Application of Cistanche deserticola extract and echinacoside in preparing medicine with gastric mucosal protective effect

Abstract

The present invention belongs to the field of medicine, and specifically relates to the use of a Cistanche deserticola extract in the preparation of a medicament having a gastric mucosal protective effect and preventing or treating gastric mucosal damage or gastric ulcers. Experimental results show that a Cistanche deserticola extract including total glycosides from Cistanche tubulosa or desert Cistanche deserticola, polysaccharides from Cistanche tubulosa or desert Cistanche deserticola, and oligosaccharides from Cistanche tubulosa or desert Cistanche deserticola, as well as monomeric compounds including salidroside, verbascoside, 2'-acetylverascoside, tubuloside A, echinacoside, and isovarascoside, all have significant gastric mucosal protective effects. The present invention has the prospect of being developed into a medicament having a gastric mucosal protective effect and preventing or treating gastric mucosal damage and gastric ulcers, and has important clinical significance and commercial value.

Description

[0001] This application is a divisional application of the patent application with application number 202310585966.5 filed on May 23, 2023. Technical Field

[0002] The invention belongs to the field of medicine and relates to a new use of a Cistanche deserticola extract, and particularly relates to the use of the Cistanche deserticola extract and echinacoside in preparing a medicine with a gastric mucosa protective effect. Background Art

[0003] Gastric ulcer is one of the most common chronic gastrointestinal disorders worldwide, with an incidence rate as high as 10%. Gastric ulcer is a multifactorial disease with multiple pathways involved. Its main clinical manifestations include long-term, cyclical, rhythmic upper abdominal pain accompanied by a series of gastrointestinal symptoms such as nausea, vomiting, regurgitation, belching, and heartburn.

[0004] Excessive drinking can also lead to gastric mucosal damage and gastric ulcers. Ethanol, the main component of alcohol, dissolves in lipids and enters the stomach wall, causing gastric mucosal damage and increased permeability. This can lead to gastric mucosal congestion, erosion, and bleeding. In severe cases, it can cause gastric perforation and massive gastrointestinal bleeding, seriously threatening the patient's life.

[0005] Gastric ulcers and gastric mucosal damage are primarily treated with medications, including chemical drugs and traditional Chinese medicines (TCMs), but these treatments are challenging. Chemical drugs primarily include metronidazole, cimetidine, aluminum sulfate, amoxicillin, and omeprazole, while TCMs are mostly compound preparations. Compared to chemical drugs, TCM compound preparations offer a higher safety profile for treating gastric ulcers. Extracting and isolating natural products from TCMs for the treatment of alcohol-induced gastric damage is an important approach for developing gastric-protective and anti-ulcer drugs and health supplements. However, the current efficacy of natural products in treating gastric ulcers and gastric mucosal damage is insufficient. Therefore, developing drugs with significant therapeutic effects for gastric ulcers and gastric mucosal damage using TCMs as raw materials has significant application value and economic benefits.

[0006] Cistanche deserticola is a perennial parasitic medicinal plant of the genus Cistanche in the family Orobanchaceae. It has extremely high medicinal value and is known as the "desert ginseng". In my country, Cistanche deserticola is mainly distributed in Xinjiang and Inner Mongolia, and is also distributed in Gansu and Ningxia. There are four species of Cistanche deserticola in my country, namely C. deserticola YCMa, C. salsa (CAMey.) G. Beck, C. tubulosa (Schenk) R. Wight, and C. sinensis G. Beck (Tu Pengfei et al., Modern Chinese Medicine, 2015, 17(4): 297-301). Among them, C. deserticola and C. tubulosa are officially recognized as the basal plants of the traditional Chinese medicine Cistanche deserticola and are included in the Chinese Pharmacopoeia. Studies have shown that the polysaccharide extract and phenylethanoid glycosides of Cistanche deserticola can improve the recovery of serum and liver indicators in mice with alcohol-induced liver damage model, increase the survival of HepG2 cells, and reduce fat microvesicles and necrotic cells in the liver tissue of model animals. It can be seen that the polysaccharide extract and phenylethanoid glycosides of Cistanche deserticola have a significant protective effect on ethanol-induced chronic liver damage. Other studies have shown that the polysaccharide-rich Cistanche deserticola extract can activate the immune system, reduce inflammatory mucosal hyperplasia and intestinal Helicobacter pylori infection in mice, and has the effect of preventing colorectal cancer and intestinal inflammation (see, Hou Lei et al., "Research Progress and Industrialization Status of Cistanche Deserticola", Shandong Agricultural Science, Vol. 52, No. 12, 2020, pp. 133-140).

