3α-hydroxy tigliane compounds and uses thereof

Abstract

The application discloses a 3alpha-OH bufotalin compound, which has a general formula of C 24 H 32 The 3alpha-OH bufotalin compound can be used for preparing medicines for resisting tumors, treating cough, treating myasthenia gravis and reducing blood sugar.

Description

Technical Field

[0001] This invention relates to the field of lactone compounds. More specifically, this invention relates to a 3α-OH bufotalin compound and its applications. Background Technology

[0002] Bufotenolactones are a class of C-type compounds with cardiotonic activity. 24 Steroid compounds, such as bufotalin, are widely distributed in plants and animals in nature. These compounds are characterized by a six-membered unsaturated lactone ring (α-pyrone) at the C-17 position, belonging to the beta-type cardiotonic steroids. These components have digitalis-like cardiotonic effects and are an important class of drugs for treating heart failure, tumors, and analgesia, primarily through inhibiting sodium... + ,K + -ATPase (NKA) can be used to treat diseases such as heart failure, respiratory depression, and traumatic shock.

[0003] Toad bile, a traditional Chinese medicine, has garnered significant attention for its antitussive, expectorant, detoxifying, and nodule-dispersing effects. It is commonly used to treat bronchitis, childhood aphonia, early-stage lymph node tuberculosis, and nasal boils. Although the medicinal properties of toad bile are documented in folk medicine, research on its chemical components, particularly bufotoxin lactones, is scarce due to the complexity of its composition. This invention, for the first time, isolates a novel 3α-OH bufotoxin lactone compound from toad bile, identified as 3-epi-bufoliene by NMR and MS methods. Pharmacological activity studies were conducted, revealing that 3-epi-bufoliene exhibits good activity in antitumor activity, cough treatment, myasthenia gravis treatment, and hypoglycemic activity, suggesting its potential for further development into related drugs. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0005] To achieve these objectives and other advantages according to the present invention, a 3α-OH bufotoxin lactone compound is provided, the general formula of which is (I):

[0006]

[0007] Preferably, the 3α-OH bufotenoid compound has the molecular formula C3. 24 H 32 O4.

[0008] This invention also provides the application of 3α-OH bufotoxin compound in the preparation of antitumor drugs.

[0009] Preferably, in the application, the tumor includes at least one of lung cancer, liver cancer, breast cancer, and colon cancer.

[0010] The present invention also provides the application of 3α-OH bufotoxin compound in the preparation of a drug for treating cough.

[0011] The present invention also provides the application of 3α-OH bufotoxin compound in the preparation of a drug for treating myasthenia gravis.

[0012] This invention also provides the application of 3α-OH bufotoxin compound in the preparation of hypoglycemic drugs.

[0013] Preferably, in the application, the drug comprises a usable salt of a 3α-OH bufotoxin compound, and pharmaceutically acceptable excipients.

[0014] Preferably, in the application, the pharmaceutical preparation is an injectable or oral preparation.

[0015] The present invention has at least the following beneficial effects:

[0016] This invention is the first to isolate and obtain 3α-OH bufotoxin lactone compound (3-epi-bufoliene) from toad bile, which has good anti-tumor, antitussive, insulin secretion-promoting, and myasthenia gravis-treating pharmacological effects.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 The 3α-OH bufotenoid compound of this invention 1 H NMR spectrum;

[0019] Figure 2 The 3α-OH bufotenoid compound of this invention 13 C10 NMR spectrum. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0023] This invention provides a 3α-OH bufotoxin lactone compound, the general formula of which is (I):

[0024]

[0025] The 3α-OH bufotenone compound has the molecular formula C3. 24 H 32 O4.

[0026] This invention also provides the application of 3α-OH bufotoxin compound in the preparation of antitumor drugs.

[0027] The application, wherein the tumor includes at least one of lung cancer, liver cancer, breast cancer, and colon cancer.

