Nasal preparation for treating rhinitis as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and provides a nasal preparation for treating rhinitis and a preparation method and application thereof. Through screening of different extraction and purification steps of the traditional Chinese medicine composition, effective active components, especially volatile oil components, of flos magnoliae, rhizoma curcumae, fructus xanthii, radix rubiae, flos lonicerae, flos chrysanthemi indici, flos caryophylli and herba menthae are reasonably extracted, through the synergistic effect of the extracted effective components, the treatment effect of the traditional Chinese medicine composition can be achieved, and the treatment effect of the traditional Chinese medicine composition is improved. The compound is prepared into a pharmaceutical dosage form, especially a nasal preparation, the effect of treating nasal cavity inflammation and infectious diseases is remarkably improved, the medicine stability is good, the preparation process is simple and convenient, quality control is convenient, direct nasal cavity administration is adopted, and the compound has the advantages of being uniform in local administration, rapid in effect taking, long in drug effect and remarkable in curative effect, has the remarkable curative effect on nasal cavity inflammation, and does not have toxic or side effect. The compliance is good.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine preparations, and particularly relates to a nasal preparation for treating rhinitis and a preparation method and application thereof. Background Art

[0002] Rhinitis is an inflammation of the nasal mucosa caused by viruses, bacteria, fungi, allergens, and allergens. It may originate from various physical and chemical factors or certain systemic diseases. Common nasal diseases include: acute rhinitis, chronic rhinitis, sinusitis, allergic rhinitis, atrophic rhinitis, and vasomotor rhinitis. The main symptoms of rhinitis include nasal congestion, sneezing, runny nose, and nasal itching. The main pathological changes are congestion, swelling, exudation, hyperplasia, atrophy, or necrosis of the nasal mucosa. As a common disease, nasal inflammation brings many inconveniences to people. For example, the incidence of chronic rhinitis and sinusitis in people over 40 years old in my country is about 10%. Although it will not threaten the life safety of patients, its easy recurrence will also cause the patient to experience a decline in olfactory function as the disease progresses, which will have a serious negative impact on the patient's daily quality of life. For example, allergic rhinitis is a chronic respiratory disease, an allergic disease to inhaled allergens, characterized by nasal itching, paroxysmal sneezing, hypersecretion and nasal congestion. Its onset is affected by both environmental and genetic factors. Allergic rhinitis is becoming increasingly prevalent around the world, and its impact is expanding. Allergic rhinitis, such as sneezing, runny nose, nasal congestion, and nasal and eye itching, may seem like mild symptoms, but they seriously affect people's sleep and normal life.

[0003] At present, surgical treatment of chronic nasal inflammation mainly includes surgical operation and sinus surgery under nasal endoscopy. In clinical practice, although surgical treatment can quickly remove inflammatory tissue and promote sinus ventilation, the invasiveness of surgical intervention and the fact that patients still have recurrent attacks after surgery, and repeated surgical treatments can cause further harm to patients, so clinically, conservative drug treatment is often recommended for CRS patients. The main drugs used in Western medicine to treat nasal inflammation diseases include antihistamines, glucocorticoids, decongestants, etc. These drugs can significantly relieve the symptoms of nasal inflammation diseases in the short term, but they cannot cure them. Moreover, due to the characteristics of recurrent nasal inflammation diseases, long-term medication is required, which will cause greater side effects to patients.

[0004] Applying the core idea of ​​holistic concept and syndrome differentiation in TCM theory to treat nasal inflammation can not only inhibit local allergic reaction and control the symptoms of rhinitis at all stages, but also effectively restore the body's immune balance and prevent recurrence, and the clinical efficacy of treating rhinitis is significant. In TCM theory, nasal inflammation is mostly caused by exogenous pathogens such as wind-cold or foreign air, so it is often treated by attacking the pathogens externally and replenishing the deficiency internally. In addition, the volatile oil components of traditional Chinese medicine have the advantages of small toxic and side effects, strong effect, and low recurrence rate. In the prior art, the volatile oil components of traditional Chinese medicine have been widely used in the treatment of nasal inflammation.

[0005] Chinese invention patent application CN114425058B discloses a Chinese medicine composition for treating rhinitis. The Chinese medicine composition provided by the present invention comprises the following components by weight: 18-36 parts of white ox gall, 15-30 parts of wild chrysanthemum, 10-20 parts of cassia twig, 9-18 parts of goose grass, and 3-6 parts of asarum. The medicine and preparation prepared by the Chinese medicine composition have fast onset, small volume, convenient use, no side effects, and effectively relieve the symptoms of rhinitis patients, and are suitable for treating various rhinitis. Although the raw material ingredients of the invention are simple, according to its preparation method, the invention ignores the volatile oil components that have strong antagonistic effects on bacteria, fungi and viruses that cause rhinitis, that is, the invention does not give full play to the active ingredients of each medicine, resulting in a low drug utilization rate.

