Esophageal cancer resisting medicine derived from fresh rabdosia rubescens and preparation method of esophageal cancer resisting medicine

Abstract

The invention provides an anti-esophageal cancer medicine derived from fresh rabdosia rubescens and a preparation method of the anti-esophageal cancer medicine, and relates to the technical field of pharmacy of fresh traditional Chinese medicines. According to the invention, fresh rabdosia rubescens is taken as a basic medicinal material, an extraction process is improved and optimized, an extract containing Oridonin (Ori) and rosmarinic acid (RA) is obtained, and the esophageal cancer resisting effect of the fresh rabdosia rubescens extract is researched. Experimental results show that the treatment effect of Ori combined with RA administration on esophageal cancer model mice is remarkable, the effect is better than that of Ori or RA independent administration, and it is shown that oridonin and rosmarinic acid are combined for use, and the esophageal cancer resisting effect can be remarkably improved. The invention provides a novel medicine and a novel treatment thought for treating esophageal cancer.

Description

Technical Field

[0001] The invention relates to the technical field of fresh Chinese medicine pharmaceutical preparation, and in particular to an anti-esophageal cancer drug derived from fresh Rubescens rubescens and a preparation method thereof. Background Art

[0002] Esophageal cancer is one of the common malignant tumors of the digestive system. Despite the continuous development of comprehensive treatment for esophageal cancer, its 5-year survival rate is still less than 20%. Therefore, the development of new drugs for the treatment of esophageal cancer is an urgent need and one of the important directions of clinical research. Winter rubescens is a traditional Chinese medicine in my country. Clinical practice has shown that it is effective in treating a variety of malignant tumors, especially for a variety of common tumors such as lung cancer, with a tumor inhibition rate of up to 41%.

[0003] Rubescens is particularly sensitive to esophageal cancer and gastric cancer, which have a high incidence in northern my country. Diterpenoids are the main active ingredients in Rubescens, among which Oridonin (Ori) is the main anti-cancer active ingredient in Rubescens extract. In addition, Rosmarinic acid (RA), as a representative of water-soluble compounds of Rubescens phenolic acid, has anti-tumor, anti-inflammatory, antioxidant and anti-microbial functions. At present, there are no reports on the use of RA in the fight against esophageal cancer, and there are no reports on the anti-esophageal cancer effect of the combined administration of Ori and RA.

[0004] Fresh Chinese medicine refers to fresh plants (including roots, stems, leaves, flowers, fruits, etc.) and fresh animals (including whole animals or organs, tissues, etc.) that have not been dried or processed. They are Chinese medicinal materials directly used to treat diseases under the guidance of traditional Chinese medicine theory, and are one of the characteristics of Chinese medicine treatment. Most of the "Donglingcao" used in clinical practice are processed products and sun-dried products, which will cause the loss of active substances during the processing. However, since fresh Chinese medicine does not undergo any drying and complex processing, the unstable substances in fresh Chinese medicine, such as volatile and heat-sensitive components, do not show degradation loss and change, the types and contents of chemical components are richer, and the natural ratio between components is not destroyed. Therefore, compared with its processed products, the original medicinal properties and efficacy of Chinese medicine are fully preserved, and its efficacy is more prominent when treating diseases. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the deficiencies in the prior art, the present invention provides a method for preparing a fresh Rubescens rubescens extract and application of the extract in preparing an anti-esophageal cancer drug.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The invention provides a medicine for resisting esophageal cancer, which consists of oridonin and rosmarinic acid.

[0010] Specifically, the oridonin and rosmarinic acid are derived from fresh oridonium rubescens.

[0011] Specifically, the mass ratio of oridonin to rosmarinic acid in the fresh Rubescens is 1:2.34.

[0012] Specifically, the oridonin is prepared and extracted according to the following steps: fresh oridonin is mixed with methanol in a ratio of 1 g: 10-70 mL, and ultrasonic extraction is performed for 20-70 min. The above steps are repeated 1-5 times, and the filtrate is collected and combined, and filtered through a microporous filter membrane to obtain an extract containing oridonin.

[0013] Preferably, the best extraction process of Rubescensine A is: ultrasonic extraction, with methanol as solvent, 30 min as extraction time, 1:30 as solid-liquid ratio, and 3 extraction times, Ori in dried and fresh Rubescens products can be completely extracted.

[0014] Specifically, the rosmarinic acid is prepared and extracted according to the following steps: fresh Rubescens rubescens and ethanol are mixed in a ratio of 1 g: 20-70 mL, and ultrasonic extraction is performed for 10-70 min. The above steps are repeated 1-5 times; the filtrate is collected and combined, and filtered through a microporous filter membrane to obtain an extract containing Rubescensine A; the mass fraction of the ethanol is 50-95%.

