Application of ethanol extract of radix tinosporae in preparation of medicine for preventing and treating gastric ulcer

Abstract

This invention belongs to the field of novel pharmaceutical uses, and more specifically, relates to the application of cinnabar extract in the preparation of drugs for the prevention and treatment of gastric ulcers. The preparation method of the extract is as follows: Cinnabar is extracted by reflux with a 60%–80% (v / v) ethanol solution. The extracts are combined, evaporated to dryness, and then mixed with water to obtain an extract. The extract is then diluted, adsorbed onto a macroporous resin, and eluted with a 35%–45% (v / v) ethanol solution. The eluent is then evaporated to dryness to obtain the final extract. Experiments show that this extract can significantly reduce gastric mucosal damage in mice, decrease the ulcer index, and increase the ulcer inhibition rate, with effects superior to the positive control drug ranitidine. Its mechanism of action includes reducing inflammatory cell infiltration, decreasing the content of pro-inflammatory factors, pepsin activity, and malondialdehyde content, and increasing antioxidant enzyme activity. This invention provides a new natural drug source for the treatment of gastric ulcers, with a clearly defined active pharmaceutical ingredient and promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the application of cinnabar ethanol extract in the preparation of drugs for the prevention and treatment of gastric ulcers. Background Technology

[0002] Gastric ulcer is a common digestive system disease. Typical symptoms include rhythmic upper abdominal discomfort, nausea, vomiting, loss of appetite, and acid reflux. Complications can include bleeding, perforation, pyloric obstruction, and even a risk of cancer. Because gastric ulcers are chronic, difficult to cure, and have numerous complications, they often negatively impact patients' daily work and studies. The causes of gastric ulcers are multifactorial, with common factors including Helicobacter pylori infection, poor dietary habits, excessive stress, and other adverse stimuli. Therefore, reducing gastric inflammation and protecting the gastric mucosa from damage are crucial treatment methods for gastric ulcers.

[0003] Cinnabar Seven is the name of the plant *Polygonum multiflorum* (hairy-veined Polygonum multiflorum), belonging to the Polygonaceae family. Fallopia multiflora var. cillinerve The dried tuberous root of [product name - likely a plant name]. This product is cool in nature, bitter and slightly astringent in taste, and has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, dispelling wind and dampness, strengthening the waist and knees, and nourishing the liver and kidneys. Clinically, it is used for tonsillitis, gastritis, enteritis, dysentery, urinary tract infection, hematemesis, epistaxis, hematochezia, dysfunctional uterine bleeding, and irregular menstruation; externally, it is used to treat bruises, traumatic bleeding, and other symptoms.

[0004] Cinnabar Gynostemma pentaphyllum, as a representative herbal medicine of "Taibai Seven Herbs", can exert its anti-gastric ulcer effect by inhibiting Helicobacter pylori, reducing oxidative damage to the gastric mucosa, and improving local blood circulation. However, its chemical composition is complex, mainly containing more than 80 compounds such as anthraquinones, stilbenes, and flavonoids. The efficacy of its effective extracts for gastric ulcers is unclear, and its regulatory mechanism is unknown, which seriously restricts the clinical application and further in-depth research of cinnabar Gynostemma pentaphyllum and its active ingredients. Summary of the Invention

[0005] The purpose of this invention is to provide the application of cinnabar heptane ethanol extract in the preparation of drugs for the prevention and treatment of gastric ulcers.

[0006] The application of cinnabar heptane ethanol extract in the preparation of drugs for treating gastric ulcers, wherein the preparation method of cinnabar heptane ethanol extract is as follows: S1. Extract Cinnabar by reflux with 60%~80% ethanol solution, evaporate the extract to dryness to obtain crude product, and mix the crude product with water at a ratio of 0.5~1.5g:2~4mL to obtain extract. S2. Mix the extract with water at a ratio of 0.5~1.5g:20~30mL, add it to a resin adsorption column and adsorb overnight. Then elute with a 35%~45% ethanol solution and evaporate the eluent to dryness to obtain the cinnabar ethanol extract.