[0006] Chinese invention patent application CN102048860B discloses a soft capsule pharmaceutical preparation for nasal sinusitis and its preparation method, which is a soft capsule preparation made by using the ratio of 672g of Chinese medicine Xanthium sibiricum, 126g of Magnolia officinalis, 42g of Rubia cordifolia, 42g of Chrysanthemum indici, and 42g of Honeysuckle as raw materials, adding a certain proportion of vegetable oil, soybean lecithin and beeswax after extraction, and pressing. In addition, the currently marketed varieties for treating "nasal sinusitis" include Biyuan Tablets, Biyuan Pills and Biyuan Syrup (see "Chinese Medicine Formula Preparations (Volume 6) of the Ministry of Health of the People's Republic of China"), wherein the prescription of "Biyuan Tablets" in "Chinese Medicine Formula Preparations (Volume 6) of the Ministry of Health of the People's Republic of China" is "672g of Xanthium sibiricum, 126g of Magnolia officinalis, 42g of Honeysuckle, 42g of Rubia cordifolia, 42g of Chrysanthemum indici, made into 1000 tablets, 6 to 8 tablets are taken each time". It can be seen that the amount of Xanthium sibiricum contained in the Biyuan soft capsule or Biyuan tablet accounts for 72.7%, and the amount of 6 to 8 tablets taken at a time is relatively large. However, because Xanthium sibiricum is hepatotoxic, a large dosage of Xanthium sibiricum is prohibited for use in pregnant and lactating women, and those with liver and kidney dysfunction.

[0007] Through searching and analyzing the Chinese medicine for rhinitis treatment that has been marketed, prescriptions containing Xanthium sibiricum or Magnolia officinalis account for more than 60%, which shows the importance of these two herbs for the treatment of rhinitis. Most prescriptions reuse Xanthium sibiricum, and clinical findings show that long-term oral administration is prone to liver and kidney toxicity. On the basis of the above Chinese medicine prescriptions, the object of the present invention is to provide a method for extracting and preparing a Chinese medicine composition with a simple process, and through in vitro tests and in vivo efficacy tests, the extraction process, preparation process and administration method are optimized, and the active ingredients in the original medicinal materials, especially the volatile oil components, are fully enriched, and the synergistic effect between the effective active ingredients of each drug is effectively exerted, while ensuring the efficacy and safety, the utilization rate of the medicinal materials is improved, the toxicity is reduced, the effect is increased, the cost is reduced, the stability of the drug is improved, and the compliance of the patient during use is improved, and the clinical value is significantly improved. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a nasal preparation of a traditional Chinese medicine composition for treating nasal inflammation and infectious diseases and a preparation method thereof. The effective active ingredients of volatile oil are extracted by water distillation to prepare a pharmaceutical dosage form. The traditional Chinese medicine composition obtained by the preparation method has high bioavailability, significant efficacy on nasal inflammation, and good compliance.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The invention provides a nasal preparation of a traditional Chinese medicine composition for treating nasal inflammatory diseases. The traditional Chinese medicine composition is prepared from raw materials of Xanthium sibiricum, Magnolia officinalis, Curcuma zedoaria, Rubia cordifolia, Honeysuckle, Wild chrysanthemum, Clove and Mint.

[0011] The magnolia includes but is not limited to magnolia, stir-fried magnolia, stewed magnolia, and honey magnolia. The zedoaria includes but is not limited to zedoaria, vinegar zedoaria, and turmeric. The Xanthium includes but is not limited to Xanthium and stir-fried Xanthium. The madder includes but is not limited to madder. The honeysuckle includes but is not limited to honeysuckle, honeysuckle, and honeysuckle vine. The wild chrysanthemum includes but is not limited to chrysanthemum and wild chrysanthemum. The clove includes but is not limited to clove and mother clove. The mint includes but is not limited to gray mint, round-leaved mint, and green mint.

[0012] A nasal preparation for treating nasal inflammatory diseases, the nasal preparation being composed of the following raw materials in parts by weight: 5-20 parts of magnolia, 5-15 parts of zedoaria, 5-10 parts of xanthium sibiricum, 5-10 parts of madder, 5-10 parts of honeysuckle, 5-10 parts of wild chrysanthemum, 3-8 parts of cloves and 2-5 parts of mint.

[0013] Furthermore, the preparation method of the nasal preparation is: weighing the prescribed amount of the whole formula of medicinal raw materials, adding water for distillation, and collecting volatile oil.

[0014] Furthermore, the nasal preparation also includes one or more pharmaceutically acceptable adjuvants and / or excipients.

[0015] Furthermore, the nasal preparation includes nasal drops, nasal wash, spray or nasal powder aerosol.

[0016] The invention provides a method for preparing a nasal preparation of a Chinese medicine composition for treating nasal inflammatory diseases, comprising the following steps: weighing a prescribed amount of whole-formula medicinal raw materials, adding water for distillation, and collecting volatile oil.

[0017] Furthermore, the solid-liquid ratio of water is 1:3-10 (W / V); the distillation time is 2 to 8 hours; and the number of distillation extractions is 1 to 3 times.

