Application of chimonanthus nitens leaf extract in preparation of medicine for treating allergic rhinitis

Abstract

The invention discloses application of chimonanthus nitens leaf extract in preparation of medicine for treating allergic rhinitis, and relates to the field of traditional Chinese medicine, based on an allergic rhinitis animal model, influences of chimonanthus nitens leaves on AR rat behavioristics, nasal mucosa histomorphology and inflammatory factor levels in serum are investigated, the pharmacodynamic effect of chimonanthus nitens leaves in treatment of AR is defined, and the application of chimonanthus nitens leaf extract in preparation of medicine for treating allergic rhinitis is provided. Namely, the chimonanthus nitens leaves can obviously reduce the content of IgE, histamine, ICAM-1 and inflammatory factors in serum of rats with allergic rhinitis and can also obviously improve nasal mucosa symptoms of the rats with allergic rhinitis, the chimonanthus nitens leaf water extract is better in effect, meanwhile, different active component groups of the chimonanthus nitens leaves are prepared, effective parts of the chimonanthus nitens for treating allergic rhinitis are screened, and the application prospect is wide. Wherein the 70% ethanol elution group and the 50% ethanol elution group have the best effect, and the traditional Chinese medicine composition has the prospect of developing and preparing the medicine for treating the allergic rhinitis.

Description

Technical Field

[0001] The 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 refers to a chronic inflammatory disease of the nasal mucosa with paroxysmal sneezing, runny nose and nasal congestion as the main symptoms, which is mainly mediated by immunoglobulin E (IgE) and involves the body's immune active cells and cytokines after susceptible individuals come into contact with allergens.

[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 treat it. The main treatment methods are as follows: (1) Western medicine: oral or nasal spray antihistamines, mast cell membrane stabilizers, anti-leukotriene drugs, corticosteroids, etc. Oral or nasal spray of anti-allergic or hormone Western medicines are effective quickly and have good symptom control effects, but they are prone to relapse after discontinuation of medication, and repeated use can easily lead to drug resistance; (2) Acupuncture therapy: Select Yingxiang and other acupoints to open up the nasal passages, and classify them according to the patient's overall condition, and cooperate with 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 patients have poor compliance; (3) Chinese herbal decoction treatment: Syndrome differentiation and treatment based on the patient's overall symptoms can regulate the five internal organs, expel evil spirits, and fundamentally treat allergic rhinitis.

[0004] Modern pharmacological studies have shown that Chimonanthus chinensis has anti-inflammatory, analgesic, anti-inflammatory, antitussive, immune-regulating, antibacterial, and anti-oxidative pharmacological effects, and mainly contains volatile oils, flavonoids, alkaloids, and other chemical components. Currently, there is no report on the use of Chimonanthus chinensis leaf extract in the preparation of drugs 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 solution of the present invention is as follows: 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: Take Chimonanthus chinensis leaves, add 12-18 times the amount of water, soak, reflux extraction, obtain an extract, reduce pressure and concentrate the extract, and then freeze-dry to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves is then separated by macroporous resin, and then rinsed with 40%-80% ethanol by volume, the rinse solution is collected, and the rinse solution is concentrated under reduced pressure and then dried.

[0007] Preferably, taking Chimonanthus chinensis leaves, adding 12-18 times the amount of water, soaking, reflux extraction, obtaining an extract, decompressing and concentrating the extract, and freeze-drying the extract to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves was then separated by macroporous resin, and then rinsed with 70% by volume ethanol, the rinse liquid was collected, concentrated under reduced pressure, and then dried.

[0008] Preferably, taking Chimonanthus chinensis leaves, adding 12-18 times the amount of water, soaking, reflux extraction, obtaining an extract, decompressing and concentrating the extract, and freeze-drying the extract to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves was then separated by macroporous resin, and then rinsed with 50% by volume ethanol, the rinse liquid was collected, concentrated under reduced pressure, and then dried.

[0009] Preferably, the Chimonanthus chinensis leaf extract mainly contains flavonoids.