[0015] Preferably, the best extraction process of rosmarinic acid is: ultrasonic extraction, using 50% ethanol as solvent, 50 minutes as extraction time, 1:50 as solid-liquid ratio, and 3 extraction times, so that RA in dried and fresh Rabdosia rubescens can be completely extracted.

[0016] The fresh Rubescens used is the stem and leaf parts of Rabdosia rubescens (Hemsl.) Hara, a plant of the Lamiaceae family.

[0017] (III) Beneficial effects

[0018] The present invention uses fresh Rubescens as the basic medicinal material, improves and optimizes the extraction process, obtains an extract containing Rubescensine A and rosmarinic acid, and studies the anti-esophageal cancer effect of the fresh Rubescens extract. Among them, the best extraction process of Ori is: ultrasonic extraction, with methanol as solvent, 30 minutes as extraction time, 1:30 as solid-liquid ratio, and 3 extraction times, Ori in the dried and fresh products of Rubescens can be completely extracted; the best extraction process of RA is: ultrasonic extraction, with 50% ethanol as solvent, 50 minutes as extraction time, 1:50 as solid-liquid ratio, and 3 extraction times, RA in the dried and fresh products of Rubescens can be completely extracted.

[0019] At the same time, we used the above optimal extraction process to extract fresh and dried products of Rabdosia rubescens, and used high performance liquid chromatography to determine the contents of Ori and RA in fresh and dried products of Rabdosia rubescens. The results showed that the contents of Ori and RA in fresh Rabdosia rubescens were higher than those in dried products.

[0020] The present invention further studies the anti-esophageal cancer effects of Ori and RA. The experimental results show that the administration of Ori combined with RA can improve the quality of life of mice with esophageal cancer, increase the weight of mice, and reduce the mortality rate; inhibit organ lesions, reduce the degree of esophageal canceration, and reduce the disorder of spleen tissue structure; reduce the levels of serum tumor markers (CEA, CYFRA21-1, SCC), reduce the levels of NLRP3, ASC, Caspase-1, and IL-1β mRNA in esophageal tissue, reduce serum TNF-α, IL-6, and COX-2 levels, and inhibit local and systemic inflammatory reactions in the esophagus; reduce the levels of N-cadherin and Bcl-2 mRNA in esophageal tissue, increase the levels of E-cadherin and Bax mRNA, inhibit the malignant proliferation and mesenchymal transformation of tumor cells, and the effect is better than that of Ori or RA alone, indicating that the combination of Rubescensine A and rosmarinic acid can significantly improve the anti-esophageal cancer effect. The present invention provides a new drug and treatment idea for the treatment of esophageal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effect on the survival time of mice with esophageal cancer.

[0022] Figure 2 Effects on body weight of mice bearing esophageal cancer.

[0023] Figure 3 Effects on serum tumor markers CEA, CYFRA21-1 and SCC in mice with esophageal cancer; Note: *Compared with M, P<0.05; **Compared with M, P<0.01; a FOR-H vs. Ori, P < 0.05; aa FOR-H vs. Ori, P < 0.01;b FOR-H vs RA, P < 0.05; bb FOR-H vs RA, P<0.01; the same below.

[0024] Figure 4 Effects on serum inflammatory factors TNF-α, IL-6 and COX-2 in mice with esophageal cancer.

[0025] Figure 5 Effects on spleen appearance, weight and index of esophageal cancer mice.

[0026] Figure 6 Effects on pathological changes of spleen in mice with esophageal cancer.

[0027] Figure 7 Effects on the esophageal epigenetic appearance of mice with esophageal cancer.

[0028] Figure 8 Effects on pathological changes in the esophagus of mice with esophageal cancer.

[0029] Fig. 9 Effects on changes in NLRP3, ASC, Caspase-1 and IL-1β mRNA in esophageal tissue of mice with esophageal cancer.

[0030] Fig.10 Effects on changes in N-cadherin and E-cadherin mRNA in esophageal tissue of mice with esophageal cancer.

[0031] Fig.11 Effects on changes in Bax and Bcl-2 mRNA in esophageal tissue of mice with esophageal cancer. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Experimental herbs

[0035] Fresh rubescens was collected in Jiyuan City, Henan Province, and was identified as the fresh aerial part of Rabdosia rubescens (Hemsl.) Hara of the Lamiaceae family. The freshly picked rubescens was divided into two parts: one part was stored in a refrigerator at -80°C, which was the fresh rubescens product and was taken as needed; the other part was dried in the sun to obtain the sun-dried rubescens product of the same batch.

[0036] 2 Methods and Results

[0037] 2.1 Chromatographic conditions

[0038] Chromatographic conditions for Ori determination: chromatographic column: phenomsil C18 (250×4.6mm, 5μm) column; mobile phase: methanol (A): water (B) = 55:45; flow rate: 0.8mL / min; column temperature: 30°C; detection wavelength: 237nm; injection volume: 10μL.

