Use of the total alkaloid extract of panga in the preparation of a medicine for preventing and / or treating arrhythmia

Abstract

The present application relates to the field of biological medicine, in particular to the application of BanGa total alkaloid extract in the preparation of drugs for preventing and / or treating arrhythmia. The present application first discovers that BanGa total alkaloid extract can prevent and treat arrhythmia, and the BanGa total alkaloid extract can prolong the latent period of mice ventricular premature beat (VPB) and reduce the incidence of ventricular tachycardia (VT), and further achieve the effect of anti-arrhythmia, and has obvious alleviating effect on the arrhythmia induced by aconitine in mice. Therefore, the BanGa total alkaloid extract provided by the present application can be applied to the preparation of drugs for preventing and / or treating ventricular premature beat, ventricular tachycardia and arrhythmia, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of the total alkaloid extract of Bangga in the preparation of drugs for the prevention and / or treatment of arrhythmias. Background Technology

[0002] Bangga is a commonly used medicinal herb in Tibetan medicine, known in Tibetan medicine as Bangaga Bao. It is derived from the dried whole herb of *Aconitum tanguticum* (Maxim.) Stapf or *Aconitum naviculare* (Bruhl.) Stapf, belonging to the Ranunculaceae family and the genus *Aconitum*. It is harvested in late summer and early autumn when the flowers are in bloom, impurities are removed, and it is dried in the shade. Its tuberous roots are small, spindle-shaped, and 2-4 cm long; the surface is brownish-brown, and the cross-section is white. The stem is cylindrical, 7-50 cm long and 1.5-3 mm in diameter; the surface is grayish-green to dark green, slightly glossy, and sparsely covered with downy hairs; it is brittle and easily broken, with a hollow cross-section; the petiole is 3-20 cm long; the leaves are mostly broken, and intact ones are kidney-shaped, 1.2-3.5 cm long and 1.2-3.8 cm wide, palmately deeply lobed, with the lobes further shallowly lobed; the racemes are terminal, the flowers are bluish-green to bluish-purple, the pedicels are short, the calyx has 5 sepals, the upper sepal is boat-shaped, there are 2 petals, and numerous stamens. Bangga was first recorded in *Yuewang Yaozhen*. It is cool in nature, bitter in taste, and slightly toxic. It has the effects of clearing heat and detoxifying, and is used to treat fever caused by infectious diseases, liver and gallbladder diseases, influenza, and food poisoning.

[0003] Bangga has wide clinical applications, but pharmacological studies are limited, and existing efficacy studies have focused on the total alkaloids of Bangga. According to literature reports, the total alkaloids of Bangga possess anti-inflammatory, analgesic, antibacterial, antiviral, antitumor, and antihypertensive activities. Reports on the activities of individual components of Bangga are scarce, and structure-activity relationship studies are even more lacking. The inventors previously discovered that a novel diterpenoid alkaloid, Tanguticurine A, isolated and identified from Bangga, exhibits good inhibitory effects on both HCV and EV71, with EC50... 50 The concentrations were 15.5 and 9.7 μM, respectively. The inventors previously used cell experiments to investigate the anti-inflammatory activity of 22 diterpenoid alkaloids isolated and identified from Bangga (including 12 diterpenoids, 4 C19 lactones, 3 C20 hytidylcholine, and 3 C20 hytidylcholine-type diterpenoids). The results showed that: ① From a structural perspective, the anti-inflammatory activity, from strongest to weakest, was: diterpenoid alkaloids > C20 hytidylcholine-type > C20 hytidylcholine-type > C19 lactone-type; ② For diterpenoid alkaloids, the more hydroxyl groups, the stronger the anti-inflammatory activity; hydroxyl groups with the same number but different positions showed different activities; the activity decreased when an oxygen atom was attached to the nitrogen atom or when the 5-OH group was removed.

[0004] Although *Bangga* belongs to the *Aconitum* genus, it differs significantly from commonly used *Aconitum* medicinal herbs such as *Aconitum carmichaelii* and *Aconitum kusnezoffii*. *Bangga* is used medicinally as a whole plant, while *Aconitum carmichaelii*, *Aconitum kusnezoffii*, and *Aconitum kusnezoffii* are used only as roots. Historical herbal texts record *Bangga* as slightly toxic, while *Aconitum carmichaelii* and *Aconitum kusnezoffii* are highly toxic. According to surveys, Tibetan medicine practitioners generally consider *Bangga* to be non-toxic or have extremely low toxicity. The inventor previously discovered that the LD50 of a 70% ethanol extract of *Bangga* administered to mice via a single oral gavage was... 50 The concentration was 107.47 g crude drug / kg, classifying it as practically non-toxic. Literature reports the LD50 of total alkaloids from Bangga administered orally to mice. 50 The LD50 for mice via intraperitoneal injection was 0.745 g / kg. 50 It is 0.336 g / kg.

[0005] Cardiac arrhythmia refers to abnormalities in the heart's rhythm, frequency, or sequence of excitation caused by disturbances in the heart's pacing and conduction functions. It mainly manifests as tachycardia, bradycardia, arrhythmia, and cardiac arrest. Ventricular arrest or fibrillation is the primary form of cardiac arrest and a significant cause of sudden cardiac death. Ventricular arrhythmias include premature ventricular contractions (PVCs) and non-sustained ventricular tachycardia occurring in the general population, as well as sudden cardiac death caused by ventricular tachycardia in patients with or without organic heart disease. The Atherosclerosis Risk In Communities (ARIC) study observed 15,792 middle-aged individuals (45-65 years old). The results showed that a 2-minute ECG screening revealed premature ventricular contractions (PVCs) in 6% of cases, frequent PVCs in 3% of cases, and polymorphic PVCs in 0.8%. Premature ventricular contractions were more common in men than women and were more prevalent in individuals with organic heart disease. Ventricular arrhythmias account for two-thirds of all arrhythmias in the elderly, with single premature ventricular contractions observed in approximately three-quarters of individuals over 70 years of age. Dynamic monitoring revealed a ventricular arrhythmia incidence rate as high as 60%-90% in asymptomatic elderly subjects; and in subjects over 90 years of age, the incidence of exercise-induced ventricular arrhythmias was as high as 60%.

[0006] A community-based cardiovascular epidemiological study conducted in Tecumseh, Michigan, USA, showed that premature ventricular contractions (PVCs) had no adverse effect on prognosis in people with normal cardiac structure and under 30 years of age, but premature ventricular contractions and short runs of non-sustained ventricular tachycardia increased the risk of death in people with normal cardiac structure and over 30 years of age.

[0007] The overall incidence of sudden cardiac death in the United States is 1-2 cases per 1,000 people per year (0.1%-0.2%), and the incidence of sudden cardiac death increases with age.

[0008] Currently, arrhythmias are controlled using medication, but antiarrhythmic drugs have the following problems:

[0009] 1. Sodium channel blockers (Class I antiarrhythmic drugs):

[0010] Class Ia (such as quinidine and procainamide) prolongs the action potential duration and slows conduction velocity; Class Ib (such as lidocaine and mexiletine) have less effect on conduction velocity and are mainly used for ventricular arrhythmias; Class Ic (such as propafenone) significantly slows conduction velocity and is used to treat supraventricular and ventricular arrhythmias.

[0011] 2. Beta-blockers (Class II antiarrhythmic drugs):

[0012] By blocking beta receptors in the heart, the influence of the sympathetic nervous system on the heart is reduced, thereby lowering heart rate and reducing the occurrence of premature ventricular contractions and ventricular tachycardia.

[0013] 3. Potassium channel blockers (Class III antiarrhythmic drugs):

[0014] Amiodarone and sotalol, for example, are used to treat supraventricular and ventricular arrhythmias by prolonging the action potential duration and effective refractory period of myocardial cells.

