CN113134009A

CN113134009A - Marsdenia tenacissima glycoside H and doxorubicin composition and application thereof

Info

Publication number: CN113134009A
Application number: CN202110442537.3A
Authority: CN
Inventors: 胡道德; 王恺纯
Original assignee: Shanghai First Peoples Hospital
Current assignee: Shanghai First Peoples Hospital
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2021-07-20

Abstract

The invention relates to the technical field of medicines, in particular to a composition of marsdenia tenacissima glycoside H and doxorubicin and application thereof, wherein the molar concentration ratio of the marsdenia tenacissima glycoside H to the doxorubicin is as follows: marsdenia tenacissima glycoside H: doxorubicin 1: 2. The invention takes human lung cancer cell A549, gastric cancer cell SGC-7901 and breast cancer cell MCF-7 as in-vitro experimental models to observe the cell proliferation inhibition effect of the combined drug of marsdenia tenacissima glycoside H and doxorubicin, and the experimental results show that: compared with a control group (single drug), the combined drug group has obvious inhibition on cell proliferation, and the synergistic effect of the two can obviously enhance the effect of the chemotherapeutic drug in tumor treatment, thereby having the prospect of developing into clinical combined drug.

Description

Marsdenia tenacissima glycoside H and doxorubicin composition and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a composition of marsdenia tenacissima glycoside H and doxorubicin and application thereof.

Background

Malignant tumor-cancer has become one of the main diseases that are common and seriously threaten human life and quality of life. In recent years, with the development of economic society, the life level of people is increasing, but the morbidity and mortality of cancer are increasing.

The currently clinically used western medicines for treating tumors have large toxic and side effects, cause great damage to the bodies of tumor patients during treatment, and also have the problems of reduced sensitivity of malignant tumor cells to the western medicines and the like. The disease is more difficult to treat, and the life quality of the patient is reduced.

Apoptosis refers to a process that factors inside and outside a body trigger a death program prestored in a cell, the death program of the cell is automatically started under the control of genes, and the life process is orderly ended, namely programmed cell death. The normal organism utilizes apoptosis to eliminate redundant, aged and damaged cells so as to keep the internal environment balance of the organism and maintain normal physiological activities. Imbalance of apoptosis may cause various diseases such as cancer and autoimmune diseases. The sensitivity of tumor cells to chemotherapeutic drugs is reduced, so that chemotherapy is difficult to progress well, and chemotherapy fails, and therefore, the research and application of mechanisms for improving the sensitivity of tumor cells to chemotherapeutic drugs is urgent. The combined medication refers to the simultaneous or sequential application of two or more drugs for achieving the purpose of treatment, and the result is mainly to increase the curative effect of the drugs or to reduce the toxic and side effects of the drugs. Research proves that the combination of the traditional Chinese medicine monomer and the chemotherapeutic drug can obviously reduce the dosage of the chemotherapeutic drug and reduce the adverse reaction of patients. Aiming at the defects of the prior art, the invention firstly proposes the combination of the marsdenia tenacissima glycoside H and the doxorubicin for resisting cancer, and the experimental result shows that the two have synergistic effect, so that the increment rate of malignant tumors can be greatly reduced, and the sensitivity of the malignant tumors to chemotherapeutic drugs can be greatly improved. The composition of the marsdenia tenacissima H and the doxorubicin and the application thereof are not reported at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a composition of marsdenin H and doxorubicin and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the invention provides a marsdenin H combined doxorubicin composition, wherein the molar concentration ratio of the marsdenin H to the doxorubicin is as follows: marsdenia tenacissima glycoside H: and (2-4) doxorubicin is 1.

Preferably, the molar concentration ratio of the marsdenia tenacissima H to the doxorubicin is as follows: marsdenia tenacissima glycoside H: and (2-3) doxorubicin is 1.

Preferably, the molar concentration ratio of the marsdenia tenacissima H to the doxorubicin is as follows: marsdenia tenacissima glycoside H: doxorubicin 1: 2.

Further, the composition comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier and the marsdenin H are processed together.

Further, the dosage form of the marsdenia tenacissima H comprises injection, tablets, capsules or granules.

In a second aspect, the present invention provides a method for preparing the composition, comprising the step of weighing the raw materials according to molar concentration ratios.

