Anti-esophageal cancer drug derived from fresh rabdosia rubescens and preparation method thereof

Abstract

The application provides an anti-esophageal cancer drug derived from fresh Rabdosia rubescens and a preparation method thereof, and relates to the technical field of traditional Chinese medicine fresh medicine pharmacy. The application takes fresh Rabdosia rubescens as a base medicinal material, improves and optimizes an extraction process, obtains an extract containing oridonin (Ori) and rosmarinic acid (RA), and studies the anti-esophageal cancer effect of the extract from fresh Rabdosia rubescens. The experimental results show that the treatment effect of Ori combined with RA on esophageal cancer model mice is remarkable, and the effect is better than that of single administration of Ori or RA, which indicates that oridonin combined with rosmarinic acid can significantly improve the anti-esophageal cancer effect. The application provides a new drug and treatment idea for the treatment of esophageal cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine fresh drug manufacturing, and particularly relates to an anti-esophageal cancer drug derived from fresh Rabdosia rubescens and a preparation method thereof. Background Art

[0002] Esophageal cancer is one of the common malignant tumors in the digestive system. Despite the continuous development of comprehensive treatment of esophageal cancer, its 5-year survival rate is still less than 20%. Therefore, developing new drugs for the treatment of esophageal cancer is an urgent need and an important direction in clinical research. Rabdosia rubescens is a traditional Chinese medicine in China. Clinical practice shows that it is effective in the treatment of various malignant tumors, especially the tumor inhibition rate of various common tumors such as lung cancer is as high as 41%.

[0003] Rabdosia rubescens is particularly sensitive to esophageal cancer and gastric cancer with a relatively high incidence in northern China. Diterpenoids are the main active components in Rabdosia rubescens. Among them, Oridonin (Ori) is the main anti-cancer active component in the extract of Rabdosia rubescens. In addition, Rosmarinic acid (RA), as a representative of phenolic acid water-soluble compounds in Rabdosia rubescens, has functions such as anti-tumor, anti-inflammatory, antioxidant and inhibiting microorganisms. At present, there is no relevant report on the use of RA for anti-esophageal cancer, and there is no report on the anti-esophageal cancer effect of the combined administration of Ori and RA.

[0004] Traditional Chinese medicine fresh drugs refer to fresh plants (including roots, stems, leaves, flowers, fruits, etc.) and fresh living bodies (including whole animals or organs, tissues, etc.) that have not been dried and processed, and are traditional Chinese medicine materials directly used for treating diseases under the guidance of traditional Chinese medicine theory, which is one of the characteristics of traditional Chinese medicine in treating diseases. Clinically, "Rabdosia rubescens" used is mostly processed products and dried products, and the active substances will be lost during the processing. Since traditional Chinese medicine fresh drugs do not go through any drying and complex processing procedures, unstable substances such as volatile and heat-sensitive components in traditional Chinese medicine fresh drugs do not degrade, lose or change, the types and contents of chemical components are more abundant, and the natural ratio between components is not destroyed. Therefore, compared with its processed products, the original medicinal properties and effects of traditional Chinese medicine are completely retained, and its curative effect is more prominent when treating diseases. Summary of the Invention

[0005] (1) Technical Problems to be Solved

[0006] In view of the deficiencies of the prior art, the present invention provides a preparation method of an extract of fresh Rabdosia rubescens and its application in the preparation of anti-esophageal cancer drugs.

[0007] (2) Technical Solutions

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

[0009] This invention provides an anti-esophageal cancer drug, which is composed of oridonin and rosmarinic acid.

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

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

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

[0013] Preferably, the optimal extraction process for oridonin A is as follows: ultrasonic extraction, using methanol as solvent, 30 minutes as extraction time, 1:30 material-to-liquid ratio, and 3 extraction times, can completely extract Ori from both dried and fresh oridonin.

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

[0015] Preferably, the optimal extraction process for rosmarinic acid is as follows: ultrasonic extraction, using 50% ethanol as solvent, 50 min as extraction time, 1:50 material-to-liquid ratio, and 3 extraction times, can completely extract RA from both dried and fresh *Rhizopus lucidus*.