[0039] Chromatographic conditions for RA determination: chromatographic column: phenomsil C18 (250×4.6 mm, 5 μm) column; mobile phase: acetonitrile (A): 0.1% acetic acid water (B) = 32:68, flow rate: 0.8 mL / min; column temperature: 30° C.; detection wavelength: 327 nm; injection volume: 10 μL.

[0040] 2.2 Preparation of reference solution

[0041] Preparation of Ori reference solution: Accurately weigh 5.0 mg of Ori reference substance into a volumetric flask, dilute to 10 mL with methanol as the mother solution, dilute to 25, 50, 100, 200, 250, 300, 400, 500, 750 μg / mL respectively, and filter with a 0.22 μm microporous filter membrane to obtain the Ori reference solution.

[0042] Preparation of RA reference solution: Accurately weigh 5.0 mg of RA reference substance into a volumetric flask, dilute to 10 mL with 50% ethanol as the mother solution, dilute to 25, 50, 100, 200, 250, 300, 400, 500 μg / mL respectively, and filter with a 0.22 μm microporous filter membrane to obtain the RA reference solution.

[0043] 2.3 Preparation of test solution

[0044] Preparation of test solutions of fresh and dried Rubescens herb for determination of Ori: using methanol as solvent, 30 min as ultrasonic extraction time, 1:30 as solid-liquid ratio, extraction times of 3 times, collecting the filtrate, combining the filtrates of 3 times, filtering with a 0.22 μm microporous filter membrane to obtain the test solution.

[0045] Preparation of test solutions of fresh and dried Rubescens rubescens for determining RA: using 50% ethanol as solvent, 50 min as ultrasonic extraction time, 1:50 as solid-liquid ratio, extraction times of 3 times, collecting the filtrate, combining the filtrates from 3 times, filtering with a 0.22 μm microporous filter membrane, and obtaining the test solution.

[0046] 3.4 Optimization of extraction conditions

[0047] 3.4.1 Optimization of Ori extraction process

[0048] Selection of extraction time: According to the properties of Ori, methanol was used as the solvent to ultrasonically extract Ori from the dried and fresh products of Rabdosia rubescens. Different extraction times were set: 20min, 30min, 50min, and 70min; 12 equal samples of dried and fresh Rabdosia rubescens were weighed, and 3 samples were prepared in parallel for each extraction time; methanol was added at 1g:30mL, and the extraction was completed. The samples were filtered with a 0.22μm microporous membrane, and the samples were injected according to the chromatographic conditions under "2.1". The peak time of Ori in the test sample was calibrated with the reference solution. After the experiment, the peak area of ​​Ori was recorded and the average peak area was calculated.

[0049] The experimental results are shown in Table 1: For the dried product of Rabdosia rubescens, there is almost no difference in the peak area of ​​Ori at each extraction time. For the fresh product of Rabdosia rubescens, the peak area of ​​Ori at 30 min is greater than that at 20 min, and is close to the peak area at 50 min and 70 min extraction times. Based on the peak areas of Ori in the dried and fresh products of Rabdosia rubescens at different extraction times, 30 min was selected as the time for subsequent extraction.

[0050] Table 1-1 The extraction effect of Ori in the dried product of Rabdosia rubescens at different extraction times

[0051]

[0052] Table 1-2 The extraction effect of Ori in fresh Rubescens rubescens at different extraction times

[0053]

[0054] Selection of material-liquid ratio: methanol was used as solvent, and 30 minutes was used as ultrasonic extraction time. The materials were extracted at 1:10, 1:30, 1:40, 1:50 and 1:70, and 15 equal samples of dried and fresh wintergrass were weighed. Three samples were prepared in parallel for each material-liquid ratio, and filtered through a 0.22μm microporous membrane after extraction, and injected according to the chromatographic conditions under "2.1". After the experiment, the peak area was recorded and converted.

[0055] The experimental results show that for the dried product of Rabdosia rubescens, the peak area at 1:10 is the lowest, and 1:30, 1:40, 1:50 and 1:70 are close. For the fresh product of Rabdosia rubescens, the peak area at 1:10 is the lowest, followed by 1:40 and 1:50, and 1:30 is close to 1:70. Based on the peak area of ​​Ori in the dried and fresh products of Rabdosia rubescens under different material-liquid ratios, 1:30 was selected as the time for subsequent extraction.

[0056] Selection of extraction times: methanol was used as solvent, extraction was performed for 30 minutes, the material-liquid ratio was 1g:30mL, and 15 equal samples of dried and fresh wintergrass were weighed. Ultrasonic extraction was performed for 1, 2, 3, 4, and 5 times, and 3 samples were prepared in parallel for each extraction. After each extraction, the corresponding filtrate was collected and the corresponding volume of solvent was added. After the experiment, the peak area of ​​RA was recorded and converted.