In a third aspect, the present invention provides the use of a composition as described above for the preparation of a medicament for the treatment of a malignant tumor.

Preferably, the malignant tumor comprises human lung cancer malignant tumor, human gastric cancer malignant tumor and human breast cancer malignant tumor.

Furthermore, the composition can inhibit the proliferation of malignant tumor cells, promote the apoptosis of the malignant tumor cells, reduce the dosage of the doxorubicin and improve the sensitivity of malignant tumor to the doxorubicin.

Term(s) for

As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents and the like. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (mackpub.co., n.j.1991). Pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as emulsifiers, fillers, binders, wetting agents, disintegrants, absorption enhancers, flavoring agents, colorants, cosolvents and the like may also be present in these carriers. The emulsifier is selected from acetylated monoglyceride, acetylated diglyceride, sucrose ester, sorbitol ester, soybean phospholipid, lauric monoglyceride, propylene glycol fatty acid ester, calcium stearoyl lactylate, diacetyl tartaric acid, glyceryl monostearate, modified soybean phospholipid, etc. Such as magnesium stearate, microcrystalline cellulose, lactose, milk sugar, high molecular weight polyethylene glycols, and the like. Such as starch, mannitol, silicic acid, dextrin, calcium hydrogen phosphate, cellulose, etc. Such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, gum arabic, starch slurry, hydroxypropyl starch, modified starch, pregelatinized starch, dextrin, microcrystalline cellulose, polyvinyl pyrrolidone mucilage, gelatin mucilage. Such as glycerin and the like. The disintegrating agent is agar, calcium carbonate, potato starch, tapioca starch, alginic acid, hydroxypropyl starch, modified starch, sodium carboxymethyl starch, microcrystalline cellulose, guar gum, xanthan gum, etc. The absorption enhancer is such as quaternary ammonium compound, effervescent agent, cyclodextrin, vitamin D and its derivatives, piperine, etc. The flavoring agent can be sour agent, sweetener, such as phosphoric acid, lactic acid, tartaric acid, malic acid, fumaric acid, acetic acid, succinic acid, xylitol, steviosin, sodium cyclamate, aspartame, oleum Menthae Dementholatum, etc. The colorant may be a plant colorant, an animal colorant or a microbial colorant, such as beet red, turmeric, chlorophyll, shellac, cochineal, red yeast colorant, and the like. Such as beta-cyclodextrin, maltodextrin, tween, ethanol, span, sodium dodecyl sulfate, propylene glycol, polyethylene glycol, glycerol, etc. However, it will be appreciated by those skilled in the art that the pharmaceutically acceptable carriers useful in the present invention are not limited to the above-mentioned types.

Dosage forms

The dosage form of the marsdenia tenacissima-H provided by the invention is not particularly limited, and can be any dosage form suitable for being taken by mammals; preferably, the preparation form is injection, tablet, capsule or granule. The preferred marsdenia tenacissima-H is an injection from the standpoint of ease of preparation, administration or administration.

Preparation method

After knowing the formulation of the composition of Marsdenia tenacissima H and doxorubicin of the present invention, one skilled in the art can use a variety of conventional methods to process the Marsdenia tenacissima H into a medicament.

Optionally, other pharmaceutically (or dietetically or nutraceutically) acceptable carriers can be added during the preparation process.

Use and method of use

The composition of the present invention may be used directly in treating malignant tumors, including human lung cancer, human stomach cancer and human breast cancer.

The amount of the composition of the present invention to be used may vary depending on the mode of administration, the dosage form and the severity of the disease to be treated. For example, a single dose may be administered several times daily in divided doses, or the dose may be reduced proportionally as required by the condition being treated. Of course, the particular dosage will also take into account such factors as the mode of administration, the physical condition of the subject being administered, and the like, which are within the skill of the art.

The invention has the advantages that:

the combination of the marsdenia tenacissima H and the doxorubicin is put forward for the first time, the optimal molar concentration ratio of the combination of the marsdenia tenacissima H and the doxorubicin is limited, the results of the combination of the marsdenia tenacissima H and the doxorubicin and the single drug combination are observed by taking the human lung cancer cells A549, the gastric cancer cells SGC-7901 and the breast cancer cells MCF-7 as in-vitro experimental models, and the results show that the combination of the marsdenia tenacissima H and the doxorubicin can obviously improve the sensitivity of malignant tumor cells to chemotherapeutic drugs, obviously reduce the proliferation rate of the malignant tumor cells, obviously improve the apoptosis rate of malignant tumors and obviously improve the survival quality of patients compared with the single drug combination.