[0016] The fresh wintergreen grass used above refers to the stems and leaves of Rabdosia rubescens (Hemsl.) Hara, a plant of the Lamiaceae family.

[0017] (III) Beneficial Effects

[0018] This invention uses fresh *Rabdosia rubescens* as the base medicinal material, improves and optimizes the extraction process to obtain an extract containing oridonin A and rosmarinic acid, and studies the anti-esophageal cancer effect of the above-mentioned extract from fresh *Rabdosia rubescens*. Specifically, the optimal extraction process for Ori is: ultrasonic extraction, using methanol as the solvent, an extraction time of 30 minutes, a material-to-liquid ratio of 1:30, and three extractions, which can completely extract Ori from both dried and fresh *Rabdosia rubescens*. The optimal extraction process for RA is: ultrasonic extraction, using 50% ethanol as the solvent, an extraction time of 50 minutes, a material-to-liquid ratio of 1:50, and three extractions, which can completely extract RA from both dried and fresh *Rabdosia rubescens*.

[0019] Meanwhile, we used the aforementioned optimal extraction process to extract the fresh and dried *Rhizoma Rabdosiae*, and then used high-performance liquid chromatography (HPLC) to determine the contents of Ori and RA in both products. The results showed that the contents of Ori and RA in the fresh product were higher than those in the dried product.

[0020] This invention further investigated the anti-esophageal cancer effects of Ori and RA. Experimental results showed that Ori combined with RA improved the quality of life, increased body weight, and reduced mortality in mice with esophageal cancer; inhibited organ lesions, reduced the degree of esophageal cancer, and alleviated splenic tissue structural disorder; decreased serum tumor marker levels (CEA, CYFRA21-1, SCC); decreased NLRP3, ASC, Caspase-1, and IL-1β mRNA levels in esophageal tissue; decreased serum TNF-α, IL-6, and COX-2 levels; and inhibited local and systemic inflammatory responses in the esophagus. Furthermore, it decreased esophageal tissue N-cadherin and Bcl-2 mRNA levels, increased E-cadherin and Bax mRNA levels, and inhibited malignant proliferation and mesenchymal transformation of tumor cells. These effects were superior to those of Ori or RA alone, indicating that the combination of oridonin and rosmarinic acid significantly improved the anti-esophageal cancer effect. This invention provides new drugs and treatment strategies for esophageal cancer. Attached Figure Description

[0021] Figure 1 Effects on survival time in mice with esophageal cancer.

[0022] Figure 2 Effects on body weight in mice with esophageal cancer.

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

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

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

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

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

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

[0031] Figure 11 Effects on changes in Bax and Bcl-2 mRNA in esophageal tissue of mice with esophageal cancer. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1

[0034] 1. Experimental medicinal materials

[0035] Fresh *Rabdosia rubescens*, harvested in Jiyuan City, Henan Province, was identified as the fresh aerial parts of *Rabdosia rubescens* (Hemsl.) Hara, a plant belonging to the Lamiaceae family. The freshly harvested *Rabdosia rubescens* was divided into two portions: one portion was stored at -80℃ as fresh *Rabdosia rubescens*, to be used as needed; the other portion was dried to obtain the same batch of dried *Rabdosia rubescens*.

[0036] 2. Methods and Results

[0037] 2.1 Chromatographic conditions

[0038] Ori assay chromatographic conditions: Column: phenomsil C18 (250×4.6mm, 5μm) column; Mobile phase: methanol (A): water (B) = 55:45; Flow rate: 0.8mL / min; Column temperature: 30℃; Detection wavelength: 237nm; Injection volume: 10μL.

[0039] Chromatographic conditions for RA determination: Column: phenomsil C18 (250×4.6mm, 5μm) column; Mobile phase: acetonitrile (A): 0.1% acetic acid water (B) = 32:68; Flow rate: 0.8mL / min; Column temperature: 30℃; Detection wavelength: 327nm; Injection volume: 10μL.