[0007] Echinacoside (ECH) is a natural compound extracted from Cistanche deserticola, and its properties are white crystalline powder. Echinacoside has many biological effects, such as neuroprotection, liver protection, anti-tumor, anti-apoptosis, anti-aging, immunomodulation and reproductive promotion. It also has the effects of lowering blood sugar, lowering blood lipids, promoting bone formation, and anti-pulmonary hypertension and preventing atherosclerosis. Previously, studies have shown that echinacoside can relieve alcohol-induced oxidative stress and cirrhosis (see, Zhi Tao, et al. "Echinacoside ameliorates alcohol-induced oxidative stress and hepaticsteatosis by affecting SREBP1c / FASN pathway viaPPARα", Food and Chemical Toxicology, Vol 148, 2021, 111956). In addition, studies have reported that an extract of Cistanche tubulosa rich in echinacoside can alleviate dextran sulfate sodium-induced colitis in mice (see, Yamin Jia, et al. "Amelioration of Dextran Sulphate Sodium-Induced Colitis in Mice by Echinacoside-Enriched Extract of Cistanche tubulosa", PHYTOTHERAPY RESEARCH, Vol 28, 2014, 110-119). Total glycosides of Cistanche tubulosa and echinacoside can also be used to prevent and treat alcoholic liver damage accompanied by intestinal damage (see CN113425764A).

[0008] From the above review of the prior art, it can be seen that there are currently no reports on the protective effects of Cistanche deserticola extract and echinacoside on gastric mucosa. The present invention just fills this technical gap. Summary of the Invention

[0009] The purpose of the present invention is to overcome the deficiencies of the existing technology, realize the development and utilization of natural products using modern pharmaceutical research methods, and provide the use of Cistanche deserticola extract and echinacoside in the preparation of a drug with gastric mucosal protective effect in combination with a large number of pharmacodynamic experimental screenings.

[0010] In order to achieve the purpose of the present invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides use of a Cistanche deserticola extract in preparing a medicament having a gastric mucosal protective effect.

[0012] In a first aspect, the present invention provides use of a Cistanche deserticola extract in the preparation of a medicament for preventing or treating gastric mucosal damage or gastric ulcer.

[0013] As an optional mode, in the above application, the Cistanche deserticola extract is extracted from Cistanche deserticola or Cistanche tubulosa.

[0014] As an optional mode, in the above application, the Cistanche deserticola extract is the total glycosides of Cistanche tubulosa or total glycosides of Cistanche deserticola, Cistanche tubulosa polysaccharide or Cistanche deserticola polysaccharide, Cistanche tubulosa oligosaccharide or Cistanche deserticola oligosaccharide extracted from Cistanche tubulosa or Cistanche deserticola.

