Application of taiwanin in preparation of anti-cancer drugs

Abstract

The invention discloses an application of tautogen in preparation of an anti-cancer drug. Studies show that the Taiwanin has an obvious anti-cancer effect, research data with Jurkat cells, Hela cells and A549 cells as objects show that the Taiwanin has an obvious inhibition effect on leukemia cells, cervical cancer cells and lung cancer cells, the Taiwanin has the value of preparing the anti-cancer drugs, and the Taiwanin is derived from natural plants, is relatively high in safety and has a good application prospect. The compound has great value in the aspect of developing and preparing anti-cancer drugs. The invention not only provides a new choice for anti-cancer drugs, but also opens up a new direction for the utilization of taiwania flousiana plant resources.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically relates to the application of tauranga in the preparation of anticancer drugs. Background Art

[0002] TSC-3 was first isolated by our research team from a plant extract of Taiwania truncatula and is disclosed in invention patent CN109232688B. Previous studies have shown that TSC-3 effectively controls a variety of weeds, providing significant theoretical guidance and practical application value for the study of botanical weed control and the development of new herbicides.

[0003] From a structural analysis, baldwood belongs to the lignan class of substances found in plants and is a phytoestrogen. Therefore, various lignans have been reported to exhibit diverse activities, including numerous applications in medicine and pesticides. However, the effects of baldwood beyond herbicidal activity are still poorly studied, leaving much room for further exploration. Summary of the Invention

[0004] The present invention aims to explore new effects of taurangaine and provide new options for the development of anti-leukemia and anti-cancer drugs.

[0005] The present invention aims at using tauranga or a plant extract rich in tauranga in the preparation of anti-leukemia and anti-cancer drugs.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The research of the present invention shows that tauranga exerts a significant anti-cancer effect. Research data using Jurkat cells, Hela cells and A549 cells show that it exhibits a significant inhibitory effect on T cell acute lymphoblastic leukemia cells, cervical cancer cells and lung cancer cells, and has value in preparing anti-cancer drugs.

[0008] Therefore, the present invention claims protection for the following solutions:

[0009] Application of tauranga or its structural analogues or pharmaceutically acceptable salts in the preparation of anticancer drugs.

[0010] The invention relates to an application of a composition in the preparation of an anticancer drug. The composition contains tauranga or a structural analogue thereof or a pharmaceutically acceptable salt thereof, and also contains other anticancer drugs.

[0011] The invention relates to an application of a composition in the preparation of an anticancer drug. The composition contains tauranga or a structural analogue thereof or a pharmaceutically acceptable salt thereof, and also contains a pharmaceutically acceptable auxiliary material.

[0012] The invention relates to an application of a plant extract in the preparation of an anticancer drug. The plant extract is a plant extract rich in tauranga or its structural analogues or its pharmaceutically acceptable salt.

[0013] The invention relates to the use of a plant extract in combination with other anticancer drugs in the preparation of an anticancer drug. The plant extract is a plant extract rich in tauranga or its structural analogues or pharmaceutically acceptable salts thereof.

[0014] The invention relates to the use of a plant extract and a pharmaceutically acceptable auxiliary material in the preparation of an anticancer drug. The plant extract is a plant extract rich in balsaminazone or its structural analogues or its pharmaceutically acceptable salt.

[0015] As a specific embodiment, the cancer is cervical cancer, leukemia or lung cancer.

[0016] More specifically, the leukemia is human acute T-cell leukemia. The lung cancer is human non-small cell lung cancer.

[0017] As a specific embodiment, the richness of tauranga means that the main active substance includes tauranga.

[0018] As a specific embodiment, the richness of tauranga means that the main active substance is tauranga.

[0019] The present invention also provides:

[0020] An anticancer drug contains tauranga or its structural analogue or its pharmaceutically acceptable salt, and also contains pharmaceutically acceptable excipients.

[0021] An anticancer drug contains tauranga or its structural analogues or pharmaceutically acceptable salts thereof, and also contains other anticancer drugs.

[0022] An anticancer drug contains a plant extract rich in balsam pear or its structural analogues or pharmaceutically acceptable salts thereof, and also contains pharmaceutically acceptable excipients.

