Application of fructus cinnamoii in preparation of medicine for treating gastric ulcer

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of fructus cinnamoii in preparation of a medicine for treating gastric ulcer. The invention discloses the application of the fructus cinnamoii oil in preparation of the gastric ulcer medicine for the first time, specifically, the fructus cinnamoii oil obviously improves the conditions of rat gastric mucosa bleeding, erosion, hyperemia, edema and the like and also obviously reduces the ulcer index of rat gastric tissues, and in addition, the fructus cinnamoii oil can be used for preparing the gastric ulcer medicine by reducing TNF-alpha, IL-6 and MDA. The contents of SOD and GSH are increased to improve the inflammatory reaction and oxidative stress of stomach tissues, so that the effect of protecting the stomach tissues of rats is achieved.

Description

Technical Field

[0001] The present application belongs to the field of medical technology, and specifically relates to the application of Litsea cubeba in the preparation of gastric ulcer medicine. Background Art

[0002] Gastric ulcer (GU) is a common digestive system disease. It is caused by excessive gastric acid secretion and elevated pepsin levels in the human body, resulting in local tissue damage to the gastric mucosa. It presents symptoms such as abdominal discomfort, nausea and vomiting, and loss of appetite. If not treated promptly and effectively, it may lead to serious ulcer complications such as intragastric bleeding, perforation, and pyloric obstruction, and may even develop into gastric cancer. Gastric ulcers not only cause physical discomfort to patients, but also bring tremendous mental stress to patients, seriously affecting their daily lives and reducing their quality of life. Currently, the main drugs used in clinical treatment of gastric ulcers, such as proton pump inhibitors, H2 receptor inhibitors, and antibiotics, may lead to side effects such as poor ulcer healing quality, high recurrence rate, and intestinal microbiome imbalance due to long-term use. As a treasure of traditional Chinese medicine, Traditional Chinese Medicine (TCM) has a profound theoretical foundation in the treatment of gastric ulcers, carrying thousands of years of wisdom and practical experience. Because of its multi-component, multi-target, multi-pathway characteristics and holistic treatment, it has unique advantages in the treatment of gastric ulcers, such as significant efficacy, few side effects and low recurrence rate. Under the guidance of traditional Chinese medicine theory, exploring drugs for the treatment of gastric ulcers has good social and economic benefits and development prospects.

[0003] Camphoramigao (HWLi) Y. Yang, Bing Liu & Zhi Yang) is the dried mature fruit of the Lauraceae plant Camphoramigao, which is one of the "Top Ten Miao Medicines" in Guizhou. It is included in the "Guizhou Traditional Chinese Medicine and Ethnic Medicinal Materials Standards" (2019 edition). At present, Camphoramigao is mainly used as medicine with the fruit essential oil extract or the whole fruit. It has the effects of warming the middle and dispersing cold, promoting qi and relieving pain. It belongs to the stomach, heart, and large intestine meridians. It is mainly used to treat stomachache, abdominal pain, chest pain and other symptoms, and has unique efficacy. The preparation of heart and stomach pain-relieving capsules developed with Camphoramigao as the raw material has been used clinically. It is mainly used to treat stomachache diseases, but there is a lack of research on the treatment of gastric ulcers. Stomachache refers to upper abdominal pain in general, which occurs irregularly. Its triggering factors are mostly related to diet, stress or indigestion, and are usually not life-threatening. Gastric ulcers are deep-seated lesions or ulcers in the gastric mucosa, characterized by burning or dull pain that recurs regularly and can last for weeks or even months. These ulcers can lead to complications such as perforation, bleeding, and cancer. In Traditional Chinese Medicine (TCM), the primary etiology and pathogenesis of gastric ulcers is the invasion of cold pathogens into the spleen and stomach, which blocks Qi and causes gastric dysfunction. Treatment should focus on warming the stomach, dispelling cold, regulating Qi, and relieving pain. Currently, there are no studies on the efficacy of Litsea cubeba oil and related preparations for gastric ulcers, either domestically or internationally. Summary of the Invention

[0004] To solve the above problems, the present application provides the use of Litsea cubeba in the preparation of a gastric ulcer medicine. This is achieved specifically through the following technical solutions:

[0005] Application of Litsea cubeba in preparing medicine for gastric ulcer.

[0006] Furthermore, the macrocarpon includes products made from the macrocarpon.