[0010] Preferably, the preparation method of the Chimonanthus chinensis leaf extract is as follows: Take Chimonanthus chinensis leaves, add 12-18 times the amount of water, soak, and extract volatile oil by steam distillation.

[0011] Preferably, the active ingredient of the drug is Chimonanthus chinensis leaf extract, and the drug also includes pharmaceutically acceptable excipients.

[0012] Preferably, the drug preparation is one of an oral preparation, an injection, an inhalant, and a nasal drop.

[0013] The beneficial effects of the present invention are: the Chimonanthus jasminoides leaves of the present invention can significantly reduce the contents 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 jasminoides leaf water extract has a better effect, and at the same time, by preparing different active component groups of Chimonanthus jasminoides leaves, the effective parts of Chimonanthus jasminoides 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

[0014] Figure 1 This is a diagram of the modeling and drug administration process of allergic rhinitis rats; Figure 2 The 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; Figure 3The 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; Figure 4 To study the effect of Chimonanthus chinensis leaf extract on nasal mucosal histopathology in rats with allergic rhinitis. 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; 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; Figure 7 To study the effects of different active parts of Chimonanthus chinensis leaves on the morphology of nasal mucosa in rats with allergic rhinitis. Among them, 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 elution group; SLM-M: medium-dose water elution group; SLM-H: high-dose water elution group; SLE-20%-L: low-dose 20% ethanol elution group, SLE-20%-M: medium-dose 20% ethanol elution group, SLE-20%-H: high-dose 20% ethanol elution 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; Figure 8 The total ion current graph of the effective parts of Chimonanthus chinensis leaves, wherein a represents the total ion current graph of the 50% alcohol elution group, b represents the total ion current graph of the 70% alcohol elution group, and c represents the total ion current graph of the volatile oil group. DETAILED DESCRIPTION

[0015] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0016] The technical solution of the present invention is further illustrated by specific experiments below.

[0017] Example 1 Pharmacodynamic study of Chimonanthus chinensis leaves in the treatment of allergic rhinitis Based on the allergic rhinitis (AR) animal model, the effect of Chimonanthus altissima leaves on the behavior, nasal mucosal tissue morphology, and serum inflammatory factor levels of AR rats was investigated to clarify the pharmacological effect of Chimonanthus altissima leaves in treating AR.

[0018] (1) Preparation of Chimonanthus chinensis leaf extract Take an appropriate amount of Chimonanthus wilt leaves, add 15 times the amount of water, soak for 1 hour, and reflux extraction twice, each time for 1 hour; the extract was concentrated under reduced pressure at 70°C and then freeze-dried, and the powder was collected for later use, namely Chimonanthus wilt leaf aqueous extract (SLMY).

[0019] (2) Animal grouping, modeling and drug administration 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 extracts of Chimonanthus altissima leaves). Specifically, the low, medium, and high doses of Chimonanthus altissima leaf water extract were 0.27 g / kg, 0.54 g / kg, and 1.09 g / kg, respectively. The ovalbumin (OVA)-induced AR model in rats was established by intraperitoneal injection sensitization and intranasal drip stimulation. The specific operation was as follows: on the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th days, once every other day for a total of 7 times, OVA 2 mg + Al(OH) 3 30 mg was mixed and dissolved with 1 mL of saline (OVA is ovalbumin) to prepare a milky white suspension. 3 It is insoluble in water. After standing for a while until the undissolved powder settles to the bottom, 1 mL of the prepared solution is drawn with a syringe and injected intraperitoneally. The normal group is injected with an equal dose of normal saline. On the second day after the completion of the systemic sensitization stage, that is, the 15th day of the experiment, 50 μL of OVA normal saline solution was drawn with a pipette and dripped into the nasal cavity, 50 μL on one side, and 100 μL in both nasal cavities for 7 consecutive days. On the 21st day, after the last drip into the rat's nasal cavity, the number of times the rat scratched its nose, the number of times it sneezed, and the range of symptoms of nasal discharge were recorded within 30 minutes, and quantitative scores were performed. The standard for model success was a total score > 5 points (Table 1). After the model was successfully established, each drug group was given oral gavage with various doses of drug intervention, once a day, for 7 consecutive days, and OVA was dripped into the nasal cavity every other day for maintenance (30 minutes after drug treatment). For details of the modeling and drug administration process, please refer to Figure 1 .

