Traditional Chinese medicine composition for treating gastric cancer and application thereof

Abstract

The invention relates to a traditional Chinese medicine composition for treating gastric cancer. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 335-415 parts of oldenlandia diffusa, 335-415 parts of solanum septemlobum bunge and 335-415 parts of chinaroot greenbrier. Cell tests prove that the traditional Chinese medicine composition can inhibit the activity of gastric cancer cells, also can significantly inhibit DNA replication activity, and reduces the proliferation and clone formation ability of the gastric cancer cells; protein expression of gastric cancer cell intermediate mesenchymal cell markers N-cadherin and Vimentin, transcription factors Snail and ZEB1 and adhesion factors CD44 and EpCAM is reduced, the EMT process is affected, and then the gastric cancer cell migration and invasion capacity is inhibited. Animal tests prove that the traditional Chinese medicine composition can inhibit tumor growth, shows good anti-gastric cancer activity and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition for treating gastric cancer and application thereof. Background Art

[0002] In 2022, there were 970,000 confirmed cases of gastric cancer and 660,000 deaths worldwide, making it the fifth most common cancer and the fifth leading cause of cancer death in the world. Although the incidence and mortality of gastric cancer have declined in recent years, gastric cancer is still an important factor in the global cancer burden. As one of the malignant tumors of the digestive tract, gastric cancer has the characteristics of low early diagnosis rate, large tumor burden, strong heterogeneity, and poor patient prognosis. The 5-year survival rate of patients diagnosed with gastric cancer in the middle and late stages after surgery is less than 30%, while the 5-year survival rate of early patients with timely treatment can be greater than 90%.

[0003] Hedyotis diffusa Willd. was first recorded in the Guangxi Chinese Materia Medica in 1959. It is the dried whole herb of Hedyotis diffusa Willd., a plant of the genus Hedyotis in the family Rubiaceae. It is cold in nature and bitter and sweet in taste. It enters the heart, liver, spleen, and large intestine meridians and has the effects of clearing away heat and detoxifying, diuresis and dehumidification. As a heat-clearing and detoxifying drug, Hedyotis diffusa Willd. has good anti-inflammatory activity. The use of Smilax china L. as a medicine was first recorded in the Famous Doctors' Records by Tao Hongjing during the Wei, Jin and Liang dynasties. It is the dried rhizome of Smilax china L., a plant of the family Liliaceae. It tastes sweet, slightly bitter, astringent, and flat, and enters the liver and kidney meridians. It has the effects of removing dampness and turbidity, dispelling wind and removing numbness, and detoxifying and dispersing blood stasis. Shuyangquan is one of the important traditional Chinese medicines in China during the pre-Qin period and is listed as a "medium-grade product" in the Shennong's Herbal Classic. The original plant is Solanumdulcamara L., a plant of the Solanaceae family. Its fruit is used as medicine. It tastes bitter and slightly cold, can clear away heat and detoxify, and treat dental caries. It is a traditional Chinese medicine mainly used to treat skin diseases. Studies have found that the effect of Shuyangquan powder plus concurrent chemoradiotherapy on the efficacy, serum indicators and survival of patients with advanced cervical cancer has been studied. It was found that Shuyangquan powder plus concurrent chemoradiotherapy has a significant effect on advanced cervical cancer, can effectively relieve patients' pain, improve patients' survival rate and quality of life, and reduce serum CYFRA21-1, SCC-Ag, TK1, and HE4 levels, which has certain clinical application value.

[0004] At present, the medical treatment for gastric cancer still mainly adopts surgery, radiotherapy and chemotherapy, but these are better for early stage patients. Surgery is also a local treatment, which is not effective for tumor cells metastasized to the blood or other tissues, and may also produce side effects. Therefore, there is an urgent need for a treatment method with significant efficacy and no side effects. Traditional Chinese medicine has gradually become an important way to treat gastric cancer. At present, there is no report on a traditional Chinese medicine composition for treating gastric cancer and its application as the present invention. Summary of the invention

[0005] The first object of the present invention is to provide a Chinese medicine composition for treating gastric cancer in view of the deficiencies in the prior art.

[0006] The second object of the present invention is to provide a composite granule.

[0007] The third object of the present invention is to provide a method for preparing the composite granules.

[0008] The fourth object of the present invention is to provide an application.

[0009] To achieve the above first purpose, the technical solution adopted by the present invention is:

[0010] A traditional Chinese medicine composition for treating gastric cancer is prepared from the following raw materials in parts by weight: 335-415 parts of oldenlandia diffusa, 335-415 parts of Shuyangquan herb, and 335-415 parts of Smilax china.

[0011] As a preferred example, the Chinese medicine composition is prepared from the following raw materials in parts by weight: 355-395 parts of Hedyotis diffusa, 355-395 parts of Shuyangquan, and 355-395 parts of Smilax china.

[0012] More preferably, the Chinese medicine composition is made from the following raw materials in parts by weight: 375 parts of Hedyotis diffusa, 375 parts of Shuyangquan, and 375 parts of Smilax china.

[0013] To achieve the above second purpose, the technical solution adopted by the present invention is:

[0014] A kind of Hedyotis diffusa granules are prepared from any one of the above traditional Chinese medicine compositions and auxiliary materials.

[0015] As a preferred example, the auxiliary materials are soluble starch, dextrin and mannitol.

[0016] To achieve the third purpose above, the technical solution adopted by the present invention is:

[0017] A method for preparing compound oldenlandia diffusa granules comprises the following steps:

[0018] 1) Extract preparation: Take any of the above-mentioned medicinal materials in weight proportions, add 8 times the amount of water and boil twice, the first time for 2 hours and the second time for 1.5 hours. After boiling, filter the decoction, combine the filtrate and let it stand for 12-16 hours, take the supernatant and concentrate it to a clear paste with a relative density of 1.30-1.35 (80-85°C), seal it in portions and store it in a -80°C low-temperature refrigerator for later use;

[0019] 2) Preparation of granules: Mix the above-mentioned paste with soluble starch, dextrin and mannitol in a certain proportion and add distilled water as a wetting agent, mix evenly and knead continuously until the soft material "lightly grasps into a ball and falls apart when pushed". Place the soft material on a 16-mesh stainless steel sieve, squeeze and sieve, and collect the sieved particles. Spread the particles flat on a tray and place them in an oven at 80°C for 3 hours to obtain granules.

[0020] As a preferred example, the mass ratio of the clear paste to the soluble starch, dextrin and mannitol is 1:1:1:1.

[0021] To achieve the fourth objective, the present invention adopts the following technical solution:

[0022] Use of any of the above-mentioned Chinese medicine compositions and compound oldenlandia diffusa granules in the preparation of drugs for treating and / or preventing gastric cancer.

[0023] As a preferred example, the drug can be used for superficial, atrophic gastritis with intestinal metaplasia and atypical hyperplasia and other precancerous lesions of gastric cancer.