[0018] Furthermore, the preparation method also includes adding one or more pharmaceutically acceptable auxiliary materials and / or excipients to prepare any pharmaceutical preparation.

[0019] Furthermore, the auxiliary materials include but are not limited to solubilizers, antibacterial agents, etc.

[0020] Furthermore, water, Tween, propylene glycol and benzalkonium bromide are added to the pharmaceutical composition to prepare a nasal spray.

[0021] The present invention also provides application of the nasal preparation obtained by the preparation method in preparing medicines for treating nasal inflammatory diseases.

[0022] Furthermore, the nasal inflammatory diseases include acute rhinitis, chronic rhinitis, sinusitis, allergic rhinitis, atrophic rhinitis, and vasomotor rhinitis.

[0023] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0024] (1) The nasal preparation of the present invention is prepared from Chinese medicinal materials with the effects of dispelling wind and cold, clearing nasal passages, clearing away heat and detoxifying, tonifying the spleen and kidney, and promoting qi and blood circulation. It has the effects of dispelling wind and cold, promoting qi and clearing the passages, clearing heat and relieving itching. The prescription conforms to the theory of traditional Chinese medicine for treating rhinitis, and while solving the symptoms of the acute phase, it considers tonifying spleen qi and benefiting kidney yang, thereby benefiting lung qi, increasing positive energy, and improving immunity. Not only is the conception unique, there is no obvious toxic and side effect, and the efficacy is remarkable, achieving the effect of treating both the symptoms and the root cause, reducing toxicity and increasing efficacy, which helps to shorten the patient's treatment cycle and reduce the recurrence rate.

[0025] (2) The present invention fully extracts the effective active ingredients and components of each medicinal material through the optimization of different extraction steps. The synergistic coordination between the effective ingredients after extraction, especially the volatile oil components, helps to fully exert the therapeutic effect of the traditional Chinese medicine composition, significantly improves the effect of treating nasal inflammation and infectious diseases, and improves the utilization rate of the drug while ensuring the efficacy.

[0026] (3) The volatile oil and volatile oil + water-extracted alcohol precipitate of the present invention have comparable effects on treating nasal inflammation in vivo and in vitro. However, the nasal spray containing water-extracted alcohol precipitate + volatile oil components has obvious precipitation or suspended matter in the drug solution after storage, and the product color is darker, and the color residue is obvious after use by patients. The color of the nasal spray containing only volatile oil components tends to be clear. Considering the adaptability and compliance of patients, as well as the stability of drug activity after storage, the present invention selects the process of water-extracting volatile oil, which has a simple extraction and preparation process, is convenient for quality control, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is the pathological section of the nasal mucosa tissue of the normal control group of Experiment 3 (HE 10×, purulent secretions and bleeding can be seen in the nasal cavity at T2);

[0028] Figure 2 This is the pathological section of the nasal mucosa tissue of the model control group of Experiment 3 (HE 10×, T2 local structural disorder of the nasal mucosa, focal mucosal epithelial cell shedding, submucosal eosinophil infiltration, tissue edema, cilia loss, goblet cell hyperplasia);

[0029] Figure 3 This is the pathological section of the nasal mucosa tissue of the positive control group of test case 3 (HE 10×, T2 focal nasal mucosal ciliary loss, tissue edema, and slight eosinophil infiltration under the mucosa);

[0030] Figure 4 This is the pathological section of the nasal mucosa tissue of the high-dose group of the test substance 1 in Test Example 3 (HE 10×, T2 focal nasal mucosal ciliary loss, goblet cell hyperplasia, and slight eosinophil infiltration under the mucosa);

[0031] Figure 5 This is the pathological section of the nasal mucosa tissue of the low-dose group of test substance 2 in test example 3 (HE 20×, T2 focal loss of nasal mucosal cilia, slight eosinophil infiltration under the mucosa);

[0032] Figure 6 These are stability test pictures of Test Example 4, A is the nasal spray of Example 4 stored at 4°C, B is the nasal spray of Comparative Example 4 stored at 4°C, and C is the nasal spray of Comparative Example 4 stored at 40°C. DETAILED DESCRIPTION

[0033] The present invention is described below by way of examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the medicinal materials and reagents described can be obtained from commercial channels unless otherwise specified; and the performance of products from different sources does not have a significant impact.

[0034] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0035] Unless otherwise specified, the various chemical reagents and raw materials used in the embodiments of the present invention are obtained through conventional commercial channels, and the products of different manufacturers have no significant effect on the effect. In addition, fluticasone propionate nasal spray (Fushuliang), specification 120 sprays, manufacturer is GlaxoSmithKline Group, import drug registration certificate number H20091100; compound fluticasone acetate propionate cream, specification 10g / box, manufacturer is Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd., national medicine standard H44024374.

