CN102688248B - Use of bufadienolide compound in preparing medicines for treating oral mucosal malignant tumors - Google Patents

Abstract

The invention discloses the application of bufadene compounds in the preparation of drugs for the treatment of oral mucosal malignant tumors. In vitro anti-cancer experiments of bufalin, cinobufafalin, deacetylbufafalin, helleboreglugenin, cinobufafagenin, and cinobufafalin on human tongue squamous cell carcinoma cells. Alkenes have obvious inhibitory effect on tongue squamous cell carcinoma cells, and cinobufalin is more effective than bufadenene extract and bufabufalin, sandbufafen, far cinobufafalin, bufabufalin, deacetylated The eight monomeric compounds of bufafetalin, helleboreaglycon, redibufagenin, and cinobufafalin have stronger anti-tumor cell activity. The bufasterene compounds provided by the invention have clear components, controllable quality, strong anticancer activity and small adverse reactions, and are expected to be developed into new drugs against malignant oral mucosal tumors.

Description

Application of Bufasterene Compounds in the Preparation of Drugs for Treating Oral Mucosa Malignant Tumors

technical field

The present invention relates to a new application of bufastenoid compounds, in particular to the application of bufastenoids compounds in the preparation of drugs for treating oral mucosal malignant tumor diseases. the

Background technique

Tongue cancer is one of the most common malignant tumors in oral cancer. Oral cancer is a general term for malignant tumors that occur in the oral cavity. It is one of the more common malignant tumors in the head and neck, ranking sixth in the world. Most of them belong to squamous cell carcinoma. Mutations. In recent years, the incidence of tongue cancer has been increasing year by year. Although the diagnosis and treatment of tongue cancer have been greatly improved, the prognosis is poor due to frequent tongue activities and rich lymphatic and blood supply in the tongue tissue. Its survival rate is still low. At present, the treatment schemes routinely adopted by domestic and foreign medical circles in the treatment of oral mucosal malignant tumors are chemical drug treatment, radiotherapy and surgical resection. Research, but also the iatrogenic problems and corresponding diseases caused by the toxic and side effects of chemical drugs, the radiation damage caused by radiotherapy, and the total or partial resection of sexual organs in surgical plans, have brought great pain to patients and seriously affected patients. health and quality of life. the

Toad Su, a traditional Chinese medicine, is the white slurry secreted by the post-auricular glands and skin glands of Bufo bufo gargarizans Cantor or Bufo gargarizans Cantor, which is processed and dried. It has the effects of clearing heat and detoxifying, reducing swelling and relieving pain, resuscitation and refreshing. It is used clinically in traditional Chinese medicine for anesthesia, boils, carbuncles, heat stroke, vomiting and diarrhea, sore throat, toothache, heart failure, cancer, etc. the

Toad venom contains a large amount of bufatoxins, which belong to steroidal compounds and are bufadienes (bufadienolactones), which are a type of six-membered unsaturated lactone ring connected at the C-17β position (α- Pyrone ring) 24C cardiotonic steroids, including bufafagenin, cinobufagenin, bufafalin, bufofabin, bufabufalin (also known as bufabufalin) and the like. In previous applications and studies, bufasterene compounds were mainly used to treat diseases of the cardiovascular system, such as enhancing myocardial contractility, and were often used clinically to treat heart failure, although some bufa steroids were used clinically. Treatment of leukemia, gastric cancer and tumor diseases, but no bufasteroids have been used for oral mucosal malignant tumor diseases, and the current toad preparations are mostly used as medicine in bold, with complex ingredients, except for bufagenin and bufatoxin In addition to indole alkaloids, it also contains various types of chemical components such as amino acids, reducing sugars, steroids, and peptides, with high adverse reactions. the

Contents of the invention

The technical problem to be solved by the present invention is to overcome the complex components of the current toad preparation and the high incidence of adverse reactions, provide clear components, controllable quality, and use bufa steroids as active components to prepare the application of the drug for treating oral mucosal malignant tumor diseases. the

The bufafalin compounds include cinobufafalin, bufabufalin, bufabufalin, bufabufalin, bufabufalin, deacetylbufafalin, hellebora aglycone, lipid Bufagenin and Cinobufagin. the

Wherein the structural formula of cinobufagin is:

The bufasterene compounds provided by the present invention and pharmaceutically acceptable carriers such as diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricating If necessary, flavoring agents, sweeteners, etc. can also be added to prepare a medicine for treating oral mucosal malignant tumors. the

The medicine of the present invention can be prepared into various dosage forms such as injection, tablet, powder injection, granule, capsule, oral liquid, ointment, cream, etc. according to conventional methods in the field of pharmacy. the

