Attenuation method for aconitum vilmorinianum

Abstract

The invention relates to a method for attenuating toxicity of Aconitum vilmorinianum Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii Kusnezoffii According to the method disclosed by the invention, the content of two diester alkaloids, namely yunaconitine and aconitine A, which take toxic components as representatives, in the co-decoction liquid can be reduced, the effect of reducing toxicity and keeping effect is achieved, and the selected meat is from a food source, so that no new toxicity risk is generated during co-decoction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine processing and traditional Chinese medicine component detection, and particularly relates to a detoxification method of Aconitum vilmorinianum Kom. BACKGROUND

[0002] Aconitum vilmorinianum Kom. is an important traditional Chinese medicinal material, which is the tuber of Ranunculaceae plant Aconitum vilmorinianum Kom. It is hot in nature, bitter and pungent in taste, and belongs to the liver, kidney and spleen channels. It has the effects of dispelling wind and dampness, and warming the channels and stopping pain. The main toxic components of Aconitum vilmorinianum Kom. are Yunnaconitine (YAC), Crassicauline A (CCA) and other double-ester type diterpenoid alkaloids, which are highly toxic and have complex mechanisms of action. The toxic effects mainly affect the central nervous system, heart and muscle tissue, and the damage to the heart is the most important and common risk factor of Aconitum vilmorinianum Kom. poisoning. Yunnaconitine is a characteristic toxic component of Aconitum vilmorinianum Kom. that is different from other Aconitum medicinal materials (such as Aconitum carmichaelii and Aconitum kusnezoffii). The toxicity of Yunnaconitine is not inferior to that of aconitine. In clinical application, the use of raw products often causes poisoning phenomena such as numbness of the lips, nausea, vomiting, palpitations and chest tightness, and even death in severe cases. Even after the traditional processing technology, Aconitum vilmorinianum Kom. may still not be completely detoxified due to improper dosage, insufficient decoction time or incorrect compatibility, leading to frequent poisoning incidents. Therefore, it is of great significance to improve the detoxification effect.

[0003] Modern pharmacological studies have shown that the components in Aconitum vilmorinianum Kom. have good therapeutic effects on rheumatoid arthritis, neuropathic pain, hypertension and other diseases, but the toxicity of Yunnaconitine is not inferior to that of aconitine, which can cause neurotoxicity and cardiotoxicity, and the safe use range is narrow, which seriously limits its clinical application. However, the effective components contained in Aconitum medicinal materials are also toxic components. Due to the significant therapeutic effect on difficult diseases, how to reduce the toxic effect while ensuring the clinical effect, that is, to reduce the toxicity and retain the effect, is the key and difficult problem that needs to be solved for such medicinal materials. However, the core of the traditional processing technology is still the conversion of double-ester type alkaloids into single-ester type alkaloids. Therefore, a new detoxification and retention technology needs to be established. SUMMARY

[0004] In view of the problems existing in the prior art, the present application provides a detoxification method of Aconitum vilmorinianum Kom.

[0005] The technical scheme for solving the above technical problems of the present application is as follows:

[0006] The present application provides a detoxification method of Aconitum vilmorinianum Kom., which comprises the following steps: co-decocting Aconitum vilmorinianum Kom. with meat, and the meat is selected from one or more of pigskin, pig lean meat, pig fat, beef, mutton and chicken.

[0007] Preferably, the meat is pork skin or mutton.

[0008] Different from the current thinking of reducing the toxicity of Aconitum medicinal materials by hydrolyzing the diester type into monoester type alkaloids, the present application proposes a new idea that "some components in meat react specifically with the toxic components in Huangcao Wu". The present application studies the "reducing toxicity and retaining efficacy" effect of meat on Huangcao Wu by "co-decoction of meat and Huangcao Wu". The selected meat is from food sources, and co-decoction with Huangcao Wu does not produce new toxic risks.

[0009] Different types of meat and different parts of meat contain different nutrients and have different effects on the reaction with toxic components. The present application compares the changes in toxic components before and after co-decoction of different types of meat and different parts of meat with Huangcao Wu, and finds that pork skin or mutton has the best reducing toxicity effect when co-decocted with Huangcao Wu.

[0010] Further, the method comprises the following steps: adding Huangcao Wu, meat, and water together, soaking, and decocting.

[0011] Further, the Huangcao Wu can be crushed and sieved Huangcao Wu.

[0012] Further, the mass ratio of Huangcao Wu to meat is 1:0.5-1:2, preferably 1:2.

[0013] Further, the volume to mass ratio of water to Huangcao Wu is 10mL:1g to 30mL:1g, preferably 30mL:1g.

[0014] Further, the decoction time is 0.5-2h, preferably 2h.

[0015] Further, the soaking time is 0.5h.

[0016] Further, the decoction times is 1-3 times, preferably 3 times.

[0017] The present application introduces orthogonal experiment to screen the primary and secondary effects of key factors such as decoction time, ratio of pork skin to Huangcao Wu, water amount, and extraction times in the co-decoction system of pork skin and Huangcao Wu, and constructs a comprehensive evaluation index of diester type alkaloids dihydrometeloidine and crude stemaconitine A. Further, the response surface method is used to analyze the interaction of multiple factors to determine the optimal process window. By quantifying the relevance of process parameters and reducing toxicity and retaining efficacy indicators, the present application breaks through the traditional experience dependence, establishes a processing quality control standard based on scientific data, provides technical support for the safe use of Huangcao Wu in clinical practice, expands the methodology system of Aconitum medicinal material processing research, and promotes the transformation and upgrading of the processing technology of ethnic medicines from an experience mode to a mathematical model.

[0018] The application provides a method for detecting components of attenuated Rhizoma Aconiti Lateralis Preparata, comprising the following steps: after the product of attenuated Rhizoma Aconiti Lateralis Preparata is treated, chromatography methanol is added, the supernatant is obtained by centrifugation, and the contents of delphinine, crude stem aconitine A, 8-deacetyldelphinine and 8-deacetylcrude stem aconitine A are detected by UHPLC-QQQ-MS / MS method.

[0019] The attenuated method of Rhizoma Aconiti Lateralis Preparata can refer to the attenuated method described above.

[0020] Further, the mobile phase is mixed by 0.1% formic acid in ultrapure water (A) and acetonitrile (B) at a volume ratio of 70:30, and the separation is completed within 6 min by using isocratic elution mode.

[0021] Further, the flow rate of the mobile phase is 0.25 mL·min -1 .

[0022] Further, the temperature of the chromatographic column thermostat is maintained at 40±1℃.

[0023] Further, the flow rate of the drying gas is 11.0 L min -1 , the temperature of the drying gas is 350℃, the atomization gas pressure is 15 psig, and the ion transmission voltage is 4000V.

[0024] Further, the ionization mass spectrum conditions of delphinine include: parent ion 660.3, daughter ion 135.1, cracking voltage 215V, collision energy 66eV, and detection mode is positive; the ionization mass spectrum conditions of crude stem aconitine A include: parent ion 644.4, daughter ion 135.1, cracking voltage 215V, collision energy 66eV, and detection mode is positive; the ionization mass spectrum conditions of 8-deacetyldelphinine include: parent ion 618.3, daughter ion 135.1, cracking voltage 205V, collision energy 62eV, and detection mode is positive; and the ionization mass spectrum conditions of 8-deacetylcrude stem aconitine A include: parent ion 602.3, daughter ion 135.1, cracking voltage 210V, collision energy 50eV, and detection mode is positive.

[0025] Further, the method further comprises the step of substituting the detected chromatographic peak area into the standard curve for calculation.

[0026] The application detects the contents of delphinine, 8-deacetyldelphinine, crude stem aconitine A and 8-deacetylcrude stem aconitine A of the same batch of Rhizoma Aconiti Lateralis Preparata and different kinds of meat, different parts of meat before and after being decocted under the same condition by liquid chromatography-mass spectrometry (UHPLC-QQQ-MS / MS), so as to evaluate the toxicity and determine the selection of meat for decocting with Rhizoma Aconiti Lateralis Preparata. The detection method has the advantages of good linear relationship, high precision, good stability, good repeatability and reliable results. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Representative EIC chromatograms of four target compounds for mixed reference solution (A) and test solution (B).

[0028] Figure 2 Response surface and contour plots of the two-factor interaction on the overall score, where a. extraction time (A) and the ratio of Aconitum carmichaeli to pigskin (B); b. extraction time (A) and extraction times (C); c. the ratio of Aconitum carmichaeli to pigskin (B) and extraction times (C).

[0029] Figure 3 Comparison of the stomach size of mice in each group after gavage, where A. blank control group; B. Aconitum carmichaeli single decoction group; C. Aconitum carmichaeli + pigskin co-decoction group.

[0030] Figure 4 Body weight changes of mice gavaged with Aconitum carmichaeli single decoction (A) and Aconitum carmichaeli + pigskin co-decoction (B) within 14 days.

[0031] Figure 5 Comparison of the death time of mice gavaged with the same dose of Aconitum carmichaeli single decoction and Aconitum carmichaeli + pigskin co-decoction (**P<0.01).

[0032] Figure 6 Comparison of the electrocardiogram of mice in each group (n=6), where A. blank control group; B. Aconitum carmichaeli single decoction group; C. Aconitum carmichaeli + pigskin co-decoction group.