[0036] Basic Example

[0037] A traditional Chinese medicine composition for treating nasal inflammatory diseases comprises the following components in parts by weight: 5-20 parts of magnolia, 5-15 parts of zedoaria, 5-10 parts of xanthium sibiricum, 5-10 parts of madder, 5-10 parts of honeysuckle, 5-10 parts of wild chrysanthemum, 3-8 parts of cloves and 2-5 parts of mint.

[0038] The raw materials and dosage of the Chinese medicine compositions of the embodiments and comparative examples are shown in Table 1.

[0039] Table 1 Prescription composition of examples and comparative examples

[0040]

[0041]

[0042] The Chinese medicine composition of Example 1 is prepared by the following method (volatile oil):

[0043] Weigh the prescribed amount of all the medicinal ingredients, add 3 times the amount of water, distill for 2 hours, and collect the volatile oil.

[0044] The Chinese medicine composition of Example 2 is prepared as follows (volatile oil):

[0045] Weigh the prescribed amount of the whole formula's medicinal ingredients, add 7 times the amount of water, distill for 4 hours, and collect the volatile oil.

[0046] The Chinese medicine composition of Example 3 is prepared as follows (volatile oil):

[0047] Weigh the prescribed amount of the whole formula's medicinal ingredients, add 10 times the amount of water, distill for 6 hours, and collect the volatile oil.

[0048] The nasal spray of Example 4 is prepared as follows:

[0049] 2 parts of the volatile oil of Example 1 were added to 5 parts of water, 3 parts of Tween, 2 parts of propylene glycol and 1 part of benzalkonium bromide to prepare a nasal spray.

[0050] The preparation method of the Chinese medicine composition of Comparative Example 1 is as follows (volatile oil + water extraction and alcohol precipitation):

[0051] S1. Weigh the whole prescription medicinal raw materials, add 5 times the amount of water and distill for 2 hours, collect the volatile oil and the extract after distillation and the medicinal residue;

[0052] S2, adding 15 times the amount of water to the medicinal residue after distillation in step S1 and distilling and extracting for 3 hours, extracting 3 times, and collecting the extract;

[0053] S3, combining the extracts in step S1 and S2, and concentrating under reduced pressure to a water extract concentrate with a relative density of 1.2;

[0054] S4, adding ethanol until the ethanol content of the water-extracted concentrate is 60%, letting it stand, taking the supernatant and concentrating it to obtain a supernatant concentrated paste with a relative density of 1.2;

[0055] S5. Add the supernatant concentrated paste in S4 to the volatile oil in step S1 and mix well to obtain the Chinese medicine composition.

[0056] The Chinese medicine composition of Comparative Example 2 is prepared by the following method (water extraction and alcohol precipitation):

[0057] S1. Weigh the whole prescription medicinal raw materials in the prescribed amount, add 7 times the amount of water and distill for 4 hours, and collect the extract and medicinal residue after distillation;

[0058] S2, adding 12 times the amount of water to the medicinal residue after distillation in step S1 and distilling and extracting for 2 hours, extracting twice, and collecting the extract;

[0059] S3, combining the extracts in step S1 and S2, and concentrating under reduced pressure to a water-extracted concentrated extract with a relative density of 1.30;

[0060] S4, adding ethanol until the ethanol content of the water-extracted concentrated solution is 60%, letting it stand, taking the supernatant and concentrating it to obtain a supernatant concentrated paste with a relative density of 1.30, that is, the Chinese medicine composition.

[0061] The Chinese medicine composition of Comparative Example 3 is prepared by the following method (alcohol extraction and water precipitation):

[0062] S1. Weigh the whole amount of the medicinal raw materials in the prescription, add 3 times the amount of 60% ethanol, and extract for 6 hours to obtain an ethanol extract;

[0063] S2, adding an equal amount of water to the ethanol extract in step S1, stirring evenly, leaving it to stand overnight at room temperature, filtering to remove the precipitate, and collecting the filtrate;

[0064] S3. The filtrate in step S2 is concentrated under reduced pressure to a concentrated extract with a relative density of 1.30 to obtain the Chinese medicine composition.

[0065] The nasal spray of Comparative Example 4 is prepared as follows:

[0066] 2 parts of the traditional Chinese medicine composition of Comparative Example 1 were added with 5 parts of water, 3 parts of Tween, 2 parts of propylene glycol and 1 part of benzalkonium bromide to prepare a nasal spray.

[0067] The nasal spray of Comparative Example 5 is prepared as follows:

[0068] 2 parts of the traditional Chinese medicine composition of Comparative Example 2 were added with 5 parts of water, 3 parts of Tween, 2 parts of propylene glycol and 1 part of benzalkonium bromide to prepare a nasal spray.

[0069] The nasal spray of Comparative Example 6 is prepared as follows:

[0070] 2 parts of the traditional Chinese medicine composition of Comparative Example 3 were added with 5 parts of water, 3 parts of Tween, 2 parts of propylene glycol and 1 part of benzalkonium bromide to prepare a nasal spray.

[0071] The Chinese medicine composition of Comparative Example 7 is prepared by the following method (volatile oil):

[0072] Weigh the prescribed amount of all the medicinal ingredients (excluding Curcuma zedoaria, cloves, and mint), add 10 times the amount of water, distill for 6 hours, and collect the volatile oil.