[0015] Preferably, the preparation method of the total glycosides of Cistanche deserticola, the polysaccharide of Cistanche deserticola or the oligosaccharide of Cistanche deserticola comprises the following steps:

[0016] The decoction pieces of Cistanche tubulosa or Cistanche deserticola are crushed into coarse powder, and 6-10 times the amount of water is added and heated under reflux for extraction three times, each time for 1.5-3 hours. The filtrates are combined, concentrated under reduced pressure to a relative density of 1.15, and measured at 50°C. 85%-95% ethanol is added until the alcohol content reaches 50-70%, and low-temperature precipitation is performed for 10-15 hours. The precipitate is filtered, and the precipitate is the total polysaccharide fraction of Cistanche deserticola. The filtrate is concentrated under reduced pressure to a relative density of 1.15, and measured at 50°C. The filtrate is purified by macroporous resin, eluted with water and different concentrations of ethanol in sequence, and the water eluate is collected. The eluate is concentrated under reduced pressure to a thick paste to obtain the total oligosaccharides of Cistanche deserticola. The filtrate is then eluted with 30-50% ethanol, and the eluate is collected. The eluate is concentrated under reduced pressure and dried to obtain the total glycosides of Cistanche deserticola.

[0017] More preferably, the preparation method of the total glycosides of Cistanche deserticola, the polysaccharide of Cistanche deserticola or the oligosaccharide of Cistanche deserticola comprises the following steps:

[0018] The Cistanche tubulosa or Cistanche deserticola slices are crushed into coarse powder, and 8 times the amount of water is added and heated under reflux for extraction three times, each time for 2 hours. The filtrates are combined and concentrated under reduced pressure to a relative density of 1.15, and measured at 50°C. 95% ethanol is added until the alcohol content reaches 60%, and low-temperature precipitation is performed for 12 hours. The precipitate is filtered, and the precipitate is the total polysaccharide fraction of Cistanche deserticola; the filtrate is concentrated under reduced pressure to a relative density of 1.15, and measured at 50°C, and then purified by macroporous resin, eluted with water and different concentrations of ethanol in sequence, and the water eluate is collected and concentrated under reduced pressure to a thick paste to obtain the total oligosaccharides of Cistanche deserticola; then eluted with 40% ethanol, and the eluate is collected, concentrated under reduced pressure, and dried to obtain the total glycosides of Cistanche deserticola.

[0019] As an optional mode, in the above application, the Cistanche deserticola extract is a natural product monomer.

[0020] Preferably, the natural product monomer is prepared from the total glycosides of Cistanche deserticola.

[0021] Preferably, the natural product monomer is selected from one or more of the following: salidroside, verbascoside, 2'-acetyl verbascoside, tubuloside A, echinacoside or isovalascoside.

[0022] Preferably, the method for preparing the natural product monomer comprises the following steps:

[0023] Total glycosides from Cistanche deserticola were dissolved in 6-10 times the amount of water and subjected to ODS column chromatography using a methanol-water gradient elution as the eluent to obtain 16 fractions, Fr.I-XVI. Fraction Fr.IV was eluted using a Sephadex LH-20 column chromatography using a methanol-water eluent to obtain 10 major fractions, Fr.1-Fr.10. Fraction Fr.5 was subjected to semi-preparative HPLC separation using a methanol-water eluent to obtain the compounds echinacoside and scutellarin A. Fraction Fr.VII was eluted using a Sephadex LH-20 column chromatography using a methanol-water eluent to obtain 12 major fractions, Fr.1-Fr.12. Fraction Fr.8 was subjected to preparative HPLC separation using a water-methanol-acetonitrile eluent to obtain the compounds verbascoside and 2'-acetylverascoside. Fraction Fr.10 was subjected to preparative HPLC separation using a methanol-water eluent to obtain the compound isoverascoside. Fr.VIII was eluted by Sephadex LH-20 column chromatography with methanol-water as the eluent to obtain 8 main fractions Fr.1-Fr.8. Fr.3 was subjected to silica gel PTLC, silica gel H, and chloroform-methanol as the developing solvent to obtain the compound salidroside.