[0015] 4. Calcium channel blockers (Class IV antiarrhythmic drugs):

[0016] It is used to treat certain supraventricular arrhythmias by blocking calcium channels in the heart, slowing the heart rate and conduction velocity.

[0017] Quinidine belongs to the class Ia antiarrhythmic drugs. It is used to suppress abnormal automaticity arrhythmias and control atrial and ventricular premature beats, atrial flutter, atrial fibrillation, and ventricular tachycardia caused by reentry. However, quinidine is poorly tolerated; up to 50% of patients experience symptomatic side effects, and 20-30% experience toxicity. The most common side effects are gastrointestinal problems, usually manifesting as drug-ineffective diarrhea. Electrolyte imbalances may exacerbate arrhythmias, in which case quinidine treatment should be discontinued. Other dose-related and reversible side effects include tinnitus, headache, dizziness, visual disturbances, nausea, and hearing loss. The most significant adverse effect of quinidine is ventricular proarrhythmia, usually due to prolonged action potential duration, conduction velocity, and tolerability changes. Patients may experience recurrent episodes of torsades de pointes (TdP), accompanied by recurrent syncope, and even sudden cardiac death. More than half of the population exhibits the rapid acetylation type of procainamide, which rapidly converts procainamide to N-acetylprocainamide, the latter acting as a class III antiarrhythmic drug. However, long-term use of procainamide may cause lupus-like syndrome, thus its use is now limited. Lidocaine is well absorbed orally, but has poor bioavailability and a strong first-pass effect. Its clearance is easily affected by hepatic blood flow, and metabolites are excreted via the kidneys. Lidocaine is mainly used to rapidly suppress symptomatic ventricular arrhythmias. When lidocaine plasma concentrations exceed therapeutic concentrations, tremors, altered mental status, and dysarthria may occur. Propafenone can prolong the PR and QRS intervals and is used to maintain sinus rhythm in patients with supraventricular tachycardia, such as atrial fibrillation. It can also be used to treat ventricular arrhythmias, but with moderate efficacy. Its metabolic pathway varies greatly among individuals; in patients with low metabolism and lack of the hepatic enzyme CYP2D6, propafenone clearance is reduced, resulting in less production of the active metabolite 5-hydroxypropafenone. Adverse reactions of propafenone include accelerating ventricular responsiveness in patients with atrial flutter, increasing the severity and frequency of ventricular tachycardia, and worsening heart failure. Amiodarone blocks potassium, sodium, and calcium channels, and also has α- and β-receptor blocking effects. This drug is lipid-soluble and is metabolized by the hepatic enzyme CYP3A4 to desethylamiodarone, the latter having an antiarrhythmic effect comparable to or stronger than amiodarone. Amiodarone is metabolized very slowly, therefore, the relief of its adverse reactions after discontinuation is also slow. Clinical use of amiodarone requires vigilance for adverse reactions such as sinus bradycardia, conduction block, torsades de pointes, and liver damage. Long-term use also requires vigilance for pulmonary fibrosis, thyroid dysfunction, and corneal pigmentation; therefore, close follow-up is necessary during treatment. Intravenous amiodarone has vasodilatory and myocardial contractile inhibitory effects, which may cause hypotension. Sotalol has the potential to cause arrhythmias and worsen heart failure. In patients with heart failure, it should be started at a low dose with close monitoring of the QT interval. Simultaneously, vigilance is needed for bradycardia, bronchospasm, and worsening of heart failure.

[0018] The above situation indicates an urgent clinical need to develop new, effective, and safe antiarrhythmic drugs.

[0019] Currently, animal models used in the development of antiarrhythmic drugs include aconitine-induced rat arrhythmia models, cardiac glycoside-induced rat arrhythmia models (isolated heart ouabain poisoning model and in vivo heart ouabain poisoning model), CaCl2-Ach-induced rat arrhythmia models, BaCl2-induced rat arrhythmia models, chloroform-induced rat arrhythmia models, and catecholamine-induced mouse arrhythmia models. There are currently very few mature isolated tissue / organ-level arrhythmia models available domestically and internationally. Based on Langendorff's isolated heart perfusion, the whole heart is stimulated using a constant current / constant voltage stimulator or chemical substances to simulate clinical arrhythmias caused by different reasons. This model is stable, rapid, and allows for self-control. After perfusion flushing, it can be used for re-evaluation with different doses or drugs, achieving high throughput.

[0020] Cardiotoxicity is the most prominent and serious clinical feature of aconite poisoning, with arrhythmia and acute heart failure being the main manifestations of cardiotoxicity caused by aconite drugs. Historical Tibetan medical texts record that Bangga (a type of Tibetan herbal medicine) can counteract aconite poisoning.

[0021] Literature reports and previous studies indicate that Bangga mainly contains diterpenoid alkaloids. Of the 76 diterpenoid alkaloids reported so far, 42 are C20 type, and 22 of these are C20-hyterigen type. Finding highly effective and low-toxicity antiarrhythmic components from Bangga would maximize its medicinal value, promote economic development in Tibetan areas, bring new hope for the development of arrhythmia drugs, and make a significant contribution to human health. Summary of the Invention

[0022] The purpose of this invention is to provide the application of the total alkaloid extract of *Bangga chinensis* in the preparation of drugs for the prevention and / or treatment of arrhythmia, thereby addressing the problems existing in the prior art. This invention is the first to discover that the total alkaloid extract of *Bangga chinensis* can prevent and treat arrhythmia. The extract exhibits high antiarrhythmic activity, low toxicity, and a large safety window, demonstrating high efficacy and low toxicity, and holds promise for development into a new, effective, and safe antiarrhythmic drug.

[0023] To achieve the above objectives, the present invention provides the following solution:

[0024] This invention provides the use of the total alkaloid extract of Bangga in the preparation of medicaments for the prevention and / or treatment of arrhythmias.

[0025] Preferably, the preparation method of the Bangga total alkaloid extract includes the following steps:

[0026] The coarse powder of the whole herb of Bangga medicinal material was mixed with an ethanol solution and refluxed to extract the extract.

[0027] The solvent in the extract was recovered until there was no alcohol odor. Then, its pH was adjusted to acidic and mixed with dichloromethane for the first extraction to obtain a dichloromethane layer and an extract water layer.

[0028] After adjusting the pH of the extraction water layer to alkaline, dichloromethane solvent was added for a second extraction to obtain the Bangga total alkaloid extract.

[0029] Preferably, the reflux extraction is performed 3 times at a temperature of 60°C; during each reflux extraction, the mass-to-volume ratio of the whole herb powder of Bangga medicinal material to the ethanol solution is 1 kg: 7-8 L; and the volume percentage of ethanol in the ethanol solution is 70%.

[0030] Preferably, the first extraction is performed three times; each time the first extraction is performed, the volume ratio of the extract to the dichloromethane is 1:1.

[0031] The second extraction is performed three times; each time the second extraction is performed, the volume ratio of the pH-adjusted extraction water layer to the dichloromethane solvent is 1:1.

[0032] This invention provides the use of the total alkaloid extract of Bangga in the preparation of medicaments for the prevention and / or treatment of premature ventricular contractions.

[0033] Preferably, the preparation method of the Bangga total alkaloid extract includes the following steps:

[0034] The coarse powder of the whole herb of Bangga medicinal material was mixed with an ethanol solution and refluxed to extract the extract.

[0035] The solvent in the extract was recovered until there was no alcohol odor. Then, its pH was adjusted to acidic and mixed with dichloromethane for the first extraction to obtain a dichloromethane layer and an extract water layer.

[0036] After adjusting the pH of the extraction water layer to alkaline, dichloromethane solvent was added for a second extraction to obtain the Bangga total alkaloid extract.

[0037] Preferably, the reflux extraction is performed 3 times at a temperature of 60°C; during each reflux extraction, the mass-to-volume ratio of the whole herb powder of Bangga medicinal material to the ethanol solution is 1 kg: 7-8 L; the volume percentage of ethanol in the ethanol solution is 70%.