[0040] 2.2 Preparation of reference solution

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

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

[0043] 2.3 Preparation of the test solution

[0044] Preparation of test solutions for fresh and dried *Rhizophora stricta* for Ori: Methanol was used as the solvent, ultrasonic extraction time was 30 min, material-to-liquid ratio was 1:30, and extraction was performed 3 times. The filtrate was collected, and the filtrates from the three extractions were combined and filtered through a 0.22 μm microporous membrane to obtain the test solution.

[0045] Preparation of test solutions for fresh and dried *Rhizophora stricta* for determining RA: Using 50% ethanol as solvent, ultrasonic extraction time of 50 min, material-to-liquid ratio of 1:50, and extraction three times, collect the filtrate, combine the three filtrates, and filter with a 0.22 μm microporous membrane to obtain the test solution.

[0046] 3.4 Optimization of Extraction Conditions

[0047] 3.4.1 Optimization of Ori extraction process

[0048] Selection of extraction time: Based on the properties of Ori, methanol was used as the solvent to perform ultrasonic extraction on both dried and fresh samples of *Rabdosia rubescens*. Different extraction times were set: 20 min, 30 min, 50 min, and 70 min. Twelve equal samples of both dried and fresh *Rabdosia rubescens* were weighed, and three replicates were prepared for each extraction time. Methanol was added at a ratio of 1 g: 30 mL. After extraction, the sample was filtered through a 0.22 μm microporous membrane and injected according to the chromatographic conditions described in section "2.1". The peak elution time of Ori in the test sample was determined using a 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*, the peak area of ​​Ori was almost the same at different extraction times. For the fresh product, the peak area of ​​Ori at 30 min was greater than that at 20 min, and close to that at 50 min and 70 min. Considering the peak areas of Ori in both the dried and fresh products at different extraction times, 30 min was selected as the subsequent extraction time.

[0050] Table 1-1 Effects of different extraction times on the extraction of Ori from dried Rhizoma Rabdosiae.

[0051]

[0052] Table 1-2 Effects of different extraction times on the extraction of Ori from fresh Rabdosia rubescens.

[0053]

[0054] Selection of solid-liquid ratio: Methanol was used as the solvent, and the ultrasonic extraction time was 30 minutes. Extraction was performed at ratios of 1:10, 1:30, 1:40, 1:50, and 1:70, with 15 equal samples each of dried and fresh *Rhizoma Isodon*. Each solid-liquid ratio was prepared in triplicate. After extraction, the samples were filtered through a 0.22 μm microporous membrane and injected according to the chromatographic conditions described in section "2.1". After the experiment, the peak areas were recorded and calculated.

[0055] Experimental results showed that for dried *Rabdosia rubescens*, the peak area was lowest at a ratio of 1:10, while the ratios of 1:30, 1:40, 1:50, and 1:70 were similar. For fresh *Rabdosia rubescens*, the peak area was lowest at 1:10, followed by 1:40 and 1:50, with 1:30 and 1:70 being similar. Considering the peak areas of Ori in both dried and fresh *Rabdosia rubescens* under different material-to-liquid ratios, 1:30 was selected as the extraction time for subsequent extractions.

[0056] Selection of extraction times: Methanol was used as the solvent, and extraction was performed for 30 minutes at a material-to-liquid ratio of 1g:30mL. Fifteen equal samples of dried and fresh *Rhizoma Rabdosiae* were weighed. Ultrasonic extraction was performed 1, 2, 3, 4, and 5 times, with each extraction number prepared in triplicate. 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 calculated.

[0057] For both dried and fresh *Isodon japonicus*, the peak area was largest after three extractions, indicating that complete extraction was achieved in three extractions. However, after five extractions, the peak area decreased, possibly due to the instability of *Ori* and its degradation with prolonged extraction time. Therefore, the final *Ori* extraction process is as follows: using methanol as solvent, ultrasonication for 30 minutes, a solid-liquid ratio of 1 g:30 mL, and extraction three times.

