Application of Chimonanthus chinensis leaf extract in preparing medicine for treating allergic rhinitis

Abstract

The present invention discloses the use of a Chimonanthus dahurica leaf extract in preparing a drug for treating allergic rhinitis, and relates to the field of traditional Chinese medicine. Based on an allergic rhinitis animal model, the present invention investigates the effects of Chimonanthus dahurica leaves on the behavior, nasal mucosal tissue morphology, and serum inflammatory factor levels of AR rats, and clarifies the pharmacodynamics of Chimonanthus dahurica leaves in treating AR. That is, Chimonanthus dahurica leaves can significantly reduce the levels of IgE, histamine, ICAM-1, and inflammatory factors in the serum of rats with allergic rhinitis, and can also significantly improve the nasal mucosal symptoms of rats with allergic rhinitis, among which the Chimonanthus dahurica leaf water extract has a better effect. At the same time, by preparing different active component groups of Chimonanthus dahurica leaves, the effective parts of Chimonanthus dahurica for treating allergic rhinitis are screened, among which the 70% ethanol elution group and the 50% ethanol elution group have the best effects. The present invention has prospects for developing and preparing drugs for treating allergic rhinitis.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to application of a Chimonanthus chinensis leaf extract in preparing a medicine for treating allergic rhinitis. Background Art

[0002] Allergic rhinitis, also known as allergic rhinitis, is a non-infectious inflammatory disease of the nasal mucosa. It is a chronic inflammatory disease of the nasal mucosa that occurs after a susceptible individual comes into contact with an allergen. It is primarily mediated by immunoglobulin E (IgE) and involves the body's immune-active cells and cytokines. Symptoms include paroxysmal sneezing, runny nose, and nasal congestion.

[0003] At present, the first point of treating allergic rhinitis with both Chinese and Western medicine is to avoid contact with allergens as much as possible, and then proceed with treatment. The main treatment methods are as follows: (1) Western medicine: oral or nasal antihistamines, mast cell membrane stabilizers, anti-leukotriene drugs, corticosteroids, etc. Oral or nasal anti-allergic or hormone Western medicines are effective quickly and have good symptom control effects, but they are prone to relapse after discontinuation of the drug, and repeated use can easily lead to drug resistance; (2) Acupuncture therapy: Select acupoints such as Yingxiang to open the nasal passages, diagnose and classify the symptoms according to the patient's overall condition, and combine other acupoints to dispel wind and evil, and nourish the lungs, spleen, and kidneys. However, many patients refuse to use this therapy due to psychological fear, and this therapy requires going to the hospital for treatment every day, and patient compliance is poor; (3) Chinese herbal decoction treatment: Diagnose and treat according to the patient's overall symptoms, which can regulate the five internal organs, expel evil spirits, and fundamentally treat allergic rhinitis.

[0004] Modern pharmacological research shows that Chimonanthus chinensis (Ciprida dahurica) possesses anti-inflammatory, analgesic, anti-tussive, immune-regulating, antibacterial, and antioxidant properties. Its main chemical components include volatile oils, flavonoids, and alkaloids. However, there are currently no reports on the use of Chimonanthus dahurica leaf extract in the preparation of medications for the treatment of allergic rhinitis. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide an application of a Chimonanthus chinensis leaf extract in the preparation of a drug for treating allergic rhinitis.

[0006] The technical solutions of the present invention are as follows:

[0007] Application of Chimonanthus chinensis leaf extract in the preparation of a drug for treating allergic rhinitis, wherein the preparation method of the Chimonanthus chinensis leaf extract is as follows:

[0008] Taking Chimonanthus chinensis leaves, adding 12-18 times the amount of water, soaking, reflux extraction, obtaining an extract, concentrating the extract under reduced pressure and then freeze-drying to obtain a Chimonanthus chinensis leaf water extract;

[0009] The aqueous extract of Chimonanthus chinensis leaves was separated by macroporous resin, and then washed with 40%-80% ethanol by volume, and the washing liquid was collected, concentrated under reduced pressure, and then dried.

[0010] Preferably, taking Chimonanthus chinensis leaves, adding 12-18 times the amount of water, soaking, reflux extraction, obtaining an extract, and concentrating the extract under reduced pressure and freeze-drying to obtain a Chimonanthus chinensis leaf water extract;

[0011] The aqueous extract of Chimonanthus chinensis leaves was separated by macroporous resin and then washed with 70% by volume ethanol. The washing liquid was collected, concentrated under reduced pressure, and then dried.