[0038] The first extraction is performed three times; each time the first extraction is performed, the volume ratio of the extract to the dichloromethane is 1:1.

[0039] The second extraction is performed three times; each time the second extraction is performed, the volume ratio of the pH-adjusted extraction water layer to the dichloromethane solvent is 1:1.

[0040] This invention provides the use of the total alkaloid extract of Bangga in the preparation of medicaments for the prevention and / or treatment of ventricular tachycardia.

[0041] Preferably, the preparation method of the Bangga total alkaloid extract includes the following steps:

[0042] The coarse powder of the whole herb of Bangga medicinal material was mixed with an ethanol solution and refluxed to extract the extract.

[0043] The solvent in the extract was recovered until there was no alcohol odor. Then, its pH was adjusted to acidic and mixed with dichloromethane for the first extraction to obtain a dichloromethane layer and an extract water layer.

[0044] After adjusting the pH of the extraction water layer to alkaline, dichloromethane solvent was added for a second extraction to obtain the Bangga total alkaloid extract.

[0045] Preferably, the reflux extraction is performed 3 times at a temperature of 60°C; during each reflux extraction, the mass-to-volume ratio of the whole herb powder of Bangga medicinal material to the ethanol solution is 1 kg: 7-8 L; the volume percentage of ethanol in the ethanol solution is 70%.

[0046] The first extraction is performed three times; each time the first extraction is performed, the volume ratio of the extract to the dichloromethane is 1:1.

[0047] The second extraction is performed three times; each time the second extraction is performed, the volume ratio of the pH-adjusted extraction water layer to the dichloromethane solvent is 1:1.

[0048] The present invention discloses the following technical effects:

[0049] This invention is the first to discover that the total alkaloid extract of *Bangga chinensis* can prevent and treat arrhythmias. The extract can prolong the latency of premature ventricular contractions (VPBs) and reduce the incidence of ventricular tachycardia (VT) in mice, thus achieving an antiarrhythmic effect. It also has a significant alleviating effect on aconitine-induced arrhythmias in mice. The total alkaloid extract of *Bangga chinensis* provided by this invention can be used to prepare drugs for the prevention and / or treatment of premature ventricular contractions, ventricular tachycardia, and arrhythmias, and has broad application prospects. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 This is a schematic diagram of the chemical structure of aconitine.

[0052] Figure 2 The image shows a thin-layer chromatogram for identifying the total alkaloids extract from Bangga; from left to right, the images are aconitine, the test solution, the test solution, and the test solution.

[0053] Figure 3 A schematic diagram of the chemical structure of the diterpenoid alkaloid Tanguticinine E;

[0054] Figure 4 The UV absorption spectrum (200-900 nm) of the blank sample is shown.

[0055] Figure 5 The ultraviolet absorption spectrum (200-900 nm) of the reference solution of the diterpenoid alkaloid Tanguticinine E;

[0056] Figure 6 The ultraviolet absorption spectrum (200-900nm) of the test solution of the total alkaloid extract of Bangga;

[0057] Figure 7 This is a standard curve of the diterpenoid alkaloid Tanguticinine E; where the horizontal axis represents the concentration of the diterpenoid alkaloid Tanguticinine E, and the vertical axis represents the absorbance.

[0058] Figure 8 BPI graph of total alkaloid extract from Bangga;

[0059] Figure 9 Mass-to-charge ratio diagram of Hordenine (MW 165) and oxidized maltine (MW 181);

[0060] Figure 10 Mass-to-charge ratio diagram for Candicinechloride (MW 214);

[0061] Figure 11 The mass-to-charge ratio diagram for Tanguticuline G (MW387);

[0062] Figure 12Mass-to-charge ratio diagram for Tangutisine / TanguticulineA (MW345);

[0063] Figure 13 Mass-to-charge ratio diagram of 2-acetyl-13-dehydro-11-epihetisine (2-acetyl-13-carbonyl-11-epihetisine) / 2-acetyl-13-dehydro-11-hetisine (2-acetyl-13-carbonyl-11-hetisine) (MW369);

[0064] Figure 14 Mass-to-charge ratio diagram for Heteratisine (MW391);

[0065] Figure 15 Mass-to-charge ratio diagram of Hetisine / Delfissinol / Tanwusine (MW329);

[0066] Figure 16 Mass-to-charge ratio plot for Hetisinone (MW327);

[0067] Figure 17 Mass-to-charge ratio plot of 11-O-acetylhetisine / 13-O-acetylhetisine (11-O-acetylhetisine / 13-O-acetylhetisine) (MW371);

[0068] Figure 18 The mass-to-charge ratio diagram for Tangutimine (MW313);

[0069] Figure 19 Mass-to-charge ratio plot for 6-acetylheteratisine (MW 433);

[0070] Figure 20 Mass-charge ratio diagram of Guan-fu base Z (MW 415);

[0071] Figure 21 Mass-to-charge ratio plot for Atisine (MW 343);

[0072] Figure 22 Mass-to-charge ratio plot of Isoatisine / Chellespontine (MW 343);

[0073] Figure 23 The mass-to-charge ratio diagram for Navigine B (MW 476);

[0074] Figure 24 Mass-to-charge ratio diagram of Tanguticinine A / B / C / D (MW 640);

[0075] Figure 25 Mass-to-charge ratio plot for Tanguticurine A (MW 622);

[0076] Figure 26 For Tanguticuline C (MW 471), see the mass-to-charge ratio diagram.

[0077] Figure 27 Mass-to-charge ratio diagram for Tanguticinine E (MW 624);

[0078] Figure 28 Here is the mass-to-charge ratio plot for Anthoroidine B (MW 624);

[0079] Figure 29 The mass-to-charge ratio diagram for N-oxide 5-deoxyanthoroidine B (MW624);

[0080] Figure 30 The mass-to-charge ratio diagram of Tanguticinine F (MW 666);

[0081] Figure 31 Mass-to-charge ratio diagram of Trichocarpinine C (MW 752);

[0082] Figure 32 Mass-to-charge ratio plot for Trichocarpinine A (MW 766);

[0083] Figures 33-35 The structural diagram of monomer components of the total alkaloid extract from Bangga obtained by LC-MS.

[0084] Figure 36The effects of aconitine solution on the electrocardiogram and conduction of isolated rat hearts were investigated. In this study, A represents the left ventricular conduction curve of the isolated rat heart before and after drug administration; Control represents the left ventricular conduction curve of the heart perfused with KH solution for 15 min; 1 nM - 15 min represents the left ventricular conduction curve of the heart perfused with 1 nM aconitine solution for 15 min; 5 nM - 15 min represents the left ventricular conduction curve of the heart perfused with 5 nM aconitine solution for 15 min; 10 nM - 1 min represents the left ventricular conduction curve of the heart perfused with 10 nM aconitine solution for 1 min; 10 nM - 2 min represents the left ventricular conduction curve of the heart perfused with 10 nM aconitine solution for 2 min; 10 nM - 3 min represents the left ventricular conduction curve of the heart perfused with 10 nM aconitine solution for 3 min; and 10 nM - 5 min represents the left ventricular conduction curve of the heart perfused with 10 nM aconitine solution for 3 min. A) Left ventricular conduction curves of the heart perfused with 10 nM aconitine solution for 5 min, 10 nM-8 min for 8 min, and 10 nM-10 min for 10 min; B) Effect of different concentrations of aconitine solution on the heart rate of isolated rat hearts; C) Effect of different concentrations of aconitine solution on the conduction velocity of isolated rat hearts; D) Electrocardiograms of isolated rat hearts before and after drug administration; E) Effect of different concentrations of aconitine solution on the QT interval of isolated rat hearts; F) Effect of different concentrations of aconitine solution on the QRS interval of isolated rat hearts; Data are expressed as mean ± standard deviation. * represents P < 0.05, ** represents P < 0.01 compared with the control.

