CN113116915A

CN113116915A - Composition of marsdenia tenacissima glycoside H and paclitaxel and application thereof

Info

Publication number: CN113116915A
Application number: CN202110442518.0A
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: 2021-07-16

Abstract

The invention relates to the technical field of medicine, in particular to a combination of sanguin H and paclitaxel and an application thereof. The molar concentration ratio of the sanguin H and paclitaxel is: sanguin H:paclitaxel=1:2. In the present invention, human lung cancer cell A549, breast cancer cell MCF7 and ovarian cancer cell SKOV3 are used as in vitro experimental models to observe the inhibitory effect on cell proliferation of the combined use of Gambrin H and paclitaxel. The experimental results of the present invention show that compared with the control group (single drug group), the combined drug group significantly inhibits cell proliferation, can significantly enhance the effect of chemotherapeutic drugs in tumor treatment, and has the prospect of developing into a clinical combined drug.

Description

Composition of marsdenia tenacissima glycoside H and paclitaxel 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 paclitaxel 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 paclitaxel 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 marsdenia tenacissima H and paclitaxel and the application thereof are not reported at present.

Disclosure of Invention

The invention aims to provide a composition of marsdenia tenacissima H and paclitaxel and application thereof aiming at the defects of the prior art.

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

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

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

Preferably, the molar concentration ratio of the marsdenia tenacissima H to the paclitaxel is as follows: marsdenia tenacissima glycoside H: paclitaxel is 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 invention provides a preparation method of the composition, which comprises the step of weighing the raw material medicines according to molar concentration ratio.

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.

Further, the malignant tumor includes human lung cancer malignant tumor, human breast cancer malignant tumor, and human ovarian 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 paclitaxel and improve the sensitivity of the malignant tumor to the paclitaxel.

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 can be 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 paclitaxel of the present invention, one skilled in the art can use a variety of conventional methods to process 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 breast cancer and human ovarian 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 invention firstly provides that the marsdenia tenacissima H and the paclitaxel are combined, the optimal molar concentration ratio of the combination of the marsdenia tenacissima H and the paclitaxel is limited, and the human lung cancer cell A549, the breast cancer cell MCF7 and the ovarian cancer cell SKOV3 are used as in-vitro experimental models in experiments to observe the cell proliferation inhibition effect of the combined application of the marsdenia tenacissima H and the paclitaxel. The experimental results of the invention show that: compared with a control group (a single medicine group), the combined medicine group has obvious inhibition on cell proliferation, can obviously enhance the effect of chemotherapeutic medicines in tumor treatment, can obviously improve the sensitivity of tumor cells to the chemotherapeutic medicines and the apoptosis rate of malignant tumor cells, further improves the life quality of patients, and has the prospect of being developed into clinical combined medicine.

Drawings

FIG. 1 shows the synergistic effect of the combination of Marsdenia tenacissima glycoside H and paclitaxel on the proliferation inhibition of lung cancer cells A549.

FIG. 2 shows the synergistic effect of the combination of Marsdenia tenacissima H and paclitaxel on the proliferation inhibition of breast cancer MCF 7.

FIG. 3 shows the synergistic effect of the combination of Marsdenia tenacissima H and paclitaxel on the proliferation inhibition of human ovarian cancer cell SKOV 3.

FIG. 4 shows the effect of Marsdenia tenacissima glycoside H and paclitaxel on the apoptosis degree of lung cancer cell A549 when used alone or in combination.

FIG. 5 shows the effect of the single use and the combined use of Marsdenia tenacissima H and paclitaxel on the apoptosis degree of human breast cancer MCF 7.

FIG. 6 shows the effect of Marsdenia tenacissima H and paclitaxel on the degree of apoptosis of human ovarian carcinoma SKOV3 when used alone or in combination.

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; paclitaxel (Paclitaxel) was purchased from MCE; DMEM high-glucose medium, fetal bovine serum, double antibody, PBS were purchased from Gibco; the BCA kits were purchased from solibao corporation; the CCK-8 kit was purchased from Dojindo; annexin V-FITC/PI apoptosis kits were 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. The aspirated cell mixture is placed in a pipettePutting the mixture into a centrifuge tube, centrifuging, removing supernatant, re-suspending a culture medium, blowing the cell mass away, uniformly blowing, and transferring the cell mass to 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 the Activity of human Lung cancer, Breast cancer and ovarian cancer cell lines

Taking logarithmic phase cells A549, MCF7 and SKOV3, 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 (marsdenine concentrations 5, 10, 15, 20 and 25 μ M) were set at different dosing concentrations. 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 process, the liquid medicine is sucked, 10 mu L of CCK-8 and 90 mu L of DMEM are added into each hole, the mixture is uniformly mixed, the mixture is respectively cultured for 2h and then placed on an enzyme-linked immunosorbent assay, and the absorbance is detected at the wavelength of 450 nm. And calculating the inhibition rate of the marsdenia tenacissima glycoside I on three tumor cells.

