Application of Xiaoxiao pill extract in preparation of medicine for treating breast cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a Xiaoxiao pill extract in preparation of a medicine for treating breast cancer, an extraction method of the Xiaoxiao pill extract comprises the following steps: taking Xiaoxiao pill pills, adding the Xiaoxiao pill pills into a 1640 culture medium, soaking for the first time, performing ultrasonic treatment, soaking for the second time, taking supernatant, and filtering to obtain filtrate which is the Xiaoxiao pill extract; the mass volume ratio of the Xiaoxiao pills to the 1640 culture medium is 3g: (30mL-40mL). The obvious effect of the Xinxiao pill extract in treating the breast cancer is found for the first time. The discovery not only breaks through the traditional treatment limitation of Xiaoxiao pills for ulcers and pyogenic infections, expands the clinical application range of the Xiaoxiao pills, but also combines the traditional Chinese medicines with the modern bioinformatics technology through the modern medicine relocation strategy, verifies the anti-breast cancer activity of the Xiaoxiao pills, can be prepared into various dosage forms such as tablets, capsules and the like with various pharmaceutic adjuvants, and has wide application prospects. The clinical applicability is good.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of an extract of Xingxiao Pills in preparing a drug for treating breast cancer. Background Art

[0002] The formula for Xingxiao Pills, originating from Wang Hongxu's "Complete Collection of Surgical Symptoms and Treatments, Volume 4" in the Qing Dynasty, boasts a 150-year history of dialectical treatment. As an exclusive product of Beijing Tong Ren Tang (National Medicine Approval Number: Z11020502), it promotes blood circulation, reduces swelling, and relieves pain. It is used for carbuncles, swelling, and pain caused by hard masses. The formula consists of realgar, musk, frankincense, and myrrh. Realgar, first mentioned in the Shennong Bencao Jing (Classic of Materia Medica), is warm in nature, pungent in flavor, and toxic. It is effective in tackling tough problems, dissipating accumulations, and clearing dampness and phlegm, making it the main ingredient. Musk is pungent and warm in nature, promoting blood circulation and unblocking meridians. With the help of musk, realgar has a profound phlegm-clearing and blood-stasis-removing power. Frankincense and myrrh, as auxiliary ingredients, promote blood circulation and qi, eliminate stasis, dissipate swelling, and relieve pain. Modern research has shown that Xingxiao Pills are effective in treating advanced non-small cell lung cancer and nodular goiter. Despite its long history, Xingxiao Pills' clinical application is limited, and reports on its use are scarce, significantly hindering its clinical application. Therefore, it is necessary to reposition the Xingxiao Pills, which will help expand their scope of application and promote their modernization research. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides the use of Xingxiao Pills extract in the preparation of a drug for treating breast cancer.

[0004] The present invention is achieved through the following technical solutions: The invention discloses an extract of Xingxiao Pills for preparing a drug for treating breast cancer. The extract of Xingxiao Pills comprises the following steps: taking Xingxiao Pills, adding them to 1640 culture medium for a first soak, ultrasonicating them, taking the supernatant after a second soak, filtering them, and obtaining the filtrate as the Xingxiao Pills extract; and the mass-to-volume ratio of the Xingxiao Pills to the 1640 culture medium is 3 g: 30 mL to 40 mL.

[0005] Preferably, the first soaking time is 12h~24h; the second soaking time is 48h~72h.

[0006] Preferably, the ultrasonic frequency is 25kHz~45kHz; and the ultrasonic time is 1h~2h.

[0007] Preferably, the filtration is performed through a 0.1 μm to 1 μm filter membrane.

[0008] Preferably, the Xingxiao Pills extract is the only active ingredient in the drug for treating breast cancer.

[0009] Preferably, the concentration of the Xingxiao Pills extract in the preparation of the drug for treating breast cancer is 0.1 mg / mL to 10 mg / mL.

