Toad cake
1、       This product is the dry secretion of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider. Catch toads more than in summer and autumn, wash them, squeeze out the white slurry of the ear gland and skin gland, process and dry them.
      [properties] this product is oblate, agglomerated or flaky. Tan or reddish brown. The lumpy shape is hard, not easy to break, and the section is brown, horny, slightly itchy; Flakes are brittle and fragile, with reddish brown and translucent sections. The smell is slightly fishy. The taste is sweet at first and then has a lasting spicy feeling. The powder smells like sneeze.
      [identification] (1) when the cross section of this product is stained with water, it is milky white and uplifted.
      (2) Take 0.1g of this product powder, add 5ml of methanol, soak for 1 hour, filter, add a small amount of p-Dimethylaminobenzaldehyde solid to the filtrate, and add a few drops of sulfuric acid to make it blue and purple.
      (3) Take 0.1g of this product powder, add 5ml of trichloromethane, soak for 1 hour, filter, evaporate the filtrate to dryness, add a small amount of acetic anhydride to dissolve the residue, add sulfuric acid dropwise, it initially appears blue purple, and gradually changes to blue-green.
      (4) Take 0.2g of this product powder, add 10ml of ethanol, heat and reflux for 30 minutes, filter, put the filtrate into a 10ml volumetric flask, add ethanol to the scale, shake well, and use it as the test solution. Another 0.2g of toad venom control medicinal material was taken, and the control medicinal material solution was prepared by the same method. Then take the resibufogenin reference substance and cinobufagin reference substance, add ethanol to make a solution containing 1 μ g per 1ml as the reference solution. . In the chromatogram of the test sample, spots of the same color appear at the position corresponding to the chromatogram of the control medicinal material; .
      [inspection] the moisture content shall not exceed 13.0% (the second method of general rule 0832).
      The total ash content shall not exceed 5.0% (general rule 2302).
      Acid insoluble ash content shall not exceed 2.0% (general rule 2302).
      [content determination] determine according to HPLC (general rule 0512).
      Chromatographic conditions and system suitability test octadecylsilane bonded silica gel was used as filler; Acetonitrile-0.5% potassium dihydrogen phosphate solution (50:50) (pH adjusted to 3.2 with phosphoric acid) was used as the mobile phase; The detection wavelength was 296nm, and the column temperature was 40 ℃. The number of theoretical plates shall not be less than 4000 according to the cinobufagin base peak and lipobufogenin ligand peak respectively.
      Preparation of reference solution take appropriate amount of cinobufagin based reference substance and lipobufogenin based reference substance, weigh accurately, add methanol to make 50 μ g of cinobufagin based and lipobufogenin based solutions per 1ml.
      Preparation of test solution take about 25mg of the fine powder of the product, accurately weigh it, place it in a conical flask with a stopper, precisely add 20ml of methanol, weigh it, heat and reflux for 1 hour, cool it, then weigh it, make up the lost weight with methanol, shake it well, filter it, and take the continued filtrate.
      The determination method is to precisely suck 2 μ 1 each of the above two reference solutions and the test solution, inject them into the liquid chromatograph, and determine.
      The total amount of cinobufagin (c26h34o6) and lipobufogenin (c24h32o4) in this product shall not be less than 6.0% according to the dry product.
      Decoction pieces
      [processing] toad cake powder take toad cake, mash it, add liquor to soak it, often stir it to a thick paste, dry it, and smash it.
      20kg of liquor is used for every 10kg of toad cake.
      [nature, taste and meridian tropism] pungent, warm; . Homing Sutra.
      [functions and indications] detoxification, pain relief, resuscitation and awakening. It is used for carbuncle, gangrene, sore throat, heat stroke, dizziness, rash, abdominal pain, vomiting and diarrhea.
      [usage and dosage] 0.015 ～ 0.03g, duoru pill powder. .
      [note] pregnant women should use it with caution.
      [storage] keep dry and damp proof.