[0024] More preferably, the method for preparing the natural product monomer comprises the following steps:

[0025] Total glycosides from Cistanche deserticola were dissolved in 8 times the amount of water and subjected to gradient elution via ODS column chromatography (100-120 mesh, methanol-water, 25:75-75:25) to obtain 16 fractions (Fr.I-XVI). Fr.IV was eluted via Sephadex LH-20 column chromatography (methanol-water, 50:50) to obtain 10 major fractions, Fr.1-Fr.10. Fr.5 was subjected to semi-preparative HPLC separation (methanol-water, 30:70) to obtain the compounds echinacoside and scutellarin A. Fr.VII was eluted with Sephadex LH-20 column chromatography (methanol-water, 50:50) to obtain 12 major fractions, Fr.1-Fr.12. Fr.8 was separated by HPLC (water-methanol-acetonitrile, 62:29:9) to obtain verbascoside and 2'-acetyl verbascoside. Fr.10 was separated by HPLC (methanol-water, 40:60) to obtain isoverascoside. Fr.VIII was eluted with Sephadex LH-20 column chromatography (methanol-water, 50:50) to obtain 8 major fractions, Fr.1-Fr.8. Fr.3 was separated by silica gel PTLC (silica gel H, chloroform-methanol, 3:1) to obtain salidroside.

[0026] More preferably, the natural product monomer is echinacoside.

[0027] As an optional method, in the above application, echinacoside dose-dependently reduces the LDH release rate, inhibits bile acid-induced autophagy overactivation, and dose-dependently significantly downregulates the levels of IL-6, IL-1β, and TNFα.

[0028] As an optional mode, in the above application, the medicine only contains the Cistanche deserticola extract as an active ingredient, and further contains a pharmaceutically acceptable carrier.

[0029] As an optional mode, in the above application, the dosage form of the drug is an oral dosage form.

[0030] As an optional mode, in the above application, the oral dosage form is a capsule, tablet, granule or oral liquid.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention leverages my country's strengths in natural product research to identify novel uses for Cistanche deserticola extract and various natural product monomers, including echinacoside, in gastric mucosal protection, as well as the prevention and treatment of gastric mucosal damage and gastric ulcers. This invention has the potential to be developed into a drug for the treatment of related indications, with significant clinical and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1:Activity of each extract component in TCA-induced GES-1 cell injury model. The differences between the two groups were analyzed by t-test. Data are expressed as Mean ± SEM, n = 6. Compared with the normal control group, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0034] Figure 2 :Activity of monomer components in TCA-induced GES-1 cell injury model. The differences between the two groups were analyzed by t-test. Data are expressed as Mean ± SEM, n = 6. Compared with the normal control group, ## P<0.01; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0035] Figure 3 :In vitro screening of the activity of echinacoside in improving gastric mucosal damage. Among them, A is the effect of ECH on the survival rate of GES-1 cells induced by ethanol; B is the effect of ECH on the survival rate of GES-1 cells induced by bile acid; C is the effect of ECH on the survival rate of cells induced by acetylsalicylic acid. ECH: echinacoside. The differences between the two groups were analyzed by t-test, and the data are expressed as Mean ± SEM, n = 6. Compared with the normal control group, ## P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0036] Figure 4 Effect of echinacoside on LDH release in a bile acid-induced GES-1 cell injury model. ECH: echinacoside. The differences between the two groups were analyzed using t-test. Data are presented as Mean ± SEM. n = 6. Compared with the normal control group, ## P<0.01; *P<0.05 compared with the model group.

[0037] Figure 5 : Effects of echinacoside on bile acid-induced autophagy in GES-1 cells.

[0038] Figure 6 Effects of echinacoside on the gross appearance of gastric tissue in each group of mice. CG: normal control group, SG: positive control sucralfate, ECH 25: 25 mg / kg echinacoside, ECH 50: 50 mg / kg echinacoside. Red arrows indicate sites of gastric mucosal bleeding.

[0039] Figure 7Effects of echinacoside on gastric histopathological morphology in mice. CG: normal control group; SG: positive control sucralfate; ECH 25: 25 mg / kg echinacoside; ECH 50: 50 mg / kg echinacoside. Black arrows indicate necrosis and disorganization of gastric mucosal cells. H&E staining results (×200); scale bar = 50 μm.