[0058] 3.4.2 Optimization of RA extraction process

[0059] Selection of extraction solvent: Based on the properties of rosmarinic acid (RA), the solvent was first selected. Equal amounts of fresh and dried *Rhizoma Rabdosiae rubescens* samples were weighed, 12 aliquots each (3 parallel samples for each solvent). Methanol, 95% ethanol, 70% ethanol, and 50% ethanol were added at a ratio of 1 g:30 mL. The mixture was ultrasonically extracted for 30 min. The filtrate was collected, filtered through a 0.22 μm microporous membrane, and injected under the chromatographic conditions described in section "2.1". The peak time of RA was determined using a standard solution. The peak area of ​​RA in the samples was recorded, and the average peak area of ​​the 3 samples was calculated.

[0060] The results are shown in Table 2: When extracted with 50% ethanol, the RA peak area was the largest in both the dried and fresh products of *Rhizoma Undaria pinnatifida*, and the extraction rate was the highest. Therefore, 50% ethanol was used as the extraction solvent in subsequent extractions.

[0061] Table 2-1 Extraction effects of different solvents on RA from dried Rhizoma Rabdosiae.

[0062]

[0063] Table 2-2 Extraction effects of different solvents on RA in fresh Rabdosia rubescens

[0064]

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

[0066] Table 3 shows that when the extraction solvent was fixed at 50% ethanol and ultrasonic extraction was performed for 50 min, the peak area of ​​RA in the dried product of *Rabdosia rubescens* was the largest. The peak areas of RA in the fresh product of *Rabdosia rubescens* were similar after ultrasonic extraction for 30 min, 50 min, and 70 min, and were all larger than the peak area at 10 min. Therefore, 50 min was subsequently used as the extraction time.

[0067] Table 3-1 Effects of different extraction times on the extraction of RA from dried Rhizoma Rabdosiae.

[0068]

[0069] Table 3-2 Effects of different extraction times on the extraction of RA from fresh Rhizoma Rabdosiae.

[0070]

[0071] Selection of the solid-liquid ratio: Equal amounts of fresh and dried *Rhizoma Rabdosiae* samples were weighed (15 aliquots each), and corresponding volumes of 50% ethanol were added to achieve solid-liquid ratios of 1:20, 1:30, 1:40, 1:50, and 1:70. Three replicates were prepared for each ratio. Ultrasonic extraction was performed for 50 min, and the filtrate was collected and filtered through a 0.22 μm microporous membrane. 10 μL of the filtrate was injected into the sample. After the experiment, the peak areas were recorded and calculated. The results showed that with 50% ethanol as the solvent and 50 min as the ultrasonic extraction time, the peak area of ​​RA in the dried *Rhizoma Rabdosiae* sample at a solid-liquid ratio of 1:50 was close to that at 1:70; the peak area of ​​RA in the fresh *Rhizoma Rabdosiae* sample was largest at a solid-liquid ratio of 1:50. Considering all factors, 1:50 was subsequently selected as the optimal solid-liquid ratio.

[0072] Selection of extraction times: Using 50% ethanol as solvent, extraction was performed for 50 minutes at a material-to-liquid ratio of 1g:50mL. Equal amounts of fresh and dried *Rhizoma Rabdosiae* samples were weighed out in 15 portions each. Ultrasonic extraction was performed for different numbers of times. After each extraction, the corresponding filtrate was collected, and the corresponding volume of solvent was added. Each extraction was performed in triplicate. At the end of the experiment, peak areas were recorded and calculated. Experimental results showed that the peak areas after 3 and 4 extractions were similar. The RA peak area was the largest in both the dried and fresh *Rhizoma Rabdosiae* samples, indicating that 3 extractions were sufficient for complete extraction. However, after 5 extractions, the peak area decreased, possibly due to the instability of RA and degradation with prolonged extraction time. Therefore, 3 extractions were selected as the optimal extraction condition for RA.

[0073] 4. Provide process optimization results

[0074] Based on the above experimental results, the optimal extraction process for Ori and RA from fresh *Isodon japonicus* was finally selected.