Drawings

FIG. 1 shows the synergistic effect of the combination of Marsdenia tenacissima H and doxorubicin on the proliferation inhibition of gastric cancer cell SGC-7901.

FIG. 2 shows the synergistic effect of the combined application of Marsdenia tenacissima glycoside H and doxorubicin on the proliferation inhibition of lung cancer cells A549.

FIG. 3 shows the synergistic effect of the combination of Marsdenia tenacissima H and doxorubicin on the proliferation inhibition of breast cancer cells MCF 7.

FIG. 4 shows the effect of Marsdenia tenacissima H and doxorubicin on the degree of gastric cancer cell SGC-7901 apoptosis alone or in combination.

FIG. 5 shows the effect of Marsdenia tenacissima H and doxorubicin on the apoptosis of lung cancer cell A549 alone or in combination.

FIG. 6 shows the effect of Marsdenia tenacissima H and doxorubicin on the degree of apoptosis of breast cancer MCF 7.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1 effects example

1 materials of the experiment

Marsdenia tenacissima glycoside H is purchased from Chenguan optical biotechnology limited of Baoji city; dimethyl sulfoxide (DMSO) was purchased from Sigma; doxorubicin hydrochloride (Doxorubicin hydrochloride) was purchased from MCE; DMEM high-glucose medium, fetal bovine serum, double antibody, PBS were purchased from Gibco; BCA kit was purchased from solibao corporation; the CCK-8 kit was purchased from Dojindo; AnnexinV-FITC/PI apoptosis kit was purchased from BD, usa.

2 method of experiment

2.1 cell culture

The cryopreservation tube containing the cells was taken out of the liquid nitrogen tank, quickly placed in a previously prepared water bath at 37 ℃, and gently shaken to rapidly dissolve the cells.Subsequently, the plate was wiped with an alcohol cotton ball and then moved to an ultraclean bench. And (3) placing the sucked cell mixed solution into a centrifuge tube by using a pipette gun, centrifuging, removing a supernatant, re-suspending a culture medium, blowing away and uniformly blowing cell blocks, and transferring the cell blocks into a culture dish, wherein the culture medium is a DMEM high-sugar medium containing 10% fetal calf serum and 1% double antibody. At 37 ℃ 5% CO₂Culturing under saturated humidity condition.

2.2 Effect of Marsdenia tenacissima glucoside H on human Lung cancer, gastric cancer and Breast cancer cell line Activity

Taking logarithmic phase cells A549, SGC-7901 and MCF-7, re-suspending with fresh culture solution, and adjusting cell concentration to 8 × 10⁴one/mL, 100. mu.L per well was inoculated into a 96-well plate and incubated in an incubator for 12h to wait for adherence. After adherence, the cell culture was discarded and the control group (without drug) and the marsdenine H group at different dosing concentrations (marsdenine H concentrations 5, 10, 15, 20 and 25 μ M) were set. Each group was set with 6 wells, each well was treated differently to ensure a final liquid volume of 100. mu.L, and incubated for 48 h. Before the end of the reaction, the liquid medicine is absorbed, 10 mu L of CCK-8 and 90 mu L of LDMEM are added into each hole, the mixture is uniformly mixed, the mixture is respectively cultured for 2h and then placed on a microplate reader, and the absorbance is detected at the wavelength of 450 nm. And calculating the inhibition rate of the marsdenia tenacissima glycoside H on three tumor cells.