When the bufastenoid compound provided by the invention is made into tablets, the bufastenoid compound and carrier lactose or corn starch are added, and if necessary, a lubricant magnesium stearate is added, mixed uniformly, and then compressed into tablets to form a tablet. When the bufasten compound provided by the invention is made into a capsule, the bufasten compound is uniformly mixed with a carrier lactose or cornstarch, granulated, and then packed into a capsule to make a capsule. When the bufastenoid compound provided by the present invention is made into granules, the bufastenes compound and the diluent lactose or cornstarch are uniformly mixed, granulated, and dried to make granules. When the bufasterene compound provided by the present invention is made into an injection, take the bufasterene compound and add physiological saline to dissolve it, then add activated carbon, stir evenly, heat at 80°C for 30 minutes, filter, adjust the pH value, and use a vertical fusing glass funnel or Filter through other filters until clear, fill, and sterilize at 100 to 115°C for 30 minutes to make an injection. the

The oral mucosal malignant tumor described in the present invention is human tongue squamous cell carcinoma. the

After in-depth research by the applicant, the bufasterene extract and nine monomer compounds cinobufafalin, bufabufalin, sandbufatoxin, bufabufalin, bufabufalin, deacetylbufatoxin In vitro anti-cancer experiments on tongue squamous cell carcinoma cells by Helleborus aglycone, resiobufagenin, and cinobufagin. The experimental results showed that bufasterene compounds had obvious anticancer effects on tongue squamous cell carcinoma cells. And it has a good dose-effect relationship; the anticancer effect of bufasteroids on tongue squamous cell carcinoma in vitro is stronger than that of lapatinib, which is widely used in clinical treatment of malignant tumors. By comparing the IC50 values, it was found that Cinobufacin has the properties of bufobufalin, bufabufagin, bufabufalin, bufobufalin, deacetylbubafalin, hellebore aglycon, and lipobuadin. better anti-oral mucosal malignancies activity of cinobufagin or cinobufagin. the

Preparation of bufasterene compound: take 50 g of Bufa venom, extract twice with ethyl acetate, and concentrate the extract under reduced pressure at 40°C to obtain 12.6 g of extract. Chromatography, elution with petroleum ether-acetone (1:1), thin-layer chromatography tracking, to obtain the effective part of the bufasterene extract, and the bufasterene extract was purified by the preparative liquid phase to obtain the monomeric compounds cinobufalin, Ribubatobumin, Sandbuad tobumin, Yuanhuabububajin, Tobubafalin, Deacetylbufabafalin, Helleborus aglycon, Repobufagenin, and Cinobubafabalin. the

The present invention takes tongue squamous cell carcinoma cells as the research object, and has the following advantages compared with the prior art:

1. The active ingredients of bufasteroids are clear, the quality is controllable, and the anti-oral mucosal malignant tumor tongue squamous cell carcinoma has strong activity;

2. The effective dose of bufasteroids to inhibit the proliferation of oral mucosal malignant tongue squamous cell carcinoma cells is small, the potential side effects are also small, and the use is safer;

3. The anti-squamous cell carcinoma of oral mucosal tumor of bufa steroids is more effective than lapatinib, which is widely used in clinical treatment of malignant tumors. It is expected to become a new drug against squamous cell carcinoma of the oral mucosa with extensive resources and easy Yes, the cost is lower;

4. The bufasterene compound can be prepared into different pharmaceutical dosage forms with different excipients, which is convenient for clinical application. the

Detailed ways:

Below in conjunction with specific embodiment, further illustrate the present invention, should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention, after having read the present invention, those skilled in the art will understand various equivalent forms of the present invention All modifications fall within the scope defined by the appended claims of the present application. the

The experimental materials used in the following examples are as follows:

Cell lines: human tongue squamous cell carcinoma cells were provided by the pharmacology laboratory of Nanjing University of Traditional Chinese Medicine; 96-well plates were purchased from Costar; RMPI1640 medium was purchased from Gibco; DMEM medium was purchased from Gibco; newborn bovine serum was purchased from Hangzhou Sijiqing Company; thiazolium blue (MTT) and DMSO were purchased from Amresco; lapatinib was purchased from (GlaxoSmithKline, batch number: 090922); MCS-1 stands for bufasterene extract, MCS-1-2 stands for Cinobufacin, MCS-1-3 stands for Sandbufafen, MCS-1-4 stands for deacetylbufafen, MCS-1- 5 stands for bufafetalin, MCS-1-6 stands for hennabubafalin, MCS-1-7 stands for cinobubafalin, MCS-1-8 stands for hellebore aglycon, MCS-1-9 stands for lipid Bufagenin and MCS-1-10 represent cinobufafin prepared in our laboratory with a purity greater than 99.8%. the

The experimental apparatus used in the following examples is as follows:

Experimental equipment: ultra-clean bench (Suzhou purification model SW-CJ-IFD), _CO2 incubator (SANYO model: XD-101), microplate reader BIO-RAD (Model NO.550Serial NO.16971).

