Application of rosmarinic acid and sinomenine in synergistic treatment of diseases

Abstract

The invention provides application of rosmarinic acid and sinomenine in synergistic treatment of diseases, and belongs to the technical field of biological medicines. The invention provides an application of rosmarinic acid and / or sinomenine in preparation of drugs for treating diseases, both rosmarinic acid and sinomenine have obvious therapeutic effects on cancers such as lung cancer, colorectal cancer and glioma, and rosmarinic acid has the effect of enhancing sinomenine analgesia. The rosmarinic acid and the sinomenine are compounded to serve as the medicine composition and the medicine for treating the diseases, the remarkable synergistic interaction effect is shown, the dosage of a single medicine can be reduced through combined use of the rosmarinic acid and the sinomenine, the curative effect is improved, and therefore the side effects of the medicine are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of rosmarinic acid and sinomenine in the synergistic treatment of diseases. Background Art

[0002] Tumor in medicine refers to abnormal cell lesions, not necessarily a lump on the body. Cancer is the most common malignant tumor. Malignant tumors are divided into "carcinomas" derived from epithelium and "sarcomas" derived from mesenchyme. In malignant tumors, these proliferating cells, in addition to aggregating into a lump, will also spread to other parts for proliferation, causing harm to the human body. Cancer is recognized as a stubborn disease worldwide, bringing pain to patients and misfortune to families. In clinical practice, the inherent and acquired drug resistance or tolerance of malignant tumors to treatment methods is a key challenge, resulting in little impact of existing treatment strategies on overall survival. The imbalance between pro-apoptotic signals and anti-apoptotic signals in cancer cells is related to therapeutic drug resistance, cancer progression and metastasis, which are the main causes of cancer-related deaths.

[0003] Treating and eradicating cancer and relieving the pain of patients are problems that people have always been eager to solve. In recent years, the treatment of cancer still includes surgical treatment, radiotherapy, chemotherapy and traditional Chinese medicine treatment in Western medicine. In addition to certain limitations, treating cancer with Western medicine also affects normal organ tissues and cells while conquering cancer cells. Therefore, using Western medicine treatment methods for patients with poor physical condition or advanced tumors is undoubtedly worse. Traditional Chinese medicine treatment can treat both the symptoms and the root causes, with small side effects and is easily accepted by patients. However, for the treatment of cancer with traditional Chinese medicine, existing traditional Chinese medicine drugs have different compositions, different effects and different situations. Summary of the Invention

[0004] The present invention provides the application of rosmarinic acid and sinomenine in the synergistic treatment of diseases. Rosmarinic acid and sinomenine both show typical synergistic effects in the treatment of cancer and analgesia, and can be used in combination.

[0005] The present invention provides the application of rosmarinic acid and / or sinomenine in the preparation of drugs for treating cancer and analgesia.

[0006] In a preferred embodiment of the present invention, when applied to cells, the working concentration of rosmarinic acid is 20-40 μM, and the working concentration of sinomenine is 20 μM.

[0007] In a preferred embodiment of the present invention, the treatment of cancer and analgesia includes at least one of the following: (1) inhibiting cancer cell proliferation;

[0008] (2) inhibiting the formation of cancer cell clone colonies;

[0009] (3) inhibiting cancer cell migration and movement;

[0010] (4) Inhibit the invasion ability of cancer cells.

[0011] In a preferred embodiment of the present invention, the cancer cells include: lung cancer cells, colorectal cancer cells, and glioma cells.

[0012] The present invention also provides a pharmaceutical composition for treating cancer and / or analgesia, comprising rosmarinic acid and sinomenine.

[0013] In a preferred embodiment of the present invention, when applied to cells, the working concentration of rosmarinic acid is 20 - 40 μM, and the working concentration of sinomenine is 20 μM.

[0014] In a preferred embodiment of the present invention, when the pharmaceutical composition is used for treating cancer, the working concentration of rosmarinic acid is 20 - 40 μM, and the working concentration of sinomenine is 10 - 20 μM;

[0015] When the pharmaceutical composition is used for analgesia, based on the body weight of the mouse, the working concentration of rosmarinic acid is 10 - 40 mg / kg, and the working concentration of sinomenine is not less than 10 mg / kg.

[0016] The present invention also provides the use of the above pharmaceutical composition in the preparation of a drug for treating cancer and / or analgesia.