2.3 Effect of Marsdenia tenacissima glycoside H in combination with Doxorubicin on tumor cell line Activity

Taking logarithmic phase cells A549, SGC-7901 and MCF-7, re-suspending with fresh culture solution, and adjusting cell concentration to 8 × 10⁴one/mL, 100. mu.L per well was inoculated into a 96-well plate and incubated in an incubator for 12h to wait for adherence. After the adherence, the cell culture solution was discarded, and a control group (no drug addition), a Marsdenia tenacissima H group (Marsdenia tenacissima H concentration of 5, 10, 15, 20 and 25. mu.M), a doxorubicin group (drug administration concentration of 10, 20, 30, 40 and 50. mu.M), and a combination drug group (doxorubicin: Marsdenia tenacissima H drug concentration 2: 1 ratio) were set. Each group was set with 6 wells, each well was treated differently to ensure a final liquid volume of 100. mu.L, and incubated for 48 h. Before the end of the reaction, the liquid medicine is absorbed, 10 mu L of CCK-8 and 90 mu L of LDMEM are added into each hole, the mixture is uniformly mixed, the mixture is respectively cultured for 2h and then placed on a microplate reader, and the absorbance is detected at the wavelength of 450 nm. Calculating Marsdenia tenacissima glycoside H, doxorubicin andthe inhibition rate of the marsdenia tenacissima glycoside H combined with doxorubicin on three tumor cells.

2.4 Marsdenia tenacissima glycoside H and doxorubicin combined use induced tumor cell apoptosis

Cells in logarithmic growth phase are taken and inoculated into a 6-well plate, 2mL of fresh culture solution is added into each well, and the cells are placed into an incubator for culture. After 12h, the medium was discarded and the drug treatment was performed. Culturing for 48h, discarding the liquid medicine, washing with PBS for 2 times, digesting with pancreatin, collecting the cell suspension, centrifuging (4 ℃, 1000rpm, 3min), resuspending 100 μ L of 1 × Binding Buffer in 1.5mL EP tube, adding 5 μ L of Lannexin V-FITC and 5 μ L of PI staining solution, standing at room temperature for 15min, and detecting on the machine with an internal flow meter for half an hour.

2.5 data processing and analysis

Data analysis using SPSS21.0, data measurement

Representing that the mean comparison among the groups adopts one-way analysis of variance (oneway ANOVA), and the comparison between every two groups adopts LSD-t test; and carrying out rank sum test on variance. P<A difference of 0.05 is statistically significant.

3 results of the experiment

After SGC-7901, SGC-7901 and MCF-7 cells are treated by each group of drugs for 48 hours, each group of drugs can inhibit the proliferation of SGC-7901, SGC-7901 and MCF-7 cells compared with a blank control group (S) ((S))^**P<0.01), the inhibition rate is concentration-dependent. The results are shown in tables 1, 2 and 3.

3.1 the proliferation inhibition effect of Marsdenia tenacissima glucoside H and doxorubicin on human gastric cancer SGC-7901 cells by single use and combined use

TABLE 1 inhibition of proliferation of human gastric carcinoma SGC-7901 cells by Marsdenia tenacissima-H and doxorubicin alone and in combination (II)

n＝3)

TABLE 2 proliferation inhibition of human lung carcinoma A549 cells by Marsdenia tenacissima glycoside H and doxorubicin alone and in combination ((

n＝3)

TABLE 3 inhibition of proliferation of human breast cancer MCF7 cells by Marsdenia tenacissima H and doxorubicin taken alone or in combination (II)

n＝3)

3.2 synergistic Effect study of Marsdenia tenacissima glucoside H and Doxorubicin combination

To verify the synergistic effect of Marsdenia tenacissima H and doxorubicin on the inhibition of tumor cell proliferation, we calculated the Combination Index (CI) values for 3 combinations using CompuSyn software, as shown in tables 4, 5 and 6, FIGS. 1, 2 and 3. The CI value function is a quantitative index for judging the existence of the synergy, the CI value <1 represents the synergy, and the smaller the value is, the stronger the synergy is. The calculation result shows that the combination of the two medicines has anticancer synergistic effect on gastric cancer, lung cancer and breast cancer cells.

TABLE 4 Combined index of Puerarin H combined with Doxorubicin for inhibition of gastric cancer cell SGC-7901 proliferation ((II))

n＝3)

TABLE 5 Combined index of Marsdenia tenacissima glycoside H combined with doxorubicin for inhibition of proliferation of lung cancer cells A549 ((

n＝3)

Table 6 combined index of the inhibition of proliferation of breast cancer cells MCF7 using marsdenin H in combination with doxorubicin: (

n＝3)

3.3 Marsdenia tenacissima glycoside H and doxorubicin are used alone or in combination to induce tumor cell apoptosis

48 hours after administration, the mean apoptosis level of gastric cancer cells SGC-7901 was significantly increased by the Marsdenia tenacissima glycoside group H, the doxorubicin group and the combined drug group, which were (7.02 + -1.46)%, (6.86 + -1.33)% and (33.78 + -3.52)%, respectively. The combination group significantly increased the level of apoptosis of cells compared to the single-dose group (^**P<0.01). The results are shown in Table 7 and FIG. 4.