[0024] The advantages of the present invention are:

[0025] The compound snake tongue grass granules, which are composed of three Chinese medicinal herbs: white flower snake tongue grass, smilax china, and Shuyangquan, are a classic ancient prescription used in our hospital for many years. It is an in-hospital preparation approved for production by the Shanghai Food and Drug Administration (approval number: SYZ-ZF-003-2007). It has the effects of clearing away heat and detoxifying, removing blood stasis and dispersing nodules. It can be used for superficial, atrophic gastritis with intestinal metaplasia, atypical hyperplasia, etc. It has a preventive and therapeutic effect on the early lesions of gastric cancer. Its efficacy has accumulated a good reputation among patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Attached Figure 1 IC50 values ​​of CHD against human gastric epithelial cells and gastric cancer cells (*p<0.05, **p<0.01 and ***p<0.001; n≥3).

[0027] Attached Figure 2 The effects of different concentrations of CHD-E on the activity of gastric cancer cells (*p<0.05, **p<0.01 and ***p<0.001; n≥3).

[0028] Attached Figure 3 The effects of different concentrations of CHD-E on the colony-forming ability of gastric cancer cells (*p<0.05and***p<0.001; n≥3).

[0029] Attached Figure 4 Edu staining was used to detect the effects of different concentrations of CHD-E on the proliferation ability of gastric cancer cells (*p<0.05, **p<0.01and ***p<0.001; n≥3).

[0030] Attached Figure 5 The scratch assay was used to detect the effects of different concentrations of CHD-E on the migration ability of gastric cancer cells (**p<0.01and***p<0.001; n≥3).

[0031] Attached Figure 6 Transwell experiment was used to detect the effect of different concentrations of CHD-E on the migration ability of gastric cancer cells (**p<0.01and***p<0.001; n≥3).

[0032] Attached Figure 7 Transwell experiment was used to detect the effect of different concentrations of CHD-E on the invasion ability of gastric cancer cells (***p<0.001; n≥3).

[0033] Attached Figure 8 Western Blot was used to detect the effect of CHD-E on the expression levels of gastric cancer cell migration-related proteins (n≥3).

[0034] Attached Fig. 9 Flow cytometry was used to detect the effects of different concentrations of CHD-E on apoptosis of gastric cancer cells (***p<0.001; n≥3).

[0035] Attached Fig.10 JC-1 staining was used to detect the effects of different concentrations of CHD-E on mitochondrial membrane potential of gastric cancer cells (***p<0.001; n≥3).

[0036] Attached Fig.11 Western Blot was used to detect the effect of CHD-E on the expression level of Wnt signaling pathway proteins in gastric cancer cells (n≥3).

[0037] Attached Fig.12 The CDX experiment was used to investigate the anti-gastric cancer efficacy of CHD (***p<0.001; n=7).

[0038] Attached Fig.13 Compound Herba Oederae Diffusae Granules were prepared from each prescription when screening mixed excipients. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0040] Example 1 Chinese medicine composition for treating gastric cancer of the present invention (I)

[0041] 375 parts of Hedyotis diffusa, 375 parts of Shuyangquan, and 375 parts of Smilax china.

[0042] Example 2 Chinese medicine composition for treating gastric cancer of the present invention (II)

[0043] 375 parts of Hedyotis diffusa, 335 parts of Shuyangquan, and 395 parts of Smilax china.

[0044] Example 3 Chinese medicine composition for treating gastric cancer of the present invention (III)

[0045] 335 parts of Hedyotis diffusa, 395 parts of Shuyangquan, and 335 parts of Smilax china.

[0046] Example 4 Chinese medicine composition for treating gastric cancer of the present invention (IV)

[0047] 395 parts of Hedyotis diffusa, 335 parts of Shuyangquan, and 415 parts of Smilax china.

[0048] Example 5 Chinese medicine composition for treating gastric cancer of the present invention (V)

[0049] 335 parts of Hedyotis diffusa, 415 parts of Shuyangquan, and 375 parts of Smilax china.

[0050] Example 6 Chinese medicine composition for treating gastric cancer of the present invention (VI)

[0051] 415 parts of Hedyotis diffusa, 375 parts of Shuyangquan, and 335 parts of Smilax china.

[0052] Example 7 Chinese medicine composition for treating gastric cancer of the present invention (VII)

[0053] 375 parts of Hedyotis diffusa, 395 parts of Shuyangquan, and 335 parts of Smilax china.

[0054] Example 8 Chinese medicine composition for treating gastric cancer of the present invention (VIII)

[0055] 335 parts of Hedyotis diffusa, 335 parts of Shuyangquan, and 415 parts of Smilax china.

[0056] Example 9 Chinese medicine composition for treating gastric cancer of the present invention (IX)

[0057] 395 parts of Hedyotis diffusa, 415 parts of Shuyangquan, and 375 parts of Smilax china.

[0058] Example 10 Chinese medicine composition for treating gastric cancer of the present invention (X)

[0059] 335 parts of Hedyotis diffusa, 375 parts of Shuyangquan, and 335 parts of Smilax china.

[0060] Example 11 Chinese medicine composition for treating gastric cancer of the present invention (XI)

[0061] 415 parts of Hedyotis diffusa, 335 parts of Shuyangquan, and 395 parts of Smilax china.

[0062] Example 12 Preparation of compound Hedyotis diffusa granules for treating gastric cancer of the present invention

[0063] 1) Preparation of extract: Take the medicinal material of any one of claims 1-3 in weight proportion, add 8 times the amount of water and boil twice, the first time for 2 hours and the second time for 1.5 hours. After boiling, filter the decoction, combine the filtrate and let it stand for 12-16 hours, take the supernatant and concentrate it to a clear paste with a relative density of 1.30-1.35 (80-85°C), divide it into small packages and seal it, and store it in a -80°C low-temperature refrigerator for later use;

[0064] 2) Preparation of granules: Mix the above-mentioned paste with soluble starch, dextrin and mannitol in a mass ratio of 1:1:1:1 and add distilled water as a wetting agent, mix evenly and knead continuously until the soft material "lightly grasps into a ball and falls apart when pushed". Place the soft material on a 16-mesh stainless steel sieve, squeeze and sieve, and collect the sieved particles. Spread the particles flat on a tray and place them in an oven at 80°C for 3 hours to obtain granules.

[0065] Example 13 Cell experiment

[0066] 1 Experimental Materials

[0067] 1.1 Cell lines

[0068] The normal human gastric epithelial cells GES-1 and human gastric cancer cell lines AGS, HGC-27, MKN-1, and MKN-28 used in this part of the experiment were all from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0069] 1.2 Main Reagents

[0070] Compound Hedyotis diffusa water extract (clear paste) (Shanghai Liantang Pharmaceutical); Tongguanteng oral liquid (Xiaoaiping oral liquid) (Henan Xinsifang Pharmaceutical); quercetin (MCE, China); phosphate buffer solution (Guangzhou Shuopu Biotechnology); RPMI 1640 medium, Ham's F-12K medium (Shanghai Yuanpei Biotechnology); JC-1 mitochondrial membrane potential kit, Edu cell proliferation detection kit (Guangzhou Ruibo Biotechnology, China); BCA protein quantification detection kit (Shanghai Biyuntian Biotechnology, China); CCK-8 kit (MCE, China).

[0071] 1.3 Main instruments

[0072] CO2 cell culture incubator (Thermo fisher scientific, USA); desktop low-temperature high-speed centrifuge (Eppendorf, Germany); Leica inverted fluorescence microscope (Leica, Switzerland); Western Blot gel clips, brackets, Western Blot transfer tank, Western Blot electrophoresis, Western Blot power supply (Bio-rad, USA), reciprocating decolorization shaker (Jiangsu Qilin Bell Instrument, China); SIM-F124 ice machine (SANYO, Japan); Tannon chemiluminescence imaging system (Shanghai Tianneng, China); multi-function microplate reader (BioTek, USA).