[0057] For the dried and fresh products of Rabdosia rubescens, the peak area was the largest when extracted three times, indicating that the extraction was complete after three times; however, the peak area decreased after five times of extraction, which may be related to the instability of Ori and its degradation as the extraction time increases. Therefore, the final Ori extraction process was: methanol as solvent, ultrasonic for 30 minutes, solid-liquid ratio of 1g:30mL, and extraction three times.

[0058] 3.4.2 Optimization of RA extraction process

[0059] Selection of extraction solvent: According to the properties of rosmarinic acid (RA), the solvent was first selected. Weigh 12 equal portions of fresh and dried samples of Rubescens rubescens (three parallel samples were prepared for each solvent). Methanol, 95% ethanol, 70% ethanol, and 50% ethanol were added at a ratio of 1g:30mL. Ultrasonic extraction was performed for 30 minutes. The filtrate was collected and filtered through a 0.22μm microporous membrane. The sample was injected according to the chromatographic conditions under "2.1". The peak time of RA was calibrated with a reference solution. The peak area of ​​RA in the sample was recorded. The average peak area of ​​the three samples was calculated.

[0060] The results are shown in Table 2: When extracted with 50% ethanol, the RA peak area in both the dried and fresh products of Rubescens rubescens was the largest, and the extraction rate was the highest, so 50% ethanol was used as the extraction solvent in the subsequent step.

[0061] Table 2-1 The extraction effect of different solvents on RA in sun-dried Rubescens rubescens

[0062]

[0063] Table 2-2 Effects of different solvents on the extraction of RA from fresh Rubescens rubescens

[0064]

[0065] Selection of extraction time: Weigh 12 equal samples of fresh and dried Rabdosia rubescens; add 50% ethanol at 1 g: 30 mL, and ultrasonically extract for 10 min, 30 min, 50 min, and 70 min, respectively. Prepare 3 samples in parallel for each extraction time, collect the filtrate, filter it with a 0.22 μm microporous filter membrane, and inject 10 μL. After the experiment, record the peak area of ​​RA in the sample and calculate the average peak area of ​​the 3 samples.

[0066] From Table 3, we know that when the extraction solvent is fixed at 50% ethanol and the ultrasonic extraction is 50 minutes, the peak area of ​​RA in the dried product of Rabdosia rubescens is the largest; when the ultrasonic extraction is 30 minutes, 50 minutes and 70 minutes, the peak area of ​​RA in the fresh product of Rabdosia rubescens is similar, and all of them are larger than the peak area at 10 minutes. Therefore, 50 minutes is used as the extraction time in the following.

[0067] Table 3-1 The extraction effect of different extraction times on RA in dried Rubescens rubescens

[0068]

[0069] Table 3-2 The extraction effect of different extraction times on RA in fresh Rubescens rubescens

[0070]

[0071] Selection of material-liquid ratio: Weigh 15 samples of fresh and dried products of Rubescens rubescens in equal amounts, add the corresponding volume of 50% ethanol, and make the material-liquid ratios 1:20, 1:30, 1:40, 1:50 and 1:70, respectively. Prepare 3 samples in parallel for each material-liquid ratio, perform ultrasonic extraction for 50 minutes, collect the filtrate, filter it with a 0.22μm microporous filter membrane, and inject 10μL. After the experiment, record the peak area and convert it. The experimental results show that when 50% ethanol is used as the solvent and 50 minutes is used as the ultrasonic extraction time, the peak area of ​​RA in the dried product of Rubescens rubescens at a material-liquid ratio of 1:50 is close to that of 1:70; when the material-liquid ratio is 1:50, the peak area of ​​RA in the fresh product of Rubescens rubescens is the largest. Considering comprehensively, 1:50 is used as the optimal material-liquid ratio in the future.

[0072] Selection of extraction times: 50% ethanol was used as solvent, extraction was performed for 50 min, the material-liquid ratio was 1 g:50 mL, and 15 samples of fresh and dried products of Rubescens rubescens were weighed. Ultrasonic extraction was performed for different times. After each extraction, the corresponding filtrate was collected and then the corresponding volume of solvent was added. Three samples were prepared in parallel for each extraction. At the end of the experiment, the peak area was recorded and converted. The experimental results showed that the peak areas of extraction 3 times and extraction 4 times were similar. The RA peak area in the dried and fresh products of Rubescens rubescens was the largest, indicating that extraction 3 times could be completely extracted; while the peak area decreased after extraction 5 times, which may be related to the instability of RA and its degradation as the extraction time was prolonged. Therefore, 3 times was selected as the extraction condition for RA.

[0073] 4Provide process optimization results

[0074] Based on the above experimental results, the optimal extraction process of Ori and RA in fresh Rubescens was finally screened out.

[0075] The best extraction process of Ori is: ultrasonic extraction, methanol as solvent, 30 minutes as extraction time, 1:30 as solid-liquid ratio, and 3 extraction times, which can completely extract Ori from dried and fresh wintergrass.