[0007] Furthermore, the Litsea cubeba is Litsea cubeba oil.

[0008] Furthermore, the Litsea cubeba oil can be any one obtained by using Litsea cubeba as a raw material through extraction steps such as pressing and distillation.

[0009] Furthermore, the gastric ulcer medicine is a medicine for treating symptoms such as gastric mucosal bleeding, erosion, congestion, edema, etc.

[0010] Furthermore, the gastric ulcer drug is a drug that improves inflammation and oxidative stress to protect the gastric tissue of gastric ulcer rats.

[0011] Furthermore, the gastric ulcer drug is a drug that reduces the content of TNF-α, IL-6, and MDA.

[0012] Furthermore, the gastric ulcer drug is a drug that increases the content of SOD and GSH.

[0013] In summary, the beneficial effects of the present application are as follows: the present application discloses for the first time the use of Litsea cubeba oil in the preparation of gastric ulcer drugs, specifically: Litsea cubeba oil significantly improves gastric mucosal bleeding, erosion, congestion, edema, etc. in rats, and also significantly reduces the ulcer index of rat gastric tissue. Moreover, Litsea cubeba oil improves the inflammatory response and oxidative stress of gastric tissue by reducing the contents of TNF-α, IL-6, and MDA, and increasing the contents of SOD and GSH, thereby playing a role in protecting rat gastric tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The gastric tissue morphology, ulcer index and ulcer inhibition rate of rats in each group (x±s, n=10). Note: A: gastric tissue morphology; B: ulcer index; C: ulcer inhibition rate; compared with the blank control group, ###p<0.001; compared with the model control group, ***p<0.001.

[0015] Figure 2HE staining and histological scoring of gastric tissue (×400, x±s, n=3). Note: A: HE staining of gastric tissue; B: Histological scoring; Compared with the blank control group, ### p<0.001; compared with the model control group, *** p<0.001.

[0016] Figure 3 Expression levels of MDA, SOD, GSH, TNF-α, and IL-6 in gastric tissue (x±s, n=6). Note: Compared with Control, ###p<0.001, compared with Model, *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0017] The specific implementation methods of the present application are further described in detail below, but the present application is not limited to these implementation methods. Any improvement or replacement based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present application.

[0018] Example 1

[0019] Study on the protective effect of Litsea cubeba oil in treating gastric ulcer

[0020] 1 Experimental Materials

[0021] 1.1 Instrument

[0022] Waterpurifier type laboratory-specific ultrapure water machine (Waters Technology Co., Ltd.); EL-240 electronic analytical balance; (Mettler-Toledo Instrument Co., Ltd.); microplate reader (Bio-Rad, USA).

[0023] 1.2 Drug testing

[0024] Litsea cubeba oil (Guizhou Yibai Pharmaceutical Co., Ltd., DGY20200715); TNF-α and IL-6 (Shanghai Zhuocai Biotechnology Co., Ltd., batch numbers: ZC-37624 and ZC-36404, respectively); MDA, SOD, and GSH (Nanjing Jiancheng Biotechnology Co., Ltd., batch numbers: A003-1-1, A001-3-1, and A006-2-1, respectively); BCA assay kit (Beijing Solaibao Biotechnology Co., Ltd., batch number: PC0020); omeprazole (Chengdu Descot Biotechnology Co., Ltd., batch number: DSTDA004601); anhydrous ethanol (Tianjin Fuyu Fine Chemical Co., Ltd., batch number: 20230917); and 4% paraformaldehyde (Lanjieke Technology Co., Ltd., batch number: BL539A).

[0025] 1.3 Experimental animals

[0026] SPF-grade male SD rats (200 ± 20 g) were purchased from the Experimental Animal Center of Guizhou Medical University (production license: SCXK-2019-0008). The rats were placed in an animal room with a temperature (18-25 °C) and humidity (50%-70%), with free access to water and food, and adaptive feeding for one week.

[0027] 2 Methods and Results

[0028] 2.1 Solution preparation

[0029] Preparation of Litsea cubeba oil solution: take an appropriate amount of Litsea cubeba oil, add an appropriate amount of 0.5% CMC-Na, ultrasonicate for 15 minutes, vortex mix for 5 minutes, mix thoroughly, and prepare a Litsea cubeba oil solution of corresponding concentration.

[0030] Preparation of omeprazole solution: The preparation operation is the same as that of Litsea cubeba oil solution.