[0073] Other reagents and instruments mentioned in the examples are commercially available common instruments and reagents, which can be purchased routinely by those skilled in the art and are not listed here one by one.

[0074] 2 Experimental methods

[0075] 2.1 Determination of the effect of compound oldenlandia diffusa on the growth activity and proliferation ability of gastric cancer cells

[0076] 2.1.1 Determination of the effect of compound oldenlandia diffusa on the vitality of gastric cancer cells

[0077] CCK-8 method was used to detect cell viability. AGS and HGC-27 cells in good growth state and in logarithmic growth phase were taken. After cell counting, cells were inoculated at a density of 5000 cells / well in a 96-well plate. 200 μL of complete culture medium was added to each well, and the cells were gently shaken in a "cross" shape to disperse the cells evenly. After marking, the cells were transferred to a 37°C, 5% CO2 incubator for culture; when the cells grew to a confluence of 60-70%, administration was prepared. After discarding the old culture medium, according to the control group (complete culture medium), the experimental group (Compound of Hedyotis diffusa-extract (Clear Paste) (Compound of Hedyotis diffusa-extract, CHD-E, hereinafter referred to as CHD-E) concentration of 2mg / mL, 4mg / mL, 8mg / mL, 16mg / mL, 32mg / mL, 64mg / mL drug-containing culture medium, prepared by gradient dilution method) was divided into groups, 200 μL of culture medium was added to each well of the 96-well plate, and 6 duplicate wells were set for each group. Transfer the 96-well plate to a 37°C, 5% CO2 incubator and incubate for the required time (24h or 36h) for the experiment; after the drug incubation is completed, discard the old culture medium, add 150μL PBS (phosphate buffered saline) to each well to wash the residual culture medium, wash 3 times and then use a pipette to suck up the residual liquid. In the dark, prepare the CCK-8 detection solution according to the ratio of CCK-8 reagent volume: complete culture medium volume = 10:100, add 110μL to each well, and set up a group of blank wells, that is, only CCK-8 detection solution is added to this group. Transfer the 96-well plate to a 37°C, 5% CO2 incubator and incubate for 2h, then transfer it away from light, and detect the OD (optical density) value of each well at a wavelength of 450nm using an enzyme reader.

[0078] IC50 (half maximal inhibitory concentration) calculation method: Create a data set under the "XY Tables" item in GraphPad Prism 8.0 software, select "Transform concentrations (X)" under the "New Analysis" item to transform the concentration, and then select "Dose-response-Inhibition / Log (inhibitor) vs. response-Variable slope (four parameters)" under the "XY Analyses (curvefit)" item for the generated data set.

[0079] IC10 (10% inhibition concentration) and IC20 (20% inhibition concentration) calculation method: Create a data set under "XY Tables" in GraphPad Prism 8.0 software, select "Transform concentrations (X)" under "New Analysis" to transform the concentration, and select "Dose-response-Special / Log (agonist) vs. response-Find ECanything" under "XY Analyses (curve fit)" for the generated data set. Fill in the F value in the pop-up dialog box. If the F value is 10, the IC10 is calculated; if the F value is 20, the IC20 is calculated.

[0080] 2.1.2 Determination of the effect of compound Hedyotis diffusa on the colony formation ability of gastric cancer cells

[0081] The plate clone formation experiment was used to detect the cell clone formation ability. AGS and HGC-27 cells in good growth state and in the logarithmic growth phase were selected. After cell counting, 1×10 3 Cells were inoculated at a density of 100 / well, and 2 mL of complete medium was added to each well. The cells were gently shaken in a "cross" shape to disperse the cells evenly. After marking, the cells were transferred to an incubator for culture; after 48 hours of adherent culture, the old culture medium was discarded, and AGS cells (3.5 mg / mL, 5 mg / mL, 9 mg / mL) and HGC-27 cells (5 mg / mL, 6.5 mg / mL, 8 mg / mL) were treated with different concentrations of CHD-E, with 3 replicates per group. The cells were cultured in the drug-containing culture medium for 2 hours; the drug-containing culture medium was discarded, rinsed with PBS 3 times, and the residual liquid was sucked dry with a pipette before replacing it with complete culture medium. Counted from the time of plating, the cells were co-cultured in a 37°C, 5% CO2 incubator for 7 days; the cell culture plate was removed, the old culture medium was discarded, the cells were rinsed with PBS 3 times, the residual liquid was sucked out with a pipette, 1 mL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 30 min; the fixative was discarded, the cells were rinsed with PBS 3 times, the residual liquid was sucked out with a pipette, 1 mL of crystal violet dye was added to each well, and the cells were incubated at room temperature for 30 min on a shaking table; after the staining was completed, the crystal violet dye was recovered, the cells were rinsed with PBS 3 times, the residual liquid was sucked out with a pipette, and the cells were dried; the number of clones in each well was counted using Image J after taking pictures. Each group of experiments was repeated three times.

[0082] 2.1.3 Effect of compound oldenlandia diffusa on the proliferation of gastric cancer cells detected by Edu staining

[0083] Take AGS and HGC-27 cells that are in good growth state and in the logarithmic growth phase, count the cells, and plate them in a 96-well plate at a rate of 1×10 4Cells were inoculated at a density of 100 μL / well, 200 μL complete medium was added to each well, and the cells were gently shaken in a "cross" shape to disperse the cells evenly. After marking, the cells were transferred to a 37°C, 5% CO2 incubator and cultured for 24 hours until the cells adhered stably; the old culture medium was discarded, and AGS cells (3.5 mg / mL, 5 mg / mL, 9 mg / mL) and HGC-27 cells (5 mg / mL, 6.5 mg / mL, 8 mg / mL) were treated with different concentrations of CHD-E, and 3 replicate wells were set for each group. The cells were cultured in the drug-containing medium for 24 hours; the Edu solution was diluted with the medium at a ratio of 1000:1 and set aside; the drug-containing medium was discarded, the cells were washed 3 times with PBS, and 200 μL of the prepared Edu medium was added to each well, and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours; the Edu (5-ethynyl-2'-deoxyuridine, 5-ethynyl-2-deoxyuridine) medium was discarded, the cells were washed 3 times with PBS, and 50 μL of 4% paraformaldehyde fixative was added to each well, and fixed for 30 minutes at room temperature; the fixative was discarded, the cells were washed 3 times with PBS, and 50 μL of 20 mg / mL glycine solution was added to each well, and the cells were incubated on a slow shaker for 5 minutes; the glycine solution was discarded, the cells were washed 3 times with PBS, and the shaker was slow for 5 minutes each time. 100 μL of permeabilization agent (0.5% Triton-X-100 in PBS solution) was added to each well, and the cells were incubated on a slow shaker for 10 minutes; the staining solution was prepared. The composition of the 500 μL staining solution system is shown in Table 1.