[0033] Figure 7 Comparison of each item of myocardial zymogram of mice gavaged with the same dose of Aconitum carmichaeli single decoction and Aconitum carmichaeli + pigskin co-decoction (n=6), where A. glutamic-oxaloacetic transaminase (AST); B. creatine kinase (CK); C. creatine kinase isoenzyme (CK-MB); D. α-hydroxybutyric acid dehydrogenase (α-HBDH); E. lactate dehydrogenase (LDH) activity. *P<0.05, **P<0.01, vs. blank group; ## P<0.01, vs. Aconitum carmichaeli single decoction group.

[0034] Figure 8 Experimental procedure of Aconitum carmichaeli single decoction and Aconitum carmichaeli + pigskin co-decoction on CIA model rats.

[0035] Figure 9 Macroscopic characterization of arthritis of the right hind foot of rats in each group (n=6).

[0036] Figure 10For the relative weight change of each group of rats (n=6), Control represents the blank control group, Model represents the CIA model group, MTX represents the positive drug methotrexate group, H-L represents the low-dose group of single decoction of Aconitum leucostomum, H-H represents the high-dose group of single decoction of Aconitum leucostomum, H+P-L represents the low-dose group of co-decoction of Aconitum leucostomum and pigskin, and H+P-H represents the high-dose group of co-decoction of Aconitum leucostomum and pigskin. **P<0.01, vs. the blank control group; # P<0.05, vs. the CIA model group.

[0037] Figure 11 For the arthritis index score of each group of rats (n=6), Control represents the blank control group, Model represents the CIA model group, MTX represents the positive drug methotrexate group, H-L represents the low-dose group of single decoction of Aconitum leucostomum, H-H represents the high-dose group of single decoction of Aconitum leucostomum, H+P-L represents the low-dose group of co-decoction of Aconitum leucostomum and pigskin, and H+P-H represents the high-dose group of co-decoction of Aconitum leucostomum and pigskin. **P<0.01, vs. the blank control group; # P<0.05, vs. the CIA model group.

[0038] Figure 12 For the toe volume of each group of rats (n=6), Control represents the blank control group, Model represents the CIA model group, MTX represents the positive drug methotrexate group, H-L represents the low-dose group of single decoction of Aconitum leucostomum, H-H represents the high-dose group of single decoction of Aconitum leucostomum, H+P-L represents the low-dose group of co-decoction of Aconitum leucostomum and pigskin, and H+P-H represents the high-dose group of co-decoction of Aconitum leucostomum and pigskin. **P<0.01, vs. the blank control group; # P<0.05, vs. the CIA model group.

[0039] Figure 13 For the three-dimensional construction image of the foot tissue of each group of rats (n=6). DETAILED DESCRIPTION

[0040] The principles and features of the present application are described below in conjunction with the accompanying drawings, and the examples are only used to explain the present application and are not used to limit the scope of the present application.

[0041] The present application adopts ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QQQ-MS / MS) to quantitatively analyze the chemical components in the co-decoction liquid of Radix Aconiti Lateralis Praeparata and pigskin, lean meat, fatty meat, beef, mutton and chicken. The results show that, compared with the single decoction liquid of Radix Aconiti Lateralis Praeparata, the contents of two diester-type alkaloids represented by dihydromesaconitine and mesaconitine A in the co-decoction liquid of Radix Aconiti Lateralis Praeparata and pigskin / mutton are the lowest. This finding indicates that pigskin / mutton can reduce the content of diester-type aconite alkaloids through a specific pathway, and its detoxification mechanism is not derived from the classic pathway of generating monoester-type alkaloids through high-temperature hydrolysis.

[0042] The present application further adopts the method of combination of orthogonal experiment and response surface method to screen the optimal reaction conditions. First, the orthogonal experiment screens the decoction time, the ratio of Radix Aconiti Lateralis Praeparata to pigskin and the extraction times as the key factors in the co-decoction system of pigskin and Radix Aconiti Lateralis Praeparata, and the response surface method further optimizes the optimal combination of these factors, so as to determine that the optimal process window is that the extraction time is 2 h, the ratio of Radix Aconiti Lateralis Praeparata to pigskin is 1:2 (mass ratio), and the extraction times is 3 times.

[0043] The LD 50 of the single decoction liquid of Radix Aconiti Lateralis Praeparata is 9.74 g / kg (95% CI: 4.52-20.97 g / kg), while the LD 50 of the co-decoction liquid of Radix Aconiti Lateralis Praeparata+pigskin is significantly increased to 19.68 g / kg (95% CI: 7.55-51.33 g / kg), which is expanded by about 2.02 times, indicating that the co-decoction liquid of Radix Aconiti Lateralis Praeparata+pigskin is less toxic than the single decoction liquid of Radix Aconiti Lateralis Praeparata; the incidence of acute poisoning symptoms (arrhythmia, respiratory depression) of the animals in the co-decoction liquid group of Radix Aconiti Lateralis Praeparata+pigskin is significantly lower than that in the single decoction liquid group of Radix Aconiti Lateralis Praeparata under the same amount of crude drug, and the death time is delayed; the comparison of the results of electrocardiogram, echocardiogram and myocardial enzyme spectrum between the co-decoction liquid of pigskin and Radix Aconiti Lateralis Praeparata+pigskin and the single decoction liquid of Radix Aconiti Lateralis Praeparata all indicates that pigskin has a certain degree of alleviating effect on the cardiotoxicity of Radix Aconiti Lateralis Praeparata.

[0044] Through the volume measurement of the toes, the inflammation index score and Micro CT scanning, the foot tissue conditions of the rats in each group are analyzed, and the results of the single decoction liquid group of Radix Aconiti Lateralis Praeparata and the co-decoction liquid group of Radix Aconiti Lateralis Praeparata+pigskin have no statistical significance, but both of them have statistical significance compared with the CIA model group, so it is determined that pigskin does not obviously weaken the anti-inflammatory effect while reducing the toxicity of Radix Aconiti Lateralis Praeparata, which clarifies the synergistic effect of pigskin on reducing the toxicity and retaining the efficacy of Radix Aconiti Lateralis Praeparata, and confirms the scientificity of the compatibility of pigskin and Radix Aconiti Lateralis Praeparata.

[0045] In the examples, the specific technologies or conditions not specified are all carried out according to the conventional methods or the technologies or conditions described in the literature in the field, or according to the product instructions. The reagents and instruments used, etc. without the manufacturer specified are all conventional products that can be purchased through regular channels.

[0046] Aconitum vilmorinianum Kom. (batch number: 200303) from Ranunculaceae. Since the detection results of different batches of Aconitum vilmorinianum may have differences, therefore, the same batch of Aconitum vilmorinianum sample should be selected for experiment.

[0047] The lean meat, fat and pigskin used in the experiment were purchased from the local market in Guangzhou. The lean meat was pure lean meat (red in color) without skin, and the fat was pure fat (white in color) without skin. The beef involved in the experiment was beef with alternating lean and fat and skin, the mutton was mutton with alternating lean and fat and skin, and the chicken was chicken with skin, all purchased from the local market in Guangzhou. Acetonitrile (ACS., U.S.A.), methanol (ACS., U.S.A.), distilled water (Guangzhou Watsons Food and Beverage Co., Ltd.); Juzunine (purity 99.10%, batch number: JOT-10999), coptisine (purity 99.30%, batch number: JOT-10274), 8-deacetyljuzunine (purity 98.46%, batch number: JOT-11842), deacetylcopisine (purity 99.74%, batch number: JOT-11854) were purchased from Chengdu Pufide Biotechnology Co., Ltd. Isoflurane was purchased from Shenzhen Ruivode Life Science and Technology Co., Ltd. Collagen type II solution (Chondrex, 20022, USA); Complete Freund's Adjuvant (CFA) (Chondrex, 7027, USA), Incomplete Freund's Adjuvant (IFA) (Chondrex, 7002, USA).

[0048] The experimental instruments involved: Agilent 6490 triple quadrupole liquid / mass spectrometer (LC / QQQ) (Agilent Technologies, Santa Clara, CA, USA), Waters ACQUITY Premier CSH C 18Chromatographic column (1.7 μm, 2.1 x 150 mm), analytical balance (Sartorius), digital temperature control electric heating jacket (Sartorius), Centrifuge5425R micro table centrifuge (Eppendorf, Germany), vortex (Woxin XH-D type). 16-channel high-speed electrocardiograph (PowerLab); RE-52A rotary evaporator (Shanghai Yalong Biochemical Instrument Factory); digital temperature control electric heating jacket (Sartorius); Centrifuge5425R micro table centrifuge (Eppendorf, Germany); small animal ultrahigh resolution B-ultrasound (Vevo2100); MS-480 automatic biochemical analyzer (Mecan Bioscience Co., Ltd.); ME204E one-thousandth balance (Mettler-Toledo). Micro-computed tomography Micro CT (Bruker skyscan 1976); BLD-ZZRJ10 foot volume measuring instrument (BILEAD BIOSCI), multi-channel small animal anesthetizing machine (Rivard).

[0049] The following is described by specific examples.