[0028] The present invention also provides the application of 3α-OH bufotoxin compound in the preparation of a drug for treating cough.

[0029] The present invention also provides the application of 3α-OH bufotoxin compound in the preparation of a drug for treating myasthenia gravis.

[0030] This invention also provides the application of 3α-OH bufotoxin compound in the preparation of hypoglycemic drugs.

[0031] The application, wherein the drug comprises a usable salt of a 3α-OH bufotoxin compound, and pharmaceutically acceptable excipients.

[0032] The application in which the pharmaceutical preparation is described is an injectable or oral preparation.

[0033] <Example 1>

[0034] Extraction and separation of 3α-OH bufotenoid compound (3-epi-bufoliene)

[0035] Step 1: Take 1 kg of toad gallbladders (wet weight, about 1000 gallbladders), extract with 75% ethanol by ultrasonic extraction 3 times at room temperature, 1 hour each time, filter and combine the extracts, and recover the ethanol under reduced pressure to obtain the concentrated extract.

[0036] Step 2: The concentrated extract was suspended in water and defatted and extracted three times with petroleum ether at a volume ratio of 1:1. The resulting aqueous layer was extracted three times with ethyl acetate at a volume ratio of 1:1. The ethyl acetate extracts were combined and concentrated to obtain 14g of thick paste.

[0037] Step 3: The viscous paste was eluted with a gradient of chloroform and methanol in volume ratios of 100:0, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1, yielding seven fractions, numbered Fr-A, Fr-B, Fr-C, Fr-D, Fr-E, Fr-F, and Fr-G. The Fr-A fraction was repeatedly purified using silica gel, dextran gel chromatography, and preparative chromatography to obtain a white solid, which is the 3-epi-bufoliene compound.

[0038] Structural identification of 3α-OH bufotenoid compound (3-epi-bufoliene)

[0039] The chemical structural formula of the 3α-OH bufotenone compound of this invention is as follows:

[0040] 3-epi-bufoliene compound: white amorphous powder (methanol), [α] 25 D = +71(c 0.10,CH3OH).

[0041] HR-ESI-MS m / z 385.2382 [M+H] + (Calculated value C) 24 H 33 O4 385.2373), the molecular formula of the compound was determined to be C 24 H 32 O4, with an unsaturation degree of nine. The UV spectrum shows maximum absorption at 305 nm, suggesting that this compound has a δ-unsaturated lactone ring structure. IR spectroscopy shows the presence of a hydroxyl group (3309 cm⁻¹). -1 ) and carbonyl (1716cm) -1 Characteristic absorption of ).