[0017] In a preferred embodiment of the present invention, the types of cancer include various common malignant tumors such as lung cancer, colorectal cancer, glioma, breast cancer, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer, melanoma, and leukemia.

[0018] The present invention also provides a drug for treating cancer and / or analgesia, the active ingredients of which include rosmarinic acid and sinomenine.

[0019] In a preferred embodiment of the present invention, the daily clinical dosage of rosmarinic acid in the drug is 10 - 1000 mg, and the daily clinical dosage of sinomenine is 10 - 1000 mg.

[0020] In a preferred embodiment of the present invention, the administration methods of the drug include oral administration, sublingual administration, injection administration, transdermal administration, or rectal instillation administration.

[0021] Beneficial effects: The present invention provides the use of rosmarinic acid and / or sinomenine in the preparation of a medicament for treating cancer and analgesia. Both rosmarinic acid and sinomenine exhibit significant therapeutic effects on cancers such as lung cancer, colorectal cancer, and glioma. After compounding rosmarinic acid and sinomenine, a significant synergistic effect is shown. The combined use of rosmarinic acid and sinomenine can reduce the dosage of a single drug and improve the curative effect, thereby reducing drug side effects. In another embodiment of the present invention, it is also confirmed that when rosmarinic acid and sinomenine are used in combination, the anti-inflammatory and antioxidant characteristics of rosmarinic acid can increase the analgesic effect of sinomenine, and the combination of the two has a synergistic analgesic effect. Description of the Drawings

[0022] Figure 1 Comparison chart of the proliferation number of A549 cells 5 days after adding drugs; * compared with the control group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ※ compared with the rosmarinic acid group, ※P < 0.05, ※※P < 0.01; # compared with the sinomenine group, #P < 0.05, ##P < 0.01, P < 0.001, #P < 0.0001, the same below;

[0023] Figure 2 Comparison chart of the proliferation number of HCT116 cells 5 days after adding drugs;

[0024] Figure 3 Statistical chart of the colony formation rate of A549 cells after 2 weeks of drug addition and culture;

[0025] Figure 4 Statistical chart of the colony formation rate of HCT116 cells after 2 weeks of drug addition and culture;

[0026] Figure 5 Migration result diagram of A549;

[0027] Figure 6 Migration cell number diagram of A549;

[0028] Figure 7 Migration result diagram of TJ905;

[0029] Figure 8 Migration cell number diagram of TJ905;

[0030] Figure 9 Invasion result diagram of A549;

[0031] Figure 10 Comparison chart of the invasion cell number of A549;

[0032] Figure 11 Invasion result diagram of TJ905;

[0033] Figure 12 It is a comparison graph of the number of cells invaded by TJ905;

[0034] Figure 13 It is the mechanical pain threshold of each treatment group in the sinomenine single-drug dose group of SNI model mice; where, #P<0.05, ##P<0.01, P<0.001, *P<0.05, ***P<0.001;

[0035] Figure 14 It is the mechanical pain threshold of each treatment group in the rosmarinic acid single-drug dose group of SNI model mice;

[0036] Figure 15 It is the mechanical pain threshold of each group in the experiment of combined administration (rosmarinic acid 20mg / kg + sinomenine 10mg / kg) of SNI model mice;

[0037] Figure 16 It is the cold pain score of each treatment group in the sinomenine single-drug dose group of SNI model mice; where, *P<0.05, **P<0.01;

[0038] Figure 17 It is the cold pain score of each treatment group in the rosmarinic acid single-drug dose group of SNI model mice;

[0039] Figure 18 It is the cold pain score of the combined administration group (rosmarinic acid 20mg / kg + sinomenine 10mg / kg) of SNI model mice in the administration experiment, where, **P<0.001. Detailed implementation manners

[0040] The present invention provides the use of rosmarinic acid and / or sinomenine in the preparation of drugs for treating cancer and relieving pain.