[0076] The best extraction process of RA is: ultrasonic extraction, 50% ethanol as solvent, 50 min as extraction time, 1:50 as solid-liquid ratio, and 3 extraction times, which can completely extract RA from both dried and fresh products of Rubescens rubescens.

[0077] 5 Determination of Ori and RA contents in fresh and dried products of Rubescens rubescens

[0078] The above optimal extraction process was used to extract fresh and sun-dried Rabdosia rubescens, and the contents of Ori and RA in fresh and sun-dried Rabdosia rubescens were determined by high performance liquid chromatography. The contents of Ori and RA in sun-dried Rabdosia rubescens were 7.79 mg / g and 6.48 mg / g, respectively, with a ratio of 1:0.83; the contents of Ori and RA in fresh Rabdosia rubescens were 2.70 mg / g and 6.32 mg / g, respectively, with a ratio of 1:2.34. The contents of Ori and RA in fresh Rabdosia rubescens were 9.52 mg / g and 22.28 mg / g after multiplying the dryness ratio by 3.525 (3.525 g of fresh product is dried to obtain 1 g of sun-dried product, and the same mass is 3.525 g of fresh product is equivalent to 1 g of sun-dried product), that is, under the same mass, the contents of Ori and RA in fresh product are higher than those in sun-dried product.

[0079] Example 2

[0080] Study on the efficacy of Rubescensine A combined with rosmarinic acid in the treatment of esophageal cancer.

[0081] 1 Experimental animals

[0082] C57BL / 6 mice, SPF grade, male, 5-6 weeks old. Purchased from: Shandong Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number: SCXK (Lu) 20190003, animal qualification certificate number: No. 370726221100163878. Animal experimental mice were raised in the SPF animal room of the Experimental Animal Center of Henan University of Traditional Chinese Medicine, and the experimental unit use license number: SYXK (Yu) 2020-0004. Animal breeding environment: temperature (20-25) ° C, relative humidity 50%-60%, 12h light, free drinking water and food.

[0083] 2 Experimental methods

[0084] 2.1 Modeling and grouping

[0085] After the mice were adaptively fed, 10 mice were randomly selected as the blank control group (Con), and the remaining mice freely drank 100 μg / mL 4-NQO solution for 1-16 weeks and freely drank drinking water without 4-NQO for 17-23 weeks to construct an esophageal cancer mouse model, and the death of mice was recorded from the start of drug administration to the end of the experiment.

[0086] Starting from the 23rd week of modeling, 70 mice with esophageal cancer were randomly divided into 5 groups, namely model group (M), low-dose group (Low, 30.56mg / kg Ori+71.54mg / kg RA), high-dose group (High, 61.12mg / kg Ori+143.08mg / kgRA), Ori group (61.12mg / kg Ori), RA group (143.08mg / kg RA), 14 mice in each group, and the drugs were administered by gavage. The Con group and the M group were gavaged with the corresponding volume of solvent, once a day, and the experiment ended at the 31st week.

[0087] 2.2 Observation indicators

[0088] 2.2.1 Growth status and death of experimental animals

[0089] Observe the mice's activity, mental state, defecation and urination every day, and pay special attention to death in the later stages.

[0090] 2.2.2 Weight, food and water intake records

[0091] At the beginning of drug administration, the body weight of mice was recorded regularly and a body weight change curve was drawn.

[0092] 2.2.3 Spleen weight, index, and appearance of spleen and esophageal tissues

[0093] After blood was drawn, the mice were killed, spleen tissue was removed, weight was recorded, and organ index was calculated: organ index = organ weight (mg) / body weight (g). The esophagus (tongue-esophagus-stomach were removed together) and spleen were removed, photographed, and apparent changes were recorded.

[0094] 2.2.4 Serum factor detection

[0095] Blood was collected from the vein, allowed to stand, and then centrifuged at 3500 rpm / min for 10 min. The upper serum was then collected and stored at -80°C to measure the levels of serum tumor markers CEA, CYFRA21-1, SCC, and inflammatory factors COX-2, TNF-α, and IL-6.

[0096] 2.2.5 Observation of pathological changes in mouse esophagus and spleen tissues by HE staining

[0097] After fixation, the esophageal and spleen tissues were dehydrated with alcohol gradient, embedded in paraffin, and sliced. After unfolding, scooping and drying, they were stained with HE, and the pathological changes were observed under a microscope.

[0098] 2.2.6 IHC measurement of Bcl-2 and Bax protein levels in esophageal tissue

[0099] IHC main operation steps: paraffin-embedded esophageal tissue was dewaxed and dehydrated with xylene and ethanol concentration gradient. The tissue was autoclaved at 120°C in citrate buffer at pH 6.0 and incubated in 3% hydrogen peroxide for 10 minutes to inactivate endogenous catalase. The sections were then blocked with 10% horse serum. Finally, the sections were incubated with Bcl-2 and Bax antibodies at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies at room temperature for 1-2 hours, and finally incubated with streptavidin HRP.