[0073] Test Example 1: In vitro test evaluation

[0074] 1. Evaluation of in vitro anti-allergic and anti-inflammatory tests

[0075] (1) Test materials

[0076] Mouse macrophages (RAW264.7), fluticasone propionate, lipopolysaccharide (LPS), trypsin, FBS, DMEM medium, ELISA kit, cell culture flask, etc.

[0077] (2) Experimental groups, dosage, and experimental sequence

[0078] After the mouse macrophages were expanded to a certain number, cells with good growth status in the logarithmic growth phase were selected and digested with trypsin to form a cell suspension. The density was adjusted to 2×10 5 The cells were randomly divided into 10 groups, namely test group 1 to group 7, blank control group, positive control group and model control group, with 3 replicates in each group. The grouping and dosage are shown in Table 2.

[0079] Table 2 Grouping and dosage table of in vitro anti-inflammatory test

[0080]

[0081]

[0082] (3) Test steps

[0083] After the cells of each group were inoculated into 12-well plates, they were cultured at 37°C and 5% CO2 and reattached to the plate. Except for the blank control group, the other model groups were intervened with 0.5 mg / L LPS for 6 hours. After 6 hours of intervention, each test substance was added for 12 hours. The cell supernatant was collected and tested according to the operating instructions of the IL-6 (interleukin-6) and TNF-α (tumor necrosis factor) kits.

[0084] (4) Test results

[0085] Table 3 In vitro anti-inflammatory test results

[0086]

[0087] Note: Compared with the model control group *P<0.05, **P<0.01; compared with the positive control group # P<0.05, ## P<0.01.

[0088] From the data in Table 3, it can be seen that: Examples 1-3 and Comparative Examples 1-3 and 7 all have anti-allergic and anti-inflammatory effects, and the in vitro anti-inflammatory effects of the volatile oils of Examples 1 to 3 and the volatile oils of Comparative Example 1 + water extraction and alcohol precipitation are better, and there is a significant difference compared with the model control group (p<0.01). In addition, the anti-inflammatory effect of test substance 7 is poor in the absence of Curcuma, cloves and mint, indicating that the prescription of the pharmaceutical composition of the present invention has a significant effect on the anti-inflammatory effect; from the results of test substances 5-6, it can be seen that the single water extraction and alcohol precipitation or alcohol precipitation water extract is less effective than the combined active ingredients of test substances 1-3, indicating that the volatile oil component in the Chinese medicine composition of the present invention has a significant anti-inflammatory effect, that is, it shows that the preparation and extraction process of the present invention has a significant effect on the anti-inflammatory effect of the final Chinese medicine composition.

[0089] 2. In vitro antibacterial test evaluation

[0090] (1) Test materials

[0091] Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger, and corresponding culture media.

[0092] (2) Test groups and test samples

[0093] The groups and test samples for in vitro antibacterial test are shown in Table 4.

[0094] Table 4 In vitro antibacterial test grouping and test samples

[0095]

[0096] (3) Test steps

[0097] The antibacterial activity of the test substance was detected by the microdilution method. The test substance with an initial concentration of 4 mg / ml was diluted to concentrations of 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL according to the serial dilution method. After co-culturing with microorganisms, it was observed under daylight. The last clear well in the co-culture well plate was judged as the MIC (minimum inhibitory concentration) by naked eyes; for the co-culture after detecting the MIC, 50 μl of the clarified culture solution was taken into a sterile petri dish after the culture ended, and the MBC (minimum bactericidal concentration) of the test substance was detected by the plate method.

[0098] (4) Test results

[0099] Table 5 Antibacterial test results (concentration unit: μg / mL)

[0100]

[0101] It can be seen from the results in Table 5 that they are basically consistent with the results of the in vitro anti-inflammatory test in Table 3. Examples 1-3 and Comparative Examples 1-3, 7 all have antibacterial and bactericidal effects, and the in vitro antibacterial and bactericidal effects of Examples 1-3 and Comparative Example 1 are relatively good. They still have certain antibacterial and bactericidal effects when diluted to a concentration of 15.625 μg / mL, which is significantly better than Test Substances 5-7, indicating that the traditional Chinese medicine prescription and the preparation process of the active substance of the present invention have a great influence on the drug activity.

[0102] Test Example 2. In vivo test evaluation

[0103] 1. Anti-inflammatory test on mice

[0104] (1) Animals: Kunming mice, a total of 60, half male and half female, 3-4 weeks old, 16.6-24.5 g, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd., and the experimental animal production license number is: SCXK(Yue)2022-0063, quarantined for 4 days.

[0105] (2) Grouping: The Kunming mice were randomly divided into 6 groups, with 10 in each group, half male and half female, namely the model control group (normal saline), the positive control group (Compound Fluticasone Propionate Cream), and Test Substance 1-4 groups. The grouping and dosage are shown in Table 6.