[0041] The rosmarinic acid referred to in the present invention is a water-soluble natural phenolic acid compound isolated from plants of the Labiatae, Boraginaceae, Cucurbitaceae, Tiliaceae or Umbelliferae families, and its molecular formula is: C 18 H 16 O 8 , CAS: 20283-92-5, and the structure is shown in Formula I:

[0042]

[0043] The molecular formula of the sinomenine described in the present invention is: C 19 H 23 NO 4 , CAS number: 115-53-7, and the structure is shown in Formula II:

[0044]

[0045] In a preferred embodiment of the present invention, experiments were conducted using rosmarinic acid at 20 - 40 μM, sinomenine at 20 μM, and a combination of 20 - 40 μM rosmarinic acid and 20 μM sinomenine. The types of cancer cells used in the experiments included lung cancer cells, colorectal cancer cells, and glioma cells, and it was confirmed that they have the effect of treating cancer, where treating cancer includes at least one of the following: (1) inhibiting the proliferation of cancer cells;

[0046] (2) inhibiting the formation of cancer cell colonies;

[0047] (3) inhibiting the migration and movement of cancer cells;

[0048] (4) inhibiting the invasive ability of cancer cells.

[0049] In another embodiment of the present invention, a spared nerve injury (SNI) model was used for verification, and it was proved that when rosmarinic acid and sinomenine are used in combination, the anti - inflammatory and antioxidant characteristics of rosmarinic acid can enhance the analgesic effect of sinomenine, and the combination of the two has a synergistic analgesic effect.

[0050] The present invention also provides a pharmaceutical composition for treating cancer and / or analgesia, comprising rosmarinic acid and sinomenine.

[0051] In a preferred embodiment of the present invention, the working concentration of rosmarinic acid is 20 - 40 μM, and the working concentration of sinomenine is 20 μM. In a preferred embodiment of the present invention, when the pharmaceutical composition is used to treat cancer, the working concentration of rosmarinic acid is 20 - 40 μM, and the working concentration of sinomenine is 10 - 20 μM; when the pharmaceutical composition is used for analgesia, based on the body weight of the mouse, the working concentration of rosmarinic acid is 10 - 40 mg / kg, and the working concentration of sinomenine is not less than 10 mg / kg.

[0052] The present invention also provides the use of the above - mentioned pharmaceutical composition in the preparation of a drug for treating cancer and / or analgesia.

[0053] In a preferred embodiment of the present invention, the types of cancer include various common malignant tumors such as lung cancer, colorectal cancer, glioma, breast cancer, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer, melanoma, and leukemia.

[0054] The present invention also provides a drug for treating cancer and / or analgesia, the active ingredients of which include rosmarinic acid and sinomenine.

[0055] The present invention does not particularly limit the dosage form and administration method of the drug, and it can be prepared based on the conventional dosage forms and preparation methods in the art. The optional administration methods include oral administration, sublingual administration, injection administration, transdermal administration, rectal drip administration, etc. When administering the drug, the daily clinical dosage of rosmarinic acid in the drug is 10-1000 mg, and the daily clinical dosage of sinomenine is 10-1000 mg. The drug of the present invention also includes pharmaceutically acceptable excipients.

[0056] To further illustrate the present invention, the following examples are used to describe in detail the application of rosmarinic acid and sinomenine provided by the present invention in synergistically inhibiting cancer and / or complications, but they should not be construed as limiting the protection scope of the present invention.

[0057] In the examples of the present invention, the rosmarinic acid used was purchased from Beijing Bailingwei Technology Co., Ltd., and the sinomenine was purchased from Beijing Bailingwei Technology Co., Ltd.

[0058] Example 1

[0059] The inhibitory effect of rosmarinic acid, sinomenine, and the combination of rosmarinic acid and sinomenine on the proliferation of cancer cells.

[0060] A549 lung cancer cells and HCT116 colorectal cancer cells were seeded in 96-well plates at a density of 3000 cells / well, with 3 wells / group, and were respectively set as the control group (DMSO), the rosmarinic acid group (final concentration of 40 μM), the sinomenine group (final concentration of 20 μM), and the combination group (40 μM rosmarinic acid + 20 μM sinomenine); on the 5th day after administration, the absorbance of the cell culture medium was measured by the CCK8 method to calculate the cell number.

[0061] The results are as Figure 1 and Figure 2 shown. When using sinomenine or rosmarinic acid alone, the cell number of A549 lung cancer cells did not significantly decrease (sinomenine), or there was only a weak decrease of about 5% (rosmarinic acid), while the cell number of A549 lung cancer cells in the combination group significantly decreased, with a reduction ratio of about 20%; for HCT116 colorectal cancer cells, when using sinomenine or rosmarinic acid alone, the cell number of HCT116 colorectal cancer cells could be reduced to a certain extent, with a reduction ratio of about 10-15%, and the combination could further reduce the cell number of HCT116 colorectal cancer cells. Compared with the control group, the inhibition rate of the combination on the proliferation of HCT116 colorectal cancer cells was about 50%. The inhibitory effects of the combination group on the proliferation of A549 lung cancer cells and HCT116 colorectal cancer cells were significantly stronger than those of using sinomenine or rosmarinic acid alone.