[0012] Preferably, taking Chimonanthus chinensis leaves, adding 12-18 times the amount of water, soaking, reflux extraction, obtaining an extract, and concentrating the extract under reduced pressure and freeze-drying to obtain a Chimonanthus chinensis leaf water extract;

[0013] The aqueous extract of Chimonanthus chinensis leaves was separated by macroporous resin and then washed with 50% by volume ethanol. The washing liquid was collected, concentrated under reduced pressure, and then dried.

[0014] Preferably, the Chimonanthus chinensis leaf extract mainly contains flavonoids.

[0015] Preferably, the preparation method of the Chimonanthus chinensis leaf extract is as follows:

[0016] Take Chimonanthus chinensis leaves, add 12-18 times the amount of water, soak, and extract volatile oil by steam distillation.

[0017] Preferably, the active ingredient of the drug is Chimonanthus chinensis leaf extract, and the drug further comprises pharmaceutically acceptable excipients.

[0018] Preferably, the drug preparation is one of oral preparation, injection, inhalation, and nasal drops.

[0019] The beneficial effects of the present invention are: the Chimonanthus wiltshire leaf of the present invention can significantly reduce the content of IgE, histamine, ICAM-1, and inflammatory factors in the serum of rats with allergic rhinitis, and can also significantly improve the nasal mucosal symptoms of rats with allergic rhinitis, among which the Chimonanthus wiltshire leaf water extract has a better effect. At the same time, by preparing different active component groups of Chimonanthus wiltshire leaf, the effective parts of Chimonanthus wiltshire for treating allergic rhinitis are screened, among which the 70% and 50% ethanol elution groups have the best effects, and the present invention has the prospect of developing and preparing drugs for treating allergic rhinitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of the modeling and drug administration process for allergic rhinitis rats;

[0021] Figure 2The effect of Chimonanthus chinensis leaf extract on the behavior of rats with allergic rhinitis; compared with the model group, express p <0.01, express p < 0.0001;

[0022] Figure 3 The effects of different extracts of Chimonanthus chinensis leaves on IgE, histamine and inflammatory factors in rats with allergic rhinitis; a: refers to the content of IGE in serum; b: refers to the content of HIS in serum; c: refers to the content of ICAM-1 in serum; d: refers to the content of TNF-ɑ in serum; e: refers to the content of IL-4 in serum; compared with the model group, express p <0.05, express p <0.01, express p < 0.001;

[0023] Figure 4 To study the effect of Chimonanthus chinensis leaf extract on nasal mucosal histopathology in rats with allergic rhinitis.

[0024] Figure 5 The effects of different active parts of Chimonanthus chinensis leaves on the behavior of rats with allergic rhinitis; compared with the model group, express p <0.05, express p <0.01, express p < 0.001;

[0025] Figure 6 The effects of different active parts of Chimonanthus chinensis leaves on serum IgE, HIS and ICAM-1 in rats with allergic rhinitis; a: refers to the content of IGE in serum; b: refers to the content of HIS in serum; c: refers to the content of ICAM-1 in serum; d: refers to the content of TNF-ɑ in serum; e: refers to the content of IL-4 in serum; compared with the model group, express p <0.05, express p <0.01, express p <0.001;

[0026] Figure 7 To study the effects of different active fractions of Chimonanthus chinensis leaves on the morphology of nasal mucosa in rats with allergic rhinitis;

[0027] Con: normal group; Mod: model group; LLTD: loratadine group; SLO-L: low-dose volatile oil group, SLO-M: medium-dose volatile oil group, SLO-H: high-dose volatile oil group; SLM-L: low-dose water-eluted group; SLM-M: medium-dose water-eluted group; SLM-H: high-dose water-eluted group; SLE-20%-L: low-dose 20% ethanol-eluted group, SLE-20%-M: medium-dose 20% ethanol-eluted group, SLE-20%-H: high-dose 20% ethanol-eluted group, SLE-50% -L: 50% ethanol elution low-dose group, SLE-50%-M: 50% ethanol elution medium-dose group, SLE-50%-H: 50% ethanol elution high-dose group, SLE-70%-L: 70% ethanol elution low-dose group, SLE-70%-M: 70% ethanol elution medium-dose group, SLE-70%-H: 70% ethanol elution low-dose group, SLE-95%-L: 95% ethanol elution low-dose group, SLE-95%-M: 95% ethanol elution medium-dose group, SLE-95%-H: 95% ethanol elution low-dose group;

[0028] Figure 8 The total ion currents of the effective parts of Chimonanthus chinensis leaves are shown in Figure 1, where a represents the total ion current of the 50% alcohol elution group, b represents the total ion current of the 70% alcohol elution group, and c represents the total ion current of the volatile oil group. DETAILED DESCRIPTION

[0029] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0030] The technical solution of the present invention is further illustrated by specific experiments below.

