Application of scopolamine alkaloid as marker for identifying adulterants in radix aucklandiae
The HPLC method was used to detect the content of hyoscyamine alkaloids in Costus root, which solved the problem of identifying adulteration in Costus root, ensured the safety of Chinese medicinal materials and met pharmacopoeia testing standards, and achieved effective control of the toxic side effects of Costus root.
Patent Information
- Application Number
- CN202511639747.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-11
- Publication Date
- 2026-02-03
AI Technical Summary
Current technology has failed to effectively identify and detect the toxic drug Sanfensan, which is adulterated in costus root, leading to safety risks in the use of traditional Chinese medicine. This is especially true in cases where costus root from Yunnan is easily confused with Sanfensan, and there is a lack of pharmacopoeia standards for detecting such toxic side effects.
The content of hyoscyamine alkaloids in Aucklandia lappa was determined by HPLC. A standard curve was established by preparing hyoscyamine standard solution and test sample solution, and the limit of quantitation of hyoscyamine was determined. This method serves as a marker for identifying adulterated products in Aucklandia lappa and provides a qualitative and quantitative detection method.
It enables accurate identification and quantitative detection of three components in Costus root, ensuring the quality of Chinese medicinal materials and the safety of clinical use, providing a testing basis for pharmacopoeia regulations, and reducing the risk of toxic side effects.
Smart Images
Figure CN121453958A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of traditional Chinese medicine identification, and particularly relates to identification of adulteration of Sanfen San in Radix Aucklandiae, and a detection method and a limited standard of anisodine as a marker for identification. BACKGROUND
[0002] Radix Aucklandiae Aucklandiae Radix is the dried root of Aucklandiae lappae Dcne. Aucklandia lappa of the Compositae family, is a commonly used traditional Chinese medicine with pungent and bitter flavors and warm nature. It has the functions of promoting qi and relieving pain, and invigorating the spleen and promoting digestion. Sanfen San belongs to the Solanaceae family and has bitter and pungent flavors and warm nature; it is highly toxic. Roots, stems and leaves all contain tropane alkaloids, and eating too much of them will cause serious consequences, such as nausea and vomiting, hallucinations, limb rigidity and respiratory depression. In market research and visits, it is found that there is a rotation of Radix Aucklandiae and Sanfen San in Yunnan, and the medicinal parts of both are dried roots, and the shapes are similar. Radix Aucklandiae is easily mixed with Sanfen San when harvested, which poses a certain safety risk to the use of Radix Aucklandiae. In recent years, there have been more and more reports of safety accidents of Radix Aucklandiae, which has attracted widespread attention from the society. It has been reported that after taking Radix Aucklandiae powder, people have toxic side effects such as dizziness, nausea, vomiting, incoherent speech, restlessness, agitation and hyperactivity. The 2025 edition of the People's Republic of China Pharmacopoeia has not yet identified and detected the index components of such toxic side effects of Radix Aucklandiae.
[0003] In order to better guide the planting, harvesting and quality inspection of medicinal materials of Radix Aucklandiae and ensure the safety of clinical use of Radix Aucklandiae, the application extracts mixed powders of Radix Aucklandiae and Sanfen San with different mass ratios, sets a quantitative limit for the detection of anisodine in the extract of Radix Aucklandiae mixed with Sanfen San, provides technical support for the quality of medicinal materials of Radix Aucklandiae and the safety of clinical use, and also provides a basis for relevant pharmacopoeia regulations and the detection of impurity limits of toxic side effects of Radix Aucklandiae, which is of great significance. SUMMARY
[0004] The purpose of the application is to screen the toxic drug Sanfen San mixed in Radix Aucklandiae. A limited detection method of the marker anisodine of the toxic drug Sanfen San mixed in Radix Aucklandiae is provided. The application can provide technical support for the quality assurance and efficacy safety of traditional Chinese medicine Radix Aucklandiae. The application uses HPLC method to determine the content of anisodine in mixed powders of Radix Aucklandiae and Sanfen San with different mass ratios, and has high feasibility and practicality in the application of Radix Aucklandiae variety adulteration identification.
[0005] Technical scheme, in order to achieve the above purpose, the technical steps adopted by the application are as follows: The application of tropane alkaloids as a marker for identifying adulteration of Sanfen San in Radix Aucklandiae is provided. The detection of whether tropane alkaloids such as anisodine are added in Radix Aucklandiae is provided to evaluate the authenticity and quality of Radix Aucklandiae.
[0006] Application of hyoscyamine alkaloids as a detection item for toxic and side effects of costus root.
[0007] Application of hyoscyamine alkaloids as markers for identifying adulteration with toxic drugs in costus root.