[0062] Example 2

[0063] Inhibitory effects of rosmarinic acid, sinomenine, and the combination of rosmarinic acid and sinomenine on the colony formation of cancer cells.

[0064] A549 lung cancer cells were seeded into 6-well plates at a density of 500 cells / well, with 2 wells / group, and were respectively set as the control group (DMSO), the rosmarinic acid group (final concentration of 20 μM), the sinomenine group (final concentration of 20 μM), and the combination group (20 μM rosmarinic acid + 20 μM sinomenine); after culturing for 2 weeks, the cells were fixed with methanol and stained with crystal violet, and the number of clone clusters with more than 50 cells per well was counted. The colony formation rate = the number of clone clusters / the number of originally seeded cells.

[0065] The results were as Figure 3 shown. The colony formation rate of A549 lung cancer cells in the control group was approximately 65%, and when using sinomenine alone, the colony formation rate of A549 lung cancer cells was reduced to 40%. When using rosmarinic acid alone, the colony formation rate of A549 lung cancer cells was reduced to about 10%. The combination group further inhibited the colony formation ability of A549 lung cancer cells, and no obvious clone clusters were formed.

[0066] HCT116 colorectal cancer cells were seeded into 6-well plates at a density of 800 cells / well, with 2 wells / group, and were respectively set as the control group (DMSO), the rosmarinic acid group (final concentration of 20 μM), the sinomenine group (final concentration of 20 μM), and the combination group (20 μM rosmarinic acid + 20 μM sinomenine); after culturing for 2 weeks, the cells were fixed with methanol and stained with crystal violet, and the number of clone clusters with more than 50 cells per well was counted. The colony formation rate = the number of clone clusters / the number of originally seeded cells.

[0067] The results were as Figure 4 shown. The colony formation rates of HCT116 colorectal cancer cells in the control group and the sinomenine group were close, approximately 80%. The colony formation rate of HCT116 colorectal cancer cells in the rosmarinic acid group decreased slightly, approximately 75%. The combination could reduce the colony formation rate of HCT116 colorectal cancer cells to about 50%, which was much lower than the colony formation rates of HCT116 colorectal cancer cells when using sinomenine or rosmarinic acid alone.

[0068] Example 3

[0069] Inhibitory effects of rosmarinic acid, sinomenine, and the combination of rosmarinic acid and sinomenine on the migration of cancer cells.

[0070] At A549 lung cancer cells were seeded in 6-well plates at a density of cells per well, and treated with DMSO, rosmarinic acid, and sinomenine, respectively. The final concentration of the rosmarinic acid group was 20 μM, the final concentration of the sinomenine group was 20 μM, and the final concentration of the combination group was 20 μM rosmarinic acid + 20 μM sinomenine. After 48 h of drug treatment, the cells in each group were digested and counted, and the cells were resuspended in serum-free medium at a density of

[0071] cells / ml. Complete medium was added to the lower chamber of the Transwell plate, and 100 μL of the cell suspension was added to the upper chamber. The cells were cultured at 37 °C for 20 h. The excess cells in the upper chamber were wiped off, the chamber was fixed with methanol and stained with crystal violet, and then pictures of the cells in the chamber were taken under a microscope. Figure 5 The migration of cells in each treatment group in the chamber is shown in Magnification

[0072] The purple color represents the migrated cells. Compared with the control group, when rosmarinic acid or sinomenine was used alone, the number of migrated A549 lung cancer cells did not increase or decrease significantly. However, the number of migrated A549 lung cancer cells in the combination group decreased significantly. Figure 6 At the same time, 4 microscopic fields were randomly selected, and the cells in the fields were counted. The average number ± SEM of the migrated cells in each treatment group is shown in

[0073] Example 4

[0074] Study on the effect of the combination of rosmarinic acid and sinomenine on the migration of TJ905 glioma cells.