[0050] Example 1

[0051] A control group and a treatment group were set up. The control group was Huangcao Wu without any meat; treatment group 1: Huangcao Wu + lean meat; treatment group 2: Huangcao Wu + fatty meat; treatment group 3: Huangcao Wu + pig skin; treatment group 4: Huangcao Wu + beef; treatment group 5: Huangcao Wu + mutton; treatment group 6: Huangcao Wu + chicken.

[0052] Each group had 6 parts, and 5g of Huangcao Wu was weighed in each part, crushed and sieved, 50mL of distilled water was added to each part, and 5g of meat was added to each part of treatment group 1 to treatment group 6, soaked for 0.5h, and then heated to boiling, and the timing started. The condensation reflux extraction method (100℃) was used to extract for 0.5h, and the extraction was repeated 3 times, and the extract was combined. The control group did not add meat, and the rest was the same as the treatment group.

[0053] The filtrate was obtained by filtering through two layers of silk cloth while hot. The filtrate was transferred to a 50mL volumetric flask, water was added to 1cm from the graduation line, and after standing, the volume was adjusted to the scale with a rubber bulb dropper. Shake well and use. 200μL of sample solution was taken, 800μL of chromatographic methanol was added, vortexed for 3min, and centrifuged at 4℃, 14000rpm for 10min, and the supernatant was placed in a sample bottle. UHPLC-QQQ-MS / MS method was used to detect the content of delphinine, crude stem aconitine A, 8-deacetyldelphinine and 8-deacetylcrudd stem aconitine A in each group.

[0054] Mass spectrometry and chromatography conditions: Agilent 6490 triple quadrupole liquid chromatography-mass spectrometry system (Agilent Technologies, Santa Clara, CA, USA) was used for chromatographic separation, and Waters ACQUITY Premier CSH C 18 chromatographic column (1.7 μm, 2.1 x 150 mm).

[0055] As shown in Table 1, the experimental system establishes a quantitative analysis method for four target components. After system optimization, the chromatographic conditions are determined: the mobile phase is a mixture of ultrapure water containing 0.1% (volume percent) formic acid (A) and acetonitrile (B) at a volume ratio of 70:30, and isocratic elution mode is used to complete separation within 6 min. The chromatographic column oven temperature is maintained at 40 ± 1℃, the flow rate of the mobile phase is set to 0.25 mL·min -1 , and the injection volume is fixed at 2 μL. The chromatographic column equilibrium stage uses the initial proportion of the mobile phase to continuously flush the column body to ensure system stability. The detection system is configured with an electrospray ionization source, and in positive ion scanning mode combined with multiple reaction monitoring technology, the synchronous analysis of the four components is completed. After optimization of the instrument parameters, the dry gas flow is 11.0 L min -1 , the dry gas temperature is 350℃, the atomization gas pressure is 15 psig, and the ion transmission voltage is 4000V. This analysis method realizes the accurate detection of target substances through system parameter optimization.

[0056] Table 1 Optimal mass spectrometry conditions for ionization of four target compounds

[0057]

[0058] Determination of the content of the four components in the sample: the chromatographic peak area of the sample obtained by detection is substituted into the standard curve to calculate the concentration of the sample, and thus the sample content is calculated according to the sample weight.

[0059] The standard curve equation of YAC is: y = 730236.28x - 1920.57, where x is the YAC concentration in μg / mL and y is the chromatographic peak area. The standard curve equation of CCA is: y = 651900.29x + 965.62, where x is the CCA concentration in μg / mL and y is the chromatographic peak area. The standard curve equation of DYA is: y = 413739.18x + 29919.47, where x is the DYA concentration in μg / mL and y is the chromatographic peak area. The standard curve equation of DCA is: y = 507100.14x + 9306.76, where x is the DCA concentration in μg / mL and y is the chromatographic peak area.

[0060] Data statistics and analysis: The experiment was detected by Agilent 6490 triple quadrupole mass spectrometry system, and the instrument operation was controlled by Mass Hunter workstation software (LC / MS data acquisition module, version B04.01). Quantitative analysis was completed by Mass Hunter quantitative analysis software (version B07.00) for data analysis.

[0061] The contents of different parts of pork and Huangcao Wu were determined, as shown in Table 2. Compared with the single decoction solution of Huangcao Wu, the contents of two kinds of diester alkaloids represented by the toxic and effective components, dienol and crude stem aconitine A, were the lowest in the co-decoction solution of Huangcao Wu and pig skin; the contents were the second lowest in the co-decoction solution of Huangcao Wu and lean meat; and the contents were the highest in the co-decoction solution of Huangcao Wu and fatty meat. However, the contents of two kinds of monoester alkaloids, 8-deacetyldienol and 8-deacetylcrudding stem aconitine A, were not only not increased, but also decreased in the co-decoction solution before and after adding different parts of pork. The above results show that the pig skin in pork is the most suitable part for chemical reaction with Huangcao Wu.

[0062] Different animal species were used for co-decoction with Huangcao Wu, and the content determination results are shown in Table 2. The meat from pigs, cows, sheep and chickens was used for co-decoction with Huangcao Wu, and the results showed that the meat from pigs, cows, sheep and chickens could achieve the effect of reducing toxicity, among which the meat from pigs and sheep had better effect of reducing toxicity, followed by the meat from cows and chickens.

[0063] Table 2 Contents of four target compounds after co-decoction of different meats with Huangcao Wu

[0064]

[0065] Example 2

[0066] (1) Linear relationship

[0067] Preparation of mixed reference solution: accurately weigh dienol (YAC), 8-deacetyldienol (DYA), crude stem aconitine A (CCA) and 8-deacetylcrudding stem aconitine A (DCA) respectively, and prepare with methanol. Then accurately pipette the stock solutions of each single reference substance, mix them, and prepare the mixed reference solution with methanol. In the mixed reference solution, the concentration of YAC is 5.22 μg / mL, the concentration of DYA is 6.55 μg / mL, the concentration of CCA is 12.25 μg / mL, and the concentration of DCA is 2.53 μg / mL. Accurately take the mixed reference solution, dilute it with chromatographic methanol to different concentrations, and store it in a 4°C refrigerator for subsequent experiments.

[0068] Before injection, the sample was filtered through a 0.22 μm microporous filter membrane into a designated injection bottle. Referring to the method of Example 1, the peak area of each component was detected by mass spectrometry parameters, and each concentration was injected 3 times.

[0069] Figure 1 Representative EIC chromatograms of four target compounds for mixed reference solution (A) and test solution (B).

[0070] The standard curve was drawn with the peak area as the ordinate (y) and the known concentration of the standard control as the abscissa (x), and the regression equation was fitted. The control was diluted step by step according to the chromatographic conditions, and then measured by machine, and the concentration corresponding to the signal-to-noise ratio of 3 (S / N = 3) was set as the detection limit (LOD), and the concentration corresponding to the signal-to-noise ratio of 10 (S / N = 10) was set as the quantification limit (LOQ).

[0071] The standard curve was established with the standard concentration of the four components as the abscissa (x) and the corresponding chromatographic peak area as the ordinate (y). The results showed that all the components showed good linear relationship in the determination concentration range, and the correlation coefficient (r) was greater than 0.999. The linear range, detection limit (LOD) and quantification limit (LOQ) and other parameters are shown in Table 3, wherein the LOD value is between 0.00163-0.0171 μg / mL, and the LOQ value is in the range of 0.00490-0.0512 μg / mL, indicating that the detection method provided by the present application has good sensitivity and can meet the detection requirements.

[0072] Table 3 Standard curve, linearity, LOD and LOQ of the standard of four target compounds

[0073]

[0074] (2) Precision

[0075] The same mixed control solution was continuously injected for determination 6 times according to the chromatographic conditions of Example 1, the peak area was calculated according to the concentration of the four components in the mixed control solution, and the relative standard deviation (RSD) was calculated. The mixed standard solution was analyzed by continuous injection for 6 times, and the relative standard deviation (RSD) of the peak area of the four target compounds was between 2.5%-3.5% (Table 4), which confirmed that the instrument precision was good.

[0076] Table 4 Precision results table of four target compounds

[0077]

[0078] (3) Stability

[0079] The test sample solution was prepared by co-decocting Huangcaowu with pigskin according to the method of Example 1, and was placed for 0-10 h, and was injected for determination according to the chromatographic conditions of Example 1 at 0 h, 2 h, 4 h, 6 h, 8 h and 10 h, respectively. The peak area was calculated according to the concentration of the four components in the sample, and the RSD value was calculated.

[0080] The same test sample solution was periodically detected under the chromatographic conditions of Example 1 within 0-10 h, and the results showed that the peak area RSD of each target compound was 4.5%-4.7%, all RSD values were less than 5%, indicating that the stability within 10 h met the requirements.

[0081] Table 5: Stability results of four target compounds

[0082]

[0083]

[0084] (4) Repeatability

[0085] Because the detection results of different batches of Radix Aconiti Lateralis Preparata samples will be different, the same batch of Radix Aconiti Lateralis Preparata sample was selected for the experiment. Six Radix Aconiti Lateralis Preparata samples were precisely weighed again, and Radix Aconiti Lateralis Preparata was decocted with pigskin. The test sample solution was prepared according to the method of Example 1, and the content of the four components in the sample was calculated according to the chromatographic conditions of Example 1, and the RSD value was calculated to evaluate the stability of the instrument. The experimental results are shown in Table 6, the content RSD of the four target compounds is 3.9%-4.8%, and the RSD is less than 5%, indicating that the repeatability of the sample preparation process meets the methodological requirements.