[0007] This invention, for the first time, clearly identifies the application value of specific active ingredients in Cinnabar Gynostemma pentaphyllum in the prevention and treatment of gastric ulcers, and reveals its intrinsic mechanism of action in combating ulcers. Using an anhydrous ethanol-induced acute gastric ulcer model, this invention screened and found that the 35%–45% ethanol-eluting fraction of Cinnabar Gynostemma pentaphyllum exhibited the best activity. Further in vivo experiments confirmed that this fraction can exert multi-target effects: on the one hand, it inhibits the inflammatory response and regulates the balance of cytokines such as IL-1β, IL-6, and IL-10; on the other hand, it enhances the antioxidant capacity of the gastric mucosa, increases the activity of SOD, GSH, and CAT, and reduces MDA content; simultaneously, it inhibits pepsin activity, reducing gastric mucosal autodigestion. This synergistic mechanism of "anti-inflammatory-antioxidant-antidigestion" constitutes the unique concept of this invention, distinguishing it from existing drugs with single mechanisms of action, and providing a new strategy for developing highly effective and low-toxicity natural anti-gastric ulcer drugs.

[0008] Furthermore, the mass-to-volume ratio of the cinnabar and the ethanol solution with a volume fraction of 60% to 80% is 0.5 to 1.5 g: 10 to 25 mL.

[0009] Furthermore, the reflux extraction is performed 1 to 3 times, at a temperature of 80 to 90°C, for a duration of 0.5 to 1.5 hours each time.

[0010] Furthermore, the drug is used to relieve gastric mucosal damage.

[0011] Furthermore, alleviating gastric mucosal damage includes reducing the infiltration of inflammatory cells in the gastric mucosa.

[0012] Furthermore, the relief of gastric mucosal damage includes reducing the content of inflammatory factors in the gastric mucosa, including IL-1β, IL-6, and IL-10.

[0013] Furthermore, the relief of gastric mucosal damage includes reducing the activity of pepsin in the gastric mucosa.

[0014] Furthermore, the relief of gastric mucosal damage includes reducing the content of malondialdehyde in the gastric mucosa.

[0015] Furthermore, the relief of gastric mucosal damage includes increasing the activity of SOD, GSH, and CAT in the gastric mucosa.

[0016] Furthermore, the drug is made from the cinnabar heptane ethanol extract and pharmaceutically acceptable excipients.

[0017] The beneficial effects of this invention are as follows: 1. This invention discloses for the first time a new use of the ethanol extract of Cinnabar Gynostemma pentaphyllum in the preparation of drugs for treating gastric ulcers, expanding the medicinal scope of Cinnabar Gynostemma pentaphyllum and providing a new natural drug source for the treatment of gastric ulcers.

[0018] 2. Experiments have confirmed that the 40% ethanol elution fraction of Cinnabar has a significant anti-gastric ulcer effect, which can significantly reduce gastric mucosal congestion, edema and damage, reduce the ulcer index, and increase the ulcer inhibition rate. It shows a significant dose-dependent effect, and the medium and high doses are more effective than the positive control drug ranitidine.

[0019] 3. The ethanol extract of Cinnabar Gynostemma pentaphyllum can reduce inflammatory cell infiltration, significantly reduce the levels of pro-inflammatory factors IL-1β and IL-6, increase the level of anti-inflammatory factor IL-10, and effectively inhibit gastric inflammatory response.

[0020] 4. The ethanol extract of Cinnabar Gynostemma can reduce pepsin activity, reduce gastric mucosal digestive damage, reduce malondialdehyde (MDA) content, increase the activity of SOD, GSH, and CAT antioxidant enzymes, enhance the antioxidant capacity of gastric mucosa, and maintain mucosal barrier function.