[0100] 2.2.7 RT-qPCR detection of NLRP3, ASC, Caspase-1, IL-1β, E-Cadherin and N-Cadherin mRNA levels in esophageal tissue

[0101] Total RNA from esophageal tissue was extracted according to the kit instructions, and 1 μg of total RNA was then reverse transcribed into cDNA using HyperScript RT SuperMix for qPCR. Finally, real-time PCR was performed using HyperScript 2×SYBR Green qPCR MasterMix. -ΔΔCt Methods Relative expression was calculated.

[0102] 2.3 Statistical analysis

[0103] SPSS 21.0 was used for statistical analysis, and one-way analysis of variance was used for inter-group comparisons. The results are expressed as mean ± standard deviation The data were analyzed in the same format, and the LSD or Games-Howell test was selected according to whether the variance was homogeneous. ImagePro_Plus software was used to analyze the immunohistochemical results, and GraphPad Prism v.8.0 was used for plotting. P < 0.05 was considered statistically significant.

[0104] 3 Results

[0105] 3.1 Effects on the growth status and mortality of 4-NQO-induced esophageal cancer model mice

[0106] 3.1.1 Effect on the growth status of esophageal cancer model mice

[0107] The Con group mice had shiny black hair, well-proportioned bodies, good mental state, and quick response when caught. The M group mice had dull, unkempt, yellowish-white hair, curled bodies, obvious hunched backs, shortened stature, thin bodies, slow movements, a sense of "trembling", blurred eyes, dullness, and obvious morbidity. There was almost no resistance when caught, and some mice had blood in the stool and yellow urine. The mice in each drug-treated group had relatively stretched hair, shiny color, more symmetrical bodies, and significantly less curling and hunched backs than the model group. They could move freely, had a better mental state, had a certain ability to resist when caught, and had less blood in the stool.

[0108] 3.1.2 Effect on the mortality of esophageal cancer model mice

[0109] By the end of the experiment, no mice in the Con group died. Six mice in the M group, Ori group, and RA group died by the end of the experiment, with a mortality rate of 42.86%. Four mice in the High group died by the end of the experiment, with a mortality rate of 28.57%. Three mice in the Low group died by the end of the experiment, with a mortality rate of 21.43%. See Table 1. Although the survival rates of mice in some groups were consistent, there were certain differences in the number of days they survived (see Figure 1 :Mouse survival curve), the results showed that compared with the administration of Ori or RA alone, the combined administration of Ori and RA had a higher survival rate and longer survival time in mice, and had a better anti-esophageal cancer effect.

[0110] Table 1 Effects of 4-NQO on the survival rate of esophageal cancer model mice

[0111]

[0112] 3.2 Effects on the body weight of esophageal model mice

[0113] like Figure 2 As shown, during the administration period, the body weight of mice in the M group was significantly lower than that in the Con group. In each administration group, compared with the single administration of Ori and RA, the combined use of the two drugs (High group) had a better effect on improving the body weight of mice. The specific changes in body weight were ( Figure 2): Compared with the Con group, the body weight of mice in the M group decreased significantly during the entire administration period (P<0.01). Compared with the M group, the body weight of mice in the High group increased significantly at 25W, 27W and 29W (P<0.05), and the body weight of mice in the High group increased significantly at 31W and the beginning of 32W (data on the day of the results at the end of 31W) (P<0.01); the body weight of mice in the Low group increased significantly at 29W and the beginning of 32W (data on the day of the results at the end of 31W) (P<0.05). At the same time, at 27W, the body weight of mice in the High group increased significantly compared with the Ori group and the RA group (P<0.05); at 31W, the body weight of mice in the High group increased significantly compared with the RA group (P<0.05). Compared with the Ori group, the body weight of mice in the High group increased significantly (P<0.01); at 32W, the body weight of mice in the High group increased significantly compared with the RA group (P<0.05).

[0114] 3.3 Effects on serum tumor markers in esophageal cancer model mice

[0115] Depend on Figure 3 It can be seen that compared with the Con group, the serum CEA, CYFRA21-1, and SCC levels of mice in the M group were significantly increased (P<0.01). Compared with the M group, the serum CEA, CYFRA21-1, and SCC levels of mice in the Low group, High group, and Ori group were significantly decreased (P<0.01), and the serum SCC and CYFRA21-1 levels of mice in the RA group were significantly decreased (P<0.01). Compared with the Ori group, the serum CEA level of mice in the High group was significantly decreased (P<0.01); compared with the RA group, the serum CEA, CYFRA21-1, and SCC levels of mice in the High group were significantly decreased (P<0.01).