[0040] Figure 8 : Effects of echinacoside on the plasma IL-1β, IL-6, and TNFα levels of ICR mice in each group. Among them, A is the plasma IL-6 level of ICR mice; B is the plasma IL-1β level of ICR mice; C is the plasma TNFα level of ICR mice. CG: normal group; MG: model group; SG: positive drug sucralfate group; ECH 25: 25 mg / kg echinacoside, ECH 50: 50 mg / kg echinacoside. The differences between the two groups were analyzed by t-test, and the data are expressed as Mean±SEM, n=7. Compared with the CG group, # P<0.05, ## P<0.01; compared with the MG group, *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0042] The pharmaceutically acceptable carrier of the present invention refers to a conventional drug carrier in the field of pharmaceutical preparations, which is selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0043] The fillers of the present invention include but are not limited to starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include but are not limited to magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include but are not limited to water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc.; the disintegrants include but are not limited to starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agents include but are not limited to polysaccharides such as acacia gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan, etc.; the solvents include but are not limited to water, balanced salt solutions, etc.

[0044] The drug can be prepared into various solid oral preparations, liquid oral preparations, and the like. Pharmaceutically acceptable oral solid preparations include conventional tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dripping pills, powders, and the like. Oral liquid preparations include oral liquids and emulsions. The above-mentioned various dosage forms can be prepared using conventional processes in the field of pharmaceutical preparations.

[0045] The Cistanche deserticola extract and various natural product monomers including echinacoside used in the present invention can be extracted and separated from Cistanche deserticola using the extraction and separation method described in the present invention or other biological extraction methods reported in the literature, or can be purchased from commercial products.

[0046] In the medical uses described above, the administration time, number of administrations, and frequency of administration of Cistanche deserticola extract and various natural product monomers including echinacoside need to be determined according to the specific diagnosis results of the disease, which is within the technical scope mastered by those skilled in the art.

[0047] In order to better understand the essence of the present invention, the following specific embodiments use pharmacodynamic experiments and their results to further illustrate the new uses of Cistanche deserticola extract and echinacoside in the pharmaceutical field.

[0048] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0049] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0050] Unless otherwise specified, all percentages and parts in the present invention are by weight.

[0051] Example 1: Extraction Method of Total Glycosides, Polysaccharides and Oligosaccharides of Cistanche Deserticola 1. Preparation of Extracts from Various Parts of Cistanche Tubulosa or Cistanche Deserticola (Example Method Only, Other Methods Can Also Be Used):

[0052] The decoction pieces of Cistanche tubulosa or Cistanche deserticola were crushed into coarse powder, and 8 times the amount of water was added and heated under reflux for extraction three times, each time for 2 hours. The filtrates were combined and concentrated under reduced pressure to a relative density of 1.15 (measured at 50°C), 95% ethanol was added until the alcohol content reached 60%, and low-temperature precipitation was performed for 12 hours. The precipitate was filtered, and the precipitate was the total polysaccharide fraction of Cistanche deserticola; the filtrate was concentrated under reduced pressure to a relative density of 1.15 (measured at 50°C), and then purified by macroporous resin, eluted with water and different concentrations of ethanol in sequence, the water eluate was collected, and concentrated under reduced pressure to a thick paste to obtain the total oligosaccharides of Cistanche deserticola; then eluted with 40% ethanol, the eluate was collected, concentrated under reduced pressure, and dried to obtain the total glycosides of Cistanche deserticola.

