Application of volatile oil from citrus aurantium in preparation of medicine for preventing and treating allergic rhinitis

Abstract

The application discloses application of volatile oil of Citrus aurantium in preparation of a medicine for preventing and treating allergic rhinitis. The application proposes that the volatile oil of Citrus aurantium can significantly reduce the levels of mast cell factors IL-1beta and TNF-alpha, and can also significantly reduce the levels of inflammatory factors IL-13 and IL-4 in mice, reduce inflammatory cell infiltration, and reduce eosinophilic granulocyte increase and gland hyperplasia. The application proposes that the volatile oil of Citrus aurantium has great potential as a medicinal and edible raw material for treating allergic rhinitis. The application also proposes that the volatile oil of Citrus aurantium can significantly improve symptoms such as sneezing, runny nose, and nasal itching of mice caused by allergic rhinitis, and the symptoms seriously affect the life of patients.

Description

Technical Field

[0001] This invention belongs to the technical field of drugs for the prevention and treatment of allergic rhinitis, and particularly relates to the application of volatile oil from Citrus reticulata peel in the preparation of drugs for the prevention and treatment of allergic rhinitis. Background Technology

[0002] Allergic rhinitis (AR) is an inflammatory disease of the nasal mucosa caused by an allergic reaction mediated by immunoglobulin E (IgE). The key to the pathogenesis of allergic rhinitis lies in damage to the epithelial barrier, allowing allergens such as dust mites, pollen, grass, and pet dander to penetrate the nasal mucosal epithelium. Once the allergen enters the nasal cavity, it is processed by antigen-presenting cells (such as dendritic cells), which in turn promotes the differentiation of naïve T cells into helper T cells 2 (Th2). Th2 cells release cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13), which promote the production of specific immunoglobulin E (IgE) against the allergen by B cells. IgE binds to high-affinity IgE receptors on the surface of mast cells, Langerhans cells, monocytes, and basophils. When exposed to the same allergen again, the allergen binds to the IgE antigen binding site on the cell surface, leading to mast cell activation and the release of bioactive mediators such as histamine, leukotrienes, and platelet-activating factor. These mediators act on blood vessels, mucus-secreting glands, and sensory nerves, triggering a series of allergic symptoms such as sneezing, itchy nose, runny nose, and nasal congestion.

[0003] Currently, the main drugs used in clinical treatment of AR include antihistamines, leukotriene inhibitors, corticosteroids, and antimastocyte drugs. These drugs can only relieve symptoms, and long-term use may lead to drowsiness, mental confusion, insomnia, and other side effects. Therefore, it is very important to develop AR treatment drugs with fewer side effects.

[0004] Inflammation of the nasal mucosa is caused by the infiltration of mast cells, eosinophils, and other inflammatory cells. Allergen exposure activates mast cells in the nasal mucosa, triggering histamine release, which is one of the causes of AR symptoms. AR is closely related to various immune cells and mediators, and to date, there is no cure for AR.

[0005] Ovalbumin (OVA) is a water-soluble globular protein extracted from egg whites. It is widely available, easily controlled in purity, and exhibits stable immunogenicity, making it one of the most commonly used allergens in constructing animal models of allergic diseases. OVA has a well-defined molecular structure, and its antigenic epitopes can be specifically recognized by the immune system, stably inducing IgE-mediated type I hypersensitivity reactions, which closely match the immunopathogenesis of human allergic diseases. OVA-induced allergic rhinitis mouse models can mimic the core pathological changes in human diseases, such as nasal mucosal congestion and edema, eosinophil infiltration, and abnormally elevated levels of inflammatory factors (e.g., IL-4, IL-13, and IgE), making it an indispensable tool in basic and translational medicine research in this field.

[0006] Huajuhong (Citrus reticulata peel) is considered the premier of the "Eight Great Southern Chinese Medicines," and has been known since ancient times as "Southern Ginseng" and "worth a thousand pieces of gold." Huajuhong is warm in nature and has a pungent and bitter taste. It has the functions of regulating qi, relieving chest congestion, drying dampness, and resolving phlegm. It is used for cough with excessive phlegm, food stagnation, alcohol poisoning, nausea, vomiting, and abdominal distension. It contains flavonoids, coumarins, volatile oils, polysaccharides, and other components. Huajuhong polysaccharides have good anti-inflammatory, antitussive, and expectorant effects.