[0086] Table 6: Repeatability results of four target compounds

[0087]

[0088] (5) Sample recovery rate

[0089] Six samples of the same batch of Radix Aconiti Lateralis Preparata were precisely weighed, and Radix Aconiti Lateralis Preparata was decocted with pigskin. The test sample solution was prepared according to the method of Example 1, and the content of the target product in the Radix Aconiti Lateralis Preparata sample was calculated according to the experimental results of the repeatability above. According to Table 7, the standard sample of the target compound was quantitatively added, and the content of the four target compounds in the sample was determined according to the chromatographic conditions of Example 1, and the sample recovery rate was calculated.

[0090] The sample recovery method was used to evaluate the accuracy of the detection method, and the sample recovery rate was calculated according to the following formula.

[0091] Sample recovery rate=(amount measured after spiking-the content of target product in Radix Aconiti Lateralis Preparata sample added) / content of target compound in standard sample added x 100%

[0092] The detection results are shown in Table 7, and the recovery rate of the four target compounds in the six parallel experiments is 99.6%-103.7%, and the RSD is 1.1%-1.9%, which verifies the reliability of the quantitative method.

[0093] Table 7: Results of the recovery rate of four target compounds

[0094]

[0095] The above results show that the new UHPLC-QQQ-MS / MS method established by the present application can be used to determine the content of four aconite alkaloids in different component samples. The verification of the above examples shows that the method is feasible, efficient and sensitive.

[0096] Example 3

[0097] In this embodiment, the extraction process of co-decoction of Radix Aconiti Lateralis + pigskin includes the following steps: the crude drug Radix Aconiti Lateralis is treated by a pulverizer and then passed through a 200-mesh standard sieve to prepare fine powder. 5.0 g of the Radix Aconiti Lateralis powder is precisely weighed and placed in a round-bottom flask with a stopper, pigskin is added, and deionized water is added for soaking for 0.5 h, and then a condensation reflux extraction method (100°C) is used for extraction. After the extraction is completed, while hot, the filtrate is transferred to a 50-mL volumetric flask, and after cooling to room temperature, water is used to precisely dilute to the calibration mark with a pipette, and mix thoroughly. Then, the content of the toxic components (dianeobine and mesaconitine) in the co-decoction of Radix Aconiti Lateralis + pigskin is detected by the method of Example 1.

[0098] In this embodiment, the data statistical analysis, orthogonal design and response surface method are all evaluated by the comprehensive scoring method. In this experiment, the comprehensive evaluation of the content weight addition of dianeobine and mesaconitine is used as an index to evaluate the extraction process of co-decoction of pigskin and Radix Aconiti Lateralis, and the toxic indicators of Radix Aconiti Lateralis and related literature are comprehensively considered. The weight of index 1 (dianeobine content) is set to 60%, and the weight of index 2 (mesaconitine content) is set to 40%. The comprehensive weighted scoring method is used to comprehensively investigate each index by using an overall desirability (OD) index. The optimized extraction process is given Y and Z coefficients, which correspond to the contents of dianeobine and mesaconitine. The comprehensive score OD is calculated as OD = Y i / Y max × 60% + Z i / Z max × 40%, and the OD value is analyzed intuitively.

[0099] (1) Orthogonal experimental design and verification

[0100] According to the previous experimental basis, under certain conditions, the extraction time, the ratio of Radix Aconiti Lateralis to pigskin, the amount of water added, and the number of extractions all have an impact on the content of toxic components in Radix Aconiti Lateralis. Therefore, in this experiment, the extraction time (A), the ratio of Radix Aconiti Lateralis to pigskin (B), the amount of water added (C), and the number of extractions (D) are selected as the four factors that have a greater impact, three levels are selected, and an L9(34) orthogonal experiment is designed. 4Orthogonal experiment was used to optimize the extraction process of pigskin and G. epiglottis. The factors and levels of orthogonal experiment were shown in Table 8.

[0101] Table 8 Orthogonal experiment design table

[0102]

[0103] Note: 15 times means the water addition amount is 15 mL and the mass of G. epiglottis is 1 g, 20 times means the water addition amount is 20 mL and the mass of G. epiglottis is 1 g, 30 times means the water addition amount is 30 mL and the mass of G. epiglottis is 1 g.

[0104] To verify the stability of the optimal process parameters, three independent repeated experiments were carried out under the same equipment conditions, and the contents of delphinine and crude sapotoxin A in G. epiglottis were determined. The results were evaluated by calculating the relative standard deviation of the comprehensive score.

[0105] According to the orthogonal factor level design L9(3 4 ) orthogonal experiment, the results of comprehensive score were shown in Table 10.

[0106] Table 9 L9(3 4 ) orthogonal table and experimental results analysis

[0107]

[0108]

[0109] Table 10 Orthogonal experiment index comprehensive score results

[0110]

[0111] The range analysis of orthogonal experiment was carried out. As shown in Table 11, R A > R B > R D > R C The four factors affecting the content of toxic components in G. epiglottis extraction were in the order of extraction time (A) > G. epiglottis and pigskin ratio (B) > extraction times (D) > water addition amount (C). Among the four factors, the extraction time had the most significant effect.

[0112] Table 11 Range analysis table of orthogonal experiment

[0113]

[0114] The optimal process group was compared with the scores of other process groups in the orthogonal table, single factor variance analysis (ANOVA) was used to determine whether there was a significant difference between the optimal group and other groups (P < 0.05), if the optimal group comprehensive score was significantly lower than other groups, and the target component content met the preset standard, it indicated its superiority. According to the variance analysis results in Table 12, A had a significant effect on the experimental results, B, C and D had no significant effect on the experimental results. Therefore, according to the results of intuitive analysis and variance analysis, the optimal extraction conditions of Radix Aconiti were A3B3C3D3, i.e. the extraction time was 2 h, the ratio of Radix Aconiti to pigskin was 1:2 (mass ratio), the water amount was 30 times, and the extraction times was 3 times.

[0115] Table 12 Variance analysis table of orthogonal experiment

[0116]

[0117] According to the conditions of A3B3C3D3, 3 independent repeated experiments were carried out, and the content of toxic components in Radix Aconiti and the comprehensive score results are shown in Table 13. The relative standard deviation (RSD) of the comprehensive score of the 3 groups of experiments was 3.82%, indicating that the process had good repeatability. The average value of the comprehensive score of the optimal process was 10.90 points, which was significantly lower than the experimental results of groups 1-9 in Table 10 (P < 0.05), indicating the superiority of the process. Therefore, A3B3C3D3 is the optimal process condition for Radix Aconiti, i.e. the extraction time is 2 h, the ratio of Radix Aconiti to pigskin is 1:2 (mass ratio), the water amount is 30 times, and the extraction times is 3 times.

[0118] Table 13 Toxic component content and comprehensive score of repeated experiment group of optimal process

[0119]

[0120] (2) Response surface method

[0121] According to the results of orthogonal experiment, the extraction time, the ratio of Radix Aconiti to pigskin and the extraction times were selected as independent variables, and the content of aconitine was selected as the response value for three-factor three-level response surface experiment. The experimental data were analyzed by Design-Expert 13.0.1.0 software, and the optimal process parameters of pigskin and Radix Aconiti decoction were obtained. The factor level analysis is shown in Table 14.

[0122] Table 14 Factor and coding value of central composite experiment

[0123]

[0124] Based on the Box-Behnken design principle, three factors including extraction time, the ratio of G. wilsonii to pigskin, and extraction times were selected to construct the experimental matrix (Table 15). The scheme included 12 groups of factor design experiments and 5 groups of central combination experiments. The first 12 groups were investigated by three-factor three-level full-factor design to examine the interaction effect, and the last 5 groups were repeated at the center point to evaluate the system error. The factor points were located at the vertex coordinates of the three-dimensional factor space, and the central point was repeated five times for calculating the experimental precision, which effectively balanced the model prediction and error control requirements.

[0125] Table 15 Response surface experimental design and results

[0126]

[0127]

[0128] The results obtained by analyzing the data in Table 16 using Design-Expert 13.0.1.0 software are shown in Table 17. As shown in Table 17, the extraction time (P<0.01) and the extraction times (P<0.01) were the comprehensive scores of the response values. After regression fitting, the regression equation Y(comprehensive score) = 263.23-2.51A-179.10B-65.48C+19.69AB+0.06AC+32.72BC-17.86A 2 +32.19B 2 +3.57C 2 Based on the statistical model verification results, the established quadratic polynomial regression equation had a significant goodness of fit (P<0.01), the lack-of-fit test P=0.2164 indicated that the model did not have a systematic deviation, and the determination coefficient R 2 =0.9469 confirmed that the mathematical model could effectively represent the quantitative relationship between the process parameters and the content of toxic components in G. wilsonii. As shown in Table 17, the extraction time (A), the extraction times (C), and their interaction terms (BC) and the quadratic terms (B 2 ) had a very significant effect on the comprehensive score (Y) of the content of toxic components (P<0.01), revealing a nonlinear relationship between the process parameters and the response value. The three-dimensional response surface and two-dimensional contour graph Figure 2 directly presented the interaction mechanism of each parameter: the steepness of the surface reflected the sensitivity of the variable, the elliptical contour represented the strength of the interaction effect, and the center region corresponded to the optimal process parameter combination. Visual analysis showed that the comprehensive score was most significantly affected by the extraction time (P<0.01), followed by the extraction times, which was consistent with the regression coefficient analysis results. By combining model prediction with graphical analysis, the optimal process parameter range for reducing the toxicity of G. wilsonii could be accurately determined, providing a theoretical basis for subsequent process optimization.