[0053] 2. Preparation of monomeric compounds of Cistanche tubulosa or Cistanche deserticola (only exemplary method, can also be obtained by other means):

[0054] Total glycosides from Cistanche deserticola were dissolved in 8 times the amount of water and subjected to gradient elution via ODS column chromatography (100-120 mesh, methanol-water, 25:75-75:25) to obtain 16 fractions (Fr.I-XVI). Fr.IV was eluted via Sephadex LH-20 column chromatography (methanol-water, 50:50) to obtain 10 major fractions, Fr.1-Fr.10. Fr.5 was subjected to semi-preparative HPLC separation (methanol-water, 30:70) to obtain the compounds echinacoside and scutellarin A. Fr.VII was eluted with Sephadex LH-20 column chromatography (methanol-water, 50:50) to obtain 12 major fractions, Fr.1-Fr.12. Fr.8 was separated by HPLC (water-methanol-acetonitrile, 62:29:9) to obtain verbascoside and 2'-acetyl verbascoside. Fr.10 was separated by HPLC (methanol-water, 40:60) to obtain isoverascoside. Fr.VIII was eluted with Sephadex LH-20 column chromatography (methanol-water, 50:50) to obtain 8 major fractions, Fr.1-Fr.8. Fr.3 was separated by silica gel PTLC (silica gel H, chloroform-methanol, 3:1) to obtain salidroside.

[0055] Example 2: Screening of the pharmacodynamic activity of various Cistanche deserticola extracts on the GES-1 cell injury model induced by taurocholic acid

[0056] A taurocholic acid (TCA)-induced gastric mucosal injury model was established in the human gastric epithelial cell line GES-1. The gastric mucosal protective activity of extracts and their major monomers from Cistanche deserticola (Cistanche deserticola) and Cistanche tubulosa (Cistanche tubulosa) was evaluated. Previous experiments showed that the IC50 for TCA damage to GES-1 cells was 1.47 mM, so this study used 1.5 mM TCA as the modeling dose for cell injury. The activity of each extract in the TCA-induced GES-1 cell injury model was evaluated at concentrations of 1, 5, and 10 μg / mL.

[0057] The experimental results showed that the cell survival rate in the model group was significantly lower than that in the control group (P < 0.01); and compared with the model group, the cell activity of total glycosides of flos tubulosa, polysaccharides of flos tubulosa, and oligosaccharides of flos tubulosa was significantly increased at a concentration of 10 μg / mL (P < 0.05), indicating that total glycosides of flos tubulosa and polysaccharides of flos tubulosa have significant gastric mucosal protective activity. The cell activity of total glycosides of flos tubulosa, oligosaccharides of flos tubulosa, and polysaccharides of flos desertificans were significantly increased at a concentration of 10 μg / mL (P < 0.05), indicating that total glycosides of flos tubulosa, oligosaccharides of flos tubulosa, and polysaccharides of flos desertificans have significant gastric mucosal protective activity ( Figure 1 ).

[0058] Example 3: Screening of the pharmacodynamic activity of Cistanche deserticola monomer components against taurocholic acid-induced GES-1 cell injury model

[0059] The activity of the main monomers of Cistanche deserticola was screened using concentrations of 1, 5, and 10 μM to evaluate their activity in a TCA-induced GES-1 cell injury model. The results showed that the cell viability in the model group was significantly decreased compared with the control group (P < 0.01). However, salidroside and verbascoside significantly increased cell viability at concentrations of 1, 5, and 10 μM compared with the model group in a dose-dependent manner, with the highest increase in cell viability at 10 μM (P < 0.01), suggesting that salidroside and verbascoside can protect gastric mucosal cells in a dose-dependent manner. 2'-acetylverascoside, echinacoside, and isovarascoside all significantly increased cell viability at 10 μM, suggesting that 2'-acetylverascoside, echinacoside, and isovarascoside have significant gastric mucosal protective activity. Tubuloside A at a concentration of 10 μM can significantly increase cell viability (P < 0.05), suggesting that Tubuloside A has significant gastric mucosal protective activity ( Figure 2 ).

[0060] Example 4: Pharmacological activity of echinacoside on different GES-1 cell injury models

[0061] Since the characteristic component of Cistanche deserticola has the most significant pharmacological activity, this study again used three in vitro GES-1 cell injury models to evaluate the in vitro gastric mucosal protective pharmacological activity of echinacoside.