[0020] Table 1 Behavioral scoring criteria

[0021] (3) Sample collection and processing After the last administration, rats in each group were fasted but not watered for 12 hours, anesthetized, and blood was collected from the abdominal aorta. Whole blood was used for routine blood tests and blood cell smears; the remaining blood was allowed to stand at room temperature for 1 hour and then centrifuged at 4°C for 10 minutes at 5000r / min to collect serum and store at -20°C for later use. The rat nasal cavity was irrigated with precooled saline, and the irrigating fluid was collected and centrifuged at 4°C for 10 minutes at 1000r / min. The supernatant was aspirated and stored in a -20°C refrigerator for later use. The maxilla of the nose of each group of rats was separated, cut along the midline of the nose, and the nasal bone and maxilla were removed to expose the nasal septum and bilateral nasal mucosa. The nasal septum with bilateral nasal mucosa was clamped and placed in 4% paraformaldehyde and fixed at room temperature.

[0022] (4) Evaluation indicators and results 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 successfully established ( 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 group of Chimonanthus chinensis leaf water extract had the best effect ( P <0.05).

[0023] 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 were significantly increased ( P <0.05); compared with the model group, the levels of various indicators in the serum of rats in the high-dose group of Chimonanthus chinensis leaves 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.

[0024] Pathological observation: The nasal septum tissue was fixed in paraformaldehyde at 4°C for 24 hours, and then decalcified with EDTA. After reaching the decalcification endpoint, it was dehydrated, embedded in paraffin, sliced, and stained with conventional HE to observe pathological changes. The results showed that compared with the normal group, the structure of the nasal mucosal epithelial tissue in the model group was incomplete, and a large number of epithelial cells were shrinking and necrotic; the thickness of the nasal mucosal epithelium increased, goblet cells proliferated significantly, 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 rats in the high-dose group of Chimonanthus altissima leaf water 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.

[0025] Example 2 Screening of effective parts of Chimonanthus chinensis leaves for treating allergic rhinitis This experiment is based on the pharmacodynamic effects of Chimonanthus altissima leaves in treating allergic rhinitis. By preparing different active component groups from Chimonanthus altissima leaves, the effective parts of Chimonanthus altissima for treating allergic rhinitis were screened.

[0026] (1) Preparation of different active parts of Chimonanthus chinensis leaves Preparation of volatile oil and non-volatile extracts from Chimonanthus chinensis leaves: Take 10 kg of Chimonanthus chinensis leaves, add 15 times the amount of water, soak for 1 hour, and extract volatile oil by steam distillation. Then take an appropriate amount of Chimonanthus chinensis leaves, add 15 times the amount of water, soak for 1 hour, and reflux extract twice, each time for 1 hour; the extract is concentrated under reduced pressure at 70°C and freeze-dried, and the powder is collected for use, that is, dry extract powder.

[0027] (2) Isolation and purification of non-volatile extracts from Chimonanthus chinensis leaves Preparation of the upper column liquid: 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 upper column liquid is obtained.

[0028] Macroporous resin separation and purification process: Take an appropriate amount of drug solution and slowly add it to the top of the resin for adsorption overnight. Rinse with 5 times the volume of pure water, 20%, 50%, 70%, and 95% ethanol respectively, and collect each part of the washing liquid. After decompression concentration, dry to obtain powder.

[0029] (3) Effects of various components of Chimonanthus chinensis leaves on rats with allergic rhinitis 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 kinase (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.

[0030] 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 successfully established ( 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, the 50% ethanol elution group and the volatile oil group showed the most obvious decreases ( P <0.05).

[0031] 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 were significantly increased ( P <0.05); compared with the model group, the levels of IgE, HIS, ICAM-1, TNF-α and IL-4 in the serum of rats in the 70% ethanol elution group, 50% ethanol elution group and volatile oil group were significantly decreased ( P <0.05), the contents of each index in the other groups of rats only showed a decreasing trend, with no statistical difference. 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.