[0031] 2.2 Establishment of gastric ulcer model

[0032] SD rats that had been adaptively fed for one week were randomly divided into 6 groups, namely normal group (Control), model group (Model), omeprazole group (Omeprazole, 20 mg / kg), low-dose CMO-L group (CMO-L, 40 mg / kg), medium-dose group (CMO-M, 80 mg / kg), and high-dose group (CMO-H, 160 mg / kg). The Control group and the Model group were gavaged with an equal amount of 0.5% CMC-Na solution, and each drug-treated group was gavaged with the corresponding drug once a day for 7 consecutive days. After the drug was administered on the 6th day, the rats in each group were fasted but not watered for 24 hours. On the 7th day, the rats in each group were gavaged with the drug once. 30 minutes later, except for the rats in the Control group, the rats in the other drug-treated groups were given anhydrous ethanol (5 mL / kg) for modeling, and the samples were collected 2 hours later.

[0033] 2.3 Sample collection

[0034] Serum samples: Blood was collected from the abdominal aorta of rats and placed in a centrifuge tube. The tube was left to stand for 1 hour at 8000 r / min. -1 Centrifuge for 15 minutes, label the serum aliquots, and store at -80°C until use.

[0035] Gastric tissue samples: Cut the removed gastric tissue along the greater curvature of the stomach, rinse with physiological saline until there is no residue in the stomach, then rinse once with DEPC water. Use filter paper to absorb the surface moisture of the gastric tissue, spread it flat on a measuring plate, observe and record the damage of the gastric mucosa with the naked eye, and then take a photo (use a bracket to fix the photo to ensure the position, brightness, and focus of the photo are consistent). After the photo is taken, take a piece of tissue of appropriate size and immerse it in 4% paraformaldehyde for fixation for histopathological observation. The remaining tissue should be aliquoted, labeled, and stored in a refrigerator at -80°C until needed.

[0036] 2.4 Evaluation of Gastric Ulcer Index and Ulcer Inhibition Rate

[0037] According to the "Guth" standard (see Table 1), the ulcer index and ulcer inhibition rate of the rat gastric tissue were calculated, and the gastric ulcer condition of each group of rats was quantitatively scored.

[0038] Gastric ulcer inhibition rate (%) = (ulcer index of model group - ulcer index of drug-treated group) / ulcer index of model group x 100%

[0039] Table 1 Ulcer index score

[0040]

[0041] The results are as follows Figure 1 As shown, the gastric mucosa of rats in the Control group was intact, with a uniform color, no edema, and no bleeding spots. Compared with the Control group, the gastric mucosal damage of rats in the Model group was more severe, with bleeding, erosion, congestion, and edema. The mucosal surface had obvious block-shaped and linear lesions, and the ulcer index was significantly increased (p < 0.001). Compared with the Model group, the gastric mucosal damage in the other treatment groups all improved to varying degrees. The CMO-L group still showed obvious linear lesions on the gastric mucosal surface, but bleeding, erosion, and congestion were alleviated, and the ulcer index decreased, but there was no statistical difference (p>0.05). In the omeprazole and CMO-M groups, gastric tissue surface damage was significantly reduced, with no linear lesions or erosions on the mucosal surface, and localized bleeding. The ulcer index was significantly reduced (p<0.001). The gastric mucosal surface of the CMO-H group was intact, with no obvious lesions or massive bleeding, and only sporadic ulcers distributed on the mucosal surface. The ulcer index was significantly reduced (p<0.001). These results indicate that Litsea cubeba oil has a protective effect on rats with ethanol-induced gastric ulcers, with the low-dose group showing some improvement, but the effect was not significant.

[0042] 2.5 HE staining of gastric tissue

[0043] Gastric tissue fixed in 4% paraformaldehyde was removed, dehydrated with gradient ethanol, and embedded in paraffin. Wax blocks were sectioned at a standard thickness of 4 μm, dewaxed in xylene, and mounted in neutral gum. Images of the sections were acquired using a microscope. Each section was first imaged at low magnification for the entire tissue, followed by acquisition of a 400x microscopic image to identify specific lesions. Histopathological findings were quantitatively scored according to a four-level lesion grading system; the scoring rules are shown in Table 2.