[0106] Table 6 Grouping and dosing table for in vivo anti-inflammatory test

[0107]

[0108] (3)Modeling method and detection method: Xylene-induced ear swelling model in mice. Xylene was evenly applied to both the inner and outer surfaces of the right auricle of each animal for 30 minutes to induce inflammation, and the left ear was not treated. After 30 minutes of inflammation induction, 0.1 mL of the test substance sample was applied to each animal in each group. After 4 hours of drug administration, both auricles were cut off. The test sample on the right auricle was washed off with physiological saline, wiped clean, the two auricles were overlapped, and two round ear pieces were punched out with a puncher and weighed separately to calculate the swelling value (the weight difference between the right ear piece and the left ear).

[0109] (4)Test results

[0110] Table 7 Results of anti-inflammatory test in vivo

[0111]

[0112] Note: Compared with the model control group, *P < 0.05, **P < 0.01.

[0113] From the results of the anti-inflammatory test in vivo in Table 7, it can be seen that test substances 1-4 all have anti-inflammatory effects in vivo to a certain extent. The anti-inflammatory effects of test substances 1-2 in vivo are comparable to those of the positive control group, and their swelling inhibition rates are both around 50%, and there are significant differences in the swelling degree compared with the model control group. In addition, for test substance 3 without volatile oil components, its swelling inhibition effect is lower than that of test substances 1-2.

[0114] 2. Allergic rhinitis experiment

[0115] (1) Animals: SPF-grade SD rats, a total of 56, half male and half female, 6-8 weeks old, weighing 175-220 g, purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. The production license number of experimental animals is: SCXK(Yue)2022-0063, and the certificate numbers are: male, 44829700034077; female, 44829700034078. After 3 days of quarantine, they were used after no abnormalities were found.

[0116] (2) Grouping: A total of 56 SPF-grade SD rats were randomly divided into 7 groups, with 8 rats in each group, namely the blank group, the model control group (physiological saline), the positive drug control group, and the test substance 1-5 groups. The grouping and dosing doses are shown in Table 8.

[0117] Table 8 Grouping and dosing doses for allergic rhinitis

[0118]

[0119]

[0120] (3) Modeling and drug administration:

[0121] Sensitization: On the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th days, the sensitization solution (10 mg / ml OVA-Al(OH)3 adjuvant solution) was intraperitoneally injected, 0.2 ml / mouse, once every other day, for a total of 7 times. Challenge: Starting from the 15th day, 10% OVA solution was administered bilaterally by nasal drops, 50 μl on each side, once a day, for 7 consecutive days. The normal group was challenged with bilateral nasal drops of normal saline. After the challenge, the rats were observed for 15 minutes, and the symptoms of allergic rhinitis in the rats were scored from three dimensions: the number of sneezes, the number of nose scratching, and the number of nasal discharge. The scoring criteria are shown in Table 9. The total score of the rat symptom score ≥ 5 points was considered a successful model.

[0122] Administration: 30 minutes before nasal drops to stimulate rhinitis, all nasal spray groups were administered bilaterally, and oral administration was by gavage, with a dosing volume of 50ul per side, once a day for 7 consecutive days.

[0123] (4) Behavioral index detection: 30 minutes after the last administration, the number of nose scratching, sneezing and runny nose was recorded and scored according to the scoring criteria in Table 9;

[0124] Table 9 Scoring criteria

[0125]

[0126] (5) Behavioral Observation Results

[0127] Table 10 Behavioral observation results

[0128]

[0129]

[0130] Note: Compared with the model control group*, P<0.05; **, P<0.01.

[0131] According to the results of Table 10, test substances 1 and 3 can significantly improve the symptoms of allergic rhinitis in the behavioral score, and the therapeutic effect is equivalent to that of the positive drug, and there is a significant difference relative to the model control group. It can be seen from test substances 2-3 that the effect of oral administration of the drug is worse than that of direct nasal spray, indicating that nasal spray can significantly improve the bioavailability of the drug. In addition, Comparative Example 3 shows that the volatile oil and prescription extraction preparation process of the present invention have a significant effect on the effect of treating rhinitis.

[0132] Test Example 3: Drug Effectiveness Evaluation Test

[0133] 1. Animals: SPF SD rats, 6-8 weeks old, weighing 175-220 g, half male and half female, purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd., production license number SCXK (Guangdong) 2022-0063, certificate numbers are: male, 44829700034077; female, 44829700034078, quarantined for 3 days and used after no abnormalities were found.

[0134] 2. Grouping: The animals that passed the quarantine were sorted by weight and randomly divided into a normal control group and a modeling group, half of which were male and half were female. There were 8 animals in the normal control group, and the rest of the animals were modeled for allergic rhinitis. The animals that successfully passed the modeling were sorted by rhinitis scores and randomly divided into 8 groups, 8 animals in each group, half of which were male and half were female. The grouping and dosing information are detailed in Table 11.