[0023] An anticancer drug contains a plant extract rich in tauranga or its structural analogues or pharmaceutically acceptable salts thereof, and also contains other anticancer drugs.

[0024] As a preferred embodiment, the plant extract is a Taiwania truncatula extract.

[0025] As an optional embodiment, the Taiwania truncatula extract is an ethyl acetate extract of Taiwania truncatula.

[0026] As a specific optional implementation scheme, the extraction method of the Taiwania truncatula extract is: extracting Taiwania truncatula with methanol, concentrating and suspending it in water, and then extracting it with petroleum ether and ethyl acetate in sequence to obtain a Taiwania truncatula extract rich in truncatulin.

[0027] More preferably, the above-obtained Taiwania truncatula extract can be purified, for example, by silica gel column chromatography using a chloroform / methanol system to obtain a crude product.

[0028] More preferably, the crude product can be further purified, for example, by sequentially performing silica gel column chromatography with petroleum ether / acetone and petroleum ether / ethyl acetate, followed by separation using dextran gel, combining the filtrate on a TLC plate, and then separating and purifying using reverse phase silica gel column chromatography.

[0029] In addition, the tauranga chinensis of the present invention can be chemically synthesized or extracted from plants, such as tauranga chinensis.

[0030] As a preferred embodiment, the method for extracting tautomerin from Thunbergia truncatula is as follows:

[0031] S1. The Taiwania baldness tree was extracted with methanol, concentrated, suspended in water, and then extracted with petroleum ether and ethyl acetate to obtain an extract;

[0032] S2. The extract obtained in S1 was subjected to silica gel column chromatography using a chloroform / methanol system, and the combined extracts were combined by TLC and then assayed for activity;

[0033] S3. The active fraction was subjected to silica gel column chromatography with petroleum ether / acetone and petroleum ether / ethyl acetate in sequence, separated by dextran gel, combined by TLC spot plate, and separated by reverse phase silica gel column chromatography to obtain alopecuroides.

[0034] In addition, preferably, the Taiwania tsugae in step S1 is Taiwania tsugae stem or bark.

[0035] Preferably, the volume of water used in step S1 is 1 to 2 times that of the concentrate. The present invention has the following beneficial effects:

[0036] The present invention provides a new anti-cancer use of baldwood. Studies conducted in the present invention have shown that baldwood exhibits significant anti-cancer effects. Data from studies using Jurkat cells, Hela cells, and A549 cells show significant inhibitory effects on leukemia cells, cervical cancer cells, and lung cancer cells. Furthermore, baldwood is derived from plants and has good safety, making it extremely valuable in the development and preparation of anti-cancer drugs.

[0037] The present invention provides a new use of thuja suspensa in the general direction of anti-cancer, which not only provides a new choice for anti-cancer drugs, but also opens up a new direction for the utilization of thuja suspensa plant resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The effect of different concentrations of TSC-3 on the viability of three tumor cells, Jurkat, A549 and Hela (24h).

[0039] Figure 2 The effect of different concentrations of TSC-3 on the viability of three tumor cells, Jurkat, A549 and Hela (48h).

[0040] Figure 3 The effect of different concentrations of TSC-3 on the viability of three tumor cells, Jurkat, A549 and Hela (72h).

[0041] Figure 4 Effects of different concentrations of TSC-3 on the viability of Jurkat tumor cells (24h, 48h, 72h). DETAILED DESCRIPTION

[0042] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form.

[0043] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0044] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0045] The compound TSC-3 is disclosed in invention patent CN109232688B:

[0046]

[0047] Example 1 Determination of the antitumor activity of the compound TSC-3

[0048] 1. Test materials:

[0049] Human leukemia tumor cells (Hela), human non-small cell lung cancer cells (A549), and human acute T-cell leukemia cells (Jurkat) were provided by Southern Medical University;

[0050] 2. Determination method:

[0051] Preparation of 1× PBS buffer: 137mmol·L -1 NaCl, 2.7mmol·L -1 KCl, 10mmol·L -1 Na2HPO4·12H2O and 2mmol·L -1 KHPO4, pH = 7.4. After high-pressure sterilization, store in a refrigerator at 4°C until use.