[0116] 3.4 Effects on serum inflammatory factors TNF-α, IL-6 and COX-2 levels in esophageal cancer model mice

[0117] like Figure 4 As shown in the results, compared with the Con group, the levels of TNF-α, IL-6 and COX-2 in the serum of mice in the M group were significantly increased (P<0.01), and the level of IFN-γ did not change significantly (P>0.05). Compared with the M group, the levels of TNF-α, IL-6 and COX-2 in the serum of mice in the Low group, High group, Ori group and RA group were significantly decreased (P<0.01). At the same time, compared with the Ori group, the level of TNF-α in the serum of mice in the High group was significantly decreased (P<0.05), and the level of IL-6 was significantly decreased (P<0.01); compared with the RA group, the levels of TNF-α and IL-6 in the serum of mice in the High group were significantly decreased (P<0.01).

[0118] 3.5 Effects on spleen and esophageal tissue lesions in esophageal cancer model mice

[0119] 3.5.1 Effects on spleen appearance, weight, index and pathological changes in esophageal cancer model mice

[0120] Effects on the appearance, weight and index of mouse spleen (see Figure 5 ): The spleen of mice in the Con group was wider, longer, and more ruddy in color. The spleen of mice in the M group was narrower, shorter, and darker in color. The spleen length of mice in each drug-treated group was between the Con group and the M group, and the color was closer to that of the Con group, and the width was wider. Compared with the Con group, the spleen weight and index of mice in the M group were significantly reduced (P<0.01). Compared with the M group, the spleen weight and index of mice in the Low group were significantly increased (P<0.01), and the spleen weight of mice in the High group was significantly increased (P<0.01).

[0121] Effects on pathological changes in the spleen of mice (see Figure 6 ): The spleen structure of mice in the Con group was normal, the spleen cord network structure was good, and there was no congestion in the splenic sinus. The spleen tissue structure of mice in the M group was disordered, the boundaries between the red pulp and white pulp were blurred, the red pulp was obviously atrophied, the white pulp was hyperplastic, and the splenic nodules were reduced. The spleen tissue structure of mice in the Low group, High group, and Ori group was significantly improved compared with the M group, with clear boundaries between the red pulp and white pulp and a good spleen cord network structure. The RA group did not significantly improve the spleen tissue pathological changes in mice with esophageal cancer.

[0122] 3.5.2 Effects on the esophageal appearance and pathological changes in esophageal cancer model mice

[0123] Effects on esophageal appearance (see Figure 7 ): Under the naked eye, the esophageal tissue of mice in the Con group was thinner and longer, with a smooth surface, and no protruding growths appeared inside the esophagus. The mouse tongue was pink and tender, and no white clusters appeared. The gastric tissue was uniform in size, and there was no ulceration, erosion, or bleeding after dissection; the esophageal tissue of mice in the M group was thicker and shorter, and the esophageal surface was rough. Under the naked eye, tumor tissue of different sizes and lengths could be seen inside the esophagus. White clusters appeared on the tongues of some mice, and the tongue was white. The gastric tissue was reduced, and there was bleeding, erosion, and ulcer bleeding after cutting. The thickness and length of the esophageal tissue of mice in the Low group, High group, Ori group, and RA group were between the Con group and the M group, the number of protruding tumors inside the esophagus decreased, and the internal growths became smaller. The tongue was pink, and the white clusters decreased or disappeared. The ulcers, bleeding, and erosions in the gastric tissue of each drug-treated group were greatly reduced.

[0124] Effects on pathological changes of esophageal tissue in mice (see Figure 8): The esophageal tissue structure of mice in the Con group was normal, and the cells were arranged regularly. Almost the entire esophageal tissue of mice in the M group became cancerous, with disordered arrangement of basal cells, loss of polarity, significant atypia, high nuclear division activity, dark staining of cell nuclei, and formation of cancer nests. A large number of inflammatory cells were observed to infiltrate, and cancer cells infiltrated and grew exophytically, with most cancer cells infiltrating into the muscular layer. The range of esophageal canceration in mice in the Low, High, Ori, and RA groups was reduced, and normal esophageal tissue and cancerous tissue could be seen alternating in the field of vision. The degree of canceration was reduced, and cancer cells could be seen infiltrating into the submucosal layer, and the muscular layer tissue was not completely invaded by cancer cells.

[0125] 3.6 Effects on NLRP3, ASC, Caspase-1, and IL-1β mRNA levels in esophageal tissues of mice with esophageal cancer

[0126] like Fig. 9 As shown in the results, compared with the Con group, the mRNA levels of NLRP3, ASC, Caspase-1 and IL-1β in the esophageal tissue of mice in the M group were significantly increased (P<0.01). Compared with the M group, the mRNA levels of NLRP3, Caspase-1 and IL-1β in the esophageal tissue of mice in the Low group, High group, Ori group and RA group were significantly decreased (P<0.01); the mRNA level of ASC in the esophageal tissue of mice in the Low group, High group and Ori group was significantly decreased (P<0.01). At the same time, compared with the Ori group, the mRNA levels of NLRP3, ASC and Caspase-1 in the esophageal tissue of mice in the High group were significantly decreased (P<0.01), and the mRNA level of IL-1β was significantly decreased (P<0.05); compared with the RA group, the mRNA levels of NLRP3, ASC, Caspase-1 and IL-1β in the esophageal tissue of mice in the High group were significantly decreased (P<0.01).