[0008] A qualitative and quantitative marker for identifying the adulteration of costus root with hyoscyamine, wherein the marker is hyoscyamine.
[0009] The detection of hyoscyamine in the extract of Costus root adulterated with three parts of the extract includes the following steps: (1) Preparation of hyoscyamine standard solution and establishment of standard curve: Preparation of hyoscyamine standard stock solution: Accurately weigh hyoscyamine powder, dissolve it in ultrapure water and make up to volume, prepare fresh each time; Preparation of hyoscyamine standard solutions: Hyoscyamine standard stock solution was taken and serially diluted to obtain hyoscyamine standard solutions of different concentrations; hyoscyamine standard solutions of different concentrations were injected into an ultra-high performance liquid chromatograph, and linear regression analysis was performed with peak area as the ordinate (Y) and hyoscyamine reference solution concentration as the abscissa (X) to plot the hyoscyamine standard curve equation; (2) Preparation of a solution of three parts test sample mixed with three parts of costus root: Take whole, mold-free, disease-free, and rot-free Saussurea costus roots and three-thirds root, and air-dry, bake, crush, and sieve them to obtain Saussurea costus powder and three-thirds powder. Mix Saussurea costus powder and three-thirds powder in a certain mass ratio, add ultrapure water, extract, filter, and obtain Saussurea costus mixed with three-thirds powder extract. Filter the extract through a microporous membrane and use the filtrate as the test solution of Saussurea costus mixed with three-thirds. (3) Determination of hyoscyamine in the test solution of costus root mixed with three parts: Take the test solution of the three-part mixture of costus root from step (2) and inject it into the ultra-high performance liquid chromatograph for analysis. Then, substitute the peak area into the standard curve equation of step (1) to calculate the content of hyoscyamine and the limit of quantitation in the three-part mixture of costus root.
[0010] As a preferred embodiment, the chromatographic conditions for steps (1) and (3) of the above-described application are as follows: the mobile phase is 0.05% potassium dihydrogen phosphate solution (containing 0.0025 mol / L sodium heptanesulfonate) as phase A, and acetonitrile (adjusted to pH 5.0 with phosphoric acid or sodium hydroxide solution) as phase B, with isocratic elution at a ratio of 84:16; the detection wavelength is 215 nm; the flow rate is 1 mL / min; the injection volume is 20 μL; and the retention time is 20 min.
[0011] As a preferred embodiment, in the application described above, step (2) preparation of the *Saussurea costus* mixed with the *Trifoliate trifoliate* test solution: Weigh *Saussurea costus* powder - *Trifoliate trifoliate* powder L1~L10 (where the mass ratio of *Trifoliate trifoliate* is L1 20%: 8 g-2 g; L2 15%: 8.5 g-1.5 g; L3 10%: 9 g-1 g; L4 5%: 9.5 g-0.5 g; L5 3%: 9.7 g-0.3 g; L6 1%: 9.9 g-0.1 g; L7 0.5%: 9.95 g-0.05 g; L8 0.3%: 9.97 g-0.03 g; L9 0.1%: 9.99 g-0.01 g; L10 0.05%: 9.995 g-0.005 g) into a 250 mL stoppered conical flask, add 50 mL of ultrapure water, 80 The mixture was sonicated at kHz for 30 min, filtered to obtain a powder extract of Costus root mixed with three parts of the powder, and filtered through a 0.45 μm microporous membrane. The filtrate was then used as the test solution of Costus root mixed with three parts of the powder.
[0012] As a preferred option, the application described above includes step (1) preparation of hyoscyamine standard stock solution and establishment of standard curve: Hyoscyamine standard stock solution: Accurately weigh 14 mg of hyoscyamine, dissolve it in ultrapure water, and dilute to 10 ml. Prepare fresh before use. Hyoscyamine standard solution: Take 5 mL of hyoscyamine standard stock solution, add ultrapure water to make up to 10 mL, and obtain a hyoscyamine standard solution with a concentration of 700 μg / mL; Accurately pipette 5.00 mL of a 700 μg / mL hyoscyamine standard solution into a 10 mL volumetric flask, and dilute with ultrapure water to 10 mL to obtain a 350 μg / mL hyoscyamine standard solution. Take another 5.00 mL of the 350 μg / mL hyoscyamine standard solution into a 10 mL volumetric flask, and dilute with ultrapure water to 10 mL to obtain a 175 μg / mL hyoscyamine standard solution. Repeat the above steps three times to obtain concentrations of 87.5 μg / mL, 43.75 μg / mL, and 21.875 μg / mL, respectively. Hyoscyamine standard solution of μg / mL was prepared; hyoscyamine standard solutions of different concentrations were injected into the ultra-high performance liquid chromatograph. Linear regression analysis was performed with peak area as the ordinate (Y) and hyoscyamine standard solution concentration as the abscissa (X). The standard curve equation for hyoscyamine was y = 186.53x - 0.1656, R0. 2 = 0.9997, with a linear range of 0.022-0.700 mg / mL.