[0129] Table 16 Results of comprehensive evaluation of response surface experiment index

[0130]

[0131] Table 17 Analysis of variance of response surface experiment

[0132]

[0133]

[0134] Verification of response surface: actual experiments were carried out using the optimal process parameters predicted by the model, and the experiments were repeated at least 3 times independently. The mean and standard deviation of the actual response value were calculated. The deviation between the predicted value and the actual value of the response value was compared. If the deviation rate between the measured value and the predicted value is <5%, the result is consistent with the model.

[0135] Based on the model verification requirements, the optimization parameters (2h of decoction time, 1:2 of the ratio of Radix Aconiti Kusnezoffii to pigskin, 3 times of extraction frequency) were selected to carry out process verification. The results of 3 independent repeated experiments are shown in Table 18. The average value of the comprehensive score is 10.45 points, the relative standard deviation is 1.38%, and the deviation rate of the actual response value from the model predicted value 10.16 is 2.85%, which meets the requirements, indicating that the experimental data is consistent with the theoretical prediction. The results confirm that the response surface method is reliable in the optimization of Radix Aconiti Kusnezoffii-pigskin co-decoction process, and the established mathematical model can effectively guide the control of toxic ingredients, and the optimization parameters have process feasibility and production applicability.

[0136] Table 18 Actual value of the results of the optimal process parameters

[0137]

[0138] The "attenuation" of pigskin and Radix Aconiti Kusnezoffii involves complex interactions of multiple components and multiple targets. Orthogonal experiment can preliminarily screen key factors through range or variance analysis, response surface method can further analyze the synergistic or antagonistic effects of key factors, and further optimize the optimal combination of these factors, which is consistent with the holistic characteristics of traditional Chinese medicine and enhances the credibility of the conclusion.

[0139] Orthogonal experiment can determine the ratio of Radix Aconiti Kusnezoffii to pigskin, decoction time and decoction frequency as key parameters, and response surface method can further reveal the synergistic promotion effect of the ratio of Radix Aconiti Kusnezoffii to pigskin and decoction time on the content of aconitine, a toxic ingredient in Radix Aconiti Kusnezoffii. After optimization by the above two methods, the process stability is significantly improved. In the early stage, orthogonal experiment is used to lock the key variables (such as decoction time, ratio of Radix Aconiti Kusnezoffii to pigskin, extraction frequency) from complex process parameters, reducing the optimization dimension; then a nonlinear prediction model is established by Box-Behnken response surface design to accurately identify the optimal process window.

[0140] In summary, the final selected optimal parameters are: decoction duration 2 h, ratio of Huangcao Wu to pigskin 1:2, and extraction times 3 times.

[0141] Example 4

[0142] In this example, the Bliss method was used to detect the median lethal dose (LD50) of mice before and after co-decoction of pigskin and Huangcao Wu. 50 The heart, liver, spleen, lung, and kidney injury conditions, serum biochemical indicators, changes in the P, Q, R waves, QRS interval, and PR interval of lead II electrocardiogram, and the heart rate (HR), ejection fraction (EF, %), fractional shortening (FS, %), stroke volume (SV, μL), and cardiac output (CO, mL min -1 ) of echocardiography were observed. Thus, the “attenuation” effect of pigskin on Huangcao Wu was determined, providing a scientific basis for the safety of Huangcao Wu in clinical use.

[0143] Experimental animals: 180 SPF healthy BALB / c mice, half male and half female, 35 days old, weighing 20 ± 2 g, provided by Guangdong Yaoke Biological Technology Co., Ltd., animal license number: SCXK (Yue) 2020-0054, quality certificate number: 44824700043931, the last quality test date: October 15, 2024. All animals were routinely bred in the SPF laboratory of the University Town Campus Experimental Animal Center of Guangzhou University of Chinese Medicine. This experiment was reviewed by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine, with the ethics number 20241111007. After 7 days of adaptive feeding, the experiment was conducted. After the experiment, all surviving mice were sacrificed by cervical dislocation, and the carcasses were transferred to the University Town Campus Animal Experiment Center of Guangzhou University of Chinese Medicine for harmless treatment.

[0144] Statistical analysis of data: In this example, the experimental data were processed by SPSS 27.0 software. The body weight, electrocardiogram, echocardiogram, and myocardial enzyme spectrum of different dose drug intervention groups were compared with the blank control group as the reference. Quantitative data were expressed as mean ± standard deviation (x ± s), and the mean values of different groups were compared by one-way ANOVA. If the data were not normally distributed, the Kruskal-Wallis test was used for non-parametric statistical analysis. The data processing process was as follows: ① Levene method was used for homogeneity of variance verification; ② when the data were normally distributed, one-way ANOVA was used to evaluate the differences between groups; ③ if the one-way ANOVA showed statistically significant differences between groups (P < 0.05), LSD method was used for multiple comparisons; ④ non-parametric statistical analysis was performed by Kruskal-Wallis test for non-homogeneous data.

[0145] (1) Preparation of Huangcao Wu single decoction and Huangcao Wu + pigskin co-decoction

[0146] Preparation of co-decoction of Radix Aconiti Lateralis Praeparata and pigskin: 30 g of Radix Aconiti Lateralis Praeparata was weighed, crushed, sieved, and added with 300 mL of distilled water and 30 g of pigskin. After soaking for 0.5 h, the mixture was heated to boiling, and the timing was started. The mixture was decocted for 0.5 h. The extraction was performed once, and the mixture was filtered through two layers of silk cloth while hot to obtain the filtrate. The filtrate was transferred to a rotary evaporator, and concentrated to 30 mL at 50°C under reduced pressure, to obtain a concentrated solution with a concentration of 1.0 g / mL (i.e. equivalent to the concentration of crude drug, 1 g of Radix Aconiti Lateralis Praeparata per mL). The concentrated solution was prepared for use.

[0147] Preparation of single decoction of Radix Aconiti Lateralis Praeparata: 30 g of Radix Aconiti Lateralis Praeparata was weighed, crushed, sieved, and added with 300 mL of distilled water. After soaking for 0.5 h, the mixture was heated to boiling, and the timing was started. The mixture was decocted for 0.5 h. The remaining steps were the same as those in the preparation of co-decoction of Radix Aconiti Lateralis Praeparata and pigskin.

[0148] (2) Formal experiment of acute toxicity

[0149] ① Single decoction of Radix Aconiti Lateralis Praeparata group: according to the results of the pre-experiment, six groups of drug doses (16.00, 10.88, 7.40, 5.03, 3.42, and 2.32 g / kg) were set between Dn and Dm at a proportion of 1:0.68 for the formal experiment of acute toxicity. Seventy BALB / c mice, half male and half female, were randomly divided into seven groups, with 10 mice in each group. The mice in groups 1-6 were orally administered with the six groups of drug solutions at different doses once, with a dose volume of 0.2 mL / 10 g. The mice in group 7 were the blank control group, which were orally administered with the same volume of distilled water. The mice could be fed 3-4 h after administration. The mice were continuously observed for 14 days, and the symptoms of poisoning, duration of poisoning, number of deaths, and mortality rate of the mice were recorded. Autopsies were performed on the dead mice in time, and the pathological changes of the main organs were observed macroscopically. The 95% confidence limit of LD 50 and LD 50 was calculated by Bliss method. The surviving mice were observed for 14 days, and the body weight of the mice was recorded every other day. All the mice were sacrificed at the end of 14 days, and the experiment was ended.

[0150] ② Co-decoction of Radix Aconiti Lateralis Praeparata and pigskin group: according to the results of the pre-experiment, six groups of drug doses (20.00, 16.00, 12.80, 10.24, 8.19, and 6.55 g of crude drug / kg) were set between Dn and Dm at a proportion of 1:0.8 for the formal experiment of acute toxicity. Seventy BALB / c mice, half male and half female, were randomly divided into seven groups, with 10 mice in each group. The mice in groups 1-6 were orally administered with the six groups of drug solutions at different doses once, with a dose volume of 0.2 mL / 10 g. The mice in group 7 were the blank control group, which were orally administered with the same volume of distilled water. The remaining steps were the same as those in item ①.