The experimental method used in the following examples is as follows:

Experimental method: Use the modified MTT method to conduct in vitro anti-tumor cell experiments on bufadenoids: human tongue squamous cell carcinoma cells (Tca-8113) were digested with trypsin, counted, and the concentration was 5×10 ⁴ cells/ml cell suspension. Add 100 μl of cell suspension (5×10 ³ cells per well) to each well of the 96-well plate, and then place it in a 37°C, 5% CO ₂ incubator for 24 hours; dilute the drug to the required level with incomplete medium Add 100 μL of corresponding drug-containing medium to each well, set up a negative control group, a vehicle control group and a positive control group at the same time, and then place the 96-well plate in a 37°C, 5% CO ₂ incubator for 72 hours . Then add 20 μL MTT (5 mg/ml) to each well, continue to cultivate for 4 hours, terminate the culture, discard the medium, add 150 μL DMSO to each well to dissolve, and shake gently for 10 minutes to mix well. Use a microplate reader to detect the absorbance or OD value of each well at two wavelengths of λ=4570nm and 620nm, and use the average value of each multiple well as the OD value of the group of cells to calculate the inhibition rate of each drug.

Example: bufafolin extract, cinobufafalin, bufabufalin, bufabufalin, bufabufalin, bufabufalin, deacetylbufafafalin, hellebore aglycon, lipid Inhibitory effect of bufagenin or cinobufagin on human tongue squamous cell carcinoma cell line Tca-8113. the

According to the MTT experimental method, the bufadene extract and nine monomer compounds cinobufalin, jabubafalin, sabubafalin, bufabufalin, bufabufalin, deacetylbufabafalin, Inhibitory effects of hellebore aglycon, redibufagenin or cinobufafalin on human tongue squamous cell carcinoma cells. the

The experimental results showed that the bufasteine extract and nine monomeric compounds cinobufalin, jabubafalin, sandbufalin, bufabufalin, bufabufalin, deacetylbubafalin, hellebore Grass aglycone, bufafagenin or cinobufafalin all had a strong inhibitory effect on human tongue squamous cell carcinoma cells, and each dose group showed a better dose-effect relationship, especially cinobufafalin showed a stronger Good anti-human tongue squamous cell carcinoma cell activity, its IC50 value is 21.33ng/ml, which is lower than other drug groups. The specific experimental results are shown in Table 1 and Table 2. the

Table 1 The effect of bufasterene extract on human tongue squamous cell carcinoma cells

Table 2 Anti-human tongue squamous cell carcinoma cell results of nine kinds of bufasterene monomer compounds

The experimental results in Table 2 show that the bufasteine extract and the monomeric compounds cinobufafalin, jabubafalin, sabubafalin, bufabufalin, bufabufalin, deacetylbufafafalin, Rhizogenin, Rebufagenin or Cinobufafalin all had a good inhibitory effect on human tongue squamous cell carcinoma cells, and the IC50 values of each compound are shown in Table 3:

Table 3 IC50 value test results of bufasterene compounds against tongue squamous cell carcinoma cells

The experimental results in Table 3 show that the compound resibufagenin IC50=4.001 μg/ml is greater than the concentration of 1 μg/ml, and its antitumor activity is a little weaker. Dujing, Yuanhuabufafenjing, Bufafen, Deacetylbubafalin, Helleborus aglycon and Cinobubafalin all showed good anti-tongue squamous cell carcinoma activity, with IC50 values reaching 1 μg /ml concentration range, and by comparison, it was found that cinobufalin showed a higher concentration than the bufasteine extract and the monomer compound bufafetoxin, sandbufafemin, distant cinobufafalin, bufabufalin, detobufafalin, Acetylbufafalin, helleboreglutinin, repobufagenin or cinobufafalin have better cell selection activity, that is, they have better anti-tongue squamous cell carcinoma activity. The IC50 value of the cells is only 0.0213 μg/ml, which indicates that the structure of cinobufalin has a better affinity for tongue squamous cell carcinoma cells, and the cell selectivity is stronger. And from the experimental results, it can also be seen that the bufasterene compound shows better anti-tongue squamous cell carcinoma activity than lapatinib, the active ingredient is clear, and the effective dose is low. 1/2 to 1/8 of the effective dose, so the potential side effects are smaller, and it is very promising to be developed as a new generation of anti-tongue squamous cell carcinoma drugs. the

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principles and concepts of the present invention, some improvements and modifications can also be made, these improvements and Retouching should also be regarded as the protection scope of the present invention. the

Claims (2)

1. bufadienolide biotransformation compounds application in preparation treatment people tongue scale cancer medicine as unique active component, is characterized in that: described bufadienolide biotransformation compounds is magnificent Toadpoison Medicine, Gamabufotalin, Arenobufagin, cinobufacin, bufotalien far away, removes acetyl bufotalien, hellebrigenin, bufogenin and Cinobufotalin.

2. bufadienolide biotransformation compounds according to claim 1 application in preparation treatment people tongue scale cancer medicine as unique active component, is characterized in that: by magnificent Toadpoison Medicine, Gamabufotalin, Arenobufagin, cinobufacin, bufotalien far away, go acetyl bufotalien, hellebrigenin, bufogenin and Cinobufotalin and pharmaceutically acceptable carrier to be prepared into the medicine of injection, tablet, injectable powder, granule, capsule, unguentum, cream or oral liquid formulation.