[0013] The present invention provides a limit of quantification of hyoscyamine in a three-part mixed sample solution of costus root with a limit of 0.2915 mg / g or higher.
[0014] Methodological review of the analysis and detection of hyoscyamine 1. Precision, repeatability and stability tests Precision test: Hyoscyamine reference solution was injected six times under the same conditions as the standard curve described above. Precision was calculated using the relative standard deviation (RSD) of the hyoscyamine peak area. The results showed that the RSD of the hyoscyamine peak area was 2.6%, indicating good precision of this method.
[0015] Repeatability test: Following the preparation method of the test solution with a 3:3 ratio of costus root and sample, six parallel groups of the test solution were prepared. The hyoscyamine content and RSD were calculated after analysis. The results showed that the RSD of the hyoscyamine content was 2.5%, indicating good repeatability of this method.
[0016] Stability test: One part of the costus root solution was mixed with three parts of the test sample solution in the repeatability test, and the peak area of hyoscyamine was measured at 0, 4, 8, 12, and 24 hours, and its content was calculated. The results showed that the peak area RSD was 3.7%, indicating that the test solution had good stability within 24 hours.
[0017] 2. Recovery test Take 5 mL of a solution of *Saussurea costus* mixed with three parts of the test sample, whose hyoscyamine content has been determined, and add hyoscyamine reference standard at three levels (80%, 100%, and 120% of the known content) to prepare a mixed test solution. Measure the solution at 215 nm and calculate the recovery rate. The results show that the average recovery rate of hyoscyamine is 98.43%, with an RSD of 1.6%, indicating that the method has good accuracy. Beneficial effects
[0018] Current Chinese Pharmacopoeia standards do not specify quality testing requirements for *Saussurea costus* adulterated with *Trichoderma solani*. In Yunnan, *Saussurea costus* is often rotated with *Trichoderma solani*, making it easy for *Trichoderma solani* containing hyoscyamine alkaloids to be mixed in during harvesting. The clinical safety of such *Saussurea costus* is therefore questionable. This invention applies hyoscyamine alkaloids as markers for identifying adulterants in *Saussurea costus*. This invention uses qualitative and quantitative determination of hyoscyamine in *Trichoderma solani* roots to identify whether *Trichoderma solani* is present in *Saussurea costus* and the amount of adulteration. This provides a scientific basis for ensuring the safety and efficacy of *Saussurea costus* in clinical use. Simultaneously, this invention can also provide a basis for limiting the amount of impurities that may cause toxic side effects in *Saussurea costus*, providing a basis for adding impurity testing items to relevant pharmacopoeia regulations.
[0019] This method was tested through precision tests, repeatability tests, stability tests, and spiking recovery tests. The results show that this method is stable, reliable, and accurate in determining hyoscyamine in a mixture of three parts of costus root. Attached Figure Description
[0020] Figure 1 The chromatograms are of the reference standards hyoscyamine and costus root, and of the three-part test solution.
[0021] Figure 2 Chromatograms of test solutions containing 20%, 15%, and 10% of Costus root in three separate solutions.
[0022] Figure 3 Chromatograms of test solutions containing 5%, 3%, and 1% of costus root.
[0023] Figure 4 Chromatograms of test solutions containing 0.5%, 0.3%, and 0.1% of Costus root.
[0024] Figure 5 Chromatogram of a 3:3 mixture of 0.05% costus root and test solution. Detailed Implementation
[0025] The following examples further illustrate the present invention. These are merely embodiments of the present experiment. Any person skilled in the art can use the above-described technical solutions to improve the present invention or modify it into an equivalent technical solution.