[0044] Table 2 Scoring criteria for gastric tissue lesions

[0045]

[0046] See the results Figure 2 The gastric tissue structure of rats in the Control group was normal, with intact mucosal layer, submucosa, muscularis and adventitia structures, and obvious stratification. A large number of closely arranged gastric glands were visible in the lamina propria. Compared with the Control group, the rats in the Model group showed extensive necrosis of the gastric mucosal layer, with cell cytoplasm lysis in the necrotic area, blurred or disappeared gastric gland structure, accompanied by hemorrhage and a large number of inflammatory cell infiltration, mainly lymphocytes with round dark-stained nuclei and neutrophils with rod-shaped segments. The submucosa was hemorrhagic and edematous, and a large number of inflammatory cells were distributed. The rats in the CMO-L group showed necrosis and hemorrhage of the mucosal layer, blurred gastric gland structure in the necrotic area, necrosis of parietal cells and chief cells, cell disintegration, and mild inflammatory cell infiltration in the necrotic area and submucosa of the mucosa. The rats in the Omeprazole and CMO-M groups showed mild degeneration and necrosis of the gastric mucosal layer in some areas, damage to a small number of gastric glands, cell cytoplasm lysis, blurred structure, and a small number of scattered inflammatory cells. The mucosal layer, submucosa, muscularis, and adventitia of the gastric tissue in the CMO-H group were relatively intact, without obvious degeneration and necrosis or inflammatory cell infiltration. The results showed that compared with the model group, the degree of gastric tissue lesions in each drug-treated group was alleviated, indicating that Litsea cubeba oil has a good protective effect on ethanol-induced gastric ulcers. Among them, the CMO-L group had a certain protective effect, but the effect was not obvious (p>0.05), while the CMO-H group had the most significant protective effect (p<0.001).

[0047] 2.6 Expression levels of MDA, SOD, GSH, TNF-α, and IL-6 in gastric tissue

[0048] According to the instructions of the enzyme-linked immunosorbent assay kit, the levels of TNF-α and IL-6 in gastric tissue were determined strictly according to the operating procedures; an appropriate amount of gastric tissue was accurately weighed and added with physiological saline at a ratio of weight (g): volume (mL) = 1:9 to prepare a tissue homogenate. The mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. A portion of the supernatant was used to detect the protein concentration using a BCA protein concentration assay kit, and the other portion was used to detect the levels of MDA, SOD, and GSH in rat gastric tissue strictly according to the operating procedures in the relevant kit instructions.

[0049] See the results Figure 3 Compared with the Control group, the expression levels of SOD and GSH in the Model group were significantly decreased (p<0.001), while the levels of TNF-α, IL-6, and MDA were significantly increased (p<0.001). Compared with the Model group, the expression levels of SOD and GSH were significantly increased in the Omeprazole, CMO-M (p<0.01), and CMO-H groups (p<0.001), while the expression levels of TNF-α and IL-6 were significantly decreased in the Omeprazole, CMO-M (p<0.05), and CMO-H groups (p<0.001). The MDA content was significantly decreased (p<0.001). Although the CMO-L group showed some improvement on the various test indicators, the improvement effect was not significant (p>0.05). These results suggest that Litsea cubeba oil can protect the gastric tissue of rats with gastric ulcer by improving inflammation and oxidative stress.

[0050] 3 Discussion and Conclusion

[0051] The formation of gastric ulcers is a complex pathophysiological process that involves multiple links such as excessive gastric acid secretion, damaged gastric mucosal barrier, inflammatory response, and oxidative stress. The occurrence of gastric ulcers may be related to a variety of factors, including excessive drinking, Helicobacter pylori infection, long-term use of nonsteroidal anti-inflammatory drugs, smoking, irregular diet, and excessive mental stress. Given the living and working habits of modern people, excessive drinking is one of the most important factors contributing to the high incidence of gastric ulcers. Studies have shown that ethanol has a high lipophilicity and fat solubility, so it can dissolve mucus in the gastric physiological environment and destroy biofilms, causing mucosal epithelial cells to begin to necrotize and shed, thereby causing superficial damage. + The increase in back diffusion leads to further development of damage, impaired gastric mucosal microcirculation, necrosis of normal tissues in and under the mucosa, and erosion and ulceration.

[39] Therefore, the ethanol-induced gastric ulcer rat model is often used to study the pathogenesis of gastric ulcer and screen therapeutic drugs. Moreover, compared with other models, this model is simple to operate and has mature technology. Therefore, this paper selected the ethanol-induced gastric ulcer rat model to explore the protective effect of Litsea cubeba oil on the gastric tissue of ulcerated rats.