[0151] After the single decoction of G. uralalensis and the co-decoction of G. uralalensis + pig skin were administered to mice by gavage, death occurred in different dose groups, but the intensity of toxic reactions was different between the single decoction and the co-decoction. After the two kinds of decoctions were administered to mice at the maximum dose, toxic reactions such as reduced activity, huddling, and abdominal breathing occurred 5 min later, and then changed to piloerection, tremor, foaming at the mouth or excessive salivation around the mouth, followed by jumping, tonic convulsions, and opisthotonos. The mice in the single decoction group died within 10 min after gavage, and all died within 30 min. The results showed that the mice in the single decoction group died 10 min after gavage, and all died within 30 min. The first death in the co-decoction group occurred 1 h later, and all died within 4 h. At a low dose, the experimental animals showed reduced voluntary activity, huddling, fluffed fur, increased salivation around the mouth, and respiratory distress. During the toxic progression period, fecal incontinence caused perianal contamination, and 1-2 h later, the animals were in a recumbent position and could not stand, accompanied by neuroinhibition and reduced reflexes. Some individuals died progressively 3-24 h later, and the non-dead mice gradually recovered from the toxic side effects within 24 h. Autopsy of the dead animals showed that the stomachs of the mice were filled with a large amount of liquid, and the stomachs of the mice in the single decoction group were 2-3 times larger than those of the control group. The stomach size of the animals in the co-decoction group was between the two groups, and no obvious abnormalities were found in the other organs. Figure 3 In summary, the toxic reaction of the single decoction of G. uralalensis occurred earliest and was the most severe.

[0152] According to the mortality rate of acute toxicity in mice (Tables 19 and 20), the median lethal dose LD 50 of the single decoction of G. uralalensis was calculated to be 9.74 g / kg, with a 95% confidence interval of 4.52-20.97 g / kg; and the median lethal dose LD 50 of the co-decoction of G. uralalensis + pig skin was 19.68 g / kg, with a 95% confidence interval of 7.55-51.33 g / kg. The results showed that the LD 50 of the co-decoction of G. uralalensis + pig skin was larger than that of the single decoction of G. uralalensis. 50

[0153] Table 19 Results of acute toxicity experiment of single decoction of G. uralalensis

[0154]

[0155] Table 20 Results of acute toxicity experiment of co-decoction of G. uralalensis + pig skin

[0156]

[0157] ​

[0158] Mice in each group were observed for 14 consecutive days after a single dose, and generally no mice died after 48 hours. Figure 4 As shown, compared with the blank control group, mice in the gavage treatment groups of 5.03 g / kg and 3.42 g / kg of Aconitum carmichaelii decoction showed reduced food intake, decreased activity, and weight loss within 3 days, returning to normal by day 5. No other significant abnormalities were observed. The 2.33 g / kg group showed normal activity, and their weight was similar to that of the blank control group. Compared with the blank control group, mice in the gavage treatment groups of 12.80 g / kg and 10.23 g / kg of pork and Aconitum carmichaelii decoction showed reduced food intake, decreased activity, and weight loss within 3 days, returning to normal by day 5. No other significant abnormalities were observed. The 8.19 g / kg and 6.55 g / kg groups showed normal food and water intake, and their weight remained synchronized with that of the blank control group.

[0159] (3) Effect of pig skin and aconite on the time of death of mice before and after decoction

[0160] In the acute toxicity formal experiment, in the group with the maximum dose (16.00 g / kg) of Huangcaowu decoction alone and the group with the same amount of raw drug in Huangcaowu + pig skin decoction, 6 dead mice were randomly selected from each group, half male and half female, so as to compare the toxic reaction process and death time data of the two groups of mice.

[0161] Mice in the group treated with a decoction of Aconitum carmichaelii (16.00 g / kg) showed signs of poisoning, including abdominal breathing, convulsions, and excessive salivation, after 5 minutes and all died within 30 minutes. Mice in the group treated with a decoction of Aconitum carmichaelii and pigskin, at the same dosage of raw drug, showed signs of poisoning, including abdominal breathing and curling up, after 30 minutes and all died within 150 minutes. This indicates that the co-decoction of pigskin and Aconitum carmichaelii delayed the progression of Aconitum carmichaelii poisoning in mice and prolonged the time to death. Figure 5 Therefore, pigskin has a certain degree of "toxicity reduction" effect on Aconitum carmichaelii.

[0162] (4) Effects of decoction of Aconitum carmichaelii and pig skin on electrocardiograms in mice

[0163] The LD50 of Aconitum carmichaelii decoction was determined in a formal acute toxicity test. 50For the dose, 18 mice were randomly selected and divided into 3 groups: blank control group, single decoction of Huangcaowu group and Huangcaowu + pigskin co-decoction group, 6 in each group, half male and half female. Except that the blank control group was given the same volume of normal saline, the single decoction group and the co-decoction group were given the same dose of liquid, 30 min later, through isoflurane anesthesia, after stabilization, the right upper limb was fixed in supine position, the positive electrode was placed in the right upper limb through the bioelectric signal acquisition device, the negative electrode was placed in the left lower limb, and the ground wire was connected to the right lower limb. The Ⅱ lead ECG parameters were continuously monitored, including P wave, QRS complex and PR interval. The heart rate was calculated by RR interval conversion method, and the standardized value was obtained according to the formula 60 / RR(s).

[0164] As shown in Figure 6 Compared with the blank control group, the ECG changes of the single decoction of Huangcaowu group were various, but according to the overall evaluation of ECG, ventricular arrhythmia was the main manifestation, and the absence of P wave and QRS widening were the basis for judging the source of arrhythmia as ventricular.

[0165] As shown in Table 21, the heart rate of the blank control group was 368.141±4.340bpm, which was extremely significantly increased in the single decoction of Huangcaowu group (P<0.01), while the co-decoction group was extremely significantly decreased (P<0.01). The R wave amplitude in the single decoction group appeared polarity inversion (-0.299±0.017mV), and the co-decoction group recovered to positive potential (0.305±0.335mV). The QRS interval in the co-decoction group was significantly prolonged (P<0.01), which was statistically different from the blank control group and the single decoction group (P<0.05). The P wave and PR interval in the single decoction of Huangcaowu group could not be detected due to severe toxic reaction, and the two parameters in the co-decoction group were 13.333±1.538ms and 17.167±1.693ms respectively, indicating that the compatibility of Huangcaowu and pork could partially improve the cardiac conduction function. The results showed that the single decoction of Huangcaowu caused characteristic changes of arrhythmia, while the co-decoction of Huangcaowu + pigskin significantly reduced the cardiac electrophysiological disorder, which supported the scientificity of the compatibility of pigskin and Huangcaowu for "attenuating toxicity". Table 21 Comparison of ECG of mice given the same dose of single decoction of Huangcaowu and co-decoction of Huangcaowu + pigskin n=6

[0166]

[0167] Note: *P<0.05, **P<0.01, vs. blank control group; ## P<0.01, vs. Huangcaowu single decoction group.

[0168] (5) Effect of Huangcaowu + pigskin co-decoction before and after on echocardiogram of mice

[0169] After the above indicators were detected, the mice anesthetized with isoflurane were subjected to echocardiography analysis (5% for induction and 1% for maintenance of anesthesia in 1 L / min oxygen). The mice were placed in a supine position on a temperature-controlled operating table to maintain the body temperature at 37°C. The echocardiography was performed by an experienced experimenter using a VEVO 2100 high-resolution imaging system (FUJIFILM, Toronto, CA) equipped with a small animal ultrahigh-resolution B-ultrasound. Vevo Lab software (FUJIFILM, Toronto, CA) was used for post-acquisition analysis. The ejection fraction (EF, %), fractional shortening (FS, %), stroke volume (SV, μL) and cardiac output (CO, mL min FUJIFILM, Toronto, CA) were determined by tracking the diastolic and systolic end areas on the longitudinal axis. -1 ) were measured in the middle level of the ventricle in M mode.

[0170] The results are shown in Table 22. The heart output and stroke volume of the co-decoction group of Radix Aconiti and pigskin were significantly lower than those of the blank control group (P < 0.05), while the fractional shortening and ejection fraction showed no statistically significant difference. Although the indicators of the single decoction group of Radix Aconiti showed a downward trend, there was no significant difference (P > 0.05).

[0171] Table 22 Comparison of echocardiography of mice gavaged with the same dose of single decoction of Radix Aconiti and co-decoction of Radix Aconiti and pigskin n = 6

[0172]

[0173] Note: * P < 0.05, vs. the blank control group.

[0174] (6) Effect of co-decoction of pigskin and Radix Aconiti before and after decoction on serum myocardial enzyme indicators of mice

[0175] After the above indicators were detected, each group of mice (n = 6) was taken blood from the orbit, and the serum samples were subjected to low-temperature high-speed centrifugation (3000 rpm, 10 min) and then the activities of aspartate aminotransferase (AST), creatine kinase (CK), creatine kinase isozyme (CK-MB), α-hydroxybutyric acid dehydrogenase (α-HBDH) and lactate dehydrogenase (LDH) were determined by using an automatic analysis system.

[0176] From the results shown in Table 23, the activities of AST, CK, CK-MB, α-HBDH and LDH of the co-decoction group of Radix Aconiti and pigskin were significantly lower than those of the blank control group (P < 0.05), while the activities of AST, CK, CK-MB, α-HBDH and LDH of the single decoction group of Radix Aconiti showed a downward trend, but there was no statistically significant difference (P > 0.05). Figure 7It was found that compared with the blank control group, the AST, CK-MB, LDH and HBDH levels of the single decoction of Huangcao Wu increased by 78.3%, 74.4%, 46.7% and 37.4% respectively (P<0.01), and the CK index also showed a significant increase of 32.6% (P<0.05). However, when the pigskin was decocted with Huangcao Wu, the above toxicity indicators showed a significant reversal: CK-MB (-44.9%), LDH (-25.1%) and HBDH (-28.3%) were significantly lower than the single decoction group (P<0.01), and the CK-MB value returned to the level without statistical difference with the blank control group (P>0.05). Although the AST index in the co-decoction group was still 27.7% higher than that in the control group, it was 28.4% lower than that in the single decoction group (P<0.01). The data showed that the co-decoction of pigskin and Huangcao Wu could effectively inhibit the abnormal increase of myocardial enzyme spectrum caused by Huangcao Wu, indicating that the co-decoction of pigskin and Huangcao Wu might change the bioavailability of toxic components through physicochemical effects.