[0075] TJ905 glioma cells were seeded in 6-well plates at a density of cells per well, and treated with DMSO, rosmarinic acid, and sinomenine, respectively. The final concentration of the rosmarinic acid group was 40 μM, the final concentration of the sinomenine group was 20 μM, and the final concentration of the combination group was 40 μM rosmarinic acid + 20 μM sinomenine. After 48 h of drug treatment, the cells in each group were digested and counted, and the cells were resuspended in serum-free medium at a density of cells / ml. Complete medium was added to the lower chamber of the Transwell plate, and 100 μL of the cell suspension was added to the upper chamber. The cells were cultured at 37 °C for 20 h. The excess cells in the upper chamber were wiped off, the chamber was fixed with methanol and stained with crystal violet, and then pictures of the cells in the chamber were taken under a microscope.

[0076] The cell migration in each treatment group in the chamber is as follows Figure 7 shown, magnification Purple represents the migrating cells. Compared with the control group, rosmarinic acid alone has no obvious effect on the migration ability of TJ905 glioma cells. Sinomenine alone or in combination with rosmarinic acid can significantly inhibit the migration of TJ905 glioma cells. The inhibitory effects of the sinomenine group and the combination group on the migration ability of TJ905 glioma cells are similar.

[0077] Randomly select 4 microscopic fields and count the cells in the fields. The average number ± SEM of migrating cells in each treatment group is as follows Figure 8 shown. Compared with the control group, rosmarinic acid alone cannot effectively reduce the number of migrating TJ905 glioma cells. Sinomenine alone can partially inhibit the migration of TJ905 glioma cells. The combination group further enhances the reduction of the number of migrating cells of TJ905 glioma cells, and the effect of the combination group on reducing the number of migrating cells of TJ905 glioma cells is significantly better than that of sinomenine or rosmarinic acid alone.

[0078] Example 5

[0079] Study on the effect of the combination of rosmarinic acid and sinomenine on the invasion ability of A549 lung cancer cells.

[0080] Seed A549 lung cancer cells into a 6-well plate at a density of cells / well, and treat them with DMSO, rosmarinic acid, and sinomenine respectively. The final concentration of the rosmarinic acid group is 40 μM, the final concentration of the sinomenine group is 20 μM, and the final concentration of the combination group is 40 μM rosmarinic acid + 20 μM sinomenine; after 48 h of drug treatment, digest the cells in each group and count them, resuspend the cells with serum-free medium at a density of cells / ml. Precoat the Transwell chamber with Matrigel in advance. After it solidifies, add complete medium to the lower chamber of the Transwell plate, and add 100 μL of cell suspension to the upper chamber. Incubate at 37 °C for 24 h. Wipe off the excess cells and Matrigel in the upper chamber, fix the chamber with methanol and stain with crystal violet, and then take pictures of the cells in the chamber under the microscope.

[0081] The cell migration in each treatment group in the chamber is as follows Figure 9 shown, magnification Purple represents the cells capable of destroying Matrigel for invasion and metastasis. Compared with the control group, using rosmarinic acid alone, sinomenine alone, or their combination can inhibit the invasion and metastasis ability of A549 lung cancer cells to varying degrees. Among them, the inhibitory effect of rosmarinic acid alone on the invasion and metastasis ability of A549 lung cancer cells is the weakest, followed by sinomenine, and the combination group has the strongest inhibitory effect on the invasion and metastasis ability of A549 lung cancer cells.

[0082] Randomly select 4 microscopic fields and count the cells in the fields. The average number of migrated cells ± SEM in each treatment group is as Figure 10 shown. Compared with the control group, using sinomenine alone or rosmarinic acid alone can effectively reduce the number of A549 lung cancer cells that undergo invasion and metastasis. The combination group further reduces the number of A549 lung cancer cells that undergo invasion and metastasis. The combination group has a better inhibitory effect on reducing the number of A549 lung cancer cells that undergo invasion and metastasis than using sinomenine alone or rosmarinic acid alone.

[0083] Example 6

[0084] Study on the effect of the combination of rosmarinic acid and sinomenine on the invasion ability of TJ905 glioma cells

[0085] At a density of cells / well, inoculate TJ905 glioma cells into 6-well plates and treat them with DMSO, rosmarinic acid, and sinomenine respectively. The final concentration of the rosmarinic acid group is 20 μM, the final concentration of the sinomenine group is 20 μM, and the final concentration of the combination group is 20 μM rosmarinic acid + 20 μM sinomenine; after 48 hours of drug treatment, digest the cells in each group and count them, resuspend the cells with serum-free medium at a density of cells / ml. Pre-lay Matrigel in the Transwell chamber in advance. After it solidifies, add complete medium to the lower chamber of the Transwell plate and add 100 μL of cell suspension to the upper chamber. Culture at 37 °C for 24 hours, wipe off the excess cells and Matrigel in the upper chamber, fix the chamber with methanol and stain with crystal violet, and then take pictures of the chamber cells under the microscope.