TABLE 7 Effect of Marsdenia tenacissima H, doxorubicin alone and in combination on the degree of gastric cancer cell SGC-7901 apoptosis (II)

n＝3)

The lung cancer cell A549 apoptosis level is remarkably increased by 48 hours after administration, and the marsdenin H group, the doxorubicin group and the combined drug group are respectively (10.12 +/-1.03)%, (13.95 +/-2.07)% and (35.84 +/-4.10)%. The combination group significantly increased the level of apoptosis of cells compared to the single-dose group (^**P<0.01). The results are shown in Table 8 and FIG. 5.

TABLE 8 Effect of Marsdenia tenacissima H, doxorubicin alone and in combination on the degree of apoptosis of Lung cancer cells A549 ((

n＝3)

The average apoptosis level of the breast cancer cells MCF7 is remarkably increased by 48 hours after administration, namely (8.81 +/-0.93)%, (13.48 +/-2.17)% and (35.20 +/-4.56)%. The combination group significantly increased the level of apoptosis of cells compared to the single-dose group (^**P<0.01). The results are shown in Table 9 and FIG. 6.

TABLE 9 Effect of Marsdenia tenacissima H, doxorubicin alone and in combination on the degree of apoptosis of breast cancer cells MCF7 ((II))

n＝3)

Discussion 4

Doxorubicin is a first-line chemotherapeutic drug for the clinical treatment of gastric cancer, however, the reduction of the sensitivity of tumor cells deteriorates the therapeutic effect. Clinical data indicate that combination therapy can improve efficacy compared to traditional chemotherapy alone. Therefore, the search for natural ingredients which can be synergistically used with conventional chemotherapeutic drugs has great clinical significance for the treatment of colorectal cancer.

In the invention, the combination of marsdenia tenacissima H and doxorubicin is adopted, and in-vitro experiments prove that the drug can effectively inhibit the proliferation of lung cancer A549 cells, gastric cancer SGC-7901 cells and breast cancer MCF7 cells, so that the sensitivity of cancer cells to chemotherapeutic drugs is enhanced, and the apoptosis of tumor cells is promoted.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims

1. The composition of the marsdenia tenacissima glycoside H combined doxorubicin is characterized in that the molar concentration ratio of the marsdenia tenacissima glycoside H to the doxorubicin is as follows: marsdenia tenacissima glycoside H: and (2-4) doxorubicin is 1.

2. The composition of the marsdenia tenacissima glycoside H combined doxorubicin is characterized in that the molar concentration ratio of the marsdenia tenacissima glycoside H to the doxorubicin is as follows: marsdenia tenacissima glycoside H: and (2-3) doxorubicin is 1.

3. The composition of the marsdenia tenacissima glycoside H combined doxorubicin is characterized in that the molar concentration ratio of the marsdenia tenacissima glycoside H to the doxorubicin is as follows: marsdenia tenacissima glycoside H: doxorubicin 1: 2.

4. The composition of any one of claims 1-3, wherein the composition comprises a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is co-processed with marsdenin H.

5. The composition according to any one of claims 1 to 3, wherein the dosage form of the marsdenia tenacissima H comprises injection, tablet, capsule or granule.

6. The method for preparing the composition as claimed in any one of claims 1 to 5, comprising the step of weighing each raw material drug in molar concentration ratio.

7. Use of a composition according to any one of claims 1 to 3 for the manufacture of a medicament for the treatment of malignant tumours.

8. The use of claim 7, wherein the malignancy comprises a human lung cancer malignancy, a human gastric cancer malignancy, a human breast cancer malignancy.

9. The use of claim 7, wherein the composition inhibits proliferation of malignant cells, promotes apoptosis of malignant cells, reduces the amount of doxorubicin administered, and increases the sensitivity of malignant cells to doxorubicin.