[0007] Volatile oils, also known as essential oils, are a class of volatile liquids that can be distilled with steam and are widely distributed in traditional Chinese medicinal materials. They are produced by plant organs through secondary metabolism and stored in secretory cells, cavities, ducts, epidermal cells, or glandular trichomes. They are widely found in Rutaceae plants such as Citrus reticulata, lemon, and citron. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide the use of tangerine peel volatile oil in the preparation of medicines for the prevention and / or treatment of allergic rhinitis.

[0009] This invention focuses on the application of Citrus reticulata volatile oil in the preparation of drugs for the prevention or treatment of allergic rhinitis. OVA was used to induce allergic rhinitis in mice, and the extraction and efficacy of Citrus reticulata volatile oil were experimentally verified, resulting in the research results that Citrus reticulata volatile oil can effectively prevent and treat allergic rhinitis.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of volatile oil from Citrus reticulata peel in the preparation of drugs for the prevention and / or treatment of allergic rhinitis.

[0011] Preferably, the volatile oil of the tangerine peel is obtained by steam distillation.

[0012] Preferably, the volatile oil of Citrus reticulata is obtained by first soaking Citrus reticulata in water for 2-3 hours, more preferably 140-160 minutes, and most preferably 145-155 minutes, and then steam distilling for 4-6 hours, more preferably 4-5 hours, more preferably 280-300 minutes, and most preferably 290-300 minutes. After extraction, the standing time is 1-2 hours, and the extraction temperature is 80-100℃, most preferably 100℃.

[0013] In this invention, it is preferred to first pulverize the tangerine peel and then perform steam extraction. This invention does not have a specific limitation on the pulverization method, and the preferred mesh size of the pulverized product is 5-80 mesh.

[0014] Preferably, the pulverized tangerine peel is soaked in water at a material-to-liquid ratio of 1:5-50g / ml, preferably 1:10g / ml.

[0015] Preferably, the volatile oil of Citrus reticulata can inhibit the levels of immunoglobulin E and histamine in the body and / or cells of animals and reduce the levels of inflammatory factors. Preferably, the inflammatory factors include IL-1β, TNF-α, IL-13 and / or IL-4.

[0016] Preferably, the drug contains volatile oil from Citrus reticulata peel as an active ingredient, and also contains pharmaceutically usable excipients. This invention does not specifically limit the type of pharmaceutical excipients; conventional pharmaceutical excipients in the art can be used. In this invention, the weight percentage of volatile oil from Citrus reticulata peel in the drug is preferably 0.01%-99%, more preferably 0.05%-10%, and even more preferably 0.1%-5%.

[0017] Preferably, the drug can be formulated into a usable dosage form, such as a wettable powder, tablet, granule, capsule, oral liquid, drop pill, injection, aerosol, controlled-release or sustained-release dosage form or nano-formulation.

[0018] The beneficial effects of this invention are: This invention proposes that the volatile oil of Citrus reticulata peel has a preventive and / or therapeutic effect on allergic rhinitis. Specifically, it proposes that the volatile oil can significantly reduce the levels of mast cell kinases IL-1β and TNF-α, and also significantly reduce the levels of inflammatory factors IL-13 and IL-4 in mice, decreasing inflammatory cell infiltration, eosinophilia, and glandular hyperplasia. Furthermore, this invention proposes that the volatile oil can effectively inhibit the level of immunoglobulin E (IgE) in mice. IgE is a key substance mediating allergic rhinitis; inhibiting IgE expression fundamentally prevents the occurrence of allergic rhinitis. In addition, this invention proposes that the volatile oil can reduce serum histamine levels. Histamine is an important signaling factor in the pathogenesis of allergic rhinitis; inhibiting histamine expression reduces the occurrence of allergic rhinitis through this pathway. Finally, this invention proposes that the volatile oil of Citrus reticulata peel, as a food-medicine homologous raw material for treating allergic rhinitis, has great potential. The present invention also proposes that the volatile oil of Citrus reticulata can significantly improve the symptoms of sneezing, runny nose, and nasal itching in mice caused by allergic rhinitis, which seriously affect the lives of patients. Attached Figure Description

[0019] Figure 1: (A) Symptom score of allergic rhinitis in mice (B) Number of sneezes in mice, n=12, *** p<0.001, ** p<0.01, and * p<0.1, compared with the model group.