[0084] Table 1 Edu staining solution system composition (500 μL)

[0085]

[0086] Discard the permeabilization agent, wash once with PBS, shake slowly for 5 minutes. Add 100μL of staining solution to each well, incubate at room temperature in the dark for 30 minutes; discard the staining solution, add 100μL of permeabilization agent to each well, incubate at slow speed on a shaker 2-3 times, 10 minutes each time; dilute reagent F, i.e. Hoechst 33342 dye, at a ratio of triple distilled water volume: reagent F volume = 100:1, add 100μL of diluted Hoechst 33342 dye to each well, incubate at room temperature in the dark, shake slowly for 30 minutes; discard the reaction solution, wash 3 times with PBS, add 100μL of PBS to each well, and take pictures under an inverted fluorescence microscope. Each group of experiments was repeated three times.

[0087] 2.2 Effects of compound Hedyotis diffusa on the migration and invasion of gastric cancer cells and down-regulation of gastric cancer metastasis-related protein expression

[0088] 2.2.1 Scratch test to detect the effect of compound Hedyotis diffusa on the migration ability of gastric cancer cells

[0089] Take AGS and HGC-27 cells that are in good growth state and in the logarithmic growth phase, count the cells, and plate them in a 12-well plate at a rate of 3×10 5 Inoculate cells at a density of 100 / well, add 1 mL of complete culture medium to each well, and gently shake in a "cross" shape to evenly disperse the cells. After marking, transfer the cells to a 37°C, 5% CO2 incubator for culture. When the cells grow to a confluence of 100%, prepare for scratching. After discarding the old culture medium, treat AGS cells (3.5 mg / mL, 5 mg / mL, 9 mg / mL) and HGC-27 cells (5 mg / mL, 6.5 mg / mL, 8 mg / mL) with different concentrations of CHD-E, with 3 replicate wells in each group. Use the lid of the 12-well plate as a ruler, make parallel scratches of uniform width for each group, and rinse with PBS 3 times until there are no obvious floating cells. After adding 1 mL of complete culture medium to each well, photos were taken under an inverted microscope, and the scratch width was recorded as time 0. After taking photos, the culture medium was discarded, the residual liquid was sucked up with a pipette, and 2 mL of culture medium was added to each well according to the grouping of the control group and the experimental group, and the 12-well plate was transferred to a 37°C, 5% CO2 incubator for 24 h. After incubation, the old culture medium was discarded, rinsed with PBS 3 times, the residual liquid was sucked up with a pipette, 1 mL of complete culture medium was added to each well, and photos were taken under an inverted microscope, and the scratch width was recorded as time 24 h.

[0090] Calculation method of wound healing rate: Quantitative statistics of experimental results were performed using Image J, wound healing rate = (wound area at time 0 - wound area at time 24 h) / wound area at time 0 * 100%. Each group of experiments was repeated three times.

[0091] 2.2.2 Transwell assay to detect the effect of compound Hedyotis diffusa on the migration ability of gastric cancer cells

[0092] Take AGS and HGC-27 cells that are in good growth state and in the logarithmic growth phase, digest them with trypsin, resuspend them in 1 mL of basal medium, and count the cells. 5Dilute the cell suspension with basal medium at a density of 100 μg / mL and set aside; prepare a Transwell chamber adapted to a 24-well plate. Treat AGS cells (3.5 mg / mL, 5 mg / mL, 9 mg / mL) and HGC-27 cells (5 mg / mL, 6.5 mg / mL, 8 mg / mL) with different concentrations of CHD-E, with 3 replicate wells in each group. Place the Transwell chamber in a 24-well plate with tweezers, add 200 μL of cell suspension to each well (pipette vertically); take the remaining wells in the well plate and add 600 μL of culture medium according to the grouping settings. Use tweezers to transfer the chamber containing the cell suspension to the well containing the culture medium, and avoid generating bubbles during the transfer process; pay attention to whether there is leakage from the chamber in the original well. If leakage occurs, replace the chamber and re-add the cell suspension; mark it, tap the side wall to evenly disperse the cells, and then transfer the 24-well plate to a 37°C, 5% CO2 incubator to culture for the required time of the experiment (according to the preliminary experimental results, AGS cells were stimulated with drugs for 8 hours; HGC-27 cells were stimulated with drugs for 4 hours); after the drug stimulation is completed, use tweezers to transfer the chamber to the well pre-added with 600 μL of complete culture medium After the incubation, the liquid in the chamber was carefully sucked out with a pipette, and transferred to a well with 600 μL 4% paraformaldehyde fixative solution added in advance with tweezers, and fixed at room temperature for 30 minutes; after the fixation, the liquid in the chamber was carefully sucked out with a pipette, and transferred to a well with 600 μL crystal violet staining solution added in advance with tweezers, and incubated slowly on a shaker at room temperature for 30 minutes; after the staining, the liquid in the chamber was carefully sucked out with a pipette, and washed in a beaker with triple distilled water with tweezers, washed 3 times and then inverted for use. Carefully wipe the cells in the chamber that have not penetrated the PC membrane of the chamber with a cotton swab; after drying, take pictures under an upright microscope, and use Image J to quantify the experimental results. Each group of experiments was repeated three times.

[0093] 2.2.3 Transwell assay to detect the effect of compound oldenlandia diffusa on the invasion ability of gastric cancer cells

[0094] Prepare the matrix gel solution required for the experiment according to the instructions. Preheat the 37℃ water bath in advance. Dilute reagent C with basal culture medium warmed in a 37℃ water bath to prepare 0.5×C buffer solution, such as adding 1mL of reagent C to 19mL of basal culture medium. After sealing, it can be stored at 4℃ for one week. Before use on the day of the experiment, seal and place in a 37℃ water bath; place reagent A in a 37℃ water bath for 10 minutes until completely dissolved. Dilute the dissolved reagent A with basal culture medium warmed in a 37℃ water bath, such as mixing 500μL of reagent A with 500μL of basal culture medium to obtain 1mL of 1× reagent A. Prepare before use, seal and place in a 37°C water bath before use to reduce the viscosity; prepare a Transwell chamber suitable for a 24-well plate; dilute 1× reagent A with 0.5× reagent C warmed in a 37°C water bath, and dilute 15 times according to the results of the preliminary experiment; treat AGS cells (3.5mg / mL, 5mg / mL, 9mg / mL) and HGC-27 cells (5mg / mL, 6.5mg / mL, 8mg / mL) with different concentrations of CHD-E, and set up 3 replicate wells in each group. Use tweezers to place the Transwell chamber in a 24-well plate, and add 100μL of diluted reagent A to each well (pipette vertically added). After sealing the 24-well plate, place it in a 4°C refrigerator for 2-3h; take AGS and HGC-27 cells in good growth condition and in the logarithmic growth phase, count the cells, adjust the cell density, and prepare a cell suspension. According to the results of the preliminary experiment, 1.5×10 AGS cells 5 / well / 100μL, i.e. 1.5×10 6 cells / mL; HGC-27 cells 1.0×10 5 / well / 100μL, i.e. 1.0×10 6 / mL; take out the 24-well plate after standing, add 100μL of cell suspension to each well (pipette vertically); take the remaining wells in the well plate, and add 800μL of culture medium respectively according to the grouping settings. Use tweezers to transfer the chamber with cell suspension to the well containing culture medium, and avoid bubbles during the transfer process; mark it, tap the side wall to evenly disperse the cells, and transfer the 24-well plate to a 37℃, 5% CO2 incubator to culture for the required time of the experiment (according to the preliminary experimental results, AGS cells and HGC-27 cells are stimulated by drugs for 6h); after the drug stimulation, use tweezers to transfer the chamber to the well with 800μL complete culture medium added in advance, and transfer it to a 37℃, 5% CO2 incubator to continue culturing for the required time of the experiment. According to the preliminary experimental results, AGS cells were cultured for 24 hours; HGC27 cells were cultured for 48 hours; at the end of incubation, the liquid in the chamber was carefully aspirated with a pipette, and transferred to a well with 800 μL 4% paraformaldehyde fixative solution added in advance with tweezers, and fixed at room temperature for 30 minutes; at the end of fixation, the liquid in the chamber was carefully aspirated with a pipette, and transferred to a well with 800 μL crystal violet staining solution added in advance with tweezers, and incubated slowly on a shaker at room temperature for 30 minutes; at the end of staining, the liquid in the chamber was carefully aspirated with a pipette, and washed in a beaker filled with triple distilled water with tweezers, washed 3 times and inverted for use. Cells that did not penetrate the PC membrane of the chamber were carefully wiped with a cotton swab; after drying, photos were taken under an upright microscope, and the experimental results were quantitatively counted using Image J. Each group of experiments was repeated three times.