[0085] Figure 37 The effects of the total alkaloid extract of Bangga on the electrocardiogram and conduction of isolated rat hearts were investigated. In this study, A represents the left ventricular conduction curve of isolated rat hearts before and after drug administration; Control represents the left ventricular conduction curve of hearts perfused with KH solution for 26 min; and 1 ng / mL-15 min, 5 ng / mL-15 min, 10 ng / mL-15 min, 50 ng / mL-15 min, 100 ng / mL-15 min, 500 ng / mL-15 min, and 1000 ng / mL-15 min are defined as 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. Left ventricular conduction maps of rat isolated hearts after sequential perfusion with 500 ng / mL and 1000 ng / mL Bangga total alkaloid extract solutions for 15 min, with Wash showing left ventricular conduction maps after elution with KH solution for 15 min; B shows the effect of different concentrations of Bangga total alkaloid extract solutions on the heart rate of isolated rat hearts; C shows the effect of different concentrations of Bangga total alkaloid extract solutions on the conduction velocity of isolated rat hearts; D shows the electrocardiograms of isolated rat hearts before and after drug administration; E shows the effect of different concentrations of Bangga total alkaloid extract solutions on the QT interval of isolated rat hearts; F shows the effect of different concentrations of Bangga total alkaloid extract solutions on the QRS interval of isolated rat hearts.

[0086] Figure 38 The study investigated the effects of combined administration of Bangga total alkaloid extract and aconitine on the electrocardiogram and conduction of isolated rat hearts. In the figures, A represents the left ventricular conduction curve of the isolated rat heart before and after administration; Control represents the left ventricular conduction curve of the heart perfused with KH solution for 27 min; 10nM aconitine + 1 ng / mL - 5 min represents the left ventricular conduction curve of the heart perfused with 10nM aconitine and 10 ng / mL Bangga total alkaloid extract for 5 min; 10nM aconitine + 10 ng / mL - 10 min represents the left ventricular conduction curve of the heart perfused with 10nM aconitine and 10 ng / mL Bangga total alkaloid extract for 10 min; 10nM aconitine + 20 ng / mL - 5 min represents the left ventricular conduction curve of the heart perfused with 10nM aconitine and 20 ng / mL Bangga total alkaloid extract for 5 min; 10nM... Aconitine + 30 ng / mL - 5 min: Left ventricular conduction curves after 5 min of perfusion of the heart with a combination of 10 nM aconitine and 30 ng / mL total alkaloid extract of *Bangga chinensis*; Aconitine + 50 ng / mL - 5 min: Left ventricular conduction curves after 5 min of perfusion of the heart with a combination of 10 nM aconitine and 50 ng / mL total alkaloid extract of *Bangga chinensis*; Aconitine + 100 ng / mL - 5 min: Left ventricular conduction curves after 5 min of perfusion of the heart with a combination of 10 nM aconitine and 100 ng / mL total alkaloid extract of *Bangga chinensis*; Aconitine + 100 ng / mL - 10 min: Left ventricular conduction curves after 10 min of perfusion of the heart with a combination of 10 nM aconitine and 100 ng / mL total alkaloid extract of *Bangga chinensis*; Wash: Left ventricular conduction curves after 15 min of KH solution elution; B: Electrocardiograms of isolated rat hearts before and after drug administration;

[0087] Figure 39The effects of combined administration of Bangga total alkaloid extract and aconitine on the electrocardiogram and conduction of isolated rat hearts were investigated. Specifically, A showed the effects of 10 nM aconitine solution perfused for 10 min on left ventricular conduction and ECG in isolated rat hearts, along with ECG results; B showed the effects of 10 nM aconitine solution and 10 ng / mL Bangga total alkaloid extract perfused for 10 min on left ventricular conduction and ECG in isolated rat hearts, along with ECG results; and C showed the effects of 10 nM aconitine solution and 10 ng / mL Bangga total alkaloid extract perfused for 20 min on left ventricular conduction and ECG in isolated rat hearts. Effects and electrocardiograms; D shows the effects of perfusion of 10 nM aconitine solution and 20 ng / mL Bangga total alkaloid extract solution for 20 min on left ventricular conduction and ECG in isolated rat hearts and electrocardiograms; E shows the effects of perfusion of 10 nM aconitine solution and 30 ng / mL Bangga total alkaloid extract solution for 20 min on left ventricular conduction and ECG in isolated rat hearts and electrocardiograms; F shows the effects of perfusion of 10 nM aconitine solution and 50 ng / mL Bangga total alkaloid extract solution for 20 min on left ventricular conduction and ECG in isolated rat hearts and electrocardiograms. Detailed Implementation

[0088] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0089] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0090] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0091] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0092] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0093] Example 1: Bangga Total Alkaloid Extract and Verification

[0094] 1. A method for extraction, separation, and purification of total alkaloids from Bangga, comprising the following steps:

[0095] (1) Take 8 kg of the whole herb of Bangga medicinal material and crush it into coarse powder.

[0096] (2) Add about 60L of 70% (volume percentage) ethanol each time, reflux and extract 3 times at 60℃, filter, and combine the filtrates.

[0097] (3) Ethanol was recovered under reduced pressure at 55℃ to obtain about 25L of black concentrate. After adding an appropriate amount of purified water and dispersing, the pH was adjusted to 2-3 with hydrochloric acid. Then, an equal volume of dichloromethane (the same volume as the pH-adjusted solution) was used for extraction 3 times. That is, an equal volume of dichloromethane was used for each extraction, and the dichloromethane layer was collected for each extraction.

[0098] (4) Combine the dichloromethane layers, recover the dichloromethane to obtain about 175g of black paste, discard it, and record the remaining solution as the extraction water layer.

[0099] (5) The pH of the aqueous layer was adjusted to about 10 with ammonia water and extracted three times with an equal volume of dichloromethane. That is, an equal volume of dichloromethane was used for each extraction. The three dichloromethane layers were combined and the dichloromethane solvent was recovered. The residue was placed in a drying oven and dried at 50°C for 8 hours to obtain about 16g of black powder. After drying and grinding, it turned brownish-brown, which is the total alkaloid extract of Bangga.

[0100] 2. Thin-layer chromatographic identification study of total alkaloid extracts from Bangga

[0101] (1) Preparation of reference solution

[0102] Weigh an appropriate amount of isoaritin reference standard and dissolve it in methanol to prepare an isoaritin solution with a concentration of 1 mg / mL. This is the reference solution. The structure of isoaritin is as follows: Figure 1 As shown.

[0103] (2) Preparation of the test solution

[0104] Take approximately 40 mg of the Bangga total alkaloid extract powder prepared in step "1. An extraction, separation and purification method for Bangga total alkaloid extract", accurately weigh it, dissolve it in methanol and dilute it to a 5 mL volumetric flask, filter it, and the product is obtained.

[0105] (3) Thin-layer chromatography development

[0106] According to the thin-layer chromatography method (General Rule 0502), 5 μL of the reference solution and 20 μL of the test solution were spotted separately onto the same silica gel G thin-layer plate. Petroleum ether (60-90℃)-dichloromethane-methanol (3:12:1) was used as the developing solvent. Before development, both the developing solvent and the thin-layer plate were simultaneously saturated with ammonia vapor for 20 min. After development, the plate was removed, dried, and sprayed with modified bismuth potassium iodide solution. The results are as follows: Figure 2 As shown, in the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.