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

Taking A549 cells, MCF7 cells and SKOV3 cells in logarithmic growth phase, re-suspending with fresh culture solution, and adjusting the cell concentration to 8 x 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 solution was discarded, and a control group (without drug), a marsdenia tenacissima-H group (the concentration of marsdenia tenacissima-H is 5, 10, 15, 20 and 25 μ M), a paclitaxel group (the concentration of drug administration is 10, 20, 30, 40 and 50 μ M) and a combined drug group (the ratio of paclitaxel to marsdenia tenacissima-H drug concentration is 2: 1) 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 rates of the marsdenia tenacissima glycoside H, the paclitaxel and the marsdenia tenacissima glycoside H combined paclitaxel on three tumor cells.

2.4 Marsdenia tenacissima glycoside H combined with paclitaxel for inducing 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 LannexinV-FITC and 5 μ LPI 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 and data measurement using SPSS21.0

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 the A549 cells, the MCF7 cells and the SKOV3 cells are treated by the medicines for 48 hours, the medicines can inhibit the proliferation of the A549 cells, the MCF7 cells and the SKOV3 cells compared with a blank control group (the medicines for each group can inhibit the proliferation of the A549 cells, the MCF7 cells and the SKOV3 cells in the blank control group)^**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 glycoside H and paclitaxel on human lung cancer A549 cells by single or combined use

TABLE 1 proliferation inhibition of human lung carcinoma A549 cells by Marsdenia tenacissima glycoside H and paclitaxel alone and in combination ((

n＝3)

TABLE 2 inhibition of proliferation of human breast cancer MCF7 cells by Marsdenia tenacissima H and paclitaxel alone and in combination ((

n＝3)

TABLE 3 inhibition of proliferation of human ovarian cancer SKOV3 cells by Marsdenia tenacissima H and paclitaxel alone and in combination (II)

n＝3)

3.2 synergistic Effect study of Marsdenia tenacissima glucoside H in combination with paclitaxel

To verify the synergistic effect of Marsdenia tenacissima H and paclitaxel on the proliferation inhibition of tumor cells, 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 is a quantitative index for judging the existence of the synergistic effect, the CI value is less than 1, the synergistic effect is realized, and the smaller the numerical value is, the stronger the synergistic effect is. The calculation result shows that the combination of the two medicines has anticancer synergistic effect on lung cancer, breast cancer and ovarian cancer cells.

TABLE 4 combination index of Marsdenia tenacissima glycoside H in combination with paclitaxel on the inhibition of proliferation of lung cancer cells A549 ((

n＝3)

TABLE 5 combination index of the combination of Marsdenia tenacissima H and paclitaxel for the inhibition of proliferation of human breast cancer cells MCF7 ((C))

n＝3)

TABLE 6 combination index of the combination of Marsdenia tenacissima H and paclitaxel on the inhibition of human ovarian cancer cell SKOV3 proliferation (II) ((III))

n＝3)

3.3 inducing apoptosis of tumor cells by using Marsdenia tenacissima glycoside H and paclitaxel alone or in combination

The lung cancer cell A549 apoptosis level is obviously increased by 48 hours after administration, namely (10.29 +/-1.48)%, (9.58 +/-1.09)% and (29.44 +/-4.15)%, 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 and paclitaxel on apoptosis degree of lung cancer cell A549 alone and in combination ((

n＝3)

The apoptosis level of the human lung cancer cell A549 is obviously increased by 48 hours after administration, namely (10.46 +/-1.53)%, (9.28 +/-1.94)% and (29.17 +/-4.55)%. Compared with the single medicine administration group, the combined medicine group can obviously increase the finenessLevel of apoptosis of cells: (^**P<0.01). The results are shown in Table 8 and FIG. 5.

TABLE 8 Effect of Marsdenia tenacissima H, paclitaxel alone and in combination on the degree of apoptosis in human breast cancer cells MCF7 (II)

n＝3)

At 48H after administration, apoptosis levels of human ovarian cancer cells SKOV3 were significantly increased by the Marsdenia tenacissima glycoside H group, the paclitaxel group and the combined drug group, which were (7.38 + -0.43)%, (9.04 + -1.01)% and (31.99 + -5.36)%, 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 9 and FIG. 6.

TABLE 9 Effect of Marsdenia tenacissima H and paclitaxel on apoptosis degree of human ovarian cancer SKOV3 alone and in combination ((

n＝3)

Discussion 4

Paclitaxel is the first line drug of clinical cancer treatment, however, the reduction of the sensitivity of tumor cells to it worsens 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 synergized with the common chemotherapeutic drugs has important clinical significance for treating the tumor.

In the invention, the combination of marsdenia tenacissima H (15 mu M) and paclitaxel (30 mu M) is adopted, and in vitro experiments prove that the drug can effectively inhibit the proliferation of lung cancer A549 cells, human breast cancer MCF7 cells and human ovarian cancer SKOV3 cells, so that the sensitivity of cancer cells to chemotherapeutic drugs is enhanced, and the apoptosis of the cancer 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 marsdenia tenacissima glycoside H combined with paclitaxel is characterized in that the molar concentration ratio of the marsdenia tenacissima glycoside H to the paclitaxel is as follows: marsdenia tenacissima glycoside H: and (2) paclitaxel ═ 1 (2-4).

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

3. The composition of marsdenia tenacissima glycoside H combined with paclitaxel is characterized in that the molar concentration ratio of the marsdenia tenacissima glycoside H to the paclitaxel is as follows: marsdenia tenacissima glycoside H: paclitaxel is 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 breast cancer malignancy, a human ovarian cancer malignancy.

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