[0010] Preferably, the medicine includes the Xingxiao Pills extract and also includes pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0012] Preferably, the dosage form of the drug includes enema, tablet, capsule, granule, oral suspension, powder or ointment.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the use of an extract from Xingxiao Pills in the preparation of a drug for treating breast cancer. The extraction method for the Xingxiao Pills extract comprises taking Xingxiao Pills, adding them to 1640 culture medium for a first soak, then sonicating them for a second soak, collecting the supernatant, and filtering the resulting filtrate to obtain the Xingxiao Pills extract. The mass-to-volume ratio of the Xingxiao Pills to the 1640 culture medium is 3 g:30 mL to 40 mL. Experimental verification demonstrates for the first time the significant efficacy of the Xingxiao Pills extract in treating breast cancer. This discovery not only transcends the traditional treatment limitations of Xingxiao Pills for carbuncle and swelling, expanding its clinical application, but also demonstrates its anti-breast cancer activity through a modern drug repositioning strategy that combines traditional Chinese medicine with modern bioinformatics technology. The standardized extraction method provided by the present invention ensures the stability and bioavailability of the active ingredient, and the extract can be formulated with a variety of pharmaceutical excipients into various dosage forms, such as tablets and capsules, demonstrating excellent clinical applicability. This invention not only provides a new drug option for breast cancer treatment, but also demonstrates its "new uses" strategy for modernizing traditional Chinese medicine research and international development, demonstrating significant scientific value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is the UPLC-MS total ion map of the aqueous extract of Xingxiao Pills; Figure 1 In the equation, A is a positive ion and B is a negative ion.

[0016] Figure 2 The figure shows the dose-dependent inhibitory effect of the Xingxiao Pill extract on three breast cancer cell lines.

[0017] Figure 3 This figure shows the effect of the Xingxiao Pill extract of the present invention on the biological behavior of 4T1 breast cancer cells; Figure 3 In the figure, A is the time-dependence of the inhibition of 4T1 cell proliferation by Xingxiaowan extract; B is the result of cloning experiment; C is the corresponding statistical graph of cloning experiment results; D is the result of migration experiment; E is the corresponding statistical graph of migration experiment results. DETAILED DESCRIPTION

[0018] To facilitate understanding of the present invention, the present invention will be described more fully below, along with preferred embodiments thereof. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0020] The beneficial effects of the present invention are described below by means of specific embodiments: The Xingxiao Pills used in the present invention were purchased from Beijing Tongrentang Co., Ltd. (Beijing, China, 21041734).

[0021] Example 1 1. Prediction of the new efficacy of Xingxiao Pills 1. Determination of differentially expressed genes Three different doses of Xingxiao Pills (0.617 g / kg, 1.233 g / kg, and 2.466 g / kg) were used to intervene in Lewis subcutaneous transplanted tumor animal models, and the obtained tumor tissues were subjected to transcriptomic sequencing. The measured transcriptomic data were normalized and standard difference analysis was performed using the limma package. p <0.05 is a differentially expressed gene, log2 FC >0.5 indicates up-regulated genes, log2 FC A p-value < -0.5 indicated downregulated genes. Differentially expressed genes were obtained for low, medium, and high doses, as well as for the active drug. The optimal dosage was determined based on the number of genes intersecting between the different doses of Xingxiao Pills and the active drug, thereby identifying differentially expressed genes for subsequent analysis. The number of genes intersecting between the low, medium, and high doses was 41, 61, and 42, respectively. Therefore, the differentially expressed genes obtained for the medium dose were selected for subsequent analysis, resulting in 81 downregulated genes and 211 upregulated genes.

[0022] 2. Using the Cmap database to predict the efficacy of new drugs The 81 downregulated genes and 211 upregulated genes were converted to human genes using an R package. The converted differentially expressed genes were used as input data for the Cmap database for gene expression similarity comparison, resulting in a list with similarity scores ranging from -100 to 100, as shown in Table 1. Four of the top five small molecule compounds are MTOR inhibitors, and MTOR inhibitors are ranked first in the Cmap Class module. Therefore, Xingxiao Pills are predicted to have similar pharmacological effects to MTOR inhibitors.