[0042] 1 H NMR spectra (specific data are shown in Table 1 and ...) Figure 1 As shown in the diagram, a set of characteristic hydrogen signals on the δ-unsaturated lactone ring can be observed in the low-field region: δ H 6.29 (1H,dd,J=9.2,0.9Hz), 7.85 (1H,dd,J=9.2,2.5Hz), 7.69 (1H,dd,J=2.5,0.9Hz)]; a double-bonded proton signal: δ H 5.5 (1H, dd, J = 2.4 Hz). Two angular methyl hydrogen signals on the steroid nucleus can be observed in the high-field region: δ H 0.98 (3H, s, H-19) and 1.03 (3H, s, H-18); in addition, two oxygen-bound methine hydrogen signals can be observed: δ H3.62(1H,m,H-3), 4.60(1H,dd,J=5.9,2.4Hz,H-16). 13 C NMR (specific data are shown in Table 1 and ...) Figure 2 As shown in the figure, the DEPT spectrum reveals 24 carbon signals: 2 methyl carbons, 7 methylene carbons, 10 methine carbons, and 5 quaternary carbons. Among these, the δ-carbons in the low-field region... C 114.5 (C-23), 119.3 (C-20), 150.7 (C-22), 152.3 (C-21), and 164.7 (C-24) are a group of characteristic carbon signals for δ-unsaturated lactone rings. C 121.3(C-15) and δ C 161.4 (C-14) represents a pair of double-bonded carbon signals, consisting of a methine carbon and a quaternary carbon. Additionally, there are two methine carbon signals bonded to oxygen: δ C 72.3 (C-3) and 77.4 (C-16). In 1 H- 1 Three sets of proton coupling systems were observed on the steroid nucleus in the HCOSY spectrum: and In HMBC, δ H 1.03(H-18) and δ C 161.4 (C-14), 58.1 (C-17) correlation, δ H 1.03(H-16) and δ C The correlation between 161.4 (C-14) and 121.3 (C-15) indicates that the double bond structure is located at positions C-14 and 15 of the D ring, and the hydroxyl group is located at position C-16. The ROESY correlation spectrum shows δ... H 2.27(H-8) and δ H 1.03 (H-18), H-19 (0.98) correlation, δ H 0.98 (H-19) and δ H 1.06 (H-1β), 2.01 (H-6β) correlation, δ H The correlations between 3.55 (H-3) and 1.06 (H-1β), and H-5 (1.44) indicate that these protons are all located at the β position. Furthermore, δ... H 1.61(H-7) and δ HThe correlation between 1.51(H-4α) and 1.51(H-9) indicates that these hydrogen atoms are located at the α position. Based on this, the compound structure can be determined to be (3α,5β,16β)-3,16-dihydroxy-bufa-14,20,22-trienolide, named 3-epi-bufoliene.

[0043] Table 1. 3α-OH bufotenoid compounds 1 H and 13 C NMR data (300MHz, CD3OD, δin ppm, J inHz)

[0044]

[0045] <Example 2>

[0046] Study on the antitumor activity of 3α-OH bufotenolone (3-epi-bufoliene)

[0047] (1) Test method

[0048] Step a: Take human liver cancer cell lines SMMC-7721 and HepG2, human lung cancer cell line A549, breast cancer cell line MDA-MB-231, and colon cancer cell line SW480 in logarithmic growth phase, prepare single-cell suspensions with culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, and seed 3000-5000 cells per well into 96-well plates, with a volume of 100 μL per well. The cells are seeded and cultured 12-24 hours in advance.

[0049] Step b: Add the test compound (3α-OH bufotalin compound) solution: The compound (3α-OH bufotalin compound) was dissolved in DMSO. The compound was screened at concentrations of 200, 100, 50, 25, and 12.5 μM, with a final volume of 200 μL per well. Each treatment was performed in triplicate. After incubation at 37°C for 48 hours, two positive compounds, cisplatin (DDP) and paclitaxel (Taxol), were included in the experiment.

[0050] Step c: After culturing for 48 hours, discard the culture medium in the wells of adherent cells, and add 20 μL of MTS solution and 100 μL of culture medium to each well; set up 3 blank replicates (a mixture of 20 μL MTS solution and 100 μL culture medium), and continue incubation for 2-4 hours to allow the reaction to proceed fully before measuring the absorbance. Using a 492 nm wavelength, read the absorbance values ​​of each well with a multi-microplate reader (MULTISKAN FC), record the results, and plot the cell growth curve with concentration on the x-axis and cell viability on the y-axis. Calculate the IC50 of the compound using the Reed and Muench method. 50 value.

[0051] (2) Antitumor activity of compound (3α-OH bufotoxin compound) IC 50 value

[0052] Based on the principle that living cells produce crystal violet during respiration while dead cells do not, the survival rate of the drug was calculated colorimetrically. A cell growth curve was plotted with concentration on the x-axis and cell survival rate on the y-axis. The IC50 of the compound was calculated using the Reed-Muench method. 50 The values ​​are shown in Table 2.