[0107] 3. Determination of alkaloid content in Bangga total alkaloid extract by acid dye colorimetric method

[0108] (1) Materials

[0109] 1.1) Instruments and Reagents

[0110] Instruments: UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.); 0.0001 g electronic analytical balance (Beijing Youce Technology Development Co., Ltd., XSE104 model); 0.0001 g electronic analytical balance (Beijing Youce Technology Development Co., Ltd., XS105 model);

[0111] Reagents: Anhydrous ethanol (analytical grade AR, Fuchen (Tianjin) Chemical Reagent Co., Ltd.); Chloroform (manufactured by Beijing Tongguang Fine Chemical Co., Ltd.); Anhydrous sodium sulfate and disodium hydrogen phosphate (Tianjin Guangfu Technology Development Co., Ltd.); Potassium dihydrogen phosphate (analytical grade AR, Sinopharm Chemical Reagent Co., Ltd.); Sodium hydroxide (analytical grade AR, Xilong Scientific Co., Ltd.); Bromothymol blue (Aladdin Reagent (Shanghai) Co., Ltd.); Water was ultrapure water.

[0112] 1.2) Drug testing

[0113] Test reagent: The total alkaloid extract of Bangga was prepared by "1. An extraction, separation and purification method for total alkaloid extract of Bangga"; the diterpenoid alkaloid Tanguticinine E (prepared in the laboratory, with a purity greater than 98% as determined by HPLC) was as follows. Figure 3 As shown. Because diterpenoid alkaloids are the characteristic components and main alkaloid types in Bangga, Tanguticinine E was chosen as the reference standard.

[0114] (2) Method

[0115] 2.1) Preparation of color developer

[0116] Accurately weigh 0.1 g of bromothymol blue, add 3.2 mL of 0.5 mol / L sodium hydroxide solution, add 200 mL of water, dissolve, and obtain bromothymol blue indicator solution.

[0117] Accurately weigh 0.6 g of potassium dihydrogen phosphate and 7.15 g of disodium hydrogen phosphate, dissolve them in 500 mL of water to obtain phosphate buffer (pH 6.8).

[0118] 2.2) Preparation of reference solution

[0119] Accurately weigh 3.02 mg of Tanguticinine E reference standard into a 5 mL volumetric flask, dissolve and dilute to volume with 70% (volume percentage) ethanol.

[0120] 2.3) Preparation of the test solution

[0121] Accurately weigh 3.06 mg of the total alkaloid extract of Bangga into a 5 mL volumetric flask, dissolve in 70% (v / v) ethanol and dilute to volume. Prepare two parallel solutions for later use.

[0122] 2.4) Determination of detection wavelength

[0123] Accurately transfer 0.5 mL each of 70% (volume percentage) ethanol, reference solution, and test solution into separate separatory funnels. Add 1.0 mL of bromothymol blue indicator solution, 10.0 mL of phosphate buffer (pH 6.8), and 10.0 mL of chloroform. Shake for 3 min, stopper tightly, and let stand for 10 min. Separately divide the chloroform solution and add 1.0 g of anhydrous sodium sulfate. Shake and let stand for 10 min. Perform full-wavelength scans at 200-900 nm. The results are as follows: Figures 4-6 As shown in the figure. The results show that both the reference solution and the test solution have maximum absorption at 415 nm, and the blank sample has no interference. Therefore, the detection wavelength was selected as 415 nm.

[0124] 2.5) Examination of linear relationships

[0125] Accurately transfer 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of Tanguticinine E standard solution into separate separatory funnels. Add 0.5, 0.4, 0.3, 0.2, 0.1, and 0.0 mL of water to each funnel, followed by 1.0 mL of bromothymol blue indicator solution, 10.0 mL of phosphate buffer (pH 6.8), and 10.0 mL of chloroform. Shake for 3 min, stopper tightly, and let stand for 10 min. Separately collect the chloroform solution and add...

[0126] 1.0 g of anhydrous sodium sulfate was shaken and allowed to stand for 10 min. The absorbance was measured at 415 nm using the first sample as a blank, and the regression equation was calculated. Results are shown below. Figure 7 The regression equation is y = 24.421x + 0.0571, R0 2 It is 0.9993.

[0127] 2.6) Determination of the test solution

[0128] Accurately transfer 0.5 mL of the test solution and perform the same operation as in step 2.5). The absorbance of the two parallel samples were measured to be 0.510 and 0.507, respectively.

[0129] (3) Results

[0130] Substituting the absorbance value of the test sample into the linear equation y = 24.421x + 0.0571, the alkaloid content in the total alkaloid extract of Bangga, calculated based on the diterpenoid alkaloid Tanguticinine E, is 60.30%.

[0131] (4) Qualitative analysis of total alkaloid extracts from Bangga by UPLC-Q-TOF-MS / MS

[0132] 4.1) Drug testing

[0133] Test drug: The total alkaloid extract of Bangga was prepared by "1. A method for extraction, separation and purification of total alkaloid extract of Bangga".

[0134] 4.2) Preparation of test solution: Accurately weigh 6 mg of Bangga total alkaloid extract and place it in a 2 mL volumetric flask. Dissolve it in chromatographic methanol and dilute to the mark. Filter through a 0.22 μm filter membrane to obtain the test solution.

[0135] 4.3) Chromatographic conditions: WatersAcquity UPLC BEH C 18 Spectroscopic column (100 mm × 2.1 mm, 1.7 μm); mobile phase: methanol (A) - 0.1 mol·L⁻¹ -1 Ammonium acetate + 0.2% glacial acetic acid (B), gradient elution, 0-12 min, 5% A-95% A, flow rate 0.4 mL / min -1 The injection volume was 2 μL, and the column temperature was 35℃.

[0136] 4.4) Mass spectrometry conditions: Electrospray ionization (ESI) source; positive ion scanning mode; capillary voltage 3.0 kV; cone voltage 20 V; ion source temperature 100 °C; desolvation gas temperature 400 °C; cone gas flow rate 50 L·h. -1 Desolvation gas flow rate 700 L·h -1The low-energy scan transport collision energy was 6 eV, and the high-energy scan transport collision energy was 25-55 eV. The scan mass range was m / z 50-1600 Da. A leucine enkephalin solution (1.0 μg / mL) was used. -1 Real-time quality correction was performed at a flow rate of 10 μL·mL. -1 Data acquisition was controlled by MassLynx V4.1 software.

[0137] 4.5) Results: The total ion chromatogram of the Bangga total alkaloid extract determined by liquid chromatography-mass spectrometry is shown in the figure. Figure 8 See the mass-to-charge ratio diagram. Figures 9-32 The deduced structures of the monomeric compounds are shown in [reference needed]. Figures 33-35 Based on the UV spectra, primary mass spectrometry, and secondary mass spectrometry information of each chromatographic peak, and comparison with literature values, 25 compounds were deduced from the total alkaloid extract of Bangga, as shown in Table 1. Except for Hordenine, oxidized maltine, and candicine chloride, the remaining 22 components were all diterpenoid alkaloids, including 8 diterpenoid alkaloids (Tanguticinine A / B / C / D, Tanguticurine A, Tanguticinine E, Anthoroidine B, N-oxide5-deoxyanthoroidine B, Tanguticinine F, Trichocarpinine). C, Trichocarpinine A), two C19 lactone-type diterpenoid alkaloids (Heteratisine and 6-acetylheteratisine), and twelve C20 type diterpenoid alkaloids (Tanguticuline G, Tangutisine / Tanguticuline A, 2-acetyl-13-dehydro-11-epihetisine / 2-acetyl-13-dehydro-11-hetisine, Hetisine / Delfissinol / Tanwusine, Hetisinone, 11-O-acetylhetisine / 13-O-acetylhetisine, Tangutimine, Guan-fubase Z, Atisine, Isoatisine / Chellespontine, Navirine B, and Tanguticuline C).

[0138] Table 1. Qualitative analysis results of total alkaloid extracts from Bangga by LC-MS

[0139]

[0140] (5) Experimental study on the anti-arrhythmic effect of total alkaloid extract from *Ranunculi Tibetici Herba* against aconitine-induced arrhythmia in mice

[0141] 5.1) Experimental materials

[0142] Test drug: the total alkaloid extract of *Ranunculi Tibetici Herba* prepared by "1. An extraction, separation and purification method for total alkaloid extract of *Ranunculi Tibetici Herba*".