[0021] 5. The extraction process of this invention is stable, easy to operate, has a clear effective part, definite efficacy, and good safety. It is suitable for industrial production and development and has high clinical application value and market prospects. Attached Figure Description

[0022] Figure 1 Images showing gastric mucosal damage in mice after treatment with different volume fractions of ethanol.

[0023] Figure 2 This is an image showing the external morphology of gastric mucosal damage.

[0024] Figure 3 This is a stained section of gastric mucosal tissue under a 200x microscope.

[0025] Figure 4 The graphs show the statistical distribution of inflammatory factors, where A represents the IL-6 content, B represents the IL-1β content, and C represents the IL-10 content.

[0026] Figure 5 This is a graph showing the results of pepsin activity assay. ## This indicates a significant difference compared to the control group. p <0.01, ** The results showed a significant difference compared to the model group. p <0.01. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0028] Example 1 I. Experimental Materials.

[0029] 1. Laboratory animals and facilities The Kunming strain of male mice was provided by Chengdu Dashuo Experimental Animal Center, animal license number: SCXK (Sichuan) 2020-030. The experimental site was the Pharmacology Laboratory of Traditional Chinese Medicine at Shaanxi University of Traditional Chinese Medicine.

[0030] 2. Experimental medicinal materials Cinnabar No. 7 was collected in May 2021 from Taibai Mountain in the Qinling Mountains of Shaanxi Province. It was identified as *Polygonum multiflorum*, a plant belonging to the genus *Polygonum*. Fallopia multiflora var. cillinerve The dried tuberous root of [the plant] is preserved in the Herbarium of Medicinal Plants, Shaanxi University of Traditional Chinese Medicine (No. 20180627).

[0031] 3. Experimental instruments and reagents The instruments and reagents used are shown in Table 1.

[0032] Table 1: Main Experimental Instruments and Reagents II. Preparation of Cinnabar Extract.

[0033] Add 50g of cinnabar to 1000mL of 70% ethanol solution and reflux at 85℃ for 1h. After filtration, add another 500mL of 70% ethanol solution and reflux at 85℃ for 1h. Combine the two extracts and evaporate to dryness to obtain crude product. Mix the crude product with water at a ratio of 1g:3mL to obtain extract.

[0034] D-101 macroporous resin (Hailanji Bioreactor Co., Ltd.) was activated by soaking in 95% ethanol solution for 24 hours. After full activation, it was packed into a column and washed with water until no alcohol odor remained. The extract was dissolved in water at a mass-to-volume ratio of 1 g: 25 mL and then loaded onto the column for overnight adsorption. Elution was performed by adding 300 mL of 40% ethanol solution and 80% ethanol solution, respectively. The eluent was evaporated to dryness to obtain the 40% ethanol fraction and the 80% ethanol fraction, respectively. The corresponding fractions were dissolved in 0.5% sodium carboxymethyl cellulose solution and stored.

[0035] III. Screening of Ethanol Fractions

[0036] Forty male Kunming mice, weighing 18-22g, were randomly divided into a control group, a gastric ulcer model group, a 40% ethanol fraction group, and an 80% ethanol fraction group. The ethanol fraction was administered by gavage at a dose of 0.1mL / 10g (2.4g / kg crude drug). The control group and the gastric ulcer model group received an equal volume of physiological saline. This was done once daily for 7 consecutive days. After the 6th administration, mice were fasted for 24 hours but allowed free access to water for 24 hours. 0.5 hours after the 7th administration, all groups except the control group were administered anhydrous ethanol (0.1mL / 10g) by gavage to induce a gastric ulcer model. One hour later, the mice were sacrificed, and the entire stomach was quickly removed. The cardia and pylorus were ligated, and 2mL of 4% paraformaldehyde fixative was injected into the stomach cavity for fixation for 10 minutes. The stomach was then cut open along the greater curvature, and the contents were washed away with pre-cooled PBS buffer. The stomach was then laid flat to observe the ulceration.