[0075] The optimal extraction process of Ori is as follows: ultrasonic extraction, using methanol as the solvent, 30 min as the extraction time, 1:30 as the solid-liquid ratio, and 3 extraction times. Under these conditions, Ori can be completely extracted from both the dried and fresh samples of Rabdosia rubescens.

[0076] The optimal extraction process of RA is as follows: ultrasonic extraction, using 50% ethanol as the solvent, 50 min as the extraction time, 1:50 as the solid-liquid ratio, and 3 extraction times. Under these conditions, RA can be completely extracted from both the dried and fresh samples of Rabdosia rubescens.

[0077] Determination of the contents of Ori and RA in fresh and dried samples of Rabdosia rubescens

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

[0079] Example 2

[0080] Pharmacodynamic study on the combined use of oridonin and rosmarinic acid in anti-esophageal cancer

[0081] 1 Experimental animals

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

[0083] 2 Experimental methods

[0084] 2.1 Modeling and grouping

[0085] After the mice were acclimatized, 10 mice were randomly selected as the blank control group (Con). The remaining mice were allowed to drink 100 μg / mL 4-NQO solution freely from 1 to 16 weeks, and free drinking water without 4-NQO from 17 to 23 weeks. An esophageal cancer mouse model was established, and the mortality of mice from the start of drug administration to the end of the experiment was recorded.

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

[0087] 2.2 Observation Indicators

[0088] 2.2.1 Growth status and mortality of experimental animals

[0089] Observe the mice's activity, mental state, and bowel movements daily, paying particular attention to mortality in the later stages.

[0090] 2.2.2 Weight, diet and water intake records

[0091] At the start of administration, mouse body weight was recorded periodically, and a curve of body weight change was plotted.

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

[0093] After blood collection, mice were euthanized, spleen tissue was removed, and its weight was recorded. The organ index was calculated as follows: 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 drawn from the vein, centrifuged at 3500 rpm for 10 min after standing, and the supernatant serum was collected and stored at -80℃. The serum levels of tumor markers CEA, CYFRA21-1, and SCC, as well as inflammatory factors COX-2, TNF-α, and IL-6 were measured.

[0096] 2.2.5 HE staining to observe pathological changes in mouse esophagus and spleen tissues

[0097] After the esophagus and spleen tissues were fixed, they were dehydrated in a gradient of alcohol, embedded in paraffin, and sectioned. After spreading, retrieving and drying the sections, they were stained with hematoxylin and eosin (HE) and observed under a microscope for pathological changes.

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

[0099] The main steps of IHC are as follows: Paraffin-embedded esophageal tissue is dewaxed and dehydrated using a gradient of xylene and ethanol concentrations. The tissue is then autoclaved at 120°C in citrate buffer (pH 6.0) and incubated in 3% hydrogen peroxide for 10 min to inactivate endogenous catalase. The sections are then blocked with 10% horse serum. Finally, the sections are incubated overnight at 4°C with Bcl-2 and Bax antibodies, respectively, followed by incubation at room temperature with HRP-conjugated secondary antibody for 1–2 h, 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 was extracted from esophageal tissue according to the kit instructions. Then, 1 μg of total RNA was reverse transcribed into cDNA using HyperScript RT SuperMix for qPCR. Finally, real-time PCR was performed using HyperScript 2×SYBR Green qPCR MasterMix. -ΔΔCt The method calculates the relative expression.

[0102] 2.3 Statistical Analysis

[0103] Statistical analysis was performed using SPSS 21.0. One-way ANOVA was used for comparisons between groups. Results are expressed as mean ± standard deviation. The format was determined by selecting either the LSD or Games-Howell test based on whether the variances were homogeneous. Immunohistochemistry results were analyzed using ImagePro_Plus software, and plotted using GraphPad Prism v.8.0. P < 0.05 was considered statistically significant.

[0104] 3 Results

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

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

[0107] Mice in group Con had glossy black fur, a well-proportioned body, and were in good spirits, reacting quickly when handled. Mice in group M had dry, tangled, yellowish-white fur, were curled up with a noticeably arched back, shorter in length, thinner, and moved slowly, appearing unsteady and shaky. Their eyes were dull and lifeless, showing obvious signs of illness. They showed almost no resistance when handled, and some mice exhibited bloody stools and yellow urine. Mice in all treatment groups had relatively smooth, glossy fur, a more well-proportioned body, and significantly less curling up and arching compared to the model group. They could move freely, were in good spirits, showed some resistance when handled, and exhibited less bloody stools.