2、
The chemical composition of toad venom is complex, mainly including 1 toad sterodiene. Toad sterodiene compounds can be divided into free type and bound type. The free type is called bufalin. So far, more than 20 kinds have been found, mainly including:Bufalin（bufalin），Yuanhua toad venom（telocinobufagin0，And bufalin（gamabufotalin），Bufalin（bufotalin）， Helleprigenin, bufotalidin,Sand toad venom（arenobufagin），Pseudoxenopus venom（φ-bufarenogin），Tebufogenin（resibufogenin），Cinobufagin（cinobufagin），Cinobufotalin（cinobufotalin）， Cinobufaginol,South American toad venom（marinobufagin），Jasmine toad essence（resibufagin）。 . Partial hydrolysates such as bufalin-3-acid octanoate, sabufagin-3-acid octanoate and lipobufalin-3-acid succinate can also be isolated from bufalin.
2. cardiac sterone toad venoms: there are salmonen-3-octanedioate arginate, salmonen-3-octanedioate arginate, salmonen-3-sulfate, salmonen-3-acid octadiester, etc.
3. indole bases, including serotonin, bufotene, bufotene, bufotene, bufothionine, and dehydrobufoteine.
,Ergosterol（ergosterol），Campesterol（campesterol），β - sitosterol(β -sitosterol) et al.
5. others, including polysaccharides, organic acids, amino acids, peptides, adrenaline, etc.

3、 Pharmacological effects of toad venom
1. cardiotonic effect: both bufogenin and bufogenin compounds have cardiotonic effect, but the effect of bufogenin compounds is more obvious, and its chemical structure has a certain relationship with cardiotonic effect. The lowest effective concentration of bufalin and ribavirin in the cardiac papillary muscle of isolated cat hearts was 10 (-8) g / ml; The lowest effective concentration of resibufogenin and cinobufagin for cardiotonic effect is 10 (-7) g/ml. The effect of cinobufagin is similar to that of cinobufagin. Perfusion of the frog heart at a suitable concentration can stop it in systole. Intravenous injection to anesthetized cats and dogs causes cardiac pulsation to slow down, the amplitude of contraction to become larger, the heart rhythm to be irregular, and then tachycardia and death; The electrocardiogram obtained from anesthetized dogs showed prolonged P-R interval, slowed ventricular rate, heterorhythmic rhythm, extrasystole, bundle branch block, flattened or inverted T wave, followed by ventricular tachycardia and death of ventricular fibers; It is believed that the effect of bufogenin on the heart is through the vagus nerve center or terminal, and can directly act on the myocardium. Compared with digitalis, it may be due to the absence of glycosyl. The cardiotonic effect of bufogenin and bufogenin is weak and lack of persistence, so it has no accumulation effect. It has also been reported that arginine can prolong its cardiotonic effect. The cardiotonic effect of Liushen Pill suggests that it can strengthen myocardial function, and with the change of dose, it presents the regulatory effect of reverse direction, which is beneficial to improve local tissue and repair. Experiments have proved that the cardiotonic effect of bufogenin and bufogenin is mainly manifested in increasing myocardial contractility, increasing cardiac output, reducing heart rate, eliminating edema and respiratory difficulties. In Japan, it is mainly used in clinic as a respiratory stimulant, while in China, it is used in clinic as a stimulant and pressor drug. Its effect of enhancing myocardial contractility is not reflexive, but the result of direct action on cardiomyocytes. Most believe that the reason why bufogenin can strengthen myocardial contractility is that bufogenin inhibits na+-k+-atpase on cardiomyocyte membrane, so that the concentration of na+ in cardiomyocytes is relatively increased, and calcium ions enter cardiomyocytes through na+-ca2+ exchange. As a result, myocardial contractility is increased. It has also been proved by experiments that toad venom can increase the level of cyclic adenosine monophosphate in human monocytes. Due to the increase of cyclic adenosine monophosphate, it can improve the activity of myocardial phosphorylase kinase. Under the action of phosphorylase kinase, it can convert inactive phosphorylase B into active phosphorylase a, so it can promote glycogenolysis and promote the production of more ATP. ATP is the driving force of myocardial contraction, making the myocardium fully play its role.