[0086] The migration of cells in the chambers of each treatment group is as Figure 11 shown, magnification Purple represents the cells capable of destroying Matrigel for invasion and metastasis. Compared with the control group, using rosmarinic acid alone, sinomenine alone, or their combination can inhibit the invasion and metastasis ability of TJ905 glioma cells to varying degrees. Among the three treatment groups, the inhibitory effect of rosmarinic acid alone on the invasion and metastasis ability of TJ905 glioma cells is the weakest, followed by sinomenine, and the combination group has the strongest inhibitory effect on the invasion and metastasis ability of TJ905 glioma cells.

[0087] Four fields of view were randomly selected and the cells in the fields of view were counted. The mean ± SEM of the number of cells that migrated in each treatment group was shown in Figure 2. Figure 12 As shown, compared with the control group, sinomenine or rosmarinic acid alone or the combination of the two can effectively reduce the number of TJ905 glioma cells that undergo invasion and metastasis, and the combination group has a better effect on reducing the number of TJ905 glioma cells that undergo invasion and metastasis than rosmarinic acid alone. Compared with the sinomenine group, the number of TJ905 glioma cells that undergo invasion and metastasis in the combination group was slightly reduced, but there was no statistical difference.

[0088] Example 7

[0089] Animal experiment-establishment of sciatic nerve branch injury (SNI) model:

[0090] Materials: SPF-grade male C57BL / 6N mice aged 6 to 8 weeks and weighing 20 to 22 g (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were raised in the Experimental Animal Center of the Institute of Materia Medica, Chinese Academy of Medical Sciences. The breeding environment was SPF-grade, with an indoor temperature of 20±1°C, a humidity of 50±5%, and a 12-h day and night alternation. Mice were free to eat and drink. Experimental operations were performed after one week of adaptive breeding. All operations followed the relevant guidelines of the International Association for the Study of Pain and were approved by the Experimental Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

[0091] (1) The mice were anesthetized by intraperitoneal injection of chloral hydrate (5%). After anesthesia, the mice were placed in the right lateral decubitus position, and the left foot was fixed to the operating table with tape. The skin of the left hind limb was prepared on the outside. The mouse skin was cut longitudinally along the femur for about 1 cm at the depression on the outside of the femur using scissors, avoiding the subcutaneous blood vessels, and the biceps femoris was cut along the femur to expose the sciatic nerve and its three terminal branches: the sural nerve, the common peroneal nerve, and the tibial nerve. The sural nerve is thinner and branches first from the upstream, which needs to be retained and should not be touched or pulled during the operation; the common peroneal nerve and the tibial nerve are thicker and run parallel to each other. Both are ligated together using No. 7 sutures, and 2 mm of nerve is removed from the distal end of the ligation point. The torn muscles and skin were replaced, and the muscles and skin were sutured layer by layer using No. 5 sutures. If there is bleeding during the operation, use sterile cotton balls to press and stop the bleeding. After the operation, the mice were placed on a heating pad to wait for awakening from anesthesia. After awakening, they were placed back in the same cage as before the operation, and the activity of the mice was observed. About 10 days after surgery, the dorsum of the mouse's operated hind paw was seen to be arched, and the operated foot tried not to touch the ground when walking, showing an obvious pain response, indicating that the model was successfully constructed and subsequent experiments could be started.

[0092] (2) randomly dividing the mice in step (1) into a solvent control group, a rosmarinic acid single-dose group, a sinomenine single-dose group, and a combined drug group;

[0093] The rosmarinic acid single-drug dose group was randomly and evenly divided into a low-dose group and a high-dose group, with 12 mice in each group;

[0094] The sinomenine single-drug dose group was randomly and evenly divided into a low-dose group and a high-dose group, with 12 mice in each group;

[0095] The remaining treatment groups had 12 mice in each group;

[0096] (3) Preparation of drug solutions

[0097] Rosmarinic acid drug solution: Weigh the corresponding mass of rosmarinic acid drug powder using an analytical balance and dissolve it in the corresponding volume of ddH 2 O;

[0098] Sinomenine drug solution: Weigh the corresponding mass of sinomenine drug powder using an analytical balance and dissolve it in the corresponding volume of ddH 2 O;

[0099] After the above solutions were prepared, administration was carried out in the manner shown in Table 1. During administration, all were by oral gavage for 7 consecutive days.