[0135] Table 11 Trial groups and dosing schedule

[0136]

[0137] 3. Modeling and Drug Administration

[0138] Modeling: On the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, 19th and 21st days, the sensitizing solution (10mg / mlOVA-Al(OH)3 adjuvant solution) was injected intraperitoneally, 0.2ml / rat, once every other day, for a total of 7 times. Challenge: Starting from the 15th day, 10% OVA solution was administered bilaterally by nasal drops, 50μl on each side, once a day, for 14 consecutive days. The normal group was challenged with bilateral nasal drops of normal saline. After the challenge, the animals were observed for 15 minutes, and the symptoms of allergic rhinitis in rats were scored from three dimensions: the number of sneezes, the number of nose scratching, and the number of nasal discharge. The scoring criteria are shown in Table 9. The total score of the rat symptom score ≥5 points was considered a successful modeling.

[0139] Drug administration: After successful modeling, all groups were given bilateral nasal spray 30 minutes before nasal drops to stimulate rhinitis, with a dosing volume of 50ul per side, once a day for 7 consecutive days.

[0140] 4.Indicator detection

[0141] 4.1 Behavioral scoring: After each stimulation, the animals were observed for 15 min, and the number of sneezes, the number of nose scratching, and the condition of nasal discharge were counted. The symptoms of allergic rhinitis in rats were scored according to Table 9.

[0142] 4.2 Biochemical test: The animals were anesthetized by intramuscular injection of 0.33 ml / kg of a mixture of Shutai 50 and Suminxin II injection (10:1). Blood samples were collected by abdominal aorta blood sampling. The collected blood was centrifuged to obtain serum, and then IgE test was performed.

[0143] 4.3 Histopathological examination: The skin on the surface of the maxillary bone, the mandibular bone, and the brain were removed, and the animal's head was cut off from the cervical vertebrae. The entire head was fixed in 10% formalin solution, sectioned in paraffin, stained with hematoxylin-eosin, and observed for pathological sections of the nasal mucosa.

[0144] 5. Data processing: All data are expressed as mean ± variance, and SPSS28.0 was used for data analysis. The independent sample t test was used for inter-group comparison of the modeling period data. The one-way analysis of variance was used for the drug administration period data. When the variances were equal, the LSD test was used for inter-group comparison, and when the variances were unequal, the Dunnett's T3 test was used for inter-group comparison. When the mean difference between the normal control group and the model control group was large, the independent sample t test was used for inter-group comparison. P<0.05 was considered statistically significant.

[0145] 6. Test results

[0146] 6.1 Modeling evaluation

[0147] Table 12 Effect of modeling agents on rhinitis scores (x±SD, n=8, before administration)

[0148]

[0149]

[0150] Note: vs normal control group, ##, P<0.01.

[0151] From the results in Table 12, it can be seen that compared with the normal control group, the rhinitis scores of the model control group were significantly increased from T15 to T21 (P<0.01); at T21, the mean rhinitis scores of the model control group and each drug group were ≥5, indicating that all the allergic rhinitis model animals have been successfully modeled and can be used for drug treatment trials.

[0152] 6.2 Behavioral Scoring

[0153] As shown in Table 13, compared with the normal control group, the rhinitis scores of the model control group D1-D7 were significantly increased (P<0.01). The comprehensive comparison of the mean rhinitis scores among the drug groups was as follows from low to high: positive control group < test substance 1 high-dose group, test substance 2 high-dose group < test substance 1 low-dose group < test substance 2 low-dose group, showing an obvious positive relationship between doses.

[0154] The results showed that at the same dose, when different test substances were compared, the improvement effects of test substance group 1 and test substance group 2 on rhinitis scores were comparable, and the high-dose test substance group and the positive drug fluticasone propionate nasal spray had a significant effect on improving rhinitis scores.

[0155] Table 13 Effect of the test substance on rhinitis score (x±SD, n=8, after administration)

[0156]

[0157]

[0158] Note: vs normal control group, ##, P < 0.01; vs model control group, *, P < 0.05; **, P < 0.01.

[0159] 6.3 Biochemical testing

[0160] As shown in Table 14, compared with the normal control group, the IgE level of the model control group was significantly increased (P<0.01). Compared with the model control group, the IgE levels of each drug group were reduced, among which the positive control group, the high-dose group of test substance 2, and the low- and high-dose groups of test substance 1 were statistically different (P<0.05 or 0.01). The mean values ​​of IgE among the drug groups were compared from low to high: high-dose group of test substance 1 < high-dose group of test substance 2 < low-dose group of test substance 1, positive control group < low-dose group of test substance 2, showing an obvious positive correlation between doses.

[0161] The results showed that at the same dose, the improvement effects of different test substances on IgE levels were as follows: test substance group 1 > test substance group 2; and at high doses, the test substances were superior to the positive drug fluticasone propionate nasal spray.

[0162] Table 14 Effect on IgE (x±SD, n=8)

[0163]

[0164] Note: vs normal control group, ##, P < 0.01; vs model control group, *, P < 0.05; **, P < 0.01.

