Application of Liao's Huifeng pill in preparation of drugs for resisting stomach cancer

Abstract

The scheme discloses the biological and medical technology field, and particularly relates to application of Liao's Huifengdan in preparation of anti-gastric cancer drugs. Through in-vitro use of gastric cancer cells AGS and SGC-7901, it is found that Liao's Huifengdan has a significant effect of inhibiting proliferation of the gastric cancer cells, and the effect is in a concentration-dependent manner, and the higher the drug concentration, the more obvious the retardation. Flow detection shows that Liao's Huifengdan has an obvious effect of promoting apoptosis of the gastric cancer cells AGS and SGC-7901, and the effect is in a concentration-dependent manner, and the higher the drug concentration, the more obvious the retardation. Through animal experiment, it is detected that Liao's Huifengdan can effectively inhibit growth of the gastric cancer cells in vivo, and can be used as a drug for preventing and treating gastric cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biology and medicine, and particularly relates to application of Liao's Huafeng Dan in preparation of anti-gastric cancer drugs. BACKGROUND

[0002] The Chinese medicine (a kind of Miao medicine) Liao's Huafeng Dan is produced and manufactured by Guizhou Wansheng Pharmaceutical Co., Ltd., and is a pill. Its existing functions are: relieving wind and relieving convulsion, resolving phlegm and opening orifice, and is used for treating wind-phlegm blockage, hemiplegia due to apoplexy, epilepsy, facial nerve palsy and facial distortion. It is used for treating wind-phlegm blockage, hemiplegia due to apoplexy, epilepsy, facial nerve palsy and facial distortion. The main components include more than 20 kinds of Chinese medicinal materials such as Radix et Rhizoma Aconiti, Rhizoma Typhonii, Rhizoma Pinelliae, Rhizoma Typhonii, Perilla Leaf, Bombyx Batryticatus, Scorpion, Rhizoma Gastrodiae, Schizonepeta, and Realgar. The water pills are of Jujube Red, and the section is brownish yellow; it has strong aroma and pungent taste. Liao's Huafeng Dan has a history of more than 370 years in treating diseases, and is recorded in Zunyi City Annals and Zunyi Prefecture Annals. Liao's Huafeng Dan has been a special medicine for treating diseases in the imperial hospital of the Qing Dynasty, and is known as "royal medicine" and "miraculous medicine".

[0003] At present, in-depth research on Liao's Huafeng Dan has shown that it has more new medical uses. For example, the application of the Miao medicine Liao's Huafeng Dan in preparation of drugs for preventing and treating melanoma is disclosed in the announcement No. CN109602859B, which discloses that Liao's Huafeng Dan has a significant effect on the cell cycle of melanoma cells, and the higher the drug concentration, the more obvious the blocking effect, and can be used as a preparation for preventing and treating melanoma. For another example, the application of Liao's Huafeng Dan in preparation of anti-leukemia drugs is disclosed in the announcement No. CN108392575B, which clearly shows that Liao's Huafeng Dan has a good inhibitory effect on the type of leukemia.

[0004] Gastric cancer is the most common malignant tumor of the digestive system, and malignant tumor metastasis is one of the main causes of death. Gastric cancer seriously threatens people's life and health. Surgery is still the main means of any curative treatment; however, about two-thirds of gastric cancer patients have a high possibility of recurrence and metastasis after surgery. At the same time, gastric cancer is not sensitive to radiotherapy, and is mainly treated by chemotherapy. Most patients with advanced gastric cancer need to receive chemotherapy as one of the main treatment methods. In recent years, platinum compounds, 5-fluorouracil and taxane drugs have been widely used in the treatment of gastric cancer. However, these chemotherapy drugs are accompanied by serious adverse reactions and toxic side effects, which limit their clinical application in the treatment of gastric cancer. Therefore, improving the early diagnosis rate, strengthening prevention and treatment, and finding effective drugs for treating gastric cancer are the primary tasks at present. SUMMARY

[0005] The purpose of the present application is to find a new drug with significant effect on treating gastric cancer in view of the difficulty in treating gastric cancer.

[0006] The present invention provides a use of Liao's Huafeng Dan in the preparation of an anti-gastric cancer drug. The effective core formula of Liao's Huafeng Dan includes Chinese medicinal materials such as Chuanwu, Arisaema, Pinellia, Rhizoma Bletillae, Folium Perillae, Bombyx Batryticatus, Scorpio, Atractylodes, Gastrodia, Schizonepeta, and Cinnabar.