[0177] In this example, the Bliss method was used to detect that the LD 50 was 9.74 g / kg (95% CI: 4.52-20.97 g / kg), while the LD 50 increased significantly to 19.68 g / kg (95% CI: 7.55-51.33 g / kg), and the dose tolerance range was expanded by about 2.02 times, indicating that the co-decoction of Huangcao Wu and pigskin was less toxic than the single decoction of Huangcao Wu; the incidence of acute poisoning symptoms (arrhythmia, respiratory depression) in the co-decoction group of Huangcao Wu and pigskin was significantly lower than that in the single decoction group of Huangcao Wu under the same amount of crude drug, and the time of death was delayed; the comparison of electrocardiogram, echocardiogram and myocardial enzyme spectrum between the co-decoction of Huangcao Wu and pigskin and the single decoction of Huangcao Wu showed that pigskin had a certain degree of alleviating effect on the cardiotoxicity of Huangcao Wu, indicating that the co-decoction of Huangcao Wu and pigskin might change the bioavailability of toxic components through physicochemical effects.

[0178] Example 5

[0179] In this example, collagen-induced arthritis (CIA) model was used to evaluate the "storage and efficacy" effect of pigskin on Huangcao Wu. LC-MS was used to quantitatively detect the levels of rheumatoid factor and inflammatory factors; HE staining, immunohistochemistry and other methods were used to detect the histopathological changes of rats; Micro CT scanning was used to analyze the tissue condition of rat feet, to determine that pigskin could reduce the toxicity of Huangcao Wu without significantly weakening its anti-inflammatory and analgesic efficacy, to clarify the synergistic effect of pigskin on reducing the toxicity of Huangcao Wu and preserving its efficacy, and to confirm the scientificity of the compatibility of pigskin and Huangcao Wu, and to provide a new idea for the development of strategies for the directional transformation of toxic components and the maximization of efficacy of Aconitum L. medicines.

[0180] Experimental animals: 42 SPF healthy SD rats, all male, 35 days old, body weight 160 ± 20 g, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd., animal license number SCXK (Yue) 2022-0063, quality certificate number: 44824700043931, the last quality test date: October 15, 2024. All animals were routinely bred in the SPF laboratory of the University Town Campus Experimental Animal Center of Guangzhou University of Chinese Medicine. This experiment was reviewed by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine, with ethics number 20241111007. After 7 days of adaptive feeding, the experiment was carried out. After the experiment, all surviving rats were sacrificed by cervical dislocation, and the carcasses were handed over to the University Town Campus Animal Experiment Center of Guangzhou University of Chinese Medicine for unified harmless treatment.

[0181] Statistical analysis of data: The experimental data were processed by SPSS 27.0 software. The body weight, inflammation index score, and foot volume measurement of different dose drug intervention groups were compared with the blank control group as the benchmark. Quantitative data were expressed as mean ± standard deviation (x ± s) , and the central tendency and dispersion degree were characterized. Data processing process: ① Homogeneity of variance verification using Levene method; ② When normal distribution is met, single factor analysis of variance is used to evaluate the difference between groups; ③ If there is a statistically significant difference between groups by single factor analysis of variance (P <0.05), LSD method is used for multiple comparisons; ④ Non-parametric statistical analysis is performed by Kruskal-Wallis test for non-homogeneous data.

[0182] Through foot volume measurement, inflammation index score, and Micro CT scanning, the foot tissue conditions of rats in each group were analyzed. There was no statistically significant difference between the single decoction of Huangcao Wu group and the co-decoction of Huangcao Wu + pigskin group, but both were significantly different from the CIA model group, indicating that pigskin can reduce the toxicity of Huangcao Wu while retaining its anti-inflammatory effect. This study elucidates the synergistic effect of pigskin on reducing the toxicity of Huangcao Wu and retaining its efficacy, and confirms the scientificity of the compatibility of pigskin and Huangcao Wu.

[0183] (1) Preparation of drugs

[0184] ① Preparation of single decoction of Huangcao Wu and co-decoction of Huangcao Wu + pigskin

[0185] According to the results of the mouse acute toxicity experiment: the LD 50 of single decoction of Huangcao Wu with an extraction time of 30 min was 9.74 g / kg, and the efficacy experiment used 1 / 20 (0.487 g / kg) of the median lethal dose measured by acute toxicity experiment as the high dose, and 1 / 40 (0.244 g / kg) as the low dose, which was equivalent to 0.338 g crude drug / kg for SD rats at the high dose and 0.169 g crude drug / kg at the low dose.

[0186] Preparation of co-decoction of Rhizoma Dioscoreae Nipponicae + pigskin: 30 g of Rhizoma Dioscoreae Nipponicae was weighed, crushed, sieved, and added with 300 mL of distilled water. 30 g of pigskin was added, soaked for 0.5 h, and then heated to boiling. Timing was started after boiling. Decoction was performed for 0.5 h. Extraction was performed once. The filtrate was obtained by hot filtration through two layers of silk cloth with a mesh size of 200. The filtrate was transferred to a rotary evaporator, and concentrated to 30 mL at 50°C under reduced pressure. The concentration was 1.0 g / mL (i.e. the concentration of crude drug, 1 g of Rhizoma Dioscoreae Nipponicae per mL).

[0187] Preparation of single decoction of Rhizoma Dioscoreae Nipponicae: 30 g of Rhizoma Dioscoreae Nipponicae was weighed, crushed, sieved, and added with 300 mL of distilled water. Soaking was performed for 0.5 h. Timing was started after heating to boiling. Decoction was performed for 0.5 h. The rest of the preparation method was the same as that of the co-decoction of Rhizoma Dioscoreae Nipponicae + pigskin.

[0188] 2. Preparation of immunogen

[0189] The immunogen was prepared on the day of modeling (day 0). The preparation process was performed on a clean bench. Equal amounts of bovine collagen II (CII) and complete Freund's adjuvant (CFA) were mixed thoroughly in an ice bath environment. Emulsification was performed by stirring with a high-speed homogenizer for about 15 min. The final emulsification degree was such that the emulsion did not disperse when dropped into water, i.e. the concentration of the bovine collagen II emulsion was 1 mg / mL.

[0190] (2) Establishment of CIA rat model

[0191] After 7 days of environmental adaptation, 6 animals were randomly selected as the blank control group, and the remaining 36 animals were used to construct the collagen-induced arthritis (CIA) model. Model construction included two stages: the first immunization was performed on day 0 of the experiment, and a mixture of bovine collagen II and complete Freund's adjuvant emulsion (0.2 mL) was injected subcutaneously at the proximal tail end; the second immunization was performed on day 7, and a bovine collagen II and incomplete Freund's adjuvant complex (0.1 mL) was injected at another area of the tail.

[0192] (3) Animal administration

[0193] On day 7 after the first immunization, 36 rats were randomly divided into 6 groups, 6 rats in each group, in addition to the normal group (normal saline, 10 mL / kg / day). The model group (normal saline, 10 mL / kg / day), methotrexate (MTX) group (1.5 mg / kg, twice a week), single decoction of Rhizoma Dioscoreae Nipponicae low-dose group (169 mg / kg, once every other day), single decoction of Rhizoma Dioscoreae Nipponicae high-dose group (338 mg / kg, once every other day), co-decoction of Rhizoma Dioscoreae Nipponicae + pigskin low-dose group (169 mg of crude drug / kg, once every other day), and co-decoction of Rhizoma Dioscoreae Nipponicae + pigskin high-dose group (338 mg of crude drug / kg, once every other day). Animals were administered continuously for 21 days. The specific experimental procedure is shown in Figure 8All the procedures involving animals were in accordance with the requirements of the Regulations for Animal Experiment Ethical Review of Guangzhou University of Chinese Medicine.

[0194] From Figure 9 Observation of the characteristics of the CIA model group, the low-dose single decoction of Huangcao Wu group and the low-dose co-decoction of Huangcao Wu and pigskin group showed that the rats' joints were red, swollen, painful and stiff, which were consistent with the characteristics of active arthritis. However, the high-dose single decoction of Huangcao Wu group, the high-dose co-decoction group and the positive drug methotrexate group improved the pathological changes of the joints of the CIA model rats.

[0195] (4) Detection index

[0196] ① General body weight changes of rats

[0197] The body weight of each group of rats was measured every 2 days after the first immunization, and the general conditions such as food intake, behavior, and hair of the rats were observed regularly.