[0143] Reagent: aconitine, HPLC grade, with purity ≧98%, supplied by Chengdu Ruifensi Deddan Biotechnology Co., Ltd.

[0144] Instrument: multi-channel physiological recorder, model MP150, manufactured by BIOPAC Inc., USA

[0145] 5.2) Experimental animals

[0146] ICR mice, male, body weight 18-22g, supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SYXK (Beijing) 2021-0006.

[0147] 5.3) Experimental methods

[0148] ICR mice were randomly divided into a single-dose administration group and a three-dose administration group according to body weight. Each test group was further randomly divided into 4 groups based on body weight (namely the control group, 15 mg / kg, 30 mg / kg and 60 mg / kg total alkaloid extract of *Ranunculi Tibetici Herba* groups, with an intragastric administration volume of 10 mL / kg, 10 mice in each group. The single-dose administration group received one dose of administration, while the control group was given pure water by intragastric administration once a day. 40 minutes after the last administration of each group, 3.8% chloral hydrate was injected intraperitoneally (0.1 mL / 10 g body weight). After the animals were anesthetized, they were fixed in the supine position, and needle electrodes were inserted into the right forelimb, left hindlimb and right hindlimb. The electrocardiogram was recorded using an MP150 physiological recorder. After the electrocardiogram stabilized, aconitine (15 μg / mL, 0.1 mL / 10 g body weight per mouse, equivalent to 150 μg / kg) was rapidly injected through the tail vein 1 hour after administration. The lead II electrocardiogram was recorded for 60 minutes, the electrocardiographic changes of each group were observed, and the onset time of ventricular premature beat (VPB), ventricular tachycardia (VT), ventricular fibrillation (VF) and the number of affected animals were recorded. The three-dose administration group received continuous administration for 3 days, and 1 hour after the third administration, aconitine (15 μg / mL, 0.0833 mL / 10 g body weight) was rapidly injected through the tail vein. The lead II electrocardiogram was recorded for 60 minutes, the electrocardiographic changes of each group were observed, and the onset time of ventricular premature beat (VPB), ventricular tachycardia (VT), ventricular fibrillation (VF) and the number of affected animals were recorded.

[0149] Statistical method: One-way analysis of variance was used for count data, and the LSD method was used for inter-group comparison; Chi-square test was used for analysis and comparison of categorical data.

[0150] 5.4) Experimental results

[0151] 5.4.1) Effect of a single administration of Bangga total alkaloid extract on aconitine-induced cardiac arrhythmia in mice.

[0152] The effects of a single administration of Bangga total alkaloid extract on aconitine-induced arrhythmias in mice are shown in Tables 2 and 3. As shown in Tables 2 and 3, a single administration of the three doses of Bangga total alkaloid extract prolonged the onset time of aconitine-induced ventricular premature beats (VPB) in mice, but the difference was not statistically significant (P>0.05 compared to the control group). Bangga total alkaloid extract at 30 mg / kg significantly prolonged the onset time of aconitine-induced ventricular tachycardia (VT) in rats (P<0.05), and the number of VT-inducing mice was also significantly reduced (P<0.01). Bangga total alkaloid extract at 60 mg / kg prolonged the onset time of aconitine-induced ventricular tachycardia (VT) in rats (P>0.05), and the number of VT-inducing animals was also significantly reduced (P<0.05).

[0153] Table 2. Effects of a single administration of Bangga total alkaloid extract on aconitine-induced cardiac arrhythmia in mice.

[0154] control group - 9 12.39±18.75 19.37±23.78 Bangga Total Alkaloid Extract 15.0 8 23.37±30.35 23.93±29.90 Bangga Total Alkaloid Extract 30.0 9 34.33±30.47 46.89±26.02* Bangga Total Alkaloid Extract 60.0 9 27.67±30.70 40.76±28.86

[0155] Note: Animals without premature ventricular contractions or tachycardia were counted from 60 minutes onwards. Compared with the control group, *P<0.05, **P<0.01; fewer than 10 animals were considered to have died during the experiment (the same applies below).

[0156] Table 3. Effects of a single administration of Bangga total alkaloid extract on aconitine-induced cardiac arrhythmia in mice.

[0157]

[0158]

[0159] Note: *P<0.05, **P<0.01 compared with the control group.

[0160] 5.4.2) Effect of three consecutive administrations of Bangga total alkaloid extract on aconitine-induced cardiac arrhythmia in mice.

[0161] The effects of three consecutive administrations of Bangga total alkaloid extract on aconitine-induced arrhythmias in mice are shown in Tables 4 and 5. Tables 4 and 5 show that three consecutive administrations of Bangga total alkaloid extract at 30 mg / kg and 60 mg / kg significantly prolonged the onset time of aconitine-induced ventricular premature beats (VPB) in rats (P<0.05 compared to the control group); three consecutive administrations of Bangga total alkaloid extract at 60 mg / kg significantly reduced the number of rats with aconitine-induced VPB (P<0.05 compared to the control group); and Bangga total alkaloid extract at 15 mg / kg, 30 mg / kg, and 60 mg / kg significantly prolonged the onset time of aconitine-induced ventricular tachycardia (VT) in rats (P<0.05 or P<0.01), and also significantly reduced the number of rats with VT (P<0.05 or P<0.01).

[0162] Table 4. Effects of three consecutive administrations of Bangga total alkaloid extract on aconitine-induced cardiac arrhythmia in mice.

[0163]

[0164] Note: Animals without premature ventricular contractions or tachycardia started with a time of 60 minutes. Compared with the control group, *P<0.05, **P<0.01.

[0165] Table 5. Effects of three consecutive administrations of Bangga total alkaloid extract on aconitine-induced cardiac arrhythmia in mice.

[0166]

[0167] Note: *P<0.05, **P<0.01 compared with the control group.

[0168] In conclusion, the total alkaloid extract of Bangga has a significant alleviating effect on aconitine-induced arrhythmia in mice.

[0169] Example 2: Study on the detection of aconitine-induced electrical conduction disorders in isolated hearts by a matrix-type multichannel electrophysiological mapping system.

[0170] 1. Experimental Methods

[0171] Clean the Langendorff perfusion system, add KH solution into the perfusion system, and adjust the perfusion flow rate to 10 mL / min. Turn on the constant temperature water bath heating system, adjust the temperature of circulating water to keep the temperature of the liquid outlet of the aortic cannula at 37(±0.5)°C. Weigh the rat (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SYXK (Jing) 2021-0006), inject 3125 U / kg of heparin sodium intraperitoneally, anesthetize with isoflurane 15 minutes later, and sacrifice by cervical dislocation. Place the animal supine on the experimental table for fixation, and disinfect with 75% (volume percentage) alcohol. Cut the chest skin at the xiphoid process, lift the xiphoid with forceps, cut open the thoracic cavity along the middle of the left and right ribs to expose the heart. Lift the lung lobes with forceps, quickly cut off the heart along the back of the lung, and place it in oxygenated (95% O₂, 5% CO₂) pre-cooled perfusate. Cut off excess tissue, free the aorta, connect the aorta to the aortic cannulation site, perfuse the heart with a syringe to pump out residual blood and restore heartbeat; pre-open the perfusion system, quickly connect the aortic cannula to the perfusion system, and start perfusion. Insert a stimulation electrode at the apex of the heart, attach the MappingLab matrix multi-channel electrode to the left ventricular position, and place ECG electrodes in the right atrium and left ventricle of the heart. After the heart stabilizes, the MappingLab matrix multi-channel electrophysiological mapping system records the signals.

[0172] Heart rate, conduction velocity, QT interval, and QRS interval are expressed as mean ± standard deviation. Statistical test is performed with SPSS 20.0 software using one-way analysis of variance (ANOVA). According to the result of homogeneity of variance test, LSD method is used for homogeneous variance, and Tamhane’s T2 method is used for uneven variance. P<0.05 is taken as the significance level of statistical test.