[0095] 2.2.4 Testing of expression levels of gastric cancer cell metastasis-related proteins

[0096] WB (western blot, protein immunoblotting) was used to detect EMT (epithelial-mesenchymal transition, epithelial-mesenchymal transition) related proteins. Combined with the above experimental results, QCT (Quercetin) was selected as a positive drug. The IC50 value of QCT for AGS cells was 130.8±22.1μM as detected by CCK-8 method; the IC50 value for HGC-27 cells was 119.7±10.2mg / mL. According to the IC20 value, the WB experimental drug concentration was set, and AGS cells were treated with 5mg / mL CHD-E and 70μM QCT; HGC-27 cells were treated with 6.5mg / mL CHD-E and 90μM QCT. Total protein was extracted; protein concentration was determined by BCA method; SDS-polyacrylamide (SDS-PAGE) gel electrophoresis; finally, the bands were scanned and developed in a chemical imaging system by gel electrophoresis, and the experimental results were quantitatively counted by Image J. Each group of experiments was repeated three times. The expression levels of mesenchymal cell markers N-cadherin and vimentin, transcription factors Snail homolog 1 (Snail) and E-box binding zinc finger protein 1 (ZEB1), adhesion factors CD44 and EpCAM (Epithelial cell adhesion molecule) were detected to reflect the effect of CHD-E on cell metastasis.

[0097] 2.3 Determination of the effect of compound Hedyotis diffusa on apoptosis of gastric cancer cells

[0098] 2.3.1 Flow cytometry to detect the effect of compound Hedyotis diffusa on apoptosis of gastric cancer cells

[0099] Take AGS and HGC-27 cells that are in good growth state and in the logarithmic growth phase, count the cells, and plate them in a 6-well plate at a rate of 5×10 5Cells were inoculated at a density of 100 / well, 2 mL of complete medium was added to each well, and the cells were gently shaken in a "cross" shape to disperse the cells evenly. After marking, the cells were transferred to a 37°C, 5% CO2 incubator for culture; when the cells grew to a confluence of 50-60%, the old medium was discarded and AGS cells (3.5 mg / mL, 5 mg / mL, 9 mg / mL) and HGC-27 cells (5 mg / mL, 6.5 mg / mL, 8 mg / mL) were treated with different concentrations of CHD-E, and 3 replicate wells were set up for each group. The cells were cultured in drug-containing medium for 24 h; the supernatant was collected in a 15 mL centrifuge tube, rinsed with PBS three times, and the residual liquid was sucked up with a pipette. 1 mL of trypsin was added to each well to digest the cells, and the digestion was terminated with the collected supernatant; after centrifugation and collection of cell pellets, the supernatant was discarded, the cells were resuspended with pre-cooled PBS, and placed in a centrifuge at 1000 rpm for 3 min; after collecting the cell pellets, the supernatant was discarded, the cells were resuspended with pre-cooled 1×BindingBuffer, placed in a 4°C pre-cooled centrifuge, 600 g, and centrifuged for 3 min, and the operation was repeated 3 times; the cell pellets were collected, 500 μL 1×Binding Buffer was added to resuspend the cells, 5 μL Annexin V-FITC dye was added, and the cells were incubated on ice in the dark for 5 min after gently mixing; 5 μL PI (propidium iodide) dye was added, and the cells were incubated on ice in the dark for 10 min after gently mixing; the data were collected by the machine. The apoptosis data were analyzed using FlowJo. Each group of experiments was repeated three times.

[0100] 2.3.2 JC-1 staining method to detect the effect of compound Hedyotis diffusa on mitochondrial membrane potential of gastric cancer cells

[0101] Take AGS and HGC-27 cells that are in good growth state and in the logarithmic growth phase, count the cells, and plate them in a 12-well plate at a rate of 3×10 5Cells were inoculated at a density of 100 / well, 1 mL of complete medium was added to each well, and the cells were gently shaken in a "cross" shape to disperse the cells evenly. After marking, the cells were transferred to a 37°C, 5% CO2 incubator for culture; when the cells grew to a confluence of 50-60%, the old culture medium was discarded, and AGS cells (3.5 mg / mL, 5 mg / mL, 9 mg / mL) and HGC-27 cells (5 mg / mL, 6.5 mg / mL, 8 mg / mL) were treated with different concentrations of CHD-E, with 3 replicates per group. The cells were cultured in the drug-containing medium for 6 hours and apoptosis was induced; the JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanine iodide) working solution was diluted. Taking a 12-well plate as an example, each well requires 500 μL of JC-1 working solution. When diluting the JC-1 working solution, add 50 μL of 200×JC-1 reagent to 8 mL of sterile triple-distilled water, shake vigorously, mix thoroughly, add 2 mL of 5×JC-1 staining buffer, mix well to get 10 mL of JC-1 staining working solution, and prepare it immediately before use; after drug stimulation, discard the old culture medium, rinse with PBS three times, add 500 μL of complete culture medium and 500 μL of JC-1 staining working solution, transfer to a 37°C, 5% CO2 incubator and incubate for 20 minutes; prepare 1×JC-1 staining buffer. That is, add 1 mL of 5×JC-1 reagent to 4 mL of sterile triple-distilled water, put it on ice for use, and prepare it immediately before use; after the cell incubation is completed, discard the old culture medium, rinse twice with pre-cooled 1×JC-1 staining buffer, add 1 mL of complete culture medium, and take photos under an inverted fluorescence microscope for observation. Place the cell culture plate on ice before taking pictures, and take pictures as soon as possible within 30 minutes. Each group of experiments was repeated three times.

[0102] 2.4 Determination of the anti-gastric cancer effect of compound Hedyotis diffusa by regulating the activity of Wnt / β-catenin signaling pathway

[0103] WB experiments were used to preliminarily explore and verify the potential pharmacological molecular mechanism of CHD against gastric cancer. Consistent with item "2.2.4", QCT was selected as the positive drug. AGS cells were treated with 5 mg / mL CHD-E and 70 μM QCT; HGC-27 cells were treated with 6.5 mg / mL CHD-E and 90 μM QCT, and the drugs were stimulated for 24 hours.