[0127] 3.7 Effects of N-cadherin and E-cadherin mRNA levels in esophageal tissue of esophageal cancer model mice

[0128] like Fig.10As shown in the figure, compared with the Con group, the N-cadherin mRNA level in the esophageal tissue of mice in group M was significantly increased, while the E-cadherin mRNA level was significantly decreased (P<0.01). Compared with the M group, the N-cadherin mRNA level in the esophageal tissue of mice in groups Low, High, Ori and RA was significantly decreased (P<0.01). Meanwhile, compared with the Ori group, the E-cadherin mRNA level in the esophageal tissue of mice in group High was significantly increased (P<0.01); compared with the RA group, the E-cadherin mRNA level in the esophageal tissue of mice in group High was significantly increased (P<0.01).

[0129] Effect of 3.8 on the mRNA levels of Bax and Bcl-2 in esophageal tissue of esophageal cancer model mice

[0130] like Fig.11 As shown: Compared with the Con group, the Bax mRNA level in the esophageal tissue of mice in the M group was significantly decreased, and the Bcl-2 mRNA level was significantly increased (P<0.01). Compared with the M group, the Bax mRNA levels in the esophageal tissue of mice in the Low group, High group and Ori group were significantly increased (P<0.01), and the Bax mRNA level in the esophageal tissue of mice in the RA group was significantly increased (P<0.05); the Bcl-2 mRNA levels in the esophageal tissue of mice in the Low group, High group, Ori group and RA group were significantly decreased (P<0.01). At the same time, compared with the Ori group, the Bax mRNA level in the esophageal tissue of mice in the High group was significantly increased (P<0.05); compared with the RA group, the Bax mRNA level in the esophageal tissue of mice in the High group was significantly increased (P<0.01), and the Bcl-2 mRNA level was significantly decreased (P<0.05).

[0131] 4 Summary

[0132] The above experimental results show that the combined administration of Ori and RA can improve the quality of life of esophageal cancer mice, increase the body weight of mice and reduce the mortality rate; inhibit organ lesions, reduce the degree of esophageal carcinogenesis, and reduce the disorder of spleen tissue structure; reduce the levels of serum tumor markers (CEA, CYFRA21-1, SCC), reduce the levels of NLRP3, ASC, Caspase-1, and IL-1β mRNA in esophageal tissue, reduce serum TNF-α, IL-6 and COX-2 levels, and inhibit local and systemic inflammatory responses in the esophagus; reduce the levels of N-cadherin and Bcl-2 mRNA in esophageal tissue, increase the levels of E-cadherin and Bax mRNA, inhibit the malignant proliferation and mesenchymal transformation of tumor cells, and the effect is better than that of Ori or RA alone, indicating that the combination of oridonin and rosmarinic acid can significantly improve the anti-esophageal cancer effect.

[0133] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A drug for treating esophageal cancer, characterized in that: The medicine consists of oridonin and rosmarinic acid.

2. The anti-esophageal cancer drug according to claim 1, characterized in that: The oridonin and rosmarinic acid are derived from fresh oridonium rubescens.

3. The anti-esophageal cancer drug according to claim 2, characterized in that: The mass ratio of oridonin to rosmarinic acid in the fresh Rubescens is 1:2.

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4. The anti-esophageal cancer drug according to claim 2, characterized in that: The oridonin is prepared and extracted according to the following steps: fresh oridonin is mixed with methanol in a ratio of 1 g: 10-70 mL, and ultrasonic extraction is performed for 20-70 min. The above steps are repeated 1-5 times, and the filtrate is collected and combined, and filtered through a microporous filter membrane to obtain an extract containing oridonin.

5. The anti-esophageal cancer drug according to claim 2, characterized in that: The rosmarinic acid is prepared and extracted according to the following steps: fresh Rubescens rubescens and ethanol are mixed in a ratio of 1g:20-70mL, ultrasonic extraction is performed, the ultrasonic time is 10-70min, and the above steps are repeated 1-5 times; the filtrate is collected and combined, and filtered with a microporous filter membrane to obtain an extract containing Rubescensine A.

6. The anti-esophageal cancer drug according to claim 5, characterized in that: The mass fraction of ethanol in the rosmarinic acid extraction step is 50-95%.

7. The anti-esophageal cancer drug according to claim 2, characterized in that: The fresh Rubescens used is the stem and leaf parts of Rabdosia rubescens (Hemsl.) Hara, a plant of the Lamiaceae family.

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