Application of Qu fructus aurantii extract in preparation of medicine for treating acute pharyngitis and cough

Abstract

The invention provides application of Qu fructus aurantii extract in preparation of medicine for treating acute pharyngitis and cough, and belongs to the technical field of traditional Chinese medicine development. The invention provides a new application of the Qu fructus aurantii extract in treatment of acute pharyngitis and cough. The Qu fructus aurantii extract can play roles in resisting acute pharyngitis and relieving cough in a multi-component, multi-target and multi-path manner. The Qu fructus aurantii is rich in source, low in price, safe and free of toxic and side effects, the preparation process route of the Qu fructus aurantii extract is simple, the production cost is low, and the Qu fructus aurantii extract can be prepared into medicines for treating acute pharyngitis and cough.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine development, and in particular relates to application of a Quzhi Fructus extract in preparing medicines for treating acute pharyngitis and cough. Background Art

[0002] Acute pharyngitis is a common upper respiratory tract disease, mainly caused by viral or bacterial infection. It has a high incidence rate, especially when the seasons change and the temperature changes greatly. Patients usually experience soreness, dryness, and burning sensation in the throat. The pain worsens when swallowing, and may also be accompanied by systemic symptoms such as fever, headache, and fatigue. If not treated in time, acute pharyngitis may turn into chronic pharyngitis, increasing the difficulty of treatment and seriously affecting the patient's quality of life. At present, the drugs used in clinical treatment of acute pharyngitis mainly include antibiotics, antiviral drugs, and traditional Chinese medicines for clearing heat and detoxification, but the abuse of antibiotics may lead to the production of drug-resistant bacteria, and some drugs have certain side effects.

[0003] Coughing is a protective reflex action of the body that helps to clear secretions or foreign bodies in the respiratory tract, but severe and frequent coughing can cause great pain to patients, seriously affecting their work, study and life. The causes of cough are complex and diverse, with common ones including respiratory tract infections, allergies, and foreign bodies in the airways. Drugs for treating cough mainly include antitussives (such as dextromethorphan), expectorants (such as ambroxol), and drugs targeting the cause of treatment, but some drugs may have adverse reactions such as addiction and gastrointestinal discomfort. Therefore, it is of great clinical significance to find safe, effective drugs with few side effects to treat acute pharyngitis and cough.

[0004] Quzhike is the immature fruit of Changshan Huyou, a plant of the Rutaceae family. It has a long history of cultivation and a wide planting area in Quzhou City. Quzhike contains a variety of chemical components, such as flavonoids, volatile oils, alkaloids, etc. These components may have anti-inflammatory, antioxidant, antibacterial and other pharmacological activities, but there are relatively few studies on the use of Quzhike extracts in the treatment of acute pharyngitis and cough. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide the use of Quzhi Fructus Extract in the preparation of medicines for treating acute pharyngitis and cough, thereby providing a new drug option for the clinical treatment of these two diseases.

[0006] The invention provides application of a Quzhi Fructus extract in preparing a medicine for treating acute pharyngitis.

[0007] Preferably, the Quzhi Fructus extract treats acute pharyngitis by inhibiting the PI3K-Akt signaling pathway.

[0008] More preferably, the Quzhi Fructus extract can reduce the number of leukocytes and neutrophils; increase SOD activity, reduce MDA content, increase GSH content; reduce TNF-α, IL-6, IL-1β levels; and reduce the mRNA expression levels of TNF-α and IL-1β.

[0009] The present invention also provides the use of the Fructus Aurantii Immaturus extract in preparing a medicine for treating cough.

[0010] Preferably, the Quzhi Fructus extract treats cough by regulating JAK-STAT, IL-17A signaling pathway and TRPV1 protein.

[0011] More preferably, the Quzhi Fructus extract can reduce the number of coughs; increase SOD activity and GSH content; reduce TNF-α, IL-1β, SP levels; reduce the mRNA expression levels of TNF-α and IL-6; increase the protein expression levels of p-STAT3 / STAT3, IL-17A and down-regulate the protein expression level of TRPV1.