2. inhibitory effect on na+, k+ - ATPase: the secretion of toad skin has a strong inhibitory effect on na+, k+ - ATPase, which is due to the fact that na+ k+ - ATPase is located on the cell membrane. Na+ binds to the enzyme on the inner side of the membrane, promoting the reaction between the enzyme and ATP and phosphorylating the enzyme on the inner side of the membrane. At this time, the enzyme produces a conformational change, and the site of binding with sodium turns to the outer side of the membrane; The phosphorylated enzyme has lower affinity for na+ and higher affinity for k+, so it releases na+ on the outer side of the membrane and binds to k+: after k+ binds to the phosphorylated enzyme, it promotes the dephosphorylation of the enzyme, so the conformation of the enzyme changes again, and the binding site with k+ turns to the inner side of the membrane, so that k+ is released on the inner side of the membrane. In this way, with the participation of na+, k+, and mg2+, na+ is discharged and k+ is brought into the cell. The reaction can be expressed as follows: atp+ enzyme na+, mg2+ enzyme-p + ADP; Enzyme-p + H2O k+, mg2+ enzyme +pi the effect of toad skin secretion on na+, k+ - ATPase lies in the phosphorylation intermediate (enzyme-p) of the enzyme. The toad venom ligand or toad toxin compounds in the secretion are competitively bound to enzyme-p with k+. After enzyme-p is bound to toad venom ligand or toad toxin, it is no longer bound to k+, so the enzyme-p is stable and not easy to hydrolyze, so that na+, k+ - ATPase cannot play its normal role, and its activity is inhibited. The inhibitory effects of bufalin and bufalin on na+, k+ - ATPase in intestinal mice were tested. In addition, in addition to the cardiotonic effect, the mechanism of surface anesthesia of toad venom may also be related to their inhibition of na+, k+ - ATPase.
. Lipobufogenin, bufalin and cinobufagin all have significant central excitatory effects such as respiratory excitation and pressor, among which lipobufalin has been used clinically as a respiratory stimulant, with the trade name respigon. The median convulsive dose (CD50) and median lethal dose (LD50) of lipobufogenin in mice were 10.52mg/kg and 20.80mg/kg, respectively (ld/cd=1.98). The ld/cd value of lipobufogenin was greater than that of other respiratory stimulants, such as Nikethamide, pentylenetetrazol and campanulaceine. Lipobufogenin has a direct excitatory effect on the patient's respiratory and vasomotor centers, as well as cardiotonic and hypertensive effects, and the effect is rapid and lasting. It is clinically used for respiratory failure, shock, dyspnea and gas inversion caused by poisoning, drowning and coma.
4. pressor effect: previously, it was believed that the pressor effect of toad venom mainly came from the cardiotonic effect of toad venom, that is, the direct effect on the heart. Now, it is believed that the pressor effect of toad venom mainly came from the contraction of peripheral blood vessels, and partly from its cardiotonic effect. From the point of view that intravenous injection of bufalin can elevate blood pressure and has no contractile effect on blood vessels when applied locally, it shows that its effect is achieved through the release of catecholamines. . It is suggested that the pressor effect may be related to stroke volume in addition to vascular factors.
5. local anesthetic effect: toad venom and its components have local anesthetic effect. 80% alcohol extract of toad venom has surface anesthetic effect. In rabbit cornea and human tongue test, the effect is slower and longer than that of Decaine. Among them, bufalin has the strongest local anesthetic effect, which is 30-60 times larger than that of cocaine, and has no stimulatory effect. Cinobufagin and cinobutalin are about 1/6 of bufalin; Ribavirin is about 1/20. Topical application of 0.1-0.5% cinobufagin solution can cause tongue numbness; The action point of local anesthesia may be sensory nerve terminal receptors, which have a weak effect on nerve fibers. The presence of unsaturated lactone ring and the hydroxyl group at C3 position seem to be necessary for local anesthesia. . The mechanism of local anesthesia is related to the slow depolarization of muscle cells and the release of acetylcholine.