[0020] Figure 2: (A) Serum IgE expression level; (B) Serum Histamine expression level; (C) Serum IL-13 expression level. n=9-12, *** p<0.001, ** p<0.01, and * p<0.1, compared with the model group.

[0021] Figure 3: IL-4 expression levels in NLF, n=12, *** p<0.001, ** p<0.01, and * p<0.1, compared with the model group.

[0022] Figure 4: HE staining scan of nasal mucosa section.

[0023] Figure 5: (A) Area of ​​glands in nasal mucosa tissue (B) Number of eosinophils in nasal mucosa tissue, n=6, *** p<0.001, ** p<0.01, and * p<0.1, compared with the model group.

[0024] Figure 6: (A) IL-1β protein level in RAW264.7, (B) IL-1β transcription level in RAW264.7, (C) TNF-α transcription level in RAW264.7, (D) CCL2 transcription level in RAW264.7, (E) INOS transcription level in RAW264.7, (F) PTGS2 transcription level in RAW264.7, n=5, *** p<0.001, ** p<0.01, and * p<0.1, compared with the model group (0.02 μM Histamine). Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the following embodiments are all conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] Data in the following examples are expressed as mean ± standard error (SEM). Statistical comparisons were performed using one-way ANOVA, followed by a multiple comparison test using the Dunnett test with the GraphPad Prism (GraphPad, La Jolla, CA, USA). Differences were considered statistically significant (P < 0.05).

[0029] Example 1 Preparation of volatile oil from Citrus reticulata: Grind 50g of Citrus reticulata peel to 5 mesh, immediately add ten times its weight of water and an appropriate amount of glass beads, place in a 1000ml round-bottom flask, shake to mix, and soak for 2 hours. Connect the volatile oil analyzer to the reflux condenser (as shown in the diagram of the Chinese Pharmacopoeia 2025 edition, 2204, Method for Determination of Volatile Oils). Add water from the top of the condenser until it fills the graduated section of the volatile oil analyzer and overflows into the flask. Place in a heating mantle and slowly heat to boiling, maintaining a gentle boil for about 5 hours, until the oil level in the analyzer no longer increases. Stop heating, let stand for a moment, then open the stopcock at the bottom of the analyzer to slowly release the water until the upper end of the oil layer reaches 5mm above the 0 mark. Let stand for 1 hour, then open the stopcock again to allow the oil layer to descend until its upper end is exactly level with the 0 mark. Read the volatile oil volume and calculate the volatile oil content (%) in the sample. Collect the volatile oil to obtain Citrus reticulata peel volatile oil, with an extraction rate of 1.3%.

[0030] Example 2 Preparation of volatile oil from Citrus reticulata: Crush 100g of Citrus reticulata peel to 10 mesh, immediately add ten times its weight of water and an appropriate amount of glass beads, place in a 2000ml round-bottom flask, shake to mix, and soak for 3 hours. Connect the volatile oil analyzer to the reflux condenser (as shown in the diagram of the volatile oil determination method in Chinese Pharmacopoeia 2025, 2204). Add water from the top of the condenser until it fills the graduated section of the volatile oil analyzer and overflows into the flask. Place in a heating mantle and slowly heat to boiling, maintaining a gentle boil for about 4 hours, until the oil level in the analyzer no longer increases. Stop heating, let stand for a moment, open the stopcock at the bottom of the analyzer, and slowly release the water until the upper end of the oil layer reaches 5mm above the 0 mark. Let stand for 1 hour, then open the stopcock again to allow the oil layer to descend until its upper end is exactly level with the 0 mark. Read the volatile oil volume and calculate the volatile oil content (%) in the sample. Collect the volatile oil to obtain Citrus reticulata peel volatile oil, with an extraction rate of 1.6%.

[0031] Example 3 Preparation of volatile oil from Citrus reticulata: Crush 100g of Citrus reticulata peel to 5 mesh, immediately add ten times its weight of water and an appropriate amount of glass beads, place in a 2000ml round-bottom flask, shake to mix, and soak for 3 hours. Connect the volatile oil analyzer to the reflux condenser (as shown in the diagram of the Chinese Pharmacopoeia 2025 edition, 2204, Method for Determination of Volatile Oils). Add water from the top of the condenser until it fills the graduated section of the volatile oil analyzer and overflows into the flask. Place in a heating mantle and slowly heat to boiling, maintaining a gentle boil for about 5 hours, until the oil level in the analyzer no longer increases. Stop heating, let stand for a moment, then open the stopcock at the bottom of the analyzer to slowly release the water until the upper end of the oil layer reaches 5mm above the 0 mark. Let stand for 2 hours, then open the stopcock again to allow the oil layer to descend until its upper end is exactly level with the 0 mark. Read the volatile oil volume and calculate the volatile oil content (%) in the sample. Collect the volatile oil to obtain Citrus reticulata peel volatile oil, with an extraction rate of 1.8%.