[0100] Table 1 Administration method

[0101]

[0102] Test Example 1

[0103] (1) Measurement of mechanical pain threshold

[0104] After administration, at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h respectively, refer to the existing technology (Gao T#, Li T#, Jiang W#, Fan W#, Xu X-J, Zhao X, Yin Z, Guo H, Wang L, Gao J*, Han Y*, Jiang J-D* and Wang D*. Antinociceptive Effects of Sinomenine Combined With Ligustrazine or Paracetamol in Animal Models of Incisional and Inflammatory Pain. Front. Physiol. 2021; 11:523769. doi:10.3389 / fphys.2020.523769.) to measure the mechanical pain threshold and draw a curve.

[0105] According to Figure 13It can be seen that in SNI model mice, when the application doses of sinomenine were 20 mg / kg and 40 mg / kg, obvious analgesic effects were produced under the detection of von Frey filaments. Compared with the solvent control group, in the sinomenine 40 mg / kg group, the PWT (paw withdrawal threshold) began to increase from 0.5 h, and significantly increased at 1 h, 3 h, 4 h, 6 h, and 8 h, reaching the highest value at 1 - 3 h and remaining until 6 h; when the application dose of sinomenine was 20 mg / kg, it had a relatively weak effect on the mechanical pain threshold of SNI model mice, and there were significant differences only at the time points of 2 - 4 hours compared with the excipient control group; when the application dose of sinomenine was 10 mg / kg, there was no significant difference in the mechanical pain threshold of SNI model mice compared with the excipient control group.

[0106] According to Figure 14 It can be seen that in SNI model mice, when the application doses of rosmarinic acid were 10 mg / kg, 20 mg / kg, and 40 mg / kg, no obvious analgesic effects were produced under the detection of von Frey filaments.

[0107] According to Figure 15 It can be seen that the basic pain threshold test was carried out 1 day before the start of the measurement, and there was no significant difference in the PWT values of mice in each group. By comprehensively comparing the rosmarinic acid 20 mg / kg group, the sinomenine 10 mg / kg group, and the rosmarinic acid 20 mg / kg + sinomenine 10 mg / kg group, it can be seen that among the SNI model mice in the three groups, only the mechanical pain threshold of the rosmarinic acid 20 mg / kg + sinomenine 10 mg / kg group was significantly different from that of the solvent control group.

[0108] (2) Measurement of cold pain threshold

[0109] After administration, the cold pain thresholds were measured at 1 h, 2 h, 4 h, and 6 h respectively, and the dose - dependent curve was plotted.

[0110] According to Figure 16 It can be seen that in SNI model mice, when the application dose of sinomenine was 40 mg / kg, obvious analgesic effects were produced in the detection of cold pain by the experimental method of dropping acetone. Compared with the solvent control group, the pain scores measured in the sinomenine 40 mg / kg group decreased at the 2 h and 4 h time points. When the application doses of sinomenine were 20 mg / kg and 10 mg / kg, there was no obvious effect on the cold pain threshold of SNI model mice;

[0111] According to Figure 17 It can be seen that in SNI model mice, when the application doses of rosmarinic acid were 10 mg / kg, 20 mg / kg, and 40 mg / kg, no obvious analgesic effects were produced in the detection of cold pain by the experimental method of dropping acetone, and no changes in the normal physiological states (respiration, heart rate) of mice were observed;

[0112] According to Figure 18 It can be seen that in the SNI model mice, the basic cold pain threshold test was conducted 1 day before the start of the measurement, and there was no significant difference in the pain scores among the mice in each group. According to Figure 6 It can be seen that the basic pain threshold test was conducted 1 day before the start of the measurement, and there was no significant difference in the cold pain scores among the mice in each group. By comprehensively comparing the groups of rosmarinic acid at 20 mg / kg, sinomenine at 10 mg / kg, and rosmarinic acid at 20 mg / kg + sinomenine at 10 mg / kg, it can be seen that among the SNI model mice in the three groups, only the cold pain score of the group of rosmarinic acid at 20 mg / kg + sinomenine at 10 mg / kg was significantly different from that of the solvent control group.