[0053] Table 2 shows the IC50 values ​​for cancer cells. 50 Value (μM)

[0054]

[0055] (3) Results Analysis

[0056] The compound can effectively inhibit lung cancer, liver cancer, breast cancer, and colon cancer cells. This indicates that compound 3-epi-bufoliene has good therapeutic effects on lung cancer, liver cancer, breast cancer, and colon cancer.

[0057] <Example 3>

[0058] Study on the antitussive activity of 3α-OH bufotenolone 3-epi-bufoliene

[0059] (1) Test method

[0060] The antitussive activity of 3-epi-bufoliene was evaluated using a concentrated ammonia-induced cough test in mice. Fifty KM mice were randomly divided into a control group, a model group, and high, medium, and low dose groups of 3-epi-bufoliene. After one week of acclimatization, the treatment groups were administered 3-epi-bufoliene at doses of 10 mg / kg, 20 mg / kg, and 40 mg / kg, respectively, while the normal and model groups were administered an equal volume of distilled water by gavage. Mice were fasted for 24 hours before the last administration but were allowed free access to water. One hour after the last administration, the ammonia-induced cough test was performed. The mouth of the beaker was sealed with plastic wrap, and 1 ml of concentrated ammonia solution was sprayed into the beaker using a metered-dose nebulizer. Mice in the treatment and model groups were placed in the beaker for 20 seconds and then transferred to another transparent plexiglass bell jar. The latency period (the time from the first cough after stimulation with concentrated ammonia) and the cough frequency within 3 minutes were recorded. Mice in the normal group were placed in a glass bell jar and inhaled saline spray for 20 seconds. The cough latency and cough frequency were recorded, and the results are shown in Table 3.

[0061] (2) Analysis of the efficacy of antitussive drugs

[0062] Antitussive experiments showed that 3-epi-bufoliene prolonged the latency period of cough in mice and significantly reduced the frequency of cough, indicating that the compound has a significant antitussive effect. As shown in the table, the cough latency period was significantly prolonged and the cough frequency was significantly reduced in each drug group. This indicates that oral administration of 3-epi-bufoliene has a significant antitussive effect in a dose-dependent manner.

[0063] Table 3 Antitussive Effect

[0064] Grouping <![CDATA[Dose / mg·kg -1 > Cough incubation period / s Number of coughs normal group / - - Model group / 8.56±2.16 59.20±6.50 low dose of drug 10 <![CDATA[12.36±2.33 * ]]> 49.67±4.35 Medium dose of drug 20 <![CDATA[20.42±5.36 ** ]]> <![CDATA[32.86±5.91 ** ]]> High dose of drug 40 <![CDATA[32.15±4.65 ** ]]> <![CDATA[22.97±3.28 ** ]]>

[0065] <Example 4>

[0066] Application of 3-epi-bufoliene in the treatment of myasthenia gravis

[0067] (1) Establishment of a mouse model of myasthenia gravis

[0068] Mice were immunized with a 1:2 mixture of AChR and Freund's adjuvant for the initial sensitization. For primary sensitization, 15 μg of AChR was prepared into a 200 μL antigen emulsion and intradermally injected at multiple sites including the back, limbs, and base of the tail. Four weeks later, mice were re-immunized with 100 μL of the antigen emulsion containing 15 μg of AChR. The adjuvant control group was immunized with an equal volume of Freund's adjuvant. The model was assessed 7–14 days after the final immunization.

[0069] Judgment criteria: (1) Clinical manifestations: EAMG mice showed varying degrees of muscle weakness, such as dry hair, hump, decreased food intake, reduced activity and lameness; (2) Swimming test: EAMG mice swam for significantly less time than the adjuvant control group.

[0070] (2) The therapeutic effect of 3-epi-bufoliene on experimental myasthenia gravis mice

[0071] Forty mice with myasthenia gravis model were randomly divided into four groups (n=10 per group) and treated as follows:

[0072] 3-epi-bufoliene treatment group: The compound was mixed with physiological saline at three ratios of 1:1, 2:1, and 4:1, dissolved in water for injection, and filtered to obtain an injection solution. The three groups of experimental mice were administered the injection solution intraperitoneally on day 1, day 8, and day 22. The dosage was 200 μg 3-epi-bufoliene per mouse.