[0032] Example 4 OVA-induced mouse model of allergic rhinitis: Seventy-two male Balb / c mice, 6-8 weeks old and weighing 25±2g (Zhuhai Baishitong Biotechnology Co., Ltd.), were used and cultured in an SPF-grade animal facility. All protocols were approved by the Laboratory Animal Ethics Committee of the School of Life Sciences, Sun Yat-sen University (SYXK (Guangdong) 2023-0313), and the application number for the use of laboratory animals and ethical review was SYSU-LS-IACUC-2025-0042.

[0033] Balb / C mice were grouped and treated according to Table 1 below: Table 1. Grouping and treatment methods of experimental mice .

[0034] On day 27, after OVA modeling, mice underwent behavioral tests. Allergic rhinitis symptom scores were recorded over two minutes, as shown in Table 2. The number of sneezes was also recorded separately as a key behavioral indicator. Results showed that the symptom score and number of sneezes in the AR group were significantly higher than those in the CTRL group. After drug treatment, both the behavioral scores and sneezing frequency in all groups significantly decreased. Figure 1 As shown.

[0035] Table 2. Scoring criteria for allergic rhinitis symptoms in mice .

[0036] Mice were euthanized after being anesthetized with sodium pentobarbital on day 28, and the following tests were performed: 1. Serum pharmacological cytokine analysis: Blood was collected from the heart of mice, allowed to stand at room temperature for 2 hours, centrifuged at 2000g for 10 min, and serum was collected. The levels of IgE, Histamine, and IL-13 in the serum were detected by ELISA.

[0037] The results showed that after OVA modeling, compared with the CTRL group, the serum protein levels of IgE, Histamine, and IL-13 in mice in the AR group were significantly increased, while the levels of each factor significantly decreased after drug administration. Figure 2 As shown.

[0038] 2. Analysis of inflammatory factors in nasal lavage fluid (NLF): The nasal cavity of mice was rinsed with 1.2 ml of sterile PBS, incubated at 4 degrees Celsius for two hours, centrifuged at 2000 g for 15 min, and the supernatant was collected and the cell pellet was discarded. The level of IL-4 in NLF was detected by ELISA.

[0039] The results showed that after OVA modeling, compared with the CTRL group, the IL-4 level in the NLF of mice in the AR group was significantly increased, while the IL-4 level showed a significant regression after administration of the positive control drug and low-dose HJH-oil, as follows. Figure 3 As shown.

[0040] 3. Histopathological analysis of nasal mucosa sections: Mouse nasal mucosa tissue was fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin. The paraffin blocks were cut into 4.5 μm sections, stained with hematoxylin and eosin (HE), and mounted. The structure and inflammatory cell infiltration of the mouse nasal mucosa were observed using a Zeiss Axioscan 7 fully automated digital slide scanning system.

[0041] The results showed that, compared with the CTRL group, the AR group mice exhibited significantly increased inflammatory cell infiltration and eosinophil count in the nasal mucosa, along with marked glandular hyperplasia, severe mucosal fragmentation, and occasional hemorrhage. After drug administration, inflammatory cell infiltration decreased, eosinophil count decreased, and glandular hyperplasia was significantly reduced. The results are as follows: Figure 4 As shown in Figure 5.