[0023] MTOR is a major cellular crossroads in the mTOR signaling pathway, playing a crucial role in diverse cellular processes, including protein synthesis, cell motility, cell proliferation and survival, gene transcription, and autophagy. As the master switch for cellular metabolism, mTOR is a highly effective target for anti-tumor drugs. Inhibiting overactivated mTOR signaling in tumors can produce a range of potent anti-tumor effects. Consequently, many mTOR inhibitors are clinically used as anti-cancer drugs. Furthermore, a search of five small molecule compounds with scores greater than 90 in the Durg bank revealed that both AZD-8055 and OSI-027 have completed clinical trials for the treatment of solid tumors. A literature review revealed that the top-scoring small molecule compound, KU-0063794, can be used in combination with docetaxel to treat triple-negative breast cancer. Therefore, Xingxiao Pills is predicted to have pharmacological activity against breast cancer.

[0024] Table 1 Cmap database score table 2. In vitro validation of Xingxiao Pills’ anti-breast cancer effect 1. Identification of the full ingredients of Xingxiao Pills Xingxiao pills were soaked in water for 24 h and ultrasonically treated for 2 h at an ultrasonic frequency of 25 kHz. The soaking was continued for 48 h, and the supernatant was removed by centrifugation. The solution was then analyzed by UPLC-Q-TOF-MS / MS through a 0.22 μm water film.

[0025] Ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS / MS) analysis was performed using an Agilent Technologies 1290 Infinity II system coupled with a 6560 Q-TOF LC / MS. The chromatographic column was a C8 (2.1 × 100 mm, 1.8 μm, water) column at 30°C. The flow rate was 0.3 mL / min, and the injection volume was 5 μL. The mobile phase consisted of a mixture of 0.1% formic acid in water (A) and acetonitrile (B), with a gradient elution rate of: 20% B (3 min); 50% B (10 min); 80% B (20 min); 100% B (25 min); 100% B (30 min); 20% B (31 min); and 20% B (35 min). The instrument parameters for the Agilent 6560 Q-TOF LC / MS analysis were as follows: nitrogen drying gas temperature 350°C, nitrogen sheath gas temperature 225°C, and nitrogen drying gas flow rate 7 L·min -1 , nitrogen sheath gas flow rate 12 L·min -1 The concentrations were 4000 V, capillary voltage 4000 V, spray pressure 25 psi, and collision energy 10 V, 20 V, and 40 V. The mass spectra covered the m / z range 50–1200. Data were analyzed using Qualitative Navigator software (version B.08.00).

[0026] A total of 42 compounds were identified in the analysis, and their details are shown in Table 2. Figure 1 shown.

[0027] Table 2 Analysis of the complete components of the aqueous extract of Xingxiao Pills 2. Verification of activity by in vitro experiments 1) Drug preparation: Take 3 g of Xingxiao Pills, add them to 30 mL of 1640 culture medium, soak for 24 hours, and ultrasonicate for 2 hours at a frequency of 25 kHz. After the second soaking for 48 hours, take the supernatant, filter it through a 0.22 filter membrane, and the filtrate obtained is the Xingxiao Pills extract, which is used for cell administration and stored in aliquots at -20°C.

[0028] 2) CCK-8 cell proliferation activity assay 100 μL of culture medium containing 10,000 cells was added to each well of a 96-well plate. After 24 hours of incubation, 100 μL of Xingxiaowan extract at different concentrations ranging from 0 mg / mL to 10 mg / mL was added. After 24 hours, 10 μL of CCK-8 reagent was added, and after 40 minutes, the absorbance was measured at 450 nm using a microplate reader. The control group was the group without Xingxiaowan interference. The blank group was the group without cells but with culture medium added only. Cell viability = (OD 给药组 –OD 空白组 ) / (OD 对照组 –OD 空白组 ) × 100%. The present invention relates to three breast cancer cell lines: MCF-7, 4T1, and MDA-MB-231. The results are as follows Figure 2 Xingxiao Pills have inhibitory effects on all three breast cancer cell lines in a dose-dependent manner. 50 The lowest 4T1 was studied for time dependence, and the results were as follows Figure 3 As shown in Figure A, the inhibitory effect of Xingxiao Pills on 4T1 cells is time-dependent.