[0198] The results showed that the relative body weight of rats in different treatment groups was statistically different, and the body weight change trend showed heterogeneity between groups over time. From the 16th day, the body weight of the blank control group continued to increase, the weight gain of the model group slowed down significantly, the weight gain of the model group, the low-dose single decoction of Huangcao Wu group and the low-dose co-decoction group was significantly inhibited, and the body weight of the high-dose single decoction group and the high-dose co-decoction group was close to that of the positive drug group. The body weight of the model group was the lowest, which was significantly lower than that of the blank control group (P<0.01), indicating that the CIA model of rats was successfully constructed. There was no statistically significant difference in body weight between groups at the beginning of intervention (D7-D 13 ), and there was a statistically significant difference between the high-dose single decoction of Huangcao Wu group, the high-dose co-decoction group, the positive drug group and the model group on the 28th day (P<0.05), indicating that the improvement effect of Huangcao Wu intervention accumulated and increased over time. There was no statistically significant difference in the improvement effect between the low-dose single decoction of Huangcao Wu group and the low-dose co-decoction of pigskin group and the model group, suggesting that their efficacy in this model was limited, which may be related to the administration period or dose. Figure 10

[0199] ② Arthritis index score of rats

[0200] After the second immunization, all CIA rats were scored according to the following standard every 2 days, and the score of each paw was added to obtain the arthritis index score of each rat. The highest score of each rat was 16 points. If the AI score was >4 points, the CIA model of rats was successfully constructed.

[0201] (a) 0 points: no signs of redness and swelling;

[0202] ​(b) 1 point: Redness and slight swelling on the tarsal bone or ankle joint;

[0203] (c) 2 points: Redness and slight swelling from the ankle to the tarsal joint;

[0204] (d) 3 points: Erythema and moderate swelling from the ankle to the metatarsal joint;

[0205] (e) 4 points: Erythema and severe swelling including ankles, feet and fingers, or rigidity of the limbs.

[0206] like Figure 11 As shown, compared with the blank control group, the arthritis index scores of rats in each group increased significantly after the second immunization, with statistically significant differences on day 10 (P<0.01). The scores of each group reached their highest point on day 18, and then began to fluctuate steadily. From day 22 onwards, compared with the CIA model group, the arthritis index scores of each drug intervention group showed a decreasing trend. On day 28, although the arthritis index scores of the high-dose Huangcaowu decoction group and the high-dose Huangcaowu + pigskin decoction group were higher than those of the methotrexate group, all three groups significantly reduced the arthritis index of rats compared with the model group (P<0.05).

[0207] ③ Assessment of paw edema in rats

[0208] After the second immunization, the toe volume of the right hind paw of each group of rats was measured every two days using a toe volume meter via the water volume method.

[0209] according to Figure 12 It was found that there were statistically significant differences in paw volume among different treatment groups, and the volume showed a dynamic trend over time. Analysis at each time point showed that on day 16, the paw volume in the model group, methotrexate group, *Aconitum carmichaelii* decoction group, and *Aconitum carmichaelii* + pigskin decoction group was significantly higher than that in the blank control group (P<0.05). Furthermore, the paw volume in the model group continued to increase over time, indicating that the rat CIA arthritis model was successfully established. Although the positive control group, high-dose *Aconitum carmichaelii* decoction group, and high-dose decoction group showed significant differences in paw volume in the early stages (D7-D...), the paw volume in the model group continued to increase over time. 19 There was no statistically significant difference between the control group and the model group, but in the later stages of the intervention (D... 19 -D 28 The toe volume decreased synchronously, showing a trend of inhibiting swelling. In particular, on day 28, the toe volume of the three groups was significantly lower than that of the model group (P<0.05), suggesting that the effects of the three groups in inhibiting the inflammatory response tended to be consistent.

[0210] ④ CT analysis of rat paw tissue condition

[0211] On the 28th day, the rats were sacrificed for sampling, and the foot tissues of the four limbs were taken out (the fur was removed), and the foot tissues of each rat were fixed in 4% paraformaldehyde. In the later stage, the right hind limb of the rat fixed in the paraformaldehyde was taken out for micro-CT detection (Guangdong Academy of Chinese Medical Sciences). From the calcaneus, continuous scanning was performed, the image information was extracted by the region of interest method, and three-dimensional image reconstruction and analysis were performed.

[0212] Three-dimensional reconstruction imaging analysis showed that the right hind limb joint structure of the blank control group experimental rats was complete, the bone cortex was continuous and smooth, the joint space was normal, and the surrounding soft tissue had no pathological changes; the articular surface of the CIA model group showed subchondral bone erosion foci, accompanied by villous bone hyperplasia, the cartilage boundary was blurred, and the joint space was abnormally narrow, which confirmed that the rat type II collagen-induced arthritis model was successfully constructed. After intervention of the high-dose single decoction of Rhizoma Aconiti Lateral Root, the high-dose co-decoction and the positive drug methotrexate, the bone erosion area was reduced, the degree of articular surface destruction was reduced, and the pathological changes of the CIA model rat joints were relieved to a certain extent. Figure 13

[0213] In this embodiment, the foot tissue conditions of the rats in each group were analyzed by macroscopic characterization, toe volume measurement, inflammation index scoring, Micro CT scanning and other methods. There was a significant difference between the CIA model group and the blank control group, which indicated that the rat type II collagen-induced arthritis model was successfully constructed. However, there was no statistically significant difference between the high-dose single decoction of Rhizoma Aconiti Lateral Root, the high-dose co-decoction of Rhizoma Aconiti Lateral Root and pigskin, and the methotrexate group, but the differences between the three groups and the CIA model group were statistically significant. The above results show that the compatibility of pigskin can reduce the toxicity of Rhizoma Aconiti Lateral Root while the anti-inflammatory effect of Rhizoma Aconiti Lateral Root is not significantly weakened, which illustrates the synergistic effect of reducing toxicity and retaining efficacy of pigskin on Rhizoma Aconiti Lateral Root, and confirms the scientificity of the compatibility of pigskin and Rhizoma Aconiti Lateral Root, which provides an experimental basis for further revealing the effect substances and mechanisms of Rhizoma Aconiti Lateral Root in treating rheumatoid arthritis.

[0214] The present application further verifies that the optimal condition decocting process (i.e. the extraction time is 2h, the mass ratio of Rhizoma Aconiti Lateral Root to pigskin / sheep meat is 1:2, the water amount is 30 times, and the extraction times is 3 times) has the effect of "reducing toxicity and retaining efficacy" after co-decocting pigskin / sheep meat and Rhizoma Aconiti Lateral Root.

[0215] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. A method for attenuating the virulence of Gaphalium chinense, characterized in that, The method comprises the following steps: The step of boiling the Radix Aconiti Lateralis with meat, which is selected from one or more of pigskin, pork lean, pork fat, beef, mutton and chicken.

2. The method of attenuating according to claim 1, wherein, The method comprises the following steps: The step of boiling the Radix Aconiti Lateralis with meat and water.

3. The method of claim 1 or 2, wherein, The mass ratio of the Radix Aconiti Lateralis to the meat is 1:0.5-1:2, preferably 1:

2.

4. The method of claim 1 or 2, wherein, The boiling time is 0.5-2h, preferably 2h.

5. The method of claim 1 or 2, wherein, The boiling times is 1-3 times, preferably 3 times.

6. The method of claim 1 or 2, wherein, The step of boiling the Radix Aconiti Lateralis with water and meat, wherein the volume to mass ratio of the water to the Radix Aconiti Lateralis is 10mL:1g to 30mL:1g, preferably 30mL:1g.

7. A method for detecting the attenuated components of Aconitum leucostomum Komar. characterized in that, The method comprises the following steps: The step of treating the attenuated product of the Radix Aconiti Lateralis, adding chromatographic methanol, centrifuging to obtain supernatant, and detecting the contents of delphinine, crude stem aconitine A, 8-deacetyldelphinine and 8-deacetylcrude stem aconitine A by UHPLC-QQQ-MS / MS.

8. The method for detecting the components of attenuated Gastrodia elata according to claim 7, characterized in that, The mobile phase was a mixture of ultrapure water containing 0.1% formic acid (A) and acetonitrile (B) at a volume ratio of 70:30, and the separation was completed within 6 min using isocratic elution mode, with a flow rate of 0.25 mL·min -1 ; the temperature of the chromatographic column thermostat was maintained at 40±1℃, the dry gas flow rate was 11.0 Lmin -1 , the dry gas temperature was 350℃, the atomization gas pressure was 15 psig, and the ion transmission voltage was 4000 V.

9. The method for detecting the components of attenuated Dioscorea bulbifera according to claim 7 or 8, characterized in that, The ionization mass spectrum conditions of delphinine include: parent ion 660.3, daughter ion 135.1, cracking voltage 215V, collision energy 66eV, and positive detection mode; the ionization mass spectrum conditions of crude stem aconitine A include: parent ion 644.4, daughter ion 135.1, cracking voltage 215V, collision energy 66eV, and positive detection mode; the ionization mass spectrum conditions of 8-deacetyldelphinine include: parent ion 618.3, daughter ion 135.1, cracking voltage 205V, collision energy 62eV, and positive detection mode; and the ionization mass spectrum conditions of 8-deacetylcrude stem aconitine A include: parent ion 602.3, daughter ion 135.1, cracking voltage 210V, collision energy 50eV, and positive detection mode.

10. The method for detecting the components of attenuated Dioscorea bulbifera according to claim 7 or 8, characterized in that, The method further comprises the step of calculating the detected chromatographic peak area by standard curve. The method further comprises the step of calculating the detected chromatographic peak area by standard curve.

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