorption peak at 230 nm, which is suitable for content determination.

[0027] 2.2.4 Drawing of standard curve Accurately weigh the aconitine reference substance, dissolve in methanol and prepare a solution containing 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.066, 0.08 mg / ml of standard solution. At 230 nm wavelength, the absorbance value is measured, and the standard curve is drawn with the reference substance concentration as the abscissa and the absorbance value as the ordinate. The standard curve equation is Y=20.767X-0.0323 (r=0.9902). The results show that in the range of 0-83.3 μg / ml, the absorbance value has a good linear relationship with the reference substance concentration. The standard curve shows good linear relationship, which meets the requirements of quantitative analysis.

[0028] 2.2.5 Repetitive experiment Take 6 portions of the same batch of Artemisia rupestris L. medicinal materials (201110-02), each 5 g, prepare the test solution by the method of 2.2.2 and measure the absorbance, calculate the average content and error. The results are shown in Table 1: Table 1 Repetitive experiment

[0029] Average content: 0.3383%, RSD=2.12% 2.2.6 Recovery experiment Take the known content of Artemisia rupestris L. (201110-02) powder, pass through No. 3 sieve, accurately weigh 9 portions, add aconitine reference solution according to 50%, 100%, 150% of the total content of diester alkaloids. Prepare the test solution by the method of 2.2.2 and measure the absorbance, calculate the recovery rate. The results are shown in Table 2: Table 2 Recovery rate experiment

[0030] Average recovery: 100.91%, RSD = 2.69% 2.2.7 Sample content determination Five batches of Aconitum brachypodum were taken to determine the content of toxic components, and the results are shown in Table 3: Table 3 Determination of toxic component content in medicinal materials

[0031] As an important medicinal material of Aconitum, Aconitum brachypodum is included in many pharmacopoeias, but most of them are listed in the toxic drug directory. Traditionally, oral drugs are processed by decoction to reduce toxicity and enhance efficacy. Modern pharmaceutical research has confirmed that the toxicity of Aconitum brachypodum and related medicinal materials mainly comes from aconitine and other bis-ester type alkaloids. Bis-ester type alkaloids have strong activity and high toxicity, with a narrow safety window, and there are safety risks in clinical application. Therefore, during the processing of medicinal materials, the bis-ester type alkaloids are converted to mono-ester or alcohol amine type alkaloids by hydrolyzing the acetyl or benzoyl groups at positions 8 and 14 (or other hydroxybenzoyl groups). These bioactive compounds have lower toxicity, a wide safety window, and the least risk of poisoning in clinical application.

[0032] Due to the lack of quality control standards, the clinical use of Aconitum brachypodum and related medicinal materials often leads to poisoning incidents. Pharmaceutical companies often over-process to ensure clinical safety, resulting in almost undetectable bioactive compound content. Through the revision of the standards for Aconitum brachypodum and related medicinal materials in the 2010 edition of the Chinese Pharmacopoeia, safety and effectiveness standards based on material basis have been established. By setting an upper limit for the concentration of bis-ester type alkaloids to control the safety of these medicinal materials, and by controlling the lower limit of mono-ester or alcohol amine type alkaloids to ensure efficacy. This quality control strategy based on material basis has been widely supported and applied, and is a major achievement in the use and maintenance of Aconitum brachypodum and related medicinal materials.

[0033] Aconitum brachypodum is also a medicinal material of Aconitum, and its chemical components are similar to those of Aconitum, Aconitum, and other Aconitum medicinal materials. These toxic components are mainly derived from bis-ester type diterpene alkaloids, including aconitine and its derivatives. However, Aconitum brachypodum is different in that its crude drug contains a large amount of alcohol amine type diterpene alkaloids, and does not need to increase the concentration of mono-ester type alkaloids through processing to improve efficacy and reduce toxicity. Therefore, the key to ensuring the clinical safety of Aconitum brachypodum lies in the qualitative and quantitative control of bis-ester type diterpene alkaloids.

[0034] Chemical studies have shown that the double ester alkaloids in Aconitum flavum include aconitine and its derivatives, which contain benzoyl groups in their chemical structures, and thus have significant absorption at 230 nm. The single ester or alcohol amine alkaloids in Aconitum flavum lack benzoyl groups and have large molecular polarity, and cannot be separated from double ester alkaloids by organic solvents due to their strong affinity for acid water. Therefore, the double ester alkaloids are enriched by acid water organic solvent extraction in the present study, and then quantified by using the absorption of the shared conjugated benzoyl system at about 230 nm. The method is specific, simple, fast, and has important significance for establishing the control standard of toxic components of Aconitum flavum and its preparations and ensuring the safe use in clinic.