[0037] IV. Screening of dosage for 40% ethanol fraction.

[0038] Sixty male Kunming mice, weighing 22g, were randomly divided into six groups of 10 mice each after one week of acclimatization. The groups were: a control group (Z40-L), a gastric ulcer model group (Z40-M), a ranitidine positive control group (Z40-H), a low-dose group (Z40-L), a medium-dose group (Z40-M), and a high-dose group (Z40-H). Mice were administered the drug via gavage for seven consecutive days. The dosages, calculated according to the human and animal body surface area ratio table in *Pharmacological Experimental Methodology*, were as follows: low-dose group 1.2g / kg, medium-dose group 2.4g / kg, high-dose group 4.8g / kg. The positive control group received ranitidine suspension at 40mg / kg. The control and model groups were administered the same volume of physiological saline via gavage. After the sixth administration, the mice were fasted but allowed to drink water for 24 hours. 0.5 hours after the seventh administration, except for the blank control group, all other groups were administered 0.1 mL / 10 g of anhydrous ethanol by gavage to induce gastric ulcers. The mice were sacrificed 1 hour later.

[0039] V. Experimental Methods.

[0040] 1. Sample collection Blood was collected from the eyeballs of mice and placed in heparinized anticoagulant tubes. After standing for 20 minutes, the tubes were centrifuged at 3000 rpm for 15 minutes at 4°C. The clear serum supernatant was collected for assay. The entire stomach was removed, a portion was cut and fixed for histopathological examination of the gastric mucosa, and the remainder was frozen at -80°C.

[0041] 2. Ulcer Index and Ulcer Inhibition Rate After enucleating the eyeballs of mice to collect blood, the entire stomach was removed, cut open along the greater curvature, and the stomach contents were flushed away with PBS buffer. The gastric mucosa was fully expanded, and the ulcer index was observed and assessed. The length of gastric congestion or diffuse bleeding bands was measured with calipers and scored. For linear lesions longer than 1 mm, the length was measured, and 1 point was awarded for each mm. If the width was greater than 1 mm, the score was doubled based on the width in mm. For lesions with both length and width less than 1 mm but greater than 0.5 mm, 0.5 points were awarded; for lesions less than 0.5 mm, 0.25 points were awarded. The scores were added together to obtain the ulcer index of the mouse. Ulcer inhibition rate (%) = (UI1 - UI2) / UI1 × 100%. Where: UI1 is the ulcer index of the model group, and UI2 is the ulcer index of each group at the 40% ethanol site.

[0042] 3. Mucosal pathological morphological examination The fixed gastric mucosal tissue was subjected to the following procedure for microscopic observation and photographic recording of morphological changes: embedding and sectioning - dewaxing of sections to water - hematoxylin staining - hydrochloric acid alcohol differentiation - tap water blueing - eosin staining - dehydration, clearing, mounting - microscopic examination.

[0043] 4. Measurement of serum IL-1β, IL-6, and IL-10 levels The levels of IL-1β, IL-6, and IL-10 in serum were determined according to the ELISA kit instructions. The steps were as follows: The provided standards were serially diluted, and 300 μL of washing buffer was added to soak the ELISA plate. 100 μL of standard solution was added to the standard wells, and 80 μL of diluent and 20 μL of serum were added to the sample wells. 50 μL of detection antibody was added to each well for sealing. The plate was incubated at room temperature for 1.5 h, washed 6 times, and 50 μL of HRP-labeled streptavidin was added to each well for sealing. The plate was incubated at room temperature for 30 min, washed 6 times, and 100 μL of chromogenic substrate was added to each well. The plate was incubated in the dark for 30 min, and 100 μL of stop solution was added to each well. The OD value was detected at 450 nm.

[0044] 5. Pepsin activity assay The gastric mucosal protease activity was determined according to the instructions of the pepsin activity kit.