[0026] The method for determining hyoscyamine in a three-part mixed sample solution of costus root provided by this invention includes the following steps: Step (1) Preparation of hyoscyamine standard stock solution and establishment of standard curve: Hyoscyamine standard stock solution: Accurately weigh 14.182 mg of hyoscyamine, dissolve it in ultrapure water, and dilute to 10 ml. Prepare fresh before use. Hyoscyamine standard solution: Take 5 mL of hyoscyamine standard stock solution, add ultrapure water to make up to 10 mL, and obtain a hyoscyamine standard solution with a concentration of 709.1 μg / mL; Accurately pipette 5.00 mL of a 709.1 μg / mL hyoscyamine standard solution into a 10 mL volumetric flask, and dilute with ultrapure water to 10 mL to obtain a hyoscyamine standard solution with a concentration of 354.55 μg / mL. Take another 5.00 mL of the 354.55 μg / mL hyoscyamine standard solution into a 10 mL volumetric flask, and dilute with ultrapure water to 10 mL to obtain a hyoscyamine standard solution with a concentration of 177.28 μg / mL. Repeat the above steps three times to obtain hyoscyamine standard solutions with concentrations of 88.645 μg / mL, 44.32 μg / mL, and 22.16 μg / mL, respectively. Hyoscyamine standard solutions of different concentrations were injected into an ultra-high performance liquid chromatograph. Linear regression analysis was performed, with peak area as the ordinate (Y) and hyoscyamine standard solution concentration as the abscissa (X). The equation for the hyoscyamine standard curve was y = 186.53x - 0.1656. R 2 = 0.9997, with a linear range of 0.022~0.709 mg / mL.
[0027] The chromatographic conditions were as follows: Octadecylsilane-bonded silica gel was used as the stationary phase; the mobile phase consisted of 0.05% potassium dihydrogen phosphate solution (containing 0.0025 mol / L sodium heptanesulfonate) (A) and acetonitrile (pH adjusted to 5.0 with phosphoric acid or sodium hydroxide solution) (B); isocratic elution was performed at an 84:16 ratio for 20 min; the detection wavelength was 215 nm. The theoretical plate number, calculated based on hyoscyamine, should be no less than 3500. The flow rate was 1.0 mL / min. -1 Column temperature 30℃.
[0028] Step (2) Preparation of the test sample solution containing three parts costus root: Select mature, unblemished, and insect-free roots of *Saussurea costus* and *Saussurea involucrata* (including *Saussurea costus* root and *Saussurea costus* root), air-dry, oven-dry, pulverize, and pass through a 50-mesh sieve. Accurately weigh the *Saussurea costus* powder – *Saussurea costus* powder – into L1~L10 portions (L1 20%: 8.0009g-2.0008g; L2 15%: 8.5004g-1.5005g; L3 10%: 9.0004g-1.0003g; L4 5%: 9.5005g-0.5006g; L5 3%: 9.7006g-0.3002g; L6 1%: 9.9003g-0.1004g; L7 0.5%: 9.9507g-0.0504g; L8 0.3%: 9.9706g-0.0303g; L9…). 0.1% (9.9902g-0.0107g; L10 0.05% (9.9951g-0.0053g)) was added to a 250 mL stoppered conical flask, along with 50 mL of ultrapure water. The mixture was sonicated at 80 kHz for 30 min, filtered to obtain a mixed extract of three parts of *Saussurea costus*, and then filtered through a 0.45 μm microporous membrane. The filtrate was used as the test solution for *Saussurea costus* mixed with three parts.
[0029] Step (3) Preparation of test solution for genuine Chinese herbal medicine Costus Root: Mature, disease-free, and insect-infested roots of *Saussurea costus* were selected, naturally air-dried, oven-dried, pulverized, and passed through a 50-mesh sieve. *Saussurea costus* powder J1~J10 (J1 10.0121g; J2 10.0279g; J3 10.0192g; J4 10.0258g; J5 10.0294g; J6 10.0177g; J7 10.0175g; J8 10.0160g; J9 10.0169g; J10 10.0233g) were accurately weighed and placed in a 250 mL stoppered conical flask. 50 mL of ultrapure water was added, and the mixture was sonicated at 80 kHz for 30 min. The extract was filtered to obtain the genuine *Saussurea costus* extract, and then filtered through a 0.45 μm microporous membrane. The filtrate was used as the test solution for genuine *Saussurea costus*.
[0030] Step (4) Determination of hyoscyamine in the test solution of the mixed three parts and the test solution of the original costus root: Take the test solution of the three-part mixed costus root from step (2) and the test solution of the genuine costus root from step (3) respectively, and inject them into the ultra-high performance liquid chromatograph for analysis under the same chromatographic conditions as in step (1); then substitute the peak area of hyoscyamine into the standard curve equation of step (1) to calculate the content of hyoscyamine in the three-part mixed costus root test solution and the genuine costus root test solution.
[0031] The hyoscyamine content in each batch of costus root was as follows: L1 0.1859 mg / mL; L2 0.1627 mg / mL; L3 0.1280 mg / mL; L4 0.0978 mg / mL; L5 0.0988 mg / mL; L6 0.0787 mg / mL; L7 0.0583 mg / mL; and hyoscyamine was not detected in L8 to L10.