[0031] Example 1 Pharmacodynamic Study on the Treatment of Allergic Rhinitis with Chimonanthus chinensis Leaves

[0032] Based on the allergic rhinitis (AR) animal model, the effects of Chimonanthus chinensis leaves on the behavior, nasal mucosal histomorphology, and serum inflammatory factor levels of AR rats were investigated to clarify the pharmacological effects of Chimonanthus chinensis leaves in treating AR.

[0033] (1) Preparation of Chimonanthus chinensis leaf extract

[0034] Take an appropriate amount of Chimonanthus wiltii leaves, add 15 times the amount of water, soak for 1 hour, and reflux extraction twice, each time for 1 hour; the extract is concentrated under reduced pressure at 70℃ and then freeze-dried, and the powder is collected for later use, namely Chimonanthus wiltii leaf aqueous extract (SLMY).

[0035] (2) Animal grouping, modeling, and drug administration

[0036] The experiment set up a normal group, a model group, a positive control group (loratadine: 1.042 mg / kg), and a drug-treated group (low-, medium-, and high-dose groups of different Chimonanthus leaf extracts). Specifically, the low, medium, and high doses of Chimonanthus leaf aqueous extract were 0.27 g / kg, 0.54 g / kg, and 1.09 g / kg, respectively. An ovalbumin (OVA)-induced AR model in rats was established by intraperitoneal injection sensitization and intranasal instillation challenge. The specific operation was as follows: on days 1, 3, 5, 7, 9, 11, and 13, once every other day for a total of 7 times, OVA 2 mg + Al(OH)3 30 mg was mixed and dissolved in 1 mL of normal saline (OVA is ovalbumin) to prepare a milky white suspension. Because Al(OH)3 is poorly soluble in water, the suspension was allowed to stand for a period of time until the undissolved powder settled to the bottom. Then, 1 mL of the prepared solution was drawn out with a syringe and injected intraperitoneally. The normal group was injected with an equal dose of normal saline. On the second day after the systemic sensitization stage, that is, on the 15th day of the experiment, 50 μL of OVA saline solution was aspirated with a pipette and dripped into the nasal cavity, 50 μL on one side, and 100 μL on both sides of the nasal cavity for 7 consecutive days. On the 21st day, after the last dripping of the rat's nasal cavity, the number of times the rat scratched its nose, the number of times it sneezed, and the range of nasal discharge were recorded within 30 minutes, and quantitative points were taken. The success standard of the model was a total score > 5 points (Table 1). After the model was successfully established, each drug group was given various doses of drug intervention by gavage, 1 time / d, for 7 consecutive days, and OVA was dripped into the nasal cavity every other day for maintenance (30 minutes after the drug treatment). For details of the modeling and drug administration process, please refer to Figure 1 .

[0037] Table 1 Behavioral scoring criteria

[0038]

[0039] (3) Sample collection and processing

[0040] After the final administration, rats in each group were fasted but not watered for 12 hours. Blood was then collected from the abdominal aorta after anesthesia. Whole blood was used for routine blood tests and blood cell smears. The remaining blood was allowed to rest at room temperature for 1 hour before centrifugation at 5000 rpm for 10 minutes at 4°C. Serum was collected and stored at -20°C until further use. The rats' nasal cavities were irrigated with pre-chilled saline. The lavage fluid was collected and centrifuged at 1000 rpm for 10 minutes at 4°C. The supernatant was aspirated and stored at -20°C until further use. The maxilla of each rat's nose was isolated and opened along the midline. The nasal bones and maxilla were removed, exposing the nasal septum and bilateral nasal mucosa. The nasal septum, along with the bilateral nasal mucosa, was clipped and fixed in 4% paraformaldehyde at room temperature.

[0041] (4) Evaluation indicators and results

[0042] Behavioral observation: Starting from the 21st day, the frequency and scores of nasal itching, sneezing, and runny nose within 30 minutes of OVA nasal drop stimulation were recorded for behavioral evaluation. The results are shown in Figure 2 The results showed that the behavioral scores of the model group increased significantly compared with the normal group, proving that the model was successful ( P <0.05); compared with the model group, the behavioral scores of each drug-treated group showed different downward trends, among which the high-dose Chimonanthus chinensis leaf water extract group had the best effect ( P <0.05).