[0007] In vitro testing using gastric cancer cells AGS and SGC-7901 revealed that Liao's Huafengdan significantly inhibited gastric cancer cell proliferation in a concentration-dependent manner, with higher drug concentrations resulting in more pronounced inhibition. Flow cytometry also demonstrated that Liao's Huafengdan significantly promoted apoptosis in gastric cancer cells AGS and SGC-7901, in a concentration-dependent manner, with higher drug concentrations resulting in more pronounced inhibition. Animal experiments also demonstrated that Liao's Huafengdan effectively inhibited gastric cancer cell growth in vivo, suggesting its potential use as a drug for the prevention and treatment of gastric cancer.

[0008] The beneficial effects of the present invention are as follows: Compared with existing uses of Liao's Huafeng Dan, the inventors have discovered that Liao's Huafeng Dan has a significant inhibitory effect on gastric cancer in vitro and in vivo, thus discovering a new use for this drug. Liao's Huafeng Dan is a marketed drug that can be directly applied to humans without requiring clinical safety evaluation, thus possessing promising application prospects and potentially rapidly increasing the product's market share.

[0009] The present invention directly prepares Liao's Huafengdan into an anti-gastric cancer preparation or mixes it with other anti-gastric cancer drugs to prepare an anti-gastric cancer compound preparation, wherein the preparation can be any one of tablets, injections, capsules, oral liquids or pills permitted in pharmacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The inhibition rate of different concentrations of Liao's Huafengdan on the proliferation of gastric cancer cells AGS and SGC-7901.

[0011] Figure 2 The flow cytometry test was used to detect the apoptosis-promoting effect of different concentrations of Liaoshi Huafengdan on gastric cancer cell SGC-7901.

[0012] Figure 3 The flow cytometry test was used to detect the apoptosis-promoting effect of different concentrations of Liao's Huafengdan on gastric cancer cell AGS.

[0013] Figure 4 This is the effect of Liao's Huafeng Dan on the growth inhibition of gastric cancer cells in vivo.

[0014] Figure 5 HE staining of the heart, liver, spleen, lungs and kidneys of mice was performed to show the results of a study on the toxic and side effects of Liao's Huafengdan. DETAILED DESCRIPTION

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0016] Example 1: Test of the in vitro anti-gastric cancer cell activity of Liao's Huifengdan

[0017] 1. Experimental materials

[0018] ① Preparation of sample solution: Dissolve Liao's Huifengdan pills into DMSO and filter under ultrasonic wave to prepare a mother solution with a concentration of 100 mg / ml. Then prepare sample solutions with different concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, etc.

[0019] ② Preparation of 5% MTT solution: Weigh 0.5 g of MTT powder and add 100 ml of normal saline. Dissolve at 60°C and store at 4°C.

[0020] ③ Cell lines: AGS and SGC-7901 cells are preserved in the laboratory of Guizhou Provincial Natural Product Research Center and are cultured in DMEM medium containing 5% FBS and 1640 medium containing 5% FBS at 37°C in a 5% CO2 incubator.

[0021] ④ DMEM medium, 1640 medium, and trypsin (Hycone Company); fetal bovine serum (FBS) (Zhejiang Tianhang Biological); tetramethylazolium salt (MTT) (Solarbio); dimethyl sulfoxide (DMSO) and amoxicillin mixture (Beijing Dingguo Changsheng Biological); Liao's Huifengdan (Guizhou Wansheng Pharmaceutical Co., Ltd.).

[0022] 2. Experimental method

[0023] 5 × 10 3 cells in the exponential growth phase were inoculated in 96-well plates at a density of 5 × 10 Figure 1 .

[0024] As Figure 1As shown, the results show that Liao's Huifeng Dan has a significant inhibitory effect on the growth of two kinds of gastric cancer cells SGC-7901-7901 and AGS. Liao's Huifeng Dan has a significant concentration-dependent inhibitory effect on the growth of gastric cancer cells SGC-7901-7901 and AGS. From the above data, it can be seen that Liao's Huifeng Dan has the potential value of developing into an anti-leukemia drug. In addition, the study shows that the effective components of Liao's Huifeng Dan prepared into tablets, injections, capsules, oral liquids or dripping pills according to the above experimental data also have the same growth inhibitory effect.

[0025] Example 2: Flow detection of Liao's Huifeng Dan in promoting apoptosis of gastric cancer cells in vitro

[0026] 1. Experimental materials

[0027] ① Preparation of sample solution: Liao's Huifeng Dan pills were ground into powder and dissolved in DMSO for filtration to prepare sample solutions with concentrations of 50 μg / ml, 100 μg / ml and 200 μg / ml, respectively.

[0028] ② Cell lines: AGS and SGC-7901 cells were preserved in the laboratory of Guizhou Province Natural Product Research Center and cultured in a 37°C, 5% CO2 incubator with DMEM and 1640 medium containing 5% FBS.