[0108] 3.1.2 Effects on mortality in esophageal cancer model mice

[0109] By the end of the experiment, no mice in the Con group had died. Six mice each in the M, Ori, and RA groups died by the end of the experiment, a mortality rate of 42.86%. Four mice in the High group died by the end of the experiment, a mortality rate of 28.57%. Three mice in the Low group died by the end of the experiment, a mortality rate of 21.43%. See Table 1. Although the survival rate was consistent in some groups, the number of days of survival varied (see Table 1). Figure 1 (See mouse survival curve). This result indicates that, compared with Ori or RA alone, the combined administration of Ori and RA resulted in higher mouse survival rates and longer survival times, demonstrating superior anti-esophageal cancer effects.

[0110] Table 1. Effect of 4-NQO on the survival rate of mice in a 4-NQO-induced esophageal cancer model.

[0111]

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

[0113] like Figure 2 As shown, during the administration period, the body weight of mice in group M was significantly lower than that in group Con. Among all treatment groups, the combined use of Ori and RA (High group) showed a better effect on improving mouse body weight compared to administration of Ori or RA alone. Specific changes in body weight were as follows: Figure 2Compared with the Con group, the body weight of mice in the M group was significantly lower throughout the entire drug administration period (P<0.01). Compared with the M group, the body weight of mice in the High group was significantly higher at 25, 27, and 29 weeks (P<0.05), and significantly higher at 31 and early 32 weeks (data from the day of results collection at the end of 31 weeks) (P<0.01); at 29 and early 32 weeks (data from the day of results collection at the end of 31 weeks), the body weight of mice in the Low group was significantly higher (P<0.05). Meanwhile, at 27 weeks, the body weight of mice in the High group was significantly higher than that of the Ori and RA groups (P<0.05); at 31 weeks, the body weight of mice in the High group was significantly higher than that of the RA group (P<0.05). Compared with the Ori group, the body weight of mice in the High group was significantly higher (P<0.01); at 32 weeks, the body weight of mice in the High group was significantly higher than that of 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 was found 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, High, and Ori groups were significantly decreased (P<0.01), while 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 levels of inflammatory factors TNF-α, IL-6, and COX-2 in esophageal cancer model mice

[0117] like Figure 4 As shown, compared with the Con group, the serum levels of TNF-α, IL-6, and COX-2 in the M group mice were significantly increased (P<0.01), while the IFN-γ level showed no significant change (P>0.05). Compared with the M group, the serum levels of TNF-α, IL-6, and COX-2 in the Low, High, Ori, and RA groups were significantly decreased (P<0.01). Meanwhile, compared with the Ori group, the serum levels of TNF-α and IL-6 in the High group mice were significantly decreased (P<0.05) and significantly decreased (P<0.01); compared with the RA group, the serum levels of TNF-α and IL-6 in the High group mice 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 the appearance, weight, index, and pathological changes of the spleen in esophageal cancer model mice

[0120] Effects on the appearance, weight and index of mouse spleen (see...) Figure 5 The spleens of mice in the Con group were wider, longer, and redder in color. The spleens of mice in the M group were narrower, shorter, and darker in color. The spleen length of mice in each treatment group was between that of the Con group and the M group, 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 In the Con group, the spleen structure of mice was normal, the splenic cord network was well-structured, and there was no congestion in the splenic sinuses. In the M group, the spleen tissue structure was disordered, the boundaries between the red and white pulp were blurred, the red pulp was significantly atrophied, the white pulp was hyperplastic, and the splenic nodules were shrunken. The spleen tissue structure of mice in the Low, High, and Ori groups was significantly improved compared to the M group, the boundaries between the red and white pulp were clear, and the splenic cord network structure was well-structured. The RA group did not show significant improvement in the splenic tissue lesions of mice with esophageal cancer.