[0029] 3) Cloning experiments Add 2 mL of complete culture medium containing 1000 cells to each well of a 6-well plate and culture for 24 hours. Add 2 mL of Xingxiao Pills extract diluted with culture medium, and the diluted concentrations are 0 mg / mL, 0.5 mg / mL, 1 mg / mL and 2 mg / mL, respectively. After 24 hours, replace all with drug-free complete culture medium. Continue to culture for 10 days, during which time the culture medium is replaced every two days. Wash off the culture medium with PBS and fix with 4% paraformaldehyde for 30 minutes. Stain with crystal violet. After drying at room temperature, count the number of cloned cells in each well. The results are as follows. Figure 3 As shown in Figures B and C, the results show that Xingxiao Pills has an inhibitory effect on the cloning of 4T1 breast cancer cells in a dose-dependent manner.

[0030] 4) Migration experiment 4T1 cells were added to a 6-well plate and cultured for 24 hours. A 10-μl pipette tip was used to create scratches, and the floating cells were washed off with PBS. The cells were photographed under an inverted fluorescence microscope and the scratch width at 0 hours was recorded. 2 mL of Xingxiaowan extract diluted with complete culture medium containing 1% serum was added, and the concentrations after dilution were 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. After 24 hours of intervention, the floating cells were washed off with PBS, and the cell scratch width 24 hours later was photographed at the same position under an inverted fluorescence microscope. Cell migration rate = (initial scratch area or width - 24-hour scratch area or width) / initial scratch area or width × 100%. The results are shown in Figure 2. Figure 3As shown in Figures D and E, the results show that Xingxiao Pills has an inhibitory effect on the migration of 4T1 breast cancer cells in a dose-dependent manner.

[0031] It should be noted that the mass volume ratio of the Xingxiao Pills of the present invention to the culture medium is 3g:30mL~40mL; the first immersion time is 12h~24h; the second immersion time is 48h~72h, the ultrasound time is 1h~2h, and the ultrasound frequency is 25kHz~45kHz; the Xingxiao Pills extract is obtained by filtration through a 0.1μm~1μm filter membrane for cell administration, and the results still show that the inhibitory effect of the Xingxiao Pills on 4T1 cells is time-dependent; the Xingxiao Pills have an inhibitory effect on the cloning of 4T1 breast cancer cells, and are dose-dependent; the Xingxiao Pills have an inhibitory effect on the migration of 4T1 breast cancer cells, and are dose-dependent.

[0032] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0033] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. The use of Xingxiao Pills extract in the preparation of a drug for treating breast cancer, characterized in that: The extraction method of the Xingxiao Pills extract comprises taking Xingxiao Pills, adding them into 1640 culture medium for a first soaking, performing a second soaking after ultrasonication, taking the supernatant, filtering, and obtaining the filtrate as the Xingxiao Pills extract; the mass volume ratio of the Xingxiao Pills to the 1640 culture medium is 3 g: 30 mL to 40 mL.

2. The use according to claim 1, characterized in that The first soaking time is 12h~24h; the second soaking time is 48h~72h.

3. The use according to claim 1, characterized in that The ultrasonic frequency is 25kHz~45kHz; the ultrasonic time is 1h~2h.

4. The use according to claim 1, wherein The use according to claim 1 is characterized in that the filtration is performed through a 0.1 μm to 1 μm filter membrane.

5. The use according to claim 1, characterized in that The Xingxiao Pills extract is the only active ingredient in a drug for treating breast cancer.

6. The use according to claim 1, wherein The concentration of the Xingxiao Pills extract in the preparation of a drug for treating breast cancer is 0.1 mg / mL to 10 mg / mL.

7. The use according to claim 1, wherein The medicine includes the Xingxiao Pills extract and pharmaceutically acceptable excipients.

8. The use according to claim 7, characterized in that The pharmaceutically acceptable excipients are one or more of diluents, disintegrants, precipitation inhibitors, glidants, binders, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives and stabilizers.

9. The use according to claim 8, characterized in that The dosage forms of the drug include enema, tablet, capsule, granule, oral suspension, powder or ointment.

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