[0042] Example 4 Culture and inflammatory factor detection of RAW264.7: RAW264.7 cells were derived from Balb / b mice and are mouse mononuclear macrophage leukemia cells obtained from Wuhan Procell Biotechnology Co., Ltd. They were cultured in RAW264.7-specific medium (DMEM high glucose + 10% FBS + 1% P / S solution + RAW264.7 cell-specific additives). The cells were divided into a control group (BLANK group), a model group, a positive control group, and an experimental group. The control group received no treatment. The model group received 0.02 μM (final concentration) Histamine to induce inflammation (Histamine dissolved in the culture medium). The positive control group received 0.02 μM (final concentration) Histamine to induce inflammation and 10 μg / mL (final concentration) DEX (Histamine and DEX dissolved in the culture medium). The experimental group consisted of high, medium, and low dose groups with HJH-oil doses of 100 μg / ml, 50 μg / ml, and 25 μg / ml, respectively. The experimental group received 0.02 μM (final concentration) Histamine to induce inflammation and HJH-oil (Histamine and HJH-oil dissolved in the culture medium). After 24 h of culture, cells were collected, cell lysates were taken, RNA was extracted, reverse transcribed into cDNA, and then detected by qPCR. This study investigated the effect of Citrus reticulata volatile oil on Histamine-induced inflammation in RAW264.7 cells. The expression levels of cytokines IL-1β, TNF-α, CCL2, INOS, and PTGS2 were detected using ELISA and real-time quantitative PCR. IL-1β dysregulation is associated with various excessive inflammation and immune diseases. TNF-α not only directly drives inflammatory responses by inducing the expression of inflammatory genes but also triggers inflammatory immune responses by inducing cell death; therefore, tumor necrosis factor (TNF) is considered a core cytokine in inflammatory responses. The ELISA experiment used the Cloud-Clone kit. The specific method for the RT-qPCR experiment is as follows: RNA was extracted using an RNA extraction kit (EZ-press RNAPurification Kit, EZBioscience), and the concentration of the extracted RNA was detected using a Nanodrop spectrophotometer. Once the RNA concentration was good, reverse transcription was performed using a Color Reverse Transcription Kit (EZBioscience). Subsequently, qPCR was performed using the SYBR GreenI chimeric fluorescence method. The reaction solution was prepared according to the reaction system in the ColorSYBR Green qPCR Master Mix (EZBioscience) manual. β-actin was used as an internal reference gene, and the relative expression levels of each cytokine were calculated. The reaction procedure is shown in Table 3 below.The primer sequences for β-actin, IL-1β, TNF-α, CCL2, INOS, and PTGS2 are shown in Table 4 below and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] Table 3 RT-qPCR reaction procedure .

[0044] surface 4 Real-time quantitative PCR primer sequences .

[0045] The results showed that after Histamine modeling, compared with the BLANK group, the transcriptional levels of IL-1β, TNF-α, CCL2, INOS, and PTGS2 in the model group cells were significantly increased. However, after administration of the positive control drug and different doses of HJH-oil, the levels of each factor showed some significant regression. Specifically, the low-dose group showed no regression in CCL2, INOS, and PTGS2 levels, the medium-dose group showed no regression in INOS levels, and the other treatment groups showed significant regression. Furthermore, tangerine peel oil also significantly reduced the protein level of IL-1β. (The results are as follows...) Figure 6 As shown.

[0046] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Citrus reticulata volatile oil in the preparation of drugs for the prevention and / or treatment of allergic rhinitis, wherein the Citrus reticulata volatile oil is prepared by first soaking Citrus reticulata in water for 2-3 hours, and then steam distilling for 4-6 hours, followed by a standing time of 1-2 hours, and an extraction temperature of 80-100℃.

2. The application according to claim 1, characterized in that, The aforementioned Citrus reticulata peel is pulverized Citrus reticulata peel, and the mesh size of the pulverization is 5-80 mesh.

3. The application according to claim 1, characterized in that, The process of soaking the tangerine peel in water has a material-to-liquid ratio of 1:5-50g / ml.

4. The application according to claim 1, characterized in that, The prevention and / or treatment of allergic rhinitis is achieved by using volatile oil from Citrus reticulata to inhibit the levels of immunoglobulin E and histamine in the body and / or cells of animals and to reduce the levels of inflammatory factors, including IL-1β, TNF-α, IL-13 and / or IL-4.

5. The application according to claim 1, characterized in that, The drug contains volatile oil from Citrus reticulata as its active ingredient, as well as pharmaceutically usable excipients.

6. The application according to claim 1, characterized in that, The weight percentage of volatile oil from Citrus reticulata in the drug is 0.01%-99%.

7. The application according to claim 6, characterized in that, The weight percentage of volatile oil from Citrus reticulata in the drug is 0.05%-10%.

8. The application according to claim 1, characterized in that, The drug described herein is available in the form of wettable powder, tablets, granules, capsules, oral liquid, droplets, aerosols, controlled-release or sustained-release formulations, or nano-formulations.

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