[0029] ③ EDTA trypsin: (Hycone Company); FITC. Annexin. V. Apoptosis Detection Kite I (BDBioscence Company); Flow cytometer (American BD Company); the rest is the same as above.

[0030] 2. Experimental method

[0031] AGS and SGC-7901 cells in logarithmic growth phase were collected and added into small dishes at a cell density of 800,000 cells / dish, 3 ml of cell suspension per dish, a total of 5 concentrations, i.e. 5 dishes, and placed in a culture box for 24 hours. When the growth density was 70-80%, 2 ml of normal saline was added for washing, and the washing liquid was sucked out. The required drug for the experiment was set according to the concentration, 3 ml was added per dish, 3 ml of culture medium was added to the control well, and it was cultured in the cell culture box for 48 h. The cell morphology was observed under a microscope. The culture medium was collected into a 15 ml centrifuge tube, washed with 2 ml of normal saline for 2 times, 300 ul of trypsin without EDTA was added, and it was placed in a cell culture box at 37°C for 20 min to stop the adhesion of adherent cells. Fresh culture medium was added to terminate digestion, transferred to a 15 ml centrifuge tube, and centrifuged. After centrifugation, the supernatant was sucked out, 2 ml of normal saline was gently washed once, and the supernatant was sucked out after centrifugation. After resuspending the cells with PBS buffer, they were transferred to a 1.5 ml EP tube, Annexin V-PI / FITC reagent was added, mixed, and protected from light. The flow cytometer was used to detect cell apoptosis, Flowjo software was used to analyze the flow cytometry detection data, and the cell apoptosis analysis results were exported. The results are shown in Figure 2 、 Figure 3 .

[0032] Figure 2 、 Figure 3 The results show that Liao's Huafeng Dan can induce apoptosis of gastric cancer cells, and has a significant concentration-dependent effect. The data were analyzed and processed by statistical software Excel and GraphPad Prism9. The measurement data were represented by mean ± standard deviation (x ± s). The numerical values among multiple groups were compared by one-way ANOVA. P<0.05 and P<0.01 were considered to have statistically significant differences.

[0033] Example 3: Detection of in vivo anti-gastric cancer cell activity of Liao's Huafeng Dan

[0034] 1. Experimental materials

[0035] ① 30 BALB / c Nude male mice, about 5 weeks old.

[0036] ② Cell line: SGC-7901 cells were cultured in a 37°C, 5% CO2 incubator with 1640 medium containing 5% FBS.

[0037] ③ Preparation of sample solution: Liao's Huafeng Dan was ground into powder and prepared into sample solutions with concentrations of 1120 mg / ml (high dose group), 560 mg / ml (medium dose group), and 280 mg / ml (low dose group) using normal saline.

[0038] ④ BALB / c Nude male mice (Guizhou Huiju Biological Technology Co., Ltd.);

[0039] 2. Experimental method

[0040] SGC-7901 cells were cultured, and SGC-7901 cells in the exponential growth phase were prepared into a cell suspension with a concentration of 1 x 10 7 The mice were divided into 4 groups, each with 6 mice, and were respectively a control group (gargling with normal saline), a low-dose group of 280 mg / ml, a medium-dose group of 560 g / ml, and a high-dose group of 1120 mg / ml. The control group and the administration group were each given 0.4 ml of intragastric administration once a day for a total of 22 days. After 22 days of intragastric administration, the mice were dissected, and important organs and subcutaneous tumors were subjected to pathological examination and tumor weight detection. The tumor weight results are shown in Table 2. Figure 4 .

[0041] Figure 4 The results show that Liao's Huafeng Dan has a significant inhibitory effect on the growth of gastric cancer cells SGC-7901 in vivo. Statistical software Excel and GraphPad Prism 9 were used for analysis, and the measurement data were expressed as mean ± standard deviation (x ± s). The numerical values among multiple groups were compared using one-way ANOVA, and P<0.05 and P<0.01 were considered to be statistically significant differences.

[0042] Figure 5 The results show that Liao's Huafeng Dan has no toxic side effects on mice through HE staining of the heart, liver, spleen, lung, and kidney of mice.

[0043] From the above results, it can be seen that Liao's Huafeng Dan has potential value in the prevention and treatment of gastric cancer drugs.

Claims

1. The use of Liao's Huifengdan in the preparation of a medicine for resisting stomach cancer.

2. The use of the rhubarb wind pill according to claim 1 in the preparation of an anti-gastric cancer drug, characterized in that: The effective components of the Liao's Huifengdan or the medicine are prepared into tablets, injections, capsules, oral liquids or drop pills.

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