[0035] The alkaloids in Aconitum flavum are extracted by acid water extraction method in the present application. In order to avoid the interference of phytosterols and other non-alkaloid compounds, the acid water extract is extracted by petroleum ether. In addition, chloroform is used to extract and enrich double ester alkaloids in acid water, so as to exclude the possible influence of other macromolecules. Finally, the obtained crude alkaloids are purified by basic aluminum oxide, which completely eliminates the interference of phenolic compounds such as flavones. The whole preparation process of the test solution basically extracts, enriches and purifies the toxic components, i.e. double ester alkaloids in Aconitum flavum, which provides a guarantee for accurate determination of the content. Moreover, the toxic alkaloid contents measured by the two methods are consistent, which provides support for the safety control of Aconitum flavum medicinal materials and its preparations in clinic.

[0036] As can be seen from the above examples, the present application provides a thin layer chromatography identification method and a UV spectrophotometric content determination method for toxic alkaloids in Aconitum flavum. The method has good repeatability (RSD=2.12%) and recovery rate (100.91%, RSD=2.69%), and can accurately determine the content of toxic components in Aconitum flavum medicinal materials, which provides a scientific basis for controlling the quality of medicinal materials and ensuring the safety of clinical medication.

[0037] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for improving the safety of *Saussurea involucrata* medicinal material or preparations, characterized in that, This includes identifying toxic alkaloids in Artemisia selengensis using thin-layer chromatography and determining the content of diester alkaloids in Artemisia selengensis using ultraviolet spectrophotometry, thereby improving safety by controlling the content of diester alkaloids.

2. The method according to claim 1, characterized in that, The thin-layer chromatography method includes preparing a reference solution and a test solution, spotting them onto a thin-layer plate, developing the solution, and developing the color.

3. The method according to claim 2, characterized in that, The reference solution contains at least one of aconitine, 3-deoxyaconitine, N-deethyl-3-deoxyaconitine, 12-epi-aconitine, and neorin, at a concentration of 0.5 mg / mL to 2 mg / mL.

4. The method according to claim 2, characterized in that, The preparation of the test solution includes ultrasonic extraction of *Artemisia selengensis* powder with hydrochloric acid solution, filtration, extraction of the aqueous layer with petroleum ether, followed by chloroform extraction, concentration of the combined chloroform extracts, dissolution with ethanol, and purification with alkaline alumina. Elute, concentrate and dry, and make up to volume with ethanol for the residue.

5. The method according to claim 4, characterized in that, The hydrochloric acid solution has a mass concentration of 0.5% to 2%, and the ultrasonic extraction is performed 2 to 4 times, with each ultrasonic extraction lasting 15 to 25 minutes. The petroleum ether extraction is performed 3 to 5 times, with each extraction using 40 to 60 ml of petroleum ether; the chloroform extraction is performed 3 to 5 times, with each extraction using 40 to 60 ml of chloroform.

6. The method according to claim 2, characterized in that, The thin-layer chromatography method uses silica gel G thin-layer plates, the developing solvent is a mixed solution of cyclohexane, ethyl acetate and methanol in a volume ratio of 6~7:3~4:0.8~1.2, the developing tank is saturated with ammonia vapor for 15~25 minutes, and the colorimetric reagent is dilute potassium bismuth iodide solution.

7. The method according to claim 1, characterized in that, The ultraviolet spectrophotometry method includes preparing a reference solution and a test solution, and measuring the absorbance at a wavelength of 225 nm to 235 nm.

8. The method according to claim 7, characterized in that, The reference solution is aconitine solution with a concentration of 20 micrograms per milliliter to 30 micrograms per milliliter. The preparation of the test solution includes diluting the thin-layer chromatography test solution by 400 to 600 times; It also includes plotting a standard curve, the equation of which is Y equal to aX plus b, where a is 20 to 22, b is -0.04 to -0.02, and the linear range is 0 micrograms per milliliter to 90 micrograms per milliliter.

9. The method according to claim 1, characterized in that, The method also includes repeatability experiments, taking multiple samples to determine the content of diester-type alkaloids, with a relative standard deviation of less than 5%; The method also includes a spiking recovery experiment, with a recovery rate of 95% to 105% and a relative standard deviation of less than 5%.

10. The method according to claim 4, characterized in that, The purification of alkaline alumina includes elution with ethanol until no alkaloid reaction occurs, followed by vacuum drying at a temperature of 70°C to 90°C for 3 to 5 hours.

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