[0045] 6. MDA content and SOD, GSH, and CAT activities in gastric mucosa tissue The determination was performed according to the instructions of each kit. The sample pretreatment procedure is as follows: After freezing and grinding an appropriate amount of gastric tissue, add 9 times the volume of NaCl solution, centrifuge at -4℃, 13000rpm for 10min, and collect the supernatant for index determination.

[0046] VI. Experimental Results.

[0047] 1. Part Selection Results The control group showed thicker, pale pink gastric mucosa with numerous folds and no bleeding points. The model group and the 80% ethanol extract group exhibited significant linear damage to the gastric mucosa, with loosening and thinning of the mucosa, indicating severe injury. In contrast, the 40% ethanol extract group showed a smooth, wrinkled gastric mucosa with only a few bleeding points. The results indicate that the 40% ethanol extract of Cinnabar effectively improved anhydrous ethanol-induced gastric mucosal damage in mice and possessed anti-gastric ulcer activity. (See attached results). Figure 1 .

[0048] 2. Observation results of morphological observation of gastric mucosal damage in mice at the 40% ethanol concentration. In the control group, the gastric mucosa of mice was intact, pale pink, and showed no congestion or cord-like damage. In the model group, the gastric mucosa of mice was thinner, looser, and showed numerous punctate hemorrhages and cord-like damage, indicating severe mucosal injury. The ranitidine group and the groups treated with different doses at the 40% ethanol fraction showed significantly improved gastric mucosal integrity, with mild mucosal damage and only a few hemorrhages. (See...) Figure 2 .

[0049] 3. Calculation results of ulcer index and ulcer inhibition rate Compared with the model group, the ulcer index of the ranitidine group and each dose group was significantly reduced, all showing good anti-ulcer activity. In mice at each dose in the 40% ethanol fraction, the ulcer index decreased and the ulcer inhibition rate increased with increasing dose, showing a dose-dependent relationship. The medium-dose group and the high-dose group were superior to ranitidine. See Table 2.

[0050] Table 2: Comparison of ulcer index and ulcer inhibition rate among different groups of mice Note: "-" indicates that the result was not measured. ## Indicates comparison with the model group p <0.01. ** Compared with the ranitidine group p <0.05.

[0051] 4. Pathological changes in mouse gastric mucosa tissue sections In the control group, the gastric mucosal epithelial cells of mice were neatly arranged, and there was no hematoma, edema, or inflammatory cell infiltration in the submucosa and lamina propria. In the model group, the gastric mucosa of mice was significantly thinner, with disordered cell arrangement, shedding and necrosis of mucosal epithelial cells, extensive infiltration of the tissue by inflammatory cells, cell shrinkage, and enlarged interglandular spaces. The tissue structure integrity was significantly improved in all dose groups of 40% ethanol and the ranitidine group, with mild mucosal damage and less accumulation and infiltration of inflammatory cells. (See...) Figure 3 .

[0052] 5. Serum inflammatory factor levels The effect of cinnabar seven-spice on improving gastric ulcers was tested by measuring the levels of relevant inflammatory factors in mouse serum. The levels of IL-1β and IL-6 were significantly higher in the model group compared to the blank group. P <0.01), significant improvement was observed in both the ranitidine group and each dose group. P <0.01). IL-10 levels were significantly lower in the model group compared to the blank group, and significantly improved in the ranitidine group and all other treatment groups. P <0.01). See Figure 4 .

[0053] 6. BCA pepsin activity assay results The pepsin detection results showed that, compared with the blank group, the pepsin activity in the model group mice was significantly increased; compared with the model group, the pepsin activity in the ranitidine group and each drug-treated group was significantly decreased. (See...) Figure 5 .