[0104] 3. Statistics and Analysis

[0105] SPSS25.0 software and GraphPadPrism 8.0 software were used for data processing, analysis and drawing. For the comparison of differences between two groups, Student t test was used; for the comparison of differences between multiple groups, one-way ANOVA single factor analysis of variance and Tukey post hoc multiple comparisons were used to determine the statistical differences; for in vivo experiments, two-way analysis of variance with repeated measures and Tukey post hoc multiple comparisons were used to determine the statistical differences. Each experiment was repeated 3 times or more. *p<0.05, **p<0.01, ***p<0.001. *p<0.05 was considered statistically significant.

[0106] 4 Experimental results

[0107] 4.1 Compound Herba Hedyotis diffusae inhibits the growth activity and proliferation ability of gastric cancer cells

[0108] Effect of compound oldenlandia diffusa on the vitality of gastric cancer cells

[0109] The results showed that at 24h administration time, the IC50 values ​​of CHD-E for each cell line were: AGS cell IC50 = 8.91 ± 0.73 mg / mL; HGC27 cell IC50 = 7.59 ± 0.15 mg / mL; MKN-1 cell IC50 = 5.30 ± 0.10 mg / mL; MKN-28 cell IC50 = 6.81 ± 0.48 mg / mL; GES-1 cell IC50 = 15.45 ± 0.23 mg / mL. The toxic effect of CHD-E on gastric cancer cell lines was greater than that on human gastric mucosal epithelial cells, and the results were statistically significant (see Figure 1 ). In addition, the experiment found that at low concentrations, CHD-E played a certain role in promoting cell proliferation. When the drug concentration increased, the cell viability of gastric cancer cells decreased significantly, showing obvious concentration dependence. The gradient setting of CHD-E drug concentration remained unchanged, and the administration time was extended. It was found that CHD-E could effectively inhibit the growth activity of 4 types of gastric cancer cells at 24h and 36h (see Figure 2 ).

[0110] Combined with the cell culture status and drug sensitivity to CHD-E, AGS and HGC-27 cells were selected as the research objects of this application for subsequent experiments, and low, medium and high doses were set according to IC10, IC20 and IC50 values.

[0111] Effect of compound oldenlandia diffusa on colony-forming ability of gastric cancer cells

[0112] The results showed that CHD-E could significantly inhibit the cloning ability of gastric cancer cells. The number of gastric cancer cell clones gradually decreased with the increase of drug concentration, and the results were statistically significant (see Figure 3 ).

[0113] Effect of compound oldenlandia diffusa on proliferation of gastric cancer cells detected by Edu staining

[0114] Consistent with the results of the plate cloning experiment, the percentage of Edu-positive cells gradually decreased, indicating that CHD-E could significantly reduce the proliferation ability of cells in a concentration-dependent manner (see Figure 4 ).

[0115] 4.2 Effects of compound Hedyotis diffusa on the migration and invasion of gastric cancer cells and down-regulation of gastric cancer metastasis-related protein expression

[0116] The results of the scratch test to detect the effect of compound Hedyotis diffusa on the migration ability of gastric cancer cells are shown in Figure 5 It was found that the "scar" healing rate decreased with the increase of drug concentration, indicating that CHD-E can significantly inhibit the horizontal migration ability of gastric cancer cells.

[0117] The results of Transwell assay to detect the effect of compound Hedyotis diffusa on the migration ability of gastric cancer cells are shown in Figure 6 It was found that the number of cells entering the lower chamber decreased significantly with the increase of drug concentration, indicating that CHD-E can significantly inhibit the vertical migration ability of gastric cancer cells.

[0118] The results of Transwell assay to detect the effect of compound oldenlandia diffusa on the invasion ability of gastric cancer cells are shown in Figure 7 It was found that the number of cells entering the lower chamber decreased significantly with the increase of drug concentration, indicating that CHD-E can significantly inhibit the invasive ability of gastric cancer cells.

[0119] The results of the effect of compound Hedyotis diffusa on the expression level of gastric cancer cell metastasis-related proteins are shown in Figure 8 After 24 hours of drug stimulation, the expressions of mesenchymal cell markers N-cadherin and Vimentin, transcription factors Snail and ZEB1, and adhesion factors CD44 and EpCAM in gastric cancer cells were significantly reduced, indicating that CHD-E may inhibit the metastasis of gastric cancer cells by affecting the EMT process and regulating the expression of cell adhesion and migration-related proteins.

[0120] 4.3 Compound Hedyotis diffusa induces apoptosis of gastric cancer cells

[0121] The results of flow cytometry detection of the effect of compound herb on apoptosis of gastric cancer cells are shown in Fig. 9 , and found that CHD-E can promote apoptosis of gastric cancer cells and has an inducing effect on both early and late apoptosis.

[0122] The results of staining method to detect the effect of compound oldenlandia diffusa on mitochondrial membrane potential of gastric cancer cells are shown in Fig.10,With the increase of drug concentration, the green fluorescence gradually increased, indicating that the mitochondrial membrane potential gradually decreased, suggesting that CHD-E-induced cell apoptosis may be related to the decrease of mitochondrial membrane potential.

[0123] 4.4 Compound Herba Hedyotis diffusae exerts anti-gastric cancer effects by regulating the activity of Wnt / β-catenin signaling pathway

[0124] Results Fig.11 After 24 hours of drug stimulation, the levels of Akt, phosphorylated glycogen synthase kinase 3β (p-GSK3β), β-catenin and c-myc proteins were significantly downregulated, indicating that the ability of Akt to phosphorylate GSK3β was reduced, and more GSK3β played a normal function, that is, phosphorylated β-catenin, causing it to degrade, and it could not enter the cell nucleus to bind to transcription factors to regulate nuclear gene expression, thereby playing a tumor suppressor role. In addition, Dickkopf-related protein 1 (DDK1) was highly expressed, indicating that DKK1 could compete with the complex on the Wnt / β-catenin pathway to bind to Wnt protein and synergistically promote Wnt / β-catenin to be in the "Off" state. The above conclusions suggest that CHD-E has the advantages of multiple therapeutic targets and can inhibit the activity of the tumor-related classical signaling pathway Wnt / β-catenin to play an anti-gastric cancer role.

[0125] Example 13 Animal Experiment

[0126] 1 Experimental Materials

[0127] 1.1 Cell lines

[0128] Same as Example 13.

[0129] 1.2 Animals

[0130] The SPF (specific pathogen free animal) grade BALB / c nude mice used in this part of the experiment were provided by the Experimental Animal Center of Shanghai First People's Hospital, and the experimental protocol was approved by the Animal Ethics Committee of Shanghai First People's Hospital (IACUC No.: 2022AW001).

[0131] 2 Experimental methods

[0132] 2.1 Animal modeling

[0133] In order to evaluate the efficacy of compound snake tongue grass against gastric cancer in an in vivo model, this experiment used nude mice to construct a CDX (cellline-derived xenograft, cell-derived xenograft tumor) model. Human gastric cancer cells AGS were injected into the right back of each nude mouse. Before injection, the cells were injected after a certain distance under the skin. After the injection, the needle was rotated and slowly withdrawn after a period of time. The injection volume was 100 μL / mouse, and the number of cells injected was 1×10 7 The tumor formation effect was observed every day. 7-10 days after the injection of tumor cells, the subcutaneous tumor grew to a volume of about 20-50mm. 3 , the modeling was considered successful.