[0173] 2. Results

[0174] 2.1) Detection of cardiac toxicity of aconitine by matrix multi-channel electrophysiological mapping system

[0175] Through the Langendorff isolated heart perfusion system, 1 nM, 5 nM and 10 nM aconitine solutions are perfused into the isolated rat heart at a flow rate of 10 mL / min respectively. The MappingLab matrix multi-channel electrophysiological mapping system is used to record parameters closely related to arrhythmia, such as single-channel ECG conduction direction, conduction velocity, QT interval, etc. The results are shown in Figure 36The results showed that after 15 minutes of perfusion with 1 nM and 5 nM aconitine, no significant abnormalities were observed in ECG or cardiac conduction thermography. After 5 minutes of perfusion with 10 nM aconitine, supraventricular ectopic pacing occurred, with the ventricles initially excited before the atria. After 8 minutes of perfusion with 10 nM aconitine, atrioventricular dissociation occurred, and after 10 minutes of perfusion with 10 nM aconitine, ventricular fibrillation occurred. The heart returned to ventricular tachycardia after 10 minutes of washout. After 15 minutes of perfusion with 1 nM aconitine, there were no significant differences in heart rate (HR), conduction velocity (CV), QT interval, and QRS interval compared to the control group. After 15 minutes of perfusion with 5 nM aconitine, the QT interval was significantly prolonged compared to the control group, with a prolongation percentage of 27% (P < 0.05); HR, CV, and QRS interval were not significantly different from the control group. Aconitine 10 nM perfusion for 8 min significantly increased heart rate (HR) by 40% compared to the control group (P < 0.05); significantly decreased conduction velocity by 63% (P < 0.01); and significantly prolonged the QT interval by 47% (P < 0.01). No significant difference was observed in the QRS interval compared to the control. In conclusion, 10 nM aconitine can induce significant electrical conduction disturbances in isolated rat hearts, with ectopic pacing beginning 5 min after administration and ventricular fibrillation beginning 10 min, partially recovering after washout.

[0176] 2.2) Detection of cardiotoxicity of Bangga alkaloids using a matrix-type multichannel electrophysiological mapping system

[0177] Using the Langendorff isolated heart perfusion system, isolated rat hearts were perfused with total alkaloid extracts of *Bangga chinensis* at concentrations of 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL at a flow rate of 10 mL / min. Single-channel ECG parameters closely related to arrhythmia, such as conduction direction, conduction velocity, and QT interval, were recorded using a MappingLab matrix multichannel electrophysiological mapping system. Results showed that compared with the control group, no significant abnormalities were observed in ECG and cardiac conduction thermograms after perfusion for 15 min at various concentrations of *Bangga chinensis*, and no significant differences were found in HR, CV, QT interval, and QRS interval. Figure 37 In conclusion, the 1000 ng / mL Bangga total alkaloid extract solution did not produce significant toxic effects on isolated rat hearts.

[0178] 2.3) Dosage-based study of the total alkaloid extract of Bangga rhizome in its effect on aconitine-induced arrhythmias in isolated rat hearts.

[0179] Based on the experimental results in 2.1) and 2.2) above, it can be seen that aconitine at a concentration of 10 nM perfused with the heart can cause significant arrhythmias in the isolated heart, while Bangga alkaloids at a dose of 1000 ng / mL have no significant toxicity to the heart.

[0180] This experiment further utilized the Langendorff isolated heart perfusion system. After the heart stabilized, 10 nM aconitine and 10 ng / mL of Bangga total alkaloid extract were combined and perfused into isolated rat hearts at a flow rate of 10 mL / min. The effects on rat electrocardiograms were observed. Once arrhythmias appeared, the dose of Bangga total alkaloid extract was gradually increased while 10 nM aconitine was continuously perfused. The antagonistic effect of Bangga total alkaloid extract was observed to explore the effective concentration of Bangga total alkaloid extract against aconitine-induced arrhythmias in isolated rat hearts. The MappingLab matrix multichannel electrophysiological mapping system was used to record single-channel ECG parameters closely related to the occurrence of arrhythmias, such as conduction direction, conduction velocity, and QT interval. The results are as follows: Figure 38 As shown in the results, when the heart was perfused with a combination of 10 nM aconitine solution and 10 ng / mL Bangga total alkaloid extract for 5 min, no significant abnormalities were observed in ECG or cardiac conduction thermogram. After 5 min of continued perfusion, atrioventricular dissociation occurred in the left ventricle. Further increases in the concentration of Bangga total alkaloid extract to 20 ng / mL, 30 ng / mL, and 50 ng / mL for 5 min of continued perfusion significantly improved left ventricular arrhythmias with increasing dose. Increasing the dose to 100 ng / mL did not show any further improvement. In conclusion, the effective concentration of Bangga total alkaloid extract against aconitine-induced arrhythmias in isolated hearts is likely around 20 ng / mL.

[0181] 2.4) Study on the effect of Bangga alkaloids on aconitine-induced arrhythmias in isolated hearts

[0182] Using the Langendorff isolated heart perfusion system, after the hearts stabilized, 10 nM aconitine was combined with 10, 20, 30, and 50 ng / mL of Bangga total alkaloid extract, respectively, and the isolated rat hearts were perfused at a flow rate of 10 mL / min. The MappingLab matrix multichannel electrophysiological mapping system was used to record parameters closely related to the occurrence of arrhythmias, such as single-channel ECG conduction direction, conduction velocity, and QT interval. Since the time for aconitine-induced arrhythmias in isolated rat hearts was within 10 min of perfusion, 20 min of perfusion was used as the observation endpoint. The results are as follows: Figure 39 As shown. The results showed that when 10 nM aconitine was used alone to perfuse the heart for 10 minutes, ventricular fibrillation occurred, as before. Figure 39(A) In combination with 10 ng / mL of Bangga total alkaloid extract, the heart was perfused for 10 min, and no obvious abnormalities were found in ECG and cardiac conduction thermogram. Figure 39 (B in the text), after 20 minutes of perfusion, ectopic pacing appeared in the left ventricle ( Figure 39 (C); 10 nM aconitine solution combined with 20, 30, and 50 ng / mL Bangga total alkaloid extract solution was used to perfuse the heart for 20 min. No significant abnormalities were observed in ECG or cardiac conduction thermogram. Figure 39 (DF in the text). In summary, Bangga alkaloids can significantly prolong the time for aconitine-induced arrhythmia in isolated hearts, and concentrations of 20 ng / mL and above can completely counteract aconitine-induced arrhythmia in isolated rat hearts.

[0183] Example 3: Acute toxicity study of total alkaloid extract from Bangga.

[0184] 1. Preparation of medicines

[0185] Solvent: Sodium dodecyl sulfate is added to a 1% CMC-Na solution and mixed thoroughly. The amount of sodium dodecyl sulfate used is 1 g / 100 mL.

[0186] Bangga total alkaloid extract: In this example, 8 kg of whole Bangga medicinal herb was used to extract 16 g of Bangga total alkaloid extract powder using the extraction method of Example 1, which is equivalent to 500 g of raw herb / g of extract. The total alkaloid content of Bangga in this Bangga total alkaloid extract is 60.3%.

[0187] Preparation of Bangga total alkaloid extract:

[0188] Preparation: Weigh 1.6g of Bangga total alkaloid extract and dissolve it in solvent to a final volume of 40mL, resulting in a concentration of 40mg extract / mL. Administer 40mL / kg orally to ICR mice. The dosage is 1.6g extract / kg, which is equivalent to 800g crude drug / kg, representing the highest concentration in this experiment. The concentration is indicated as ①.

[0189] Take 9 mL of concentration ① + 4.1 mL of solvent, the total volume is 13.1 mL, the concentration is 27.5 mg extract / mL, which is equivalent to 500 g crude drug / kg. This is labeled as concentration ②.