[0052] Preparation of MTT solution: Weigh 0.5 g of MTT, add 100 mL of PBS buffer to completely dissolve it, and then filter it through a 0.22 μm filter membrane to obtain a concentration of 5 mg mL -1Wrap the MTT solution in tin foil and store it in a refrigerator at 4°C away from light for later use. Also pay attention to keep it away from light during the preparation of the solution.

[0053] Preparation of complete culture medium: 450 mL of cell culture medium (RPMI-1640) was added with 50 mL of fetal bovine serum (FBS) and 5 mL of antibiotics (100 U mL -1 Penicillin and 100 mg·L -1 Streptomycin), mix well, and store in refrigerator at 4℃ for later use.

[0054] Cell recovery: Remove the frozen tumor cells from the liquid nitrogen tank and confirm that the cryopreservation tube lid is tightly sealed and intact. Place the cryopreservation tube in a water bath at 37°C and shake the cryopreservation tube evenly back and forth to thaw quickly; transfer the completely dissolved cell cryopreservation solution to a sterile centrifuge tube containing 3mL of complete culture medium (incubated at 37°C), centrifuge at 1000rpm for 5 minutes, discard the supernatant, and then add 3mL of fresh complete culture medium. Gently pipette to fully suspend the cells into single cells. Transfer the cell suspension to a culture flask and place it in an incubator at 37°C and a CO2 concentration of 5%. After about 24 hours, replace the cell culture medium with fresh one after the cells have attached.

[0055] Cell culture and subculture: When the cell adherent growth area reaches 90%, discard the original culture medium, wash the cells 2-3 times with 1× PBS, add 0.5 mL of trypsin to digest the cells, wait until the cells are completely rounded, discard the trypsin, add 2 mL of fresh complete culture medium, gently pipette the cells to form a single-cell suspension, add fresh complete culture medium to 3 mL, pipette evenly, and continue culturing.

[0056] MTT assay for cell viability: The cell viability assay was performed according to the literature by Xu Zengguang and Rello-Varona et al. (Xu Zengguang, 2012; Rello-Varona et al., 2006). Cells in good condition were used for the experiment. A cell suspension with a density of 1 × 105 cells / mL was pipetted evenly and inoculated into a 96-well plate at 200 μL / well. A certain amount of TSC-3 was added to give a final concentration of 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 μM for the treated cells. The control contained only DMSO, and the organic solvent content was 1 / 1000. Each treatment was repeated three times. Toxicity tests were performed at 24, 48, and 72 h after drug treatment. After adding 20 μL of MTT to each well and incubating the cells for 4 hours, 150 μL of DMSO was added and the mixture was shaken for 10 minutes to completely dissolve the blue-purple crystalline formazan. The absorbance of all experimental groups was measured at 570 nm on a microplate reader. Succinate dehydrogenase in the mitochondria of living cells reduces MTT to water-insoluble blue-purple crystalline formazan, which precipitates in the cells; however, this phenomenon does not occur in dead cells. Adding DMSO dissolves the crystals, and the measured absorbance indirectly reflects the number of viable cells. The cell viability is the ratio of the absorbance of the treated group to that of the control group, calculated as follows:

[0057]

[0058] 3. Measurement results:

[0059] Activity of different concentrations of TSC-3 against Jurkat, A549 and Hela tumor cells:

[0060] Different concentrations of TSC-3 had a significant inhibitory effect on the viability of three tumor cells, Jurkat, A549 and Hela cells, and the inhibitory effect on Jurkat cells was particularly significant. With the increase of TSC-3 concentration and the extension of treatment time, cell viability continued to decrease, showing an obvious time-dose effect.

[0061] Specifically by Figure 1-4It can be seen that when A549 cells were treated with 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 μM TSC-3 for 24 h, the cell viabilities were 91.79%, 91.64%, 92.53%, 87.21%, 82.55%, 79.43%, 81.44%, 84.97% and 137.92%, respectively; when treated with 0.195-50 μM TSC-3 for 48 h, the cell viabilities were 91.79%, 91.64%, 92.53%, 87.21%, 82.55%, 79.43%, 81.44%, 84.97% and 137.92%, respectively. The viabilities of the cells were 84.36%, 82.13%, 75.14%, 71.03%, 69.05%, 65.36%, 68.56%, 76.03% and 104.53% respectively. At 72 hours of treatment, the viabilities of the cells in the 0.195-50 μM treatment groups were 87.33%, 69.24%, 54.42%, 51.37%, 52.21%, 51.58%, 49.66%, 56.43% and 42.37% respectively. It can be seen that TSC-3 has an inhibitory effect on the viability of A549 cells, and the inhibitory effect is most significant after 72 hours of treatment. When A549 cells were treated with TSC-3 for 24h and 48h, the cell viability of the 0.195-25μM treatment group decreased with the increase of drug concentration, but the cell viability of the high concentration (50μM) treatment group increased instead, which indicates that the effect of high concentration of the drug on cell viability needs further study.