6. anti tumor and anti radiation effects: bufalin has inhibitory effects on mouse sarcoma 180, rabbit BP tumor, cervical cancer14, ascitic liver cancer, etc. In the body, it can inhibit the respiration of human zygomatic maxillary and mandibular undifferentiated cancer, mesothelial cancer, gastric cancer, splenic sarcoma, liver tumor and other tumor cells. It can prolong the survival time of mice suffering from seminoma, ascites cancer and liver cancer, and inhibit leukemia cells in test tubes. . Toad venom can prevent and treat the leukopenia caused by chemotherapy and radiotherapy to varying degrees. The application of toad venom to those who have declined can rise, and will not decline. Toad venom preparation has adrenaline like effect, can enhance the body's tolerance to radiotherapy and chemotherapy, and has protective effect on X-ray local irradiation, which may be one of the important mechanisms of this product's anti-tumor. The antitumor effect of bufogenin-3 acetic acid on rat W256 was 75%, but the therapeutic index was low; . It is also reported that cardiac glycosides and aglycones, which are similar in structure to bufalin, isolated from plants, have inhibitory effects on hala-s3 tumor cells and ubenin on Ehrlich ascites cancer. This anti-tumor effect is believed to be caused by the inhibition of na+ -k+ -atpase, which reduces the intracellular K concentration, thus failing to maintain the K concentration necessary for nucleic acid synthesis. . This kind of increasing B cell effect may be an important mechanism of bufonin anti-tumor. The total water-soluble components of toad venom still have inhibitory effect on malignant glioma cells at the concentration of 1 μ g/ml in vitro. More than 10 μ g/ml concentration had obvious inhibitory effect on Chinese golden hamster malignant transformation cell line (BHLB4). 1 μ g/ml had inhibitory effect on BHLB4 cells when initially added, but BHLB4 cells soon adapted to growth. The median lethal dose in the acute toxicity test was 60.71mg, without irritation and hemolysis. No lesions were found in the subacute toxicity test. . This product is superior to whole toad venom and its fat soluble components, and is safe and non-toxic. It has obvious curative effect on anti-inflammatory, anti tuberculosis and anti-cancer in clinic, and can replace whole toad venom.
7. anti inflammatory effect: bufalin sterols can inhibit vascular permeability. For rabbits infected with Staphylococcus aureus and Streptococcus haemolyticus a, subcutaneous injection of Bufalin Injection 9mg/kg can prevent the spread of lesions, make the surrounding swelling subside, and the bacteriostatic effect is obvious and rapid. This product also has an inhibitory effect on suppurative diseases that are insensitive to some antibiotics or have developed resistance to antibiotics. The effect of toad venom on inhibiting granulation formation was observed by formaldehyde liquid filter paper method. The half effective inhibitory dose (ID50) of its subcutaneous injection was 159mg/kg, and the ID50 of hydrocortisone acetate was 35.7mg/kg, which had an inhibitory effect on tissue vascular hyperpermeability stimulated by acetic acid. Cardiotonic glycosomes can constrict blood vessels, so it can receive good anti-inflammatory effect for burns and other wounds when combined with anti infectives. . It is related to the improvement of the function of the adrenal cortical system, which needs further study. It is also reported that toad venom preparation can activate peritoneal macrophages in mice and improve their phagocytic capacity. It is known that activated macrophages and antigen processing can directly kill bacteria and inhibit bacterial growth.
8. effect on smooth muscle: toad venom can contract coronary artery blood vessels, reduce coronary blood flow, excite intestinal smooth muscle, increase its contraction amplitude and speed up its frequency. Atropine and morphine can resist this effect; . Bufalin can excite striated muscle, the neuromuscular blocking effect of bufalin psionic part against magnesium ion, and the intensity of contraction of rectus abdominis caused by bufalin, which changes the membrane potential, so it is a direct effect.