[0073] Control group: On day 1 of the experiment, animals were intraperitoneally injected with pH 7.4, 0.01 mol / L PBS buffer. On day 8 of the experiment, animals were intraperitoneally injected again with pH 7.4, 0.01 mol / L PBS buffer. On day 22 of the experiment, animals were intraperitoneally injected again with pH 7.4, 0.01 mol / L PBS buffer. The injection dose was 100 μL per animal each time.

[0074] 1) Weight monitoring: Starting from day 1 of modeling, the weight of mice was monitored and recorded every other day.

[0075] 2) Treatment Efficacy Scoring: Two observers performed a blinded clinical assessment of EAMG severity in mice. 0 points: Normal muscle strength; 1 point: Mildly reduced activity, weak grasping or whimpering, easy fatigue; 2 points: Significantly reduced activity, significant weight loss, arched back, head down, flexed forelegs, and trembling at rest; 3 points: Severe generalized weakness, no whimpering, no grasping movements, near-death state or death. Mice with intermediate symptoms were scored 0.5, 1.5, and 2.5 points respectively. Clinical scoring was performed at week 4 post-treatment.

[0076] 3) Monitoring the gripping force of mice in each group

[0077] Starting from day 1 of modeling, the gripping force of the mice was monitored every other day using a mouse gripping force meter. Before monitoring the gripping force, the mice were first allowed to perform a cage-grabbing experiment, grasping the cage continuously for 30 seconds, and then the force was measured three times consecutively. The average value was taken as the final record value.

[0078] Table 4 shows the results of the effect of 3-epi-bufoliene on mouse body weight. The results indicate that, compared with the control group, mice treated with 3-epi-bufoliene had significantly higher body weight.

[0079] Table 4. Effects of 3-epi-bufoliene on mouse body weight (mean ± SD value)

[0080] Group n Before treatment After treatment control group 10 14.53±1.12 15.94±1.23 Experimental group 1 10 14.36±1.59 16.58±1.25* Experimental group 2 10 14.02±1.89 17.32±1.97* Experimental group 3 10 14.31±1.94 18.98±1.67*

[0081] Note: *P<0.01 compared with the control group.

[0082] The effects of 3-epi-bufoliene on the treatment efficacy score in mice with myasthenia gravis are shown in Table 5. The results indicate that, compared with the control group, the treatment efficacy score of mice immunized with 3-epi-bufoliene was significantly reduced.

[0083] Table 5. Effect of 3-epi-bufoliene immunotherapy on clinical scores in mice (mean ± SD value)

[0084] Group n Before treatment After treatment control group 10 2.57±2.35 3.64±1.42 Experimental group 1 10 2.13±2.02 1.32±1.83* Experimental group 2 10 2.45±1.85 1.03±2.51* Experimental group 3 10 2.61±1.05 0.56±1.25*

[0085] Note: *P<0.01 compared with the control group.

[0086] Table 6 shows the results of the detection of the effect of 3-epi-bufoliene on the grip strength of mice. The results indicate that, compared with the control group, the grip strength of mice treated with 3-epi-bufoliene was significantly enhanced.

[0087] Table 6. Effects of 3-epi-bufoliene immunotherapy on grip strength in mice (mean ± SD value)

[0088] Group n Before treatment After treatment control group 10 256±6 123±32 Experimental group 1 10 262±12 156±18* Experimental group 2 10 258±10 183±20* Experimental group 3 10 266±16 245±11*

[0089] Note: *P<0.01 compared with the control group.

[0090] The above results indicate that immunizing mice with 3-epi-bufoliene can effectively treat or alleviate the symptoms of myasthenia gravis.