[0190] Take 10 mL of concentration ① + 10 mL of solvent, the total volume is 20.0 mL, the concentration is 20 mg extract / mL, which is 400 g crude drug / kg.

[0191] The concentration is indicated as ③.

[0192] Take 4.5 mL of concentration ① + 7.5 mL of solvent, for a total volume of 12.0 mL. The concentration is 15 mg extract / mL, which is equivalent to 300 g crude drug / kg. This is labeled as concentration ④.

[0193] Take 8 mL of concentration ③ + 8 mL of solvent, the total volume is 16.0 mL, the concentration is 10 mg extract / mL, which is 200 g crude drug / kg.

[0194] The concentration is indicated as ⑤;

[0195] Take 5 mL of concentration ⑤ + 5 mL of solvent, the total volume is 10.0 mL, the concentration is 5 mg extract / mL, which is 100 g crude drug / kg.

[0196] 2. Experimental Methods

[0197] Seventy ICR mice, weighing 18-21g, were randomly divided into a control group and six dosage groups (100, 200, 300, 400, 550, and 800g crude drug / kg) of Bangga total alkaloid extract, with 10 mice in each group (half male and half female). After a 15-hour fast, the mice were administered the extract via gavage at a single dose of 40mL / kg body weight. The concentrations of Bangga total alkaloid extract were 5.0, 10.0, 15.0, 20.0, 27.5, and 40.0g extract / mL. The control group received the same volume of solvent. Immediately after administration, the animals were observed for toxic reactions, their severity, and the time of onset and resolution of toxicity. Adverse reactions were recorded. After the 7-day observation period, the mice were fasted again. On the 8th day, surviving animals were necropsized, and major organs such as the heart, liver, spleen, lungs, kidneys, adrenal glands, brain, stomach, intestines, testes, prostate, ovaries, and uterus were examined for any visible changes in volume, color, and texture.

[0198] 3. Test Results

[0199] The main toxicity of the total alkaloid extract from Bangga was acute death. Animal deaths occurred after administration in the 400 g crude drug / kg and above dosage groups, while no animal deaths occurred in the 300 g crude drug / kg and below dosage groups. The time of animal death occurred within 4 min-22 min after administration. The mortality rates of animals in the six dosage groups of 100, 200, 300, 400, 550, and 800 g crude drug / kg were 0%, 0%, 0%, 10%, 20%, and 100%, respectively. The results are shown in Table 6.

[0200] Table 6. Mortality of animals in single-dose toxicity tests of Bangga total alkaloid extract (n=10)

[0201]

[0202] Note: The day of administration is denoted as D0, the first day after administration is denoted as D1, the second to fourth days after administration are denoted as D2-4, and so on.

[0203] Besides acute death, other visible adverse reactions mainly manifested as lethargy, unsteady gait, rapid breathing, and convulsions, occurring in the 400 and 550 g crude drug / kg dosage groups, with an incidence of 20% in both groups. Adverse symptoms appeared approximately 20 minutes after administration and resolved by the following day. Thereafter, until the end of the observation period, no abnormalities were observed in the general condition, activity, gait, respiration, feeding, drinking, urination, defecation, or fur of the surviving animals. The results are shown in Table 7.

[0204] Table 7 Summary of Adverse Reactions in Single-Dose Toxicity Tests of Bangga Total Alkaloid Extract

[0205]

[0206] After the 8th day of observation, all surviving animals were dissected and visually examined. No exudate, hemorrhage, or adhesions were found in the thoracic and abdominal cavities. No visible lesions such as congestion, ecchymosis, hemorrhage, exudate, adhesions, erosion, or ulcers were found in the heart, liver, spleen, lungs, kidneys, adrenal glands, gastrointestinal tract, bladder, testes, or epididymis.

[0207] Conclusion: When different doses of Bangga total alkaloid extract were administered to mice via a single oral gavage at a dose of 40 mL / kg, the main observable toxicity was acute death. The LD50 was calculated using the Bliss method. 50 The effective dose was 584.666 g crude drug / kg, with a 95% confidence limit of 504.694-711.101 g crude drug / kg; the maximum no-lethal dose and the no-toxicity dose were both 300 g crude drug / kg. In the experimental study of the anti-aconitine-induced arrhythmia in mice using the total alkaloid extract of Bangga, the effective doses were 15, 30, and 60 mg extract / kg. The no-toxicity dose was 10 times the maximum effective dose, indicating a large safety window and low toxicity of the test substance.

[0208] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the total alkaloid extract of Bangga in the preparation of drugs for the prevention and / or treatment of arrhythmia, characterized in that, The preparation method of the Bangga total alkaloid extract includes the following steps: The coarse powder of the whole herb of Bangga medicinal material was mixed with an ethanol solution and refluxed to extract the extract. The solvent in the extract was recovered until there was no alcohol odor. Then, its pH was adjusted to acidic and mixed with dichloromethane for the first extraction to obtain a dichloromethane layer and an extract water layer. After adjusting the pH of the extraction water layer to alkaline, dichloromethane solvent was added for a second extraction to obtain the Bangga total alkaloid extract. The reflux extraction was performed three times at a temperature of 60°C. During each reflux extraction, the mass-to-volume ratio of the coarse powder of the whole Bangga medicinal herb to the ethanol solution was 1 kg: 7-8 L. The ethanol solution contained 70% ethanol by volume. The first extraction is performed three times; each time the first extraction is performed, the volume ratio of the extract to the dichloromethane is 1:

1. The second extraction is performed three times; each time the second extraction is performed, the volume ratio of the pH-adjusted extraction water layer to the dichloromethane solvent is 1:

1.

2. The application of Bangga total alkaloid extract in the preparation of drugs for the prevention and / or treatment of premature ventricular contractions, wherein the preparation method of Bangga total alkaloid extract includes the following steps: The coarse powder of the whole herb of Bangga medicinal material was mixed with an ethanol solution and refluxed to extract the extract. The solvent in the extract was recovered until there was no alcohol odor. Then, its pH was adjusted to acidic and mixed with dichloromethane for the first extraction to obtain a dichloromethane layer and an extract water layer. After adjusting the pH of the extraction water layer to alkaline, dichloromethane solvent was added for a second extraction to obtain the Bangga total alkaloid extract. The reflux extraction was performed three times at a temperature of 60°C. During each reflux extraction, the mass-to-volume ratio of the coarse powder of the whole Bangga medicinal herb to the ethanol solution was 1 kg: 7-8 L. The ethanol solution contained 70% ethanol by volume. The first extraction is performed three times; each time the first extraction is performed, the volume ratio of the extract to the dichloromethane is 1:

1. The second extraction is performed three times; each time the second extraction is performed, the volume ratio of the pH-adjusted extraction water layer to the dichloromethane solvent is 1:

1.

3. The application of the total alkaloid extract of Bangga in the preparation of drugs for the prevention and / or treatment of ventricular tachycardia, characterized in that, The preparation method of the Bangga total alkaloid extract includes the following steps: The coarse powder of the whole herb of Bangga medicinal material was mixed with an ethanol solution and refluxed to extract the extract. The solvent in the extract was recovered until there was no alcohol odor. Then, its pH was adjusted to acidic and mixed with dichloromethane for the first extraction to obtain a dichloromethane layer and an extract water layer. After adjusting the pH of the extraction water layer to alkaline, dichloromethane solvent was added for a second extraction to obtain the Bangga total alkaloid extract. The reflux extraction was performed three times at a temperature of 60°C. During each reflux extraction, the mass-to-volume ratio of the coarse powder of the whole Bangga medicinal herb to the ethanol solution was 1 kg: 7-8 L. The ethanol solution contained 70% ethanol by volume. The first extraction is performed three times; each time the first extraction is performed, the volume ratio of the extract to the dichloromethane is 1:

1. The second extraction is performed three times; each time the second extraction is performed, the volume ratio of the pH-adjusted extraction water layer to the dichloromethane solvent is 1:1.

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