[0134] 2.2 Grouping and Dosing

[0135] According to the principle of random grouping, the tumor-bearing mice were divided into a control group (drinking water, intragastric administration), a Tongguanteng positive drug administration group (TGT, 1 times the clinical equivalent dose, 3.90 mL / kg nude mouse body weight, intragastric administration), a CHD-E low-dose administration group (1 times the clinical equivalent dose, 450.72 mg / kg nude mouse body weight, intragastric administration), and a CHD-E high-dose administration group (2 times the clinical equivalent dose, 901.44 mg / kg nude mouse body weight, intragastric administration). The drug was administered continuously for 14 days, and the intragastric volume was 0.1 mL / 10 g nude mouse body weight, that is, the maximum intragastric volume for a 20 g nude mouse was 200 μL.

[0136] 2.3 Measurement indicators

[0137] From the day of drug administration, the weight of nude mice and the growth of subcutaneous tumors were recorded. The formula for calculating the volume of subcutaneous tumors is: Volume (mm 3 )=Length(mm)×Width 2 (mm 2 ) / 2; After the administration on the 14th day, the nude mice were killed by CO2 asphyxiation on the 15th day. The tumor tissue was completely removed, and the tumor size was recorded and photographed.

[0138] 3. Statistics and Analysis

[0139] Same as Example 13.

[0140] 4 Results

[0141] During the drug administration period, the nude mice in each group grew well, without abnormal manifestations such as arched back, convulsions, and erect hair. Fig.12 As shown in Figures AC, compared with the control group, the administration of positive drugs Tongguanteng oral liquid (TGT) and CHD-E can significantly inhibit the growth of xenograft tumors, indicating that CHD-E also exhibits good anti-tumor activity in in vivo experiments and has a significant anti-gastric cancer effect. Fig.12As shown in D, there was no significant change in body weight of nude mice in all groups during the drug administration period, indicating that the drug intervention did not produce obvious toxicity or adverse reactions to the body.

[0142] Example 14 Optimization of the preparation process of compound Hedyotis diffusa granules

[0143] 1 Experimental Materials

[0144] 1.1 Experimental Reagents

[0145] Compound Hedyotis diffusa water extract (Shanghai Liantang Pharmaceutical, China); dextrin, soluble starch, mannitol, sodium chloride (Sinopharm Group, China).

[0146] 1.2 Experimental consumables

[0147] Glass vacuum desiccator, weighing paper, weighing bottle, funnel (Beekman Biotechnology, China); standard sieve (16 mesh) (Shaoxing Shengchao Instrument, China); national standard pharmacopoeia sieves (No. 1 sieve, No. 5 sieve) (Dongmai Technology Instrument, China); 500mL, 1000mL beakers, glass rods, 100mL, 500mL, 1000mL measuring cylinders (Sichuan Shubo Group, China).

[0148] 1.3 Experimental instruments

[0149] Refrigerator (4°C, -20°C, -80°C) (Haier, China); electric blast drying oven (Shanghai Jinghong Experimental Equipment, China); AL04 electronic analytical balance (Mettler Toledo, USA); water purifier (Millipore, USA); (water bath Qunan Experimental Instrument, China).

[0150] 2 Experimental methods

[0151] 2.1 Preparation of particles

[0152] Same as Example 12.

[0153] 2.2 Optimization of formulation process

[0154] 2.2.1 Selection of auxiliary materials

[0155] Soluble starch, dextrin and mannitol were selected as candidate excipients, and subsequent single-factor investigation and mixed excipient screening were carried out to determine the optimal type and amount of excipients. Depending on the sweetness of the final granules, it was decided whether to add stevioside as a sweetener.

[0156] 2.2.2 Single factor investigation of excipients

[0157] Following the principle of minimum auxiliary materials, four groups were set with extract: auxiliary material = 1:1, extract: auxiliary material = 1:2, extract: auxiliary material = 1:3, and extract: auxiliary material = 1:4. The particles were prepared using the above method to investigate the effect of a single auxiliary material on the soft material and particle formability.

[0158] The results are shown in Table 2. When dextrin and soluble starch are used as single excipients, the ratio of extract: excipient = 1:2 can successfully make soft materials and granules, and the granules are uniform in size and have good formability. When the amount of excipients is further increased, there are too many small particles and fine powders after the soft materials are sieved. When mannitol is used as a single excipient, the ratio of extract: mannitol = 1:4 still cannot make granules, indicating that when it is used alone as an excipient for CHD granules, its performance is poor.

[0159] Table 2 Results of single factor investigation experiment on auxiliary materials

[0160]

[0161]

[0162] 2.2.3 Screening of mixed excipients and investigation of granules

[0163] With reference to the above single auxiliary material screening results, different groups were set up according to Table 3 to prepare granules, and the obtained granules were evaluated for molding rate, angle of repose, bulk density, solubility, hygroscopicity and water content to screen the best mixed auxiliary material types and ratios.

[0164] Table 3 Prescription for mixed excipient screening

[0165]

[0166] Forming rate

[0167] Refer to the particle size determination method of the Pharmacopoeia of the People's Republic of China: Volume 4 (2020 Edition) (hereinafter referred to as the Pharmacopoeia), and use the manual double screening method. Accurately weigh 5g of granules, with No. 1 sieve on top and No. 5 sieve on the bottom, first pass through No. 1 sieve, then pass through No. 5 sieve, keeping the sieve holes level. Place the granules on No. 1 sieve and tap for 3 minutes. Granules that can pass through No. 1 sieve but not No. 5 sieve are qualified granules, and the molding rate is calculated after weighing.

[0168] Forming rate = (mass of particles after sieving / mass of particles before sieving) × 100%.

[0169] The results are shown in Table 4. Prescription 2 has the best molding rate, while prescriptions 14 and 17 have lower molding rates. There are too many small particles or fine powders that can pass through the No. 5 sieve, and the molding properties are slightly poor.

[0170] Table 4 Forming rate measurement results

[0171]

[0172] Angle of repose

[0173] The fixed funnel method is used to measure the angle of repose. The funnel is fixed at a height of H (cm) from the tabletop, and the particles are slowly poured along the wall of the funnel until the tip of the particle cone formed on the tabletop touches the funnel mouth. The radius of the bottom of the particle cone is measured and recorded as R (cm). The angle formed by the cone and the tabletop is the angle of repose α. tgα=H / R.

[0174] The results are shown in Table 5. The angles of repose of each formulation are all less than 30°, indicating that the obtained particles have good fluidity. Among them, the angle of repose of formulation 14 is the smallest, and its particle fluidity is the best.

[0175] Table 5 Repose angle measurement results

[0176]

[0177]

[0178] Bulk density

[0179] According to the pharmacopoeia, a certain amount of granules is accurately weighed and placed in a dry measuring cylinder. The mass of the granules is taken as M (g). After gentle vibration, the value of the surface granules near the scale is read and recorded as V (mL). The ratio of the two is the bulk density. Bulk density / (g / mL) = M / V.

[0180] The bulk density measurement results of each prescription are shown in Table 6. The results show that the bulk density value of prescription 13 is the largest.