[0032] After conversion, the limit of quantification for the adulteration of costus root with tris(tris(hyoscyamine)) is not to exceed 0.2915 mg / g.
[0033] Hyoscyamine was not detected in any batch of genuine Costus root products.
[0034] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. Application of hyoscyamine alkaloids as markers for identifying adulterants in costus root.
2. Application of hyoscyamine alkaloids as a detection item for toxic and side effects of costus root.
3. Application of hyoscyamine alkaloids as markers for identifying adulteration with toxic drugs in costus root.
4. The application according to claim 3, for detecting hyoscyamine in the extract of costus root adulterated with trifoliate extract, characterized in that, Includes the following steps: (1) Preparation of hyoscyamine standard solution and establishment of standard curve: Preparation of hyoscyamine standard stock solution: Accurately weigh hyoscyamine powder, dissolve it in ultrapure water and make up to volume, prepare fresh each time; Preparation of hyoscyamine standard solutions: Hyoscyamine standard stock solution was taken and serially diluted to obtain hyoscyamine standard solutions of different concentrations; hyoscyamine standard solutions of different concentrations were injected into an ultra-high performance liquid chromatograph, and linear regression analysis was performed with peak area as the ordinate (Y) and hyoscyamine reference solution concentration as the abscissa (X) to plot the hyoscyamine standard curve equation; (2) Preparation of a solution of three parts test sample mixed with three parts of costus root: Take whole, mold-free, disease-free, and rot-free Saussurea root and three-thirds root, and air-dry, bake, crush, and sieve them to obtain Saussurea powder and three-thirds powder. Mix Saussurea powder and three-thirds powder in a certain mass ratio, add water, extract, filter, and obtain Saussurea mixed with three-thirds powder extract. Filter the extract through a microporous membrane and use the filtrate as the test solution of Saussurea mixed with three-thirds. (3) Determination of hyoscyamine in the test solution of costus root mixed with three parts: Take the test solution of the three-part mixture of costus root from step (2) and inject it into the ultra-high performance liquid chromatograph for analysis. Then, substitute the peak area into the standard curve equation of step (1) to calculate the content of hyoscyamine and the limit of quantitation in the three-part mixture of costus root.
5. The application according to claim 4, characterized in that, The chromatographic conditions for steps (1) and (3) are as follows: 0.05% potassium dihydrogen phosphate solution (containing 0.0025 mol / L sodium heptanesulfonate) is phase A, acetonitrile (pH adjusted to 5.0 with phosphoric acid or sodium hydroxide solution) is phase B, elution is 84:16 isocratic; detection wavelength is 215 nm; flow rate is 1 mL / min; injection volume is 20 μL, retention time is 20 min.
6. The application according to claim 2, characterized in that, Step (2) Preparation of the test solution of costus root mixed with three parts: Weigh costus root powder and three parts powder L1~L10, where the mass ratio of three parts is L1 20%; L2 15%; L3 10%; L4 5%; L5 3%; L6 1%; L7 0.5%; L8 0.3%g; L9 0.1%; L10 0.05%, place them in a stoppered conical flask, add ultrapure water, extract by ultrasonication, filter to obtain costus root mixed with three parts, and filter through a 0.45 μm microporous membrane. Take the filtrate as the test solution of costus root mixed with three parts.
7. The application according to claim 2, characterized in that, Step (1) Preparation of hyoscyamine standard stock solution and establishment of standard curve: Hyoscyamine standard stock solution: Accurately weigh 14 mg of hyoscyamine, dissolve it in ultrapure water, and dilute to 10 ml. Prepare fresh before use. Hyoscyamine standard solutions: A certain amount of hyoscyamine standard stock solution was taken and diluted with ultrapure water to obtain hyoscyamine standard solutions with concentrations of 700 μg / mL, 350 μg / mL, 175 μg / mL, 87.5 μg / mL, 43.75 μg / mL and 21.875 μg / mL, respectively. Hyoscyamine standard solutions of different concentrations were injected into an ultra-high performance liquid chromatograph. Linear regression analysis was performed with peak area as the ordinate (Y) and hyoscyamine standard solution concentration as the abscissa (X). The standard curve equation for hyoscyamine was y = 186.53x - 0.1656, R² = 0.9997, and the linear range was 0.022–0.700 mg / mL.
8. The application according to any one of claims 1 to 7, characterized in that, The limit of quantification for the adulteration of costus root with tris(tris(hyoscyamine)) alkaloids is not to exceed 0.2915 mg / g.
9. The application according to claim 8, characterized in that, Hyoscyamine is a type of alkaloid.