[0054] 7. Results of gastric mucosal MDA content and SOD, GSH, and CAT activity assays The results of the determination of gastric mucosal MDA content and SOD, GSH and CAT activities showed that, compared with the blank group mice, the gastric mucosal MDA content of the model group mice was significantly increased, and the SOD, GSH and CAT activities were significantly decreased. The gastric mucosal MDA content of the ranitidine group and each drug administration group mice was significantly reduced, and the SOD, GSH and CAT activities were significantly increased. The results are shown in Table 3.

[0055] Table 3: Results of MDA content and SOD, GSH, and CAT activity determination Note: # This indicates a comparison with the blank group. p <0.05. ## This indicates a comparison with the blank group. p <0.01. * This indicates a comparison with the model group. p <0.05. ** This indicates a comparison with the model group. p <0.01.

[0056] VII. Conclusion.

[0057] This invention explores the anti-gastric ulcer effect of Cinnabar Gynostemma pentaphyllum by observing anhydrous ethanol-induced gastric mucosal damage in mice, calculating the ulcer index and ulcer inhibition rate, and measuring relevant indicators in serum and gastric mucosal tissue. The results of various experiments show that the low, medium and high dose groups of 40% ethanol fraction of Cinnabar Gynostemma pentaphyllum all have a dose-dependent inhibitory effect on anhydrous ethanol-induced gastric ulcers in mice, and the anti-ulcer activity of the medium and high dose groups is better than that of the positive control drug ranitidine.

[0058] The 40% ethanol fraction of cinnabar can significantly inhibit ulcer formation, possibly by regulating the levels of factors such as IL-1β, IL-6, and IL-10 in mouse serum, reducing the MDA content in gastric mucosa, alleviating the inflammatory response in vivo, and maintaining the protective barrier of gastric mucosal cells by increasing the activity of protective factors such as SOD, GSH, and CAT in gastric mucosal cells, thereby exerting an anti-gastric ulcer effect.

[0059] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0060] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of cinnabar heptane ethanol extract in the preparation of drugs for the prevention and treatment of gastric ulcers, characterized in that, The preparation method of the cinnabar heptaethanol extract is as follows: S1. Extract Cinnabar by reflux with 60%~80% ethanol solution, evaporate the extract to dryness to obtain crude product, and mix the crude product with water at a ratio of 0.5~1.5g:2~4mL to obtain extract. S2. Mix the extract with water at a ratio of 0.5~1.5g:20~30mL, add it to a resin adsorption column and adsorb overnight. Then elute with a 35%~45% ethanol solution and evaporate the eluent to dryness to obtain the cinnabar ethanol extract.

2. The application according to claim 1, characterized in that, The mass-to-volume ratio of the cinnabar and the ethanol solution with a volume fraction of 60% to 80% is 0.5 to 1.5 g: 10 to 25 mL.

3. The application according to claim 1, characterized in that, The reflux extraction is performed 1 to 3 times at a temperature of 80 to 90°C, with each extraction lasting 0.5 to 1.5 hours.

4. The application according to claim 1, characterized in that, The drug is used to relieve gastric mucosal damage.

5. The application according to claim 4, characterized in that, The relief of gastric mucosal damage includes reducing the infiltration of inflammatory cells in the gastric mucosa.

6. The application according to claim 4, characterized in that, The relief of gastric mucosal damage includes reducing the content of inflammatory factors in the gastric mucosa, including IL-1β, IL-6 and IL-10.

7. The application according to claim 4, characterized in that, The relief of gastric mucosal damage includes reducing the activity of pepsin in the gastric mucosa.

8. The application according to claim 4, characterized in that, The relief of gastric mucosal damage includes reducing the content of malondialdehyde in the gastric mucosa.

9. The application according to claim 4, characterized in that, The relief of gastric mucosal damage includes increasing the activity of SOD, GSH, and CAT in the gastric mucosa.

10. The application according to claim 1, characterized in that, The drug is made from the ethanol extract of cinnabar heptadecanol and pharmaceutically acceptable excipients.

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