[0113] (3) Determination of Jin Zhengjun q value

[0114] The Jin Zhengjun q value is a calculated value used to evaluate whether there is a synergistic inhibitory effect between two different drugs.

[0115] The calculation formula is: q = E a+b / (E a +E b -E a ×E b ); where E a , E b are the inhibition rates of the two inhibitors acting alone, and E a+b is the inhibition rate of the combined action of the two inhibitors (i.e., the inhibition rate of combined medication). The numerator in the calculation formula represents the "measured combined effect", and the denominator represents the "expected combined effect"; different numerical ranges of the Jin Zhengjun q value represent three results respectively:

[0116] q < 0.85: There is an antagonistic effect between the inhibitory effects of the two drugs;

[0117] 0.85 ≤ q < 1.15: The inhibitory effects of the two drugs are a simple additive effect;

[0118] q ≥ 1.15: There is a synergistic effect between the inhibitory effects of the two drugs.

[0119] After calculation (Tables 2 and 3), the Jin Zhengjun q values of the mechanical pain experiment and the cold pain experiment were 4.82 and 3.53 respectively, indicating that sinomenine and rosmarinic acid showed an obvious analgesic synergistic effect on the mouse peripheral neuropathic pain model, and the two can be prepared into a drug combination with synergistic analgesia.

[0120] Table 2 Inhibitory effect of the combination of sinomenine and rosmarinic acid on mechanical pain in the peripheral neuropathic pain model

[0121] Group Foot-withdrawal threshold (g) Pain inhibition rate (%) Synergy index Sinomenine 10 mg / kg 0.30±0.18 8±10 Rosmarinic acid 20 mg / kg 0.44±0.16 16±9 Sinomenine 10 mg / kg + Rosmarinic acid 20 mg / kg 1.00±0.23 54±12 4.82 Solvent control group 0.14±0.04 0

[0122] Table 3 Inhibitory effect of the combination of sinomenine and rosmarinic acid on cold pain in the peripheral neuropathic pain model

[0123] Group Cold pain score Pain inhibition rate (%) Synergy index Sinomenine 10 mg / kg 2.33±0.47 -3±21 Rosmarinic acid 20 mg / kg 1.91±0.64 15±29 Sinomenine 10 mg / kg + Rosmarinic acid 20 mg / kg 1.25±0.43 44±19 3.53 Solvent control group 2.25±0.43 0

[0124] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of rosmarinic acid and / or sinomenine in the preparation of drugs for treating cancer and / or relieving pain.

2. The application according to claim 1, characterized in that: When applied to cells, the working concentration of rosmarinic acid is 20-40 μM, and the working concentration of sinomenine is 10-20 μM.

3. The use according to claim 1 or 2, characterized in that: The cancer treatment includes at least one of the following: (1) inhibiting cancer cell proliferation; (2) Inhibit the formation of cancer cell clones; (3) Inhibit cancer cell migration; (4) Inhibit the invasive ability of cancer cells.

4. The use according to claim 3, characterized in that: The cancer cells include lung cancer cells, colorectal cancer cells and glioma cells.

5. A pharmaceutical composition for treating cancer and / or relieving pain, characterized in that: Includes rosmarinic acid and sinomenine.

6. The pharmaceutical composition according to claim 5, characterized in that When applied to cells, the working concentration of rosmarinic acid is 20-40 μM, and the working concentration of sinomenine is 10-20 μM.

7. Use of the pharmaceutical composition according to claim 5 or 6 in the preparation of drugs for treating cancer and / or relieving pain.

8. The use according to claim 7, characterized in that: The types of cancer include lung cancer, colorectal cancer, glioma, breast cancer, prostate cancer, gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovarian cancer, cervical cancer, endometrial cancer, kidney cancer, bladder cancer, thyroid cancer, melanoma and leukemia.

9. A drug for treating cancer and / or relieving pain, characterized in that: Active ingredients include rosmarinic acid and sinomenine.

10. The drug according to claim 9, characterized in that: The daily clinical dosage of rosmarinic acid in the medicine is 10 to 1000 mg, and the daily clinical dosage of sinomenine is 10 to 1000 mg.

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