[0165] 6.4 Histopathology

[0166] As shown in Table 15, compared with the normal control group, the total score of nasal mucosal lesions in the model control group was significantly increased (P<0.01); compared with the model control group, the total score of nasal mucosal lesions in the positive control group and each drug group was significantly reduced (P<0.05 or 0.01). The mean total score of nasal mucosal lesions among the drug groups was compared from low to high: high-dose group of test substance 1 < high-dose group of test substance 2 < low-dose group of test substance 1 < low-dose group of test substance 2 < positive control group, showing an obvious positive relationship between doses.

[0167] Table 15 Pathological index score total score table ( n=8)

[0168]

[0169] Note: vs normal control group, ##, P < 0.01; vs model control group, **, P < 0.01; *, P < 0.05.

[0170] The results showed that at the same dose, the improvement effects of different test substances on nasal mucosal lesions were as follows: test substance group 1 > test substance group 2; both test substance groups 1 and 2 were superior to the positive drug fluticasone propionate nasal spray.

[0171] Based on the above test results, the active drugs in Example 1 (volatile oil) of the present invention and Comparative Example 1 (water extraction and alcohol precipitation + volatile oil) can improve rhinitis scores, IgE levels and the total score of nasal mucosal lesions, and have significant effects on the treatment of nasal inflammation and infectious diseases. Therefore, in order to investigate the adaptability and compliance of patients during use, the appearance and stability of the active substances extracted by different processes after being prepared into nasal sprays were further investigated.

[0172] Test Example 4: Stability Study

[0173] Test object: nasal spray prepared in Example 4 and Comparative Example 4.

[0174] Determination method: Place the above nasal spray liquid in a colorless transparent container and seal it. Place it at 4°C, room temperature and 40°C for 10 days respectively, and observe the changes in the appearance, properties and color of the liquid.

[0175] result:

[0176] Table 16 Nasal spray stability test results

[0177]

[0178]

[0179] From Table 16 and Figure 6 The results (A is the nasal spray of Example 4 stored at 4°C, B is the nasal spray of Comparative Example 4 stored at 4°C, and C is the nasal spray of Comparative Example 4 stored at 40°C) show that the color of the nasal spray of Example 4 is lighter, and there is no obvious change in properties after being placed under refrigeration, room temperature and 40°C for 10 days, no discoloration or generation of suspended matter, precipitation, and clarity, and its appearance and properties are stable. The color of the nasal spray of Comparative Example 4 is darker, and precipitation or suspended matter can be seen under refrigeration and 40°C conditions, and obvious solids can be seen hanging on the wall after the container is inverted.

[0180] Based on the above, in the case where both the nasal spray of Example 4 and the nasal spray of Comparative Example 4 have volatile oil components and have a certain effect in preventing and treating rhinitis, considering the stability of the drug during the later storage of the product, and the fact that the color is too dark, which will lead to color residue during actual use, thereby reducing the patient's compliance with use, the nasal spray of Example 4 is preferred in the present invention, that is, a nasal preparation made by extracting the volatile oil components of the drug prescription.

[0181] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A nasal preparation for treating nasal inflammatory diseases, characterized in that: The nasal preparation is composed of the following raw materials in parts by weight: 5-20 parts of magnolia, 5-15 parts of zedoaria, 5-10 parts of xanthium sibiricum, 5-10 parts of madder, 5-10 parts of honeysuckle, 5-10 parts of wild chrysanthemum, 3-8 parts of cloves and 2-5 parts of mint.

2. The nasal preparation according to claim 1, characterized in that The preparation method of the nasal preparation comprises: weighing a prescribed amount of the whole prescription medicinal raw materials for extraction and collecting volatile oil.

3. The nasal preparation according to claim 1, characterized in that One or more pharmaceutically acceptable adjuvants and / or excipients are also included.

4. The nasal preparation according to claim 2, characterized in that The nasal preparations include nasal drops, nasal washes, sprays or nasal powder mists.

5. The method for preparing the nasal preparation according to any one of claims 1 to 4, characterized in that: The preparation method of the nasal preparation comprises: weighing a prescribed amount of the whole formula of medicinal raw materials, adding water for distillation, and collecting volatile oil.

6. The preparation method according to claim 5, characterized in that: The solid-liquid ratio of water is 1:3-10 (W / V).

7. The preparation method according to claim 5, characterized in that: The distillation time is 2 to 8 hours, and the distillation extraction times are 1 to 3 times.

8. The preparation method according to claim 5, characterized in that: It also includes adding one or more pharmaceutically acceptable adjuvants and / or excipients to prepare a clinically acceptable dosage form.

9. Use of the nasal preparation according to any one of claims 1 to 4 or the nasal preparation obtained by the preparation method according to any one of claims 5 to 8 in the preparation of a drug for treating nasal inflammatory diseases.

10. The use according to claim 8, characterized in that: The nasal inflammatory diseases include acute rhinitis, chronic rhinitis, sinusitis, allergic rhinitis, atrophic rhinitis, and vasomotor rhinitis.

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