[0052] The gastric ulcer index and ulcer inhibition rate are quantitative scores based on visual or microscopic observation of gastric mucosal damage. They directly reflect the severity of gastric ulcers in experimental models and serve as a preliminary assessment of the effectiveness of therapeutic interventions. HE staining is the most basic histopathological staining method and the "gold standard" for confirming the success of a gastric ulcer model. It can visually demonstrate the effects of drugs on mucosal repair and inflammation suppression. Combining the gastric ulcer index, ulcer inhibition rate, and HE staining for evaluating drug efficacy avoids the limitations of a single indicator. Furthermore, numerous studies have shown that the development of ethanol-induced gastric ulcers is closely associated with inflammation and oxidative stress. TNF-α is a key proinflammatory cytokine in initiating inflammatory responses. By activating the NF-κB signaling pathway, it promotes the release of other inflammatory factors, induces apoptosis of gastric mucosal cells, and increases neutrophil infiltration, leading to tissue damage. IL-6, secreted by macrophages and T cells, promotes the recruitment of inflammatory cells, exacerbates local inflammation, and inhibits gastric mucosal repair. The inflammatory factors TNF-α and IL-6 are core mediators of the inflammatory cascade and can directly reflect the intensity of the gastric mucosal inflammatory response. Both have been extensively validated in various gastric ulcer models. MDA, the end product of lipid peroxidation, reflects the extent of oxidative damage to cell membranes caused by free radicals. SOD is a key antioxidant enzyme that scavenges superoxide anion radicals, protecting cells from oxidative damage. GSH is an important non-enzymatic antioxidant in cells that directly neutralizes free radicals and participates in the repair of oxidative damage. MDA reflects the intensity of oxidative damage, while SOD and GSH reflect antioxidant defense capacity. The combination of these three provides a comprehensive assessment of oxidative stress status. By measuring the gastric ulcer index, ulcer inhibition rate, pathological conditions, and the levels of the inflammatory factors TNF-α and IL-6 and the oxidative stress factors MDA, SOD, and GSH in gastric tissue of rats with gastric ulcers, the efficacy of Litsea cubeba oil in treating gastric ulcers can be more comprehensively evaluated.

[0053] In summary, this paper established an ethanol-induced gastric ulcer model, observed the differences in gastric tissue morphology in each group of rats, calculated the ulcer index and ulcer inhibition rate of rat gastric tissue according to the "Guth" standard, observed the pathological changes of rat gastric tissue by HE staining, and then determined the contents of TNF-α, IL-6, MDA, SOD, and GSH in the gastric tissue of each group of rats by ELISA kits. The results showed that Litsea cubeba oil significantly improved the bleeding, erosion, congestion, and edema of the gastric mucosa in rats, and significantly reduced the ulcer index of the gastric tissue in rats. Not only that, Litsea cubeba oil improved the inflammatory response and oxidative stress of the gastric tissue by reducing the contents of TNF-α, IL-6, and MDA and increasing the contents of SOD and GSH, thereby playing a role in protecting the gastric tissue of rats.

[0054] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Application of Litsea cubeba in the preparation of gastric ulcer medicine.

2. The use of Litsea cubeba in preparing a gastric ulcer medicine according to claim 1, characterized in that: The macrocarpon Litsea cubeba includes products made from the macrocarpon Litsea cubeba.

3. The use of Litsea cubeba in preparing a gastric ulcer medicine according to claim 2, characterized in that: The macrocarpon is macrocarpon oil.

4. The use of Litsea cubeba in preparing a gastric ulcer medicine according to claim 1, characterized in that: The gastric ulcer medicine is a medicine for treating symptoms such as gastric mucosal bleeding, erosion, congestion, edema, etc.

5. The use of Litsea cubeba in preparing a gastric ulcer medicine according to claim 1, characterized in that: The gastric ulcer medicine is a medicine that improves inflammation and oxidative stress to protect the gastric tissue of gastric ulcer rats.

6. The use of Litsea cubeba in preparing a gastric ulcer medicine according to claim 1, characterized in that: The gastric ulcer medicine is a medicine for reducing the contents of TNF-α, IL-6 and MDA.

7. The use of Litsea cubeba in preparing a gastric ulcer medicine according to claim 1, characterized in that: The gastric ulcer medicine is a medicine that increases the content of SOD and GSH.

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