[0032] 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 of the rats in the normal group was basically normal, with a relatively regular arrangement, no obvious degeneration such as shedding, edema, necrosis, etc. in the mucosal epithelium, and no obvious inflammatory cell infiltration in the nasal septum tissue; the overall structure of the nasal mucosal tissue in the model group was abnormal, the mucosal epithelial cells were arranged in disorder, and blood vessels were obviously congested in the interstitial tissue, and a large number of inflammatory cells such as eosinophils were infiltrated; the nasal mucosa structure of the rats in the 70% ethanol elution group, 50% ethanol elution group and volatile oil group was relatively complete and tightly arranged, and no obvious degeneration such as shedding, edema, necrosis, etc. in the mucosal epithelium was observed, and no obvious inflammatory cell infiltration was observed in the tissue. 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.

[0033] Table 2 Analysis of nasal mucosal tissue morphology in rats with allergic rhinitis

[0034]

[0035] Example 3 Identification of the effective components of Chimonanthus chinensis leaves for treating allergic rhinitis Based on the above-mentioned allergic rhinitis animal model, the effective parts of Chimonanthus chinensis leaves for treating allergic rhinitis were screened as 50% and 70% ethanol elution parts and volatile oils. 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 Table 3-Table 5. The results show that the 50% and 70% ethanol elution parts are mostly flavonoid components, the components of the two are slightly different, but the proportions are quite different; volatile oils contain volatile terpenoid components, indicating that the components that exert the medicinal effects of volatile oils are mostly volatile terpenoid components.

[0036] Table 3 Identification of components of 50% and 70% alcohol elution groups of Chimonanthus chinensis leaves

[0037]

[0038] Table 4 Quantitative table of components of 50% and 70% alcohol elution groups of Chimonanthus chinensis leaves

[0039]

[0040] Table 5 Identification of volatile components of Chimonanthus chinensis leaves

[0041] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. The use of Chimonanthus chinensis leaf extract in the preparation of a drug for treating allergic rhinitis, characterized in that: The preparation method of the Chimonanthus chinensis leaf extract is as follows: Take Chimonanthus chinensis leaves, add 12-18 times the amount of water, soak, reflux extraction, obtain an extract, reduce pressure and concentrate the extract, and then freeze-dry to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves is then separated by macroporous resin, and then rinsed with 40%-80% ethanol by volume, the rinse liquid is collected, and the rinse liquid is concentrated under reduced pressure and then dried.

2. The use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a drug for treating allergic rhinitis, characterized in that: Take Chimonanthus chinensis leaves, add 12-18 times the amount of water, soak, reflux extraction, obtain an extract, reduce pressure and concentrate the extract, and then freeze-dry to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves was then separated by macroporous resin, and then rinsed with 70% by volume ethanol, the rinse liquid was collected, concentrated under reduced pressure, and then dried.

3. The use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a drug for treating allergic rhinitis, characterized in that: Take Chimonanthus chinensis leaves, add 12-18 times the amount of water, soak, reflux extraction, obtain an extract, reduce pressure and concentrate the extract, and then freeze-dry to obtain a Chimonanthus chinensis leaf water extract; The aqueous extract of Chimonanthus chinensis leaves was then separated by macroporous resin, and then rinsed with 50% by volume ethanol, the rinse liquid was collected, concentrated under reduced pressure, and then dried.

4. The use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a drug for treating allergic rhinitis, characterized in that: The Chimonanthus chinensis leaf extract includes flavonoid compounds.

5. The use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a drug for treating allergic rhinitis, characterized in that: The preparation method of the Chimonanthus chinensis leaf extract is as follows: Take Chimonanthus chinensis leaves, add 12-18 times the amount of water, soak, and extract volatile oil by steam distillation.

6. Use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a drug 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.

7. Use of the Chimonanthus chinensis leaf extract according to claim 1 in preparing a drug 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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