[0091] <Example 5>

[0092] Cellular level testing of the effect of 3-epi-bufoliene on insulin secretion

[0093] Using in vitro cultured pancreatic β-cell lines, the inventors evaluated the activity of 3-epi-bufoliene by using insulin secretion levels as a bioindicator.

[0094] (1) Experimental materials:

[0095] 1) Cell culture: MIN6 cells were cultured in 24-well plates in DMEM medium (with 10% serum) (culture conditions: 37°C, 5% CO2).

[0096] 2) Reagents used in the experiment: a. Kit: Insulin level detection kit (Millipore). b. Bovine serum albumin: BSA (5g / Qianchen Biotechnology Co., Ltd.). c. KRB buffer: 124mM NaCl, 5mM KCl, 1.3mM MgSO4, 26mM NaHCO3, 1.2mM KH2PO4, 1.8mM CaCl2.

[0097] (2) Insulin secretion level test experiment

[0098] MIN6 cells were cultured in DMEM medium (with 10% serum) in 24-well plates. When the cells reached 80-90% confluence, they were washed twice with glucose-free KRB buffer containing 0.2% BSA. 500 μL of this buffer was added to each well and incubated for 2 hours. 3-epi-bufoliene was prepared in a high-glucose KRB buffer containing 16.8 mM glucose. The glucose-free KRB buffer was then aspirated, and the cells were incubated with 0.2% BSA KRB buffer containing 3-epi-bufoliene and 16.8 mM glucose for 2 hours. The buffer was then carefully transferred to 1.5 mL EP tubes and incubated at 3000 rpm for 5 min at room temperature. The supernatant was used to detect insulin levels using an enzyme-linked immunosorbent assay (ELISA) kit.

[0099] Key experimental parameters for enzyme-linked immunosorbent assay (ELISA): The reagents used are as described in the experimental materials. Take 10 μL of each of the above samples for ELISA. After binding for 2 hours, wash away the bound samples three times with washing buffer. Then add the enzyme and incubate for 30 minutes. Remove the enzyme and wash six times with washing buffer. Then add the chromogenic substrate and incubate for 20 minutes. Once a blue color appears, add the stop reagent to terminate the reaction. Immediately use a microplate reader to detect changes in light absorption. Calculate the insulin content by measuring OD450.

[0100] (3) Experimental Results

[0101] Using DMSO as a control, this invention investigated the effect of 3-epi-bufoliene on insulin secretion in the MIN6 cell line using an enzyme-linked immunosorbent assay (ELISA). The ratio of the insulin-increasing effect of 3-epi-bufoliene to that of DMSO was 5.5, indicating that 3-epi-bufoliene can increase insulin secretion levels at the cellular level. Subsequently, the concentration-dependent effect of 3-epi-bufoliene on insulin secretion was examined. The results showed that 3-epi-bufoliene significantly increased insulin levels in a concentration-dependent manner, with an EC50 of [missing value]. 50 The value is 55.6 μM.

[0102] <Example 6>

[0103] Effects of 3-epi-bufoliene on fasting blood glucose, insulin tolerance (ITT), triglycerides (TG), and serum insulin in db / db mice

[0104] (1) Animal source: Genetically spontaneously diabetic db / db mice were purchased from Jackson Company, USA.

[0105] (2) Animal culture conditions: SPF-grade animal housing; temperature: 22-24℃; humidity: 45-80%; light: 150-300Lx, 12-hour day-night cycle. Feeding, medication, blood glucose monitoring, and euthanasia were all strictly in accordance with animal experimental guidelines.

[0106] (3) Animal grouping and administration: db / db mice were housed in an SPF-grade animal facility and acclimatized for one week. Based on the mean fasting blood glucose levels measured after 6 hours of fasting, the mice were divided into a blank control group, a positive control group, and a test substance group, with 9 mice in each group. Each group of mice was administered the following daily by gavage: solvent (5% Tween 80, blank control); 10 mg / kg positive compound (rosiglitazone maleate tablets, positive control group); and 40 mg / kg 3-epi-bufoliene (test substance group).