[0062] In the 8% ethanol-induced cell injury model, echinacoside at three concentrations of 1, 5, and 10 μM can significantly increase the survival rate of ethanol-damaged cells in a dose-dependent manner (P < 0.01), among which 10 μM echinacoside has the best efficacy. In the 1.5 mM bile acid (BA)-induced GES-1 cell injury model, echinacoside at three concentrations of 1, 5, and 10 μM can also significantly increase the survival rate of bile acid-induced GES-1 cell injury. In the 5 mM acetylsalicylic acid-induced GES-1 cell injury model, echinacoside has significant gastric mucosal cell protective activity at a concentration of 10 μM (P < 0.05). In three different gastric mucosal cell injury models, echinacoside showed good pharmacodynamic activity, laying the foundation for further mechanism exploration and in vitro efficacy verification of this study ( Figure 3 ).

[0063] Example 5: Effect of echinacoside on LDH release rate in bile acid-induced GES-1 cell injury model

[0064] Cell damage will release a large amount of lactate dehydrogenase, so detecting the LDH release rate can evaluate the cytoprotective effect of echinacoside.

[0065] The results showed that bile acid treatment significantly increased the intracellular LDH release rate (P < 0.01), while echinacoside treatment reduced the LDH release rate in a dose-dependent manner (P < 0.05), indicating that echinacoside has a certain cytoprotective effect ( Figure 4 ).

[0066] Example 6: Effect of echinacoside on bile acid-induced autophagy in GES-1 cells

[0067] Autophagy is a common stress mechanism of cell damage and the pathological basis of various digestive system diseases. Autophagy is a programmed self-catabolic regulation process. Once activated, cell debris, damaged organelles, and abnormal proteins to be decomposed are encapsulated into vesicles. These vesicles gradually mature and acidify during cytoplasmic transport, then bind to lysosomes to form autolysosomes, which are ultimately digested, degraded, and reused. Acridine orange dye can penetrate into autolysosomes, exhibiting red-yellow fluorescence under a fluorescence microscope, thereby reflecting the level of autophagy in the cell.

[0068] To investigate the effect of echinacoside on bile acid-induced autophagy in GES-1 cells, acridine orange staining was used for analysis. The results showed that bile acid treatment increased the red and green fluorescence levels in the cells, indicating that autophagy was significantly activated. After echinacoside treatment, the red and green fluorescence levels improved, indicating that echinacoside inhibited the excessive activation of autophagy induced by bile acid. The gastroprotective effect of echinacoside is related to the regulation of autophagy ( Figure 5 ).

[0069] Example 7: Effects of echinacoside on the gross appearance of gastric tissue in each group of mice

[0070] Healthy male SPF-grade ICR mice, weighing 20-25 g, were housed in a barrier environment and fed a normal diet for 1 week. They were then randomly divided into five groups (10 mice each): control group (CG), model group (MG), positive drug sucralfate group (SG), ECH 25 group (25 mg / kg dose of ECH-treated group, ECH 25), and ECH 50 group (50 mg / kg dose of ECH-treated group, ECH50). Except for the CG group, the mice in the other groups were gavaged with reflux solution (2.5% TCA, 1.5% trypsin, 0.25% lecithin) twice daily at a dose of 15 mL / kg to establish a bile reflux gastritis model. Three days after mice were gavaged with reflux fluid, each group began drug treatment, and the drug treatment lasted for 2 weeks. Every day, 6 hours after gavage with reflux fluid, the ECH group was gavaged with different doses of ECH 25 mg / kg and 50 mg / kg, and the positive drug sucralfate group was gavaged with 120 mg / kg.

[0071] When bile reflux causes stomach damage, symptoms of chronic gastritis or gastric ulcer can be seen under gastroscopy, such as uneven color of the gastric mucosa, congestion and brittleness, bleeding spots or ulcer spots, and the presence of green or yellow bile-like fluid in the gastric mucus.