[0043] Determination of serum IgE, histamine, ICAM-1, and inflammatory factors: The levels of serum IgE, histamine (HIS), intercellular adhesion molecule (ICAM-1), TNF-α, and IL-4 were detected according to the instructions of the Elisa kit. Figure 3 The results showed that compared with the normal group, the levels of serum IgE, HIS, ICAM-1, TNF-α, and IL-4 in the model group rats increased significantly ( P <0.05); compared with the model group, the serum levels of various indicators in the rats in the high-dose Chimonanthus chinensis leaf group were significantly reduced ( P <0.05). The results showed that Chimonanthus chinensis leaves could significantly reduce the levels of IgE, histamine, ICAM-1, and inflammatory factors in the serum of rats with allergic rhinitis.

[0044] Pathological observation: The nasal septum tissue was fixed in paraformaldehyde at 4°C for 24 hours, decalcified with EDTA, and after reaching the decalcification endpoint, it was dehydrated, embedded in paraffin, sliced, and stained with conventional HE to observe the pathological changes. The results showed that compared with the normal group, the nasal mucosal epithelial tissue structure of the model group was incomplete, with a large number of epithelial cells shrinking and necrotizing; the thickness of the nasal mucosal epithelium increased, goblet cells significantly proliferated, and a large number of inflammatory cells, mainly eosinophils, infiltrated. Compared with the model group, the mucosal epithelium and the lamina propria of the nasal mucosa tissue of the rats in the high-dose group of Chimonanthus chinensis leaves aqueous extract were clearly arranged and demarcated, the ciliated epithelium was neatly arranged, and the number of eosinophils was significantly reduced (see Figure 4 Chimonanthus chinensis leaves can significantly improve the nasal mucosal symptoms of rats with allergic rhinitis.

[0045] Example 2 Screening of effective fractions of Chimonanthus chinensis leaves for treating allergic rhinitis

[0046] This experiment is based on the pharmacodynamic effects of Chimonanthus chinensis leaves in treating allergic rhinitis. By preparing different active component groups of Chimonanthus chinensis leaves, the effective parts of Chimonanthus chinensis in treating allergic rhinitis were screened.

[0047] (1) Preparation of different active fractions from Chimonanthus chinensis leaves

[0048] Preparation of volatile oil and non-volatile extracts from Chimonanthus chinensis leaves: 10 kg of Chimonanthus chinensis leaves were added to 15-fold water, soaked for 1 hour, and the volatile oil was extracted by steam distillation. An appropriate amount of Chimonanthus chinensis leaves was then added to 15-fold water, soaked for 1 hour, and refluxed for extraction twice, each for 1 hour. The extract was concentrated under reduced pressure at 70°C and freeze-dried. The powder was collected and used as the dry extract powder.

[0049] (2) Isolation and purification of non-volatile extracts from Chimonanthus chinensis leaves

[0050] Preparation of the supernatant: Take an appropriate amount of the above-mentioned Chimonanthus chinensis leaf dry extract powder, add water to dilute it to contain 0.2-0.4g of crude drug per milliliter, and the supernatant is obtained.

[0051] Macroporous resin separation and purification process: Take an appropriate amount of drug solution and slowly add it to the top of the resin. Allow to adsorb overnight. Rinse with 5 volumes of pure water, 20%, 50%, 70%, and 95% ethanol, respectively. Collect each washing fraction. After vacuum concentration, dry to obtain a powder.

[0052] (3) Effects of various components of Chimonanthus chinensis leaves on rats with allergic rhinitis

[0053] The experimental settings included normal group, model group, positive control group (loratadine: 1.042 mg / kg), volatile oil group (low, medium, and high doses (SLO-L / M / H): 10.4 mg / kg, 20.8 mg / kg, and 41.60 mg / kg), water elution group (low, medium, and high doses (SLW-L / M / H), 20% ethanol elution group (low, medium, and high doses (SLE-20%-L / M / H), 50% ethanol elution group (low, medium, and high doses (SLE-50%-L / M / H), 70% ethanol elution group (low, medium, and high doses (SLE-70%-L / M / H), and 95% ethanol elution group (low, medium, and high doses (SLE-95%-L / M / H). The dosage for each elution site was 1 g / kg to 4 g / kg), modeling and administration were the same as in Example 1 (2). The behavioral characteristics of rats with allergic rhinitis, serum levels of IgE, histamine (HIS), intercellular adhesion molecule (ICAM-1), TNF-α and IL-4, and nasal mucosal tissue morphology were used as indicators to screen the effective parts of Chimonanthus chinensis leaves for the treatment of allergic rhinitis.

[0054] Behavioral results of rats with allergic rhinitis Figure 5 Compared with the normal group, the behavioral scores of the model group increased significantly, proving that the model was successful ( P <0.05); compared with the model group, the behavioral scores of each drug-treated group showed different downward trends, among which the 70% ethanol elution group, 50% ethanol elution group and volatile oil group showed the most obvious decreases ( P <0.05).