[0107] (4) Observation indicators:

[0108] A. Long-term effect on blood glucose in mice: Fasting blood glucose was monitored once a week during the drug administration period. Fasting blood glucose was the blood glucose value of mice 6 hours after fasting but with unlimited water. The average blood glucose of each group was calculated. The results are detailed in Table 7.

[0109] Table 7. Average blood glucose levels (mM) in each group of mice.

[0110] Time (week) Blank group Positive control group test group 0 11 12 12 1 14 9 10 2 16 8 10 3 18 7 9 4 18 8 8 5 19 6 8

[0111] B. Insulin Tolerance Test (ITT): Mice in each group underwent an insulin tolerance test at week 5 after administration. After fasting for 6 hours, mice were intraperitoneally injected with 1.5 U / kg insulin. Blood glucose levels were measured at 15, 30, 45, 60, 90, and 120 minutes after insulin administration. The results are detailed in Table 8.

[0112] Table 8. Mean blood glucose levels (mM) for each group of mice after insulin administration.

[0113] Time (min) Blank group Positive control group test group 0 14 9 15 15 27 10 23 30 25 5 20 45 22 8 20 60 21 6 19 90 20 4 19 120 18 4 17

[0114] C. Analysis of the effect on serum triglyceride and insulin levels: Mice in each group were sacrificed 5 weeks after drug administration, dissected, blood was collected from the orbital cavity, centrifuged, and the level of serum triglycerides (TG) was detected by a biochemical analyzer and the level of serum insulin was detected by an insulin detection kit. The contents of triglycerides (TG) and insulin were measured respectively. The results are detailed in Table 9.

[0115] Table 9. Mean triglyceride (mM) and serum insulin levels (mM) in each group of mice.

[0116] Blank group Positive control group test group Triglycerides (mM) 0.95 0.63 0.42 Insulin (mM) 1.14 0.43 1.28

[0117] (5) Analysis of experimental results:

[0118] As shown in Table 7, the fasting blood glucose level of the control group mice remained relatively high throughout the experiment. The fasting blood glucose level of the positive control group showed a significant decrease from the first week, a phenomenon that persisted until the end of the experiment. The test group also showed a significant decrease in fasting blood glucose from the first week, a phenomenon that continued until the end of the experiment. Therefore, the fasting blood glucose level of the test group was significantly lower than that of the control group (*P < 0.05).

[0119] As shown in Table 8, the blood glucose levels of the 3-epi-bufoliene group mice before insulin administration and at 15, 30, 45, 60, 90 and 120 min after insulin administration showed significant differences in insulin sensitivity compared with the blank control group (*P<0.05).

[0120] As shown in Table 9, compared with the blank control group, 3-epi-bufoliene administration significantly reduced triglycerides (*P<0.05) and significantly increased plasma insulin levels (*P<0.05, compared with the blank control group).

[0121] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0122] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

The application of 1,3α-OH bufotoxin compounds in the preparation of drugs for treating cough, characterized in that, The general formula of the 3α-OH bufotoxin compound is ( ): （ ）。 2.3 The application of α-OH bufotoxin compounds in the preparation of drugs for treating myasthenia gravis, characterized in that, The general formula of the 3α-OH bufotoxin compound is ( ): （ ）。 The application of 3.3α-OH bufotalin compounds in the preparation of hypoglycemic drugs, characterized in that, The general formula of the 3α-OH bufotoxin compound is ( ): （ ）。 4. The application as described in any one of claims 1 to 3, characterized in that, The drug comprises a pharmaceutically acceptable salt of the 3α-OH bufotoxin compound, and pharmaceutically acceptable excipients.

5. The application as described in claim 4, characterized in that, The drug is available in either injectable or oral formulation.

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