[0072] When the animals were sampled 2 weeks after administration, the gastric mucosa of the mice was photographed and observed grossly to preliminarily determine the extent of gastric mucosal damage. Figure 6 As shown, the gastric mucosa of mice in the normal group was intact and rosy, with normal wrinkles and no redness or swelling. There were no ulcers or bleeding spots on the surface. In the model group, the gastric mucosa showed obvious bleeding spots, redness and swelling, and no wrinkles. The bleeding spots and redness of the gastric mucosa in the high-dose ECH group and the positive drug sucralfate group were improved.

[0073] Example 8: Effects of echinacoside on gastric tissue pathology in mice of each group

[0074] The animal experiment method is as shown in Example 8. The pathological morphology of gastric tissue sections of bile reflux gastric mucosal injury model mice was observed by H&E staining, and the pathological morphology of gastric mucosal epithelial cells in each group was observed under a microscope. Figure 7 As shown, the gastric mucosal structure in the CG group was intact, with epithelial cells arranged in a single columnar pattern and few defects or shedding. Compared with the CG group, the MG group showed shedding and defects of gastric epithelial cells, as well as disordered arrangement of the intrinsic glands. These features are similar to those observed in patients with bile reflux gastritis. After ECH treatment, these changes were significantly milder than those in the model group. The gastric mucosal structure was largely intact, with relatively regular arrangement of gastric epithelial cells, indicating that ECH treatment significantly alleviated pathological damage to gastric tissue.

[0075] Example 9: Effects of echinacoside on plasma IL-1β, IL-6, and TNFα levels in mice of each group

[0076] The animal experimental method is described in Example 7. The gastric mucosa is attacked by stimuli such as bile, which damages its defense factors, leading to chronic inflammation and tissue damage. IL-1β, IL-6, and TNFα are all markers of inflammatory responses. To further investigate the role of ECH in bile reflux gastritis, the plasma levels of the inflammatory factors IL-6, IL-1β, and TNFα were measured.

[0077] like Figure 8 As shown in the data, after 2 weeks of bile reflux, the above three inflammatory factors were significantly upregulated (P < 0.01), while ECH significantly downregulated the levels of IL-6, IL-1β, and TNFα in a dose-dependent manner.

[0078] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. The use of a Cistanche deserticola extract in the preparation of a medicament having a gastric mucosal protective effect, characterized in that: The Cistanche deserticola extract is a natural product monomer, echinacoside, which is prepared from the total glycosides of Cistanche deserticola.

2. Use of a Cistanche deserticola extract in the preparation of a medicament for preventing or treating gastric mucosal damage, characterized in that: The Cistanche deserticola extract is a natural product monomer, echinacoside, which is prepared from the total glycosides of Cistanche deserticola.

3. The use according to claim 2, characterized in that: The gastric mucosal injury is gastric ulcer.

4. The use according to claim 1 or claim 2, characterized in that: The preparation method of the natural product monomer comprises the following steps: Total glycosides from Cistanche deserticola were dissolved in 6-10 times the amount of water and chromatographed on an ODS column using methanol-water as the eluent. Gradient elution afforded 16 fractions, Fr. I-XVI. Fr. IV was chromatographed on a Sephadex LH-20 column using methanol-water as the eluent to afford 10 major fractions, Fr.1-Fr.

10. Fr.5 was semi-preparatively separated by HPLC using methanol-water as the eluent to afford the compound echinacoside.

5. The use according to claim 1 or claim 2, characterized in that: Echinacoside dose-dependently reduced the LDH release rate, inhibited bile acid-induced autophagy overactivation, and dose-dependently downregulated the levels of IL-6, IL-1β, and TNFα.

6. The use according to claim 1 or claim 2, characterized in that: The medicine contains only the Cistanche deserticola extract as an active ingredient and also contains a pharmaceutically acceptable carrier.

7. The use according to claim 6, characterized in that: The dosage form of the drug is an oral dosage form.

8. The use according to claim 7, characterized in that: The oral dosage form is capsule, tablet, granule or oral liquid.

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