[0055] The results of serum IgE, histamine (HIS), intercellular adhesion molecule (ICAM-1), TNF-α and IL-4 levels in rats with allergic rhinitis are shown in Figure 6 The results showed that compared with the normal group, the serum IgE, HIS and ICAM-1 levels of the model group rats increased significantly ( P <0.05); compared with the model group, the serum IgE, HIS, ICAM-1, TNF-α and IL-4 levels of rats in the 70% ethanol elution group, 50% ethanol elution group and volatile oil group were significantly decreased ( P <0.05). The levels of each indicator in the other groups of rats showed a decreasing trend, with no statistically significant differences. The results showed that the 70% ethanol elution group had the best effect, followed by the 50% ethanol elution group and the volatile oil group.

[0056] The morphological results of nasal mucosa in rats with allergic rhinitis are shown in Figure 7 and Table 2. The results showed that the overall structure of the nasal mucosa in the normal group was essentially normal, with a relatively regular arrangement. No significant desquamation, edema, necrosis, or other degeneration of the mucosal epithelium was observed, and no significant inflammatory cell infiltration was observed in the nasal septum. In the model group, the overall structure of the nasal mucosa was abnormal, with disordered arrangement of mucosal epithelial cells, significant vascular congestion in the interstitial tissue, and a large number of inflammatory cells, such as eosinophils, infiltrating. In the 70% ethanol elution group, the 50% ethanol elution group, and the volatile oil group, the nasal mucosa was relatively intact and tightly arranged. No significant desquamation, edema, necrosis, or other degeneration of the mucosal epithelium was observed, and no significant inflammatory cell infiltration was observed in the tissue. The results showed that the 70% ethanol elution group was the most effective, followed by the 50% ethanol elution group and the volatile oil group.

[0057] Table 2 Histomorphological analysis of nasal mucosa in rats with allergic rhinitis

[0058]

[0059]

[0060] Example 3 Identification of the active components of Chimonanthus chinensis leaves for the treatment of allergic rhinitis

[0061] Based on the above-mentioned allergic rhinitis animal model screening, the effective parts of Chimonanthus chinensis leaves for treating allergic rhinitis were 50% and 70% ethanol elution parts and volatile oil. The components and content proportions of each effective part were identified by LC-MS, GC-MS, etc. The results are as follows Figure 8 and Tables 3-5. The results showed that the 50% and 70% ethanol elution fractions were mostly flavonoids, with slightly different components but a large difference in proportion; the volatile oil contained mostly volatile terpenoids, indicating that the components that exert the medicinal effects of the volatile oil are mostly volatile terpenoids.

[0062] Table 3 Identification of components of 50% and 70% alcohol elution groups of Chimonanthus chinensis leaves

[0063]

[0064]

[0065] Table 4 Quantitative table of components of 50% and 70% alcohol elution groups of Chimonanthus chinensis leaves

[0066]

[0067]

[0068] Table 5 Identification of volatile components of Chimonanthus chinensis leaves

[0069]

[0070] The above-described embodiments merely represent preferred implementations of the present invention. While the descriptions thereof are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the scope of protection of the claims of the present invention.

Claims

1. Application of Chimonanthus chinensis leaf extract in preparing a drug for treating allergic rhinitis, characterized in that: The preparation method of the Chimonanthus chinensis leaf extract is as follows: Taking Chimonanthus chinensis leaves, adding 12-18 times the amount of water, soaking, reflux extraction, obtaining an extract, concentrating the extract under reduced pressure and then freeze-drying to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves was separated by macroporous resin, and then washed with 50% ethanol by volume, and the washing liquid was collected, concentrated under reduced pressure, and dried; or, Taking Chimonanthus chinensis leaves, adding 12-18 times the amount of water, soaking, reflux extraction, obtaining an extract, concentrating the extract under reduced pressure and then freeze-drying to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves was separated by macroporous resin and then washed with 70% by volume ethanol. The washing liquid was collected, concentrated under reduced pressure, and then dried.

2. The use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a medicament for treating allergic rhinitis, characterized in that: The Chimonanthus chinensis leaf extract includes flavonoids.

3. The use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a medicament for treating allergic rhinitis, characterized in that: The active ingredient of the medicine is Chimonanthus chinensis leaf extract, and the medicine also includes pharmaceutically acceptable excipients.

4. The use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a medicament for treating allergic rhinitis, characterized in that: The preparation of the medicine is one of oral preparation, injection, inhalation and nasal drops.

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