[0062] When Hela cells were treated with TSC-3 for 24 h, the cell viabilities of the 0.195-50 μM treatment groups were 100.04%, 90.82%, 90.91%, 91.97%, 90.39%, 86.67%, 86.99%, 93.25% and 129.20%, respectively. When treated for 48 h, the cell viabilities of the 0.195-50 μM treatment groups were 76.70%, 73.1 The cytotoxicity of TSC-3 to Hela and A549 cells was similar, with longer treatment times producing significant cytotoxicity. When TSC-3 was used to treat Jurka cells for 24 h, the cell viabilities of the 0.195-50 μM treatment groups were 100.84%, 96.46%, 83.52%, 81.02%, 78.10%, 72.94%, 63.81%, 40.45% and 25.24%, respectively. When TSC-3 was used to treat Jurka cells for 48 h, the cell viabilities of the 0.195-50 μM treatment groups were 73.15%, 62.5 The cell viability of the 0.195-50 μM treatment groups was 35.87%, 24.93%, 15.57%, 13.38%, 14.04%, 14.40%, 12.25%, 11.29% and 10.25% after 72 hours of treatment. These data indicate that TSC-3 has the strongest cytotoxicity against Jurkat cells. Cell viability decreases with increasing concentration and prolonged treatment time. When TSC-3 is treated with Jurkat cells for 24, 48 and 72 hours, the cell viability of the high concentration (50 μM) treatment group is reduced by 75.60%, 54.59% and 25.62% compared with the low concentration (0.195 μM) treatment group, respectively. In addition, when TSC-3 acted on Jurkat cells for 48 hours, the cell viability of all treatment groups at 0.195-50 μM was no higher than 73.15%; when treated for 72 hours, the cell viability of all treatment groups at 0.195-50 μM was no higher than 35.87%.

[0063] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of tauranga or its structural analogues or pharmaceutically acceptable salts in the preparation of anticancer drugs.

2. Use of a composition in the preparation of an anticancer drug, wherein the composition contains tauranga or a structural analogue thereof or a pharmaceutically acceptable salt thereof, and also contains other anticancer drugs.

3. Use of a composition in the preparation of an anticancer drug, wherein the composition contains tauranga or a structural analogue thereof or a pharmaceutically acceptable salt thereof, and further contains a pharmaceutically acceptable excipient.

4. Use of a plant extract in the preparation of an anticancer drug, wherein the plant extract is a plant extract rich in tauranga or its structural analogues or pharmaceutically acceptable salts thereof.

5. Use of a plant extract in combination with other anticancer drugs in the preparation of anticancer drugs, wherein the plant extract is a plant extract rich in balsaminarin or its structural analogues or pharmaceutically acceptable salts thereof.

6. Use of a plant extract and a pharmaceutically acceptable excipient in the preparation of an anticancer drug, wherein the plant extract is a plant extract rich in balsaminarin or its structural analogues or pharmaceutically acceptable salts thereof.

7. The use according to any one of claims 1 to 6, characterized in that: The cancer is cervical cancer, leukemia or lung cancer.

8. The use according to any one of claims 4 to 6, characterized in that: The plant extract is a Taiwania truncatula extract.

9. An anticancer drug, characterized in that: The invention contains tauranga or its structural analogue or its pharmaceutically acceptable salt, and also contains pharmaceutically acceptable excipients and / or other anticancer drugs.

10. An anticancer drug, characterized in that: The invention contains a plant extract rich in balsaminarin or its structural analogues or its pharmaceutically acceptable salts, and also contains pharmaceutically acceptable excipients and / or other anticancer drugs.

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