9. effects on myocardial ischemia: in vitro experiments show that toad venom can prolong the coagulation time of fibrinogen solution, and its anticoagulant effect is similar to that of urokinase, which can activate fibrinolytic enzyme, thereby increasing coronary perfusion. Toad venom can increase myocardial nutrient blood flow, improve microcirculation, and increase myocardial oxygen supply for ischemic heart disorders such as myocardial infarction caused by coronary stenosis caused by thrombosis. Both bufalin preparation and pilocarpin K can enhance the myocardial contractility and action time of anesthetized dogs. At the early stage of administration, the rise rate of various parameters in the bufalin group was significantly higher than that in the toxic K group. The results suggest that bufalin has a dual positive inotropic effect on the myocardium. .
10. anti pathogenic microorganisms: experimental reports sterols can inhibit vascular permeability. For rabbits infected with Staphylococcus aureus and Hemolytic Streptococcus A, subcutaneous injection of toad venom injection can prevent the spread of lesions, so that the surrounding swelling subsides. The bacteriostatic effect is obvious and rapid. This product also has inhibitory effect on suppurative diseases that are insensitive to some antibiotics or have developed resistance to antibiotics. The effect of toad venom on the formation of granulation and the hyperpermeability of blood vessels stimulated by acetic acid was observed by formaldehyde liquid filter paper method.
11. anti endotoxic shock: a large dose of crude endotoxin from Escherichia coli is injected intravenously into dogs to cause endotoxic shock. Both toad venom and anisodamine have certain anti shock effects, and the combination of the two (toad venom) can significantly improve the curative effect. The curative effect of chanshanhe liquid may be due to the same effect of the two, which can better inhibit disseminated intravascular coagulation, reduce the consumption of fibronectin, and improve the hemorheology and erythrocyte membrane function. ; However, after 9 hours, only the Chanshan Heye group was significantly higher than the saline group (p< 0.05). According to endotoxic shock experiments in rabbits and dogs, the curative effect of chanshanhe liquid was better than that of simple toad venom or anisodamine compared with the control group, no matter from the aspects of animal survival rate, survival time, arterial blood pressure maintenance degree after infusion and femoral artery blood flow velocity (p< 0.05). . It was found that the survival time of endotoxic shock dogs was significantly related to the basic level of cellulose. The plasma cellulose and total complement content were reduced 10 hours after endotoxin injection, and the reduction degree of plasma cellulose was significantly different from that of the blank control.
. The cell rabbit skin test, macrophage viability assay, rosette test and lymphocyte transformation rate were improved to varying degrees. The study on the monomer fraction of water-soluble total components of toad venom and its immunopharmacology and molecular biology can enhance the phagocytic function of reticuloendothelial system and improve the non-specific immunity of the body.
Toad venom has the effect of similar immunity or improving immune function, which may lie in the regulation of the whole body function, indicating that the anti tuberculosis mechanism of this product seems to be related to the regulation of cellular immunity. However, there was no obvious regularity in the changes of serum immunoglobulin G and m.
13. antitussive effect: according to animal experiments, pre subcutaneous injection of Bufo tryptamine can significantly protect guinea pigs from tracheal spasm caused by serotonin spray. For the isolated uterus or ileum of guinea pigs sensitized by egg white, Bufo tryptamine has anti allergic effect. Chansu decoction has antitussive effect on experimental cough caused by sulfur dioxide in mice, but its expectorant effect is poor.
14. effect on platelet aggregation function: for the experimental rabbit IP toad venom, there was no significant change in microanalytical reagents and standards before and after injection in the saline group (p> 0.05), but there was a significant difference after injection compared with that before injection (p< 0.05), and the maximum inhibition rate of aggregation and the inhibition rate of aggregation speed in the toad venom group were higher than those in the saline group. This indicates that toad venom can inhibit the degree and speed of platelet aggregation. It has been reported abroad that toad venom can reduce the occurrence of disseminated intravascular coagulation, which may also be related to the inhibition of platelet aggregation and release.