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

[0123] Effects on esophageal appearance (see) Figure 7 Under macroscopic observation, the esophageal tissue of mice in the Con group was thinner and longer, with a smooth surface and no protruding growths inside the esophagus. The mice's tongues were pink and without white clusters. The stomach tissue was uniform in size, and no ulceration, erosion, or bleeding was observed after dissection. The esophageal tissue of mice in the M group was thicker and shorter, with a rough surface. Under macroscopic observation, tumor tissue of varying sizes and lengths was visible inside the esophagus. Some mice had white clusters on their tongues, which were whitish. The stomach tissue was shrunken, and bleeding, erosion, and ulcer bleeding were observed after dissection. The esophageal tissue of mice in the Low, High, Ori, and RA groups was between that of the Con and M groups in terms of thickness and length. The number of protruding tumors inside the esophagus was reduced, and the internal growths were smaller. The tongues were pink, and the white clusters were reduced or disappeared. Ulcers, bleeding, and erosion in the stomach tissue were significantly reduced in all treatment groups.

[0124] Effects on pathological changes in mouse esophageal tissue (see...) Figure 8In the Con group, the esophageal tissue structure of mice was normal, and the cells were arranged in a regular manner. In the M group, almost the entire esophageal tissue of mice was cancerous, with disordered arrangement of basal cells, loss of polarity, significant atypia, high mitotic activity, deeply stained nuclei, formation of cancer nests, and extensive infiltration of inflammatory cells. Cancer cells infiltrated and exhibited exophytic growth, with most cancer cells infiltrating into the muscle layer. In the Low, High, Ori, and RA groups, the extent of esophageal cancer was reduced, and normal esophageal tissue and cancerous tissue could be seen alternating in the field of view. The degree of cancer was reduced, and cancer cells could be seen infiltrating into the submucosa, while the muscle layer was not completely invaded by cancer cells.

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

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

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

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

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

[0130] like Figure 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 level in the esophageal tissue of mice in the Low, High, and Ori groups was 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 level in the esophageal tissue of mice in the Low, High, Ori, and RA groups was significantly decreased (P<0.01). Meanwhile, 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 indicate that Ori combined with RA administration can improve the quality of life of esophageal cancer mice, increase mouse weight, and reduce mortality; inhibit organ lesions, reduce the degree of esophageal cancer, and alleviate splenic tissue structural disorder; reduce serum tumor marker (CEA, CYFRA21-1, SCC) levels, reduce NLRP3, ASC, Caspase-1, and IL-1β mRNA levels in esophageal tissue, reduce serum TNF-α, IL-6, and COX-2 levels, and inhibit local and systemic inflammatory responses in the esophagus; reduce esophageal tissue N-cadherin and Bcl-2 mRNA levels, increase E-cadherin and Bax mRNA levels, and inhibit malignant proliferation and mesenchymal transformation of tumor cells. Furthermore, the effects are better than those of Ori or RA alone, indicating that the combination of oridonine 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, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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 drug is composed of oridonin A and rosmarinic acid, derived from fresh *Rabdosia rubescens* in a mass ratio of 1:2.

34. Oridonin A is prepared and extracted according to the following steps: fresh *Rabdosia rubescens* is mixed with methanol at a ratio of 1 g:30 mL, and ultrasonic extraction is performed for 30 min. This step is repeated three times. The filtrates are collected and combined, and filtered using a microporous membrane to obtain an extract containing oridonin A. Rosmarinic acid is prepared and extracted according to the following steps: fresh *Rabdosia rubescens* is mixed with 50% ethanol at a ratio of 1 g:50 mL, and ultrasonic extraction is performed for 50 min. This step is repeated three times. The filtrates are collected and combined, and filtered using a microporous membrane to obtain an extract containing rosmarinic acid.

2. The anti-esophageal cancer drug according to claim 1, characterized in that, The fresh wintergreen grass mentioned refers to the stems and leaves of Rabdosia rubescens (Hemsl.) Hara, a plant belonging to the Lamiaceae family.

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