[0181] Table 6 Bulk density measurement results

[0182]

[0183] Solubility

[0184] Accurately weigh 0.5 g of particles and place them in a beaker. Add 10 mL of boiling water and stir for 5 min. Transfer to a 50 mL centrifuge tube and centrifuge at 3000 rpm for 10 min. Discard the supernatant and dry the residue in an oven at 80 °C to constant weight. Calculate the solubility rate after weighing.

[0185] Dissolution rate value = (mass of residue after dissolution - mass of particles before dissolution) / mass of particles before dissolution × 100%.

[0186] In this experiment, after stirring, most of the soluble particles of the prescriptions were completely dissolved, prescriptions 13 and 15 showed slight turbidity, and prescriptions 2 and 14 showed obvious turbidity. Subsequently, after centrifugation and drying to constant weight, the solubility test results of each prescription calculated after weighing the residue are shown in Table 7. Consistent with the observation results, the solubility rates of prescriptions 2, 14 and 13 were low, and the solubility rate of prescription 17 was the highest, indicating that its particles had the best solubility.

[0187] Table 7 Solubility test results

[0188]

[0189] Hygroscopicity

[0190] Add a supersaturated sodium chloride solution to the bottom of the glass desiccator, place the desiccator in a 25°C constant temperature incubator for 24 hours, and make the relative humidity in the desiccator 75%. Accurately weigh a certain amount of particles and a glass weighing bottle that has been dried to a constant weight, spread the particles on the bottom of the glass weighing bottle, keep the weighing bottle open, place it in a glass desiccator, and place it in a 25°C constant temperature incubator for 72 hours. Calculate the moisture absorption rate after weighing.

[0191] Moisture absorption rate = (mass of particles after moisture absorption - mass of particles before moisture absorption) / mass of particles before moisture absorption × 100%.

[0192] The results of the hygroscopicity test of each prescription are shown in Table 8. The results show that the hygroscopicity of each prescription is less than 1%, among which prescription 15 has the weakest hygroscopicity and the highest score.

[0193] Table 8 Hygroscopicity measurement results

[0194]

[0195] Water content

[0196] Referring to the Pharmacopoeia moisture determination method, the drying method is used to determine the moisture content of the granules. Accurately weigh 2-5g of granules, the weight is recorded as W1 (g), and the granules are spread flat in a glass weighing bottle that has been dried to constant weight. The weight of the weighing bottle is recorded as W2 (g), and the thickness does not exceed 5mm. Keep the weighing bottle open and dry it in a constant temperature box at 100-105℃ for 5h. After drying, cover the weighing bottle cap, move it to a dryer with a supersaturated sodium chloride solution at the bottom, cool it for 30 minutes, and then accurately weigh it. Dry it under the above conditions for another 1h, cool it, and weigh it until the difference between two consecutive weighings does not exceed 5mg. At this time, the sum of the weight of the glass weighing bottle and the granules is recorded as W3 (g). Calculate the moisture content of the granules based on the lost weight.

[0197] Water content = (W1 + W2 - W3) / W1 x 100%.

[0198] The results of the water content determination of each prescription are shown in Table 9. The results show that the water content of each prescription is less than 5%, which meets the requirements of the pharmacopoeia. Among them, prescription 18 has the lowest water content and the highest score.

[0199] Table 9 Water content determination results

[0200]

[0201] 2.3 Evaluation of particle forming process

[0202] The optimized molding process was comprehensively evaluated from the uniformity, stability and solubility of the granules, and the molding rate, angle of repose, bulk density, solubility, hygroscopicity and water content measured above were used as evaluation indicators, and the optimal preparation process of compound snake tongue grass granules was determined according to the comprehensive scoring results. The weight coefficients of each evaluation indicator were 15%, 15%, 15%, 15%, 20%, and 20%, respectively. The comprehensive score was the multiplication of each value by the weight coefficient, that is, the comprehensive score Y = molding rate / maximum molding rate × 15 + minimum angle of repose value / angle of repose value × 15 + bulk density value / maximum bulk density value × 15 + dissolution rate / maximum dissolution rate × 15 + minimum hygroscopicity value / hygroscopicity value × 20 + minimum water content value / water content × 20.

[0203] The granules obtained from each prescription are shown in Fig.13 The comprehensive evaluation results of the molding process are shown in Table 10. The granules obtained by prescription 18 are yellow-brown and have a good molding rate; the granules have a small bulk density, small gaps between particles, good fluidity, and are easy to pack, which can reduce the difference in loading volume; the granules have good solubility; the granules have weak hygroscopicity and low water content, which can ensure the stability of the drug as much as possible and are not easy to deteriorate. After the volunteers tried it, the granules were fragrant, sweet, and slightly bitter, and no additional sweeteners were needed. Therefore, prescription 18, that is, extract: soluble starch: dextrin: mannitol = 1:1:1:1 is the optimal molding process for compound snake tongue grass granules.

[0204] Table 10 Comprehensive scoring results of particle forming process

[0205]

[0206] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for treating gastric cancer, characterized in that: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 335-415 parts of Hedyotis diffusa, 335-415 parts of Shuyangquan, and 335-415 parts of Smilax china.

2. The Chinese medicine composition according to claim 1, characterized in that The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 355-395 parts of Hedyotis diffusa, 355-395 parts of Shuyangquan, and 355-395 parts of Smilax china.

3. The Chinese medicine composition according to claim 2, characterized in that The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 375 parts of Hedyotis diffusa, 375 parts of Shuyangquan, and 375 parts of Smilax china.

4. A compound Hedyotis diffusa granule, characterized in that: The invention is prepared from the traditional Chinese medicine composition according to any one of claims 1 to 3 and auxiliary materials.

5. The compound oldenlandia tongue grass granules according to claim 4, characterized in that The auxiliary materials are soluble starch, dextrin and mannitol.

6. A method for preparing the compound Hedyotis diffusa granules according to claim 5, characterized in that: The following steps are involved: 1) Extract Preparation: Take the medicinal material according to any of claims 1-3 in the proportions by weight, add 8 times the amount of water, and boil twice, decocting for 2 hours the first time and 1.5 hours the second time. After decoction, filter the decoction, combine the filtrates, and let them stand for 12-16 hours. Concentrate the supernatant to a clear paste with a relative density of 1.30-1.35 (80-85°C), aliquot, seal, and store in a -80°C freezer until ready for use. 2) Granule Preparation: Mix the above-mentioned paste with soluble starch, dextrin, and mannitol in a specific proportion. Add distilled water as a wetting agent. Mix thoroughly and knead the mixture until the soft material forms a ball that falls apart when gently gripped. Place the soft material on a 16-mesh stainless steel sieve, squeeze it through the sieve, and collect the sieved granules. Spread the granules evenly on a tray and dry them in an 80°C oven for 3 hours to obtain granules.

7. The preparation method according to claim 6, characterized in that The mass ratio of the clear paste to the soluble starch, dextrin and mannitol is 1:1:1:

1.

8. Use of the traditional Chinese medicine composition according to any one of claims 1 to 3 and the compound herbaceous snake tongue grass granules according to claim 4 or 5 in the preparation of a drug for treating and / or preventing gastric cancer.

9. The use according to claim 8, characterized in that The drug can be used for superficial and atrophic gastritis accompanied by intestinal metaplasia, atypical hyperplasia and other precancerous lesions of gastric cancer.

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