[0012] Preferably, the preparation process of the Quzhi Fructus extract comprises the following steps: (1) extracting the Quzhi Fructus slices with slightly boiling hot water by reflux, filtering, and combining the filtrate; (2) concentrating the filtrate under reduced pressure, vacuum drying, and pulverizing to obtain the Quzhi Fructus extract.

[0013] More preferably, the amount of water added in the water reflux extraction in step (1) is 8-12 times the amount of the Quzhi Fructus decoction pieces, the number of extractions is 1-3 times, and the extraction time is 0.5-1.5h.

[0014] More preferably, the temperature for the reduced pressure concentration in step (2) is 60-80°C, and the temperature for the vacuum drying is 50-70°C.

[0015] More preferably, in the Fructus Aurantii Immaturus extract, the content of naringin is 8.7-16.2 mg / g, the content of naringin is 88.7-164.7 mg / g, the content of hesperidin is 4.5-8.3 mg / g, and the content of neohesperidin is 57.4-106.6 mg / g.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention has discovered a new use of Quzhi Fructus extract in preparing medicines for treating acute pharyngitis and cough, providing a new drug option for clinical treatment of these two diseases. Quzhi Fructus is a dried slice of Changshan Huyou fruit, which is rich in sources and widely planted in Quzhou, Zhejiang and other places. It is cheap and easy to obtain, which is conducive to the large-scale production and application of drugs and reduces medical costs.

[0018] The extract of Quzhi Fructus can play an anti-acute pharyngitis and antitussive role through multiple components, multiple targets and multiple pathways. In the treatment of acute pharyngitis, it can inhibit the PI3K-Akt signaling pathway, reduce the number of white blood cells and neutrophils in the whole blood, and reduce the inflammatory response; increase the activity of SOD in serum, reduce the content of MDA, increase the content of GSH, and regulate the level of oxidative stress; reduce the levels of TNF-α, IL-6, and IL-1β in serum, and reduce the mRNA expression levels of TNF-α and IL-1β in pharyngeal tissues, thus improving the pathological state of acute pharyngitis from multiple aspects. In the treatment of cough, it exerts an antitussive effect by regulating the JAK-STAT, IL-17A signaling pathways and TRPV1 protein, which can reduce the number of coughs, increase SOD activity and GSH content in BALF, reduce serum TNF-α, IL-1β, and SP levels, reduce the mRNA expression levels of TNF-α and IL-6 in lung tissue, increase the protein expression levels of p-STAT3 / STAT3 and IL-17A in lung tissue, and down-regulate the protein expression level of TRPV1 in lung tissue, comprehensively regulating the physiological and pathological processes related to cough.

[0019] Quzhi Fructus Extract is derived from fruit (Changshan Pomelo), is safe and has no toxic side effects. Compared with some chemical drugs, it has less adverse effects on the body when used for a long time. It is more suitable for patients, especially children, the elderly and other special groups, and improves the safety and tolerance of medication. In addition, the preparation process of Quzhi Fructus Extract is simple. It can be obtained only through conventional operations such as water reflux extraction, reduced pressure concentration, vacuum drying, and crushing. It does not require complex equipment and technology and is easy to industrialize. And the production cost is low, which is conducive to the promotion and application of the product and improves the accessibility of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of QAFE on the symptoms of acute pharyngitis in rats. In the figure, A is the score of hair loss around the mouth and nose, B is the score of oral secretions, C is the score of cough and asthma, and D is the score of activity ability;

[0021] Figure 2 The effect of QAFE on the pathological morphology of pharyngeal tissue in rats with acute pharyngitis. In the figure, A is the gross observation of pharynx, and B is HE staining;

[0022] Figure 3 The effect of QAFE on the number of white blood cells and neutrophils in the blood of rats with acute pharyngitis. In the figure, A is the number of white blood cells, and B is the number of neutrophils;

[0023] Figure 4 The effect of QAFE on serum oxidative stress indexes in rats with acute pharyngitis, in which A is SOD activity, B is MDA content, and C is GSH content;

[0024] Figure 5 The effect of QAFE on the levels of TNF-α, IL-6 and IL-1β in the serum of rats with acute pharyngitis. In the figure, A is the level of TNF-α in the serum of rats with acute pharyngitis, B is the level of IL-6 in the serum of rats with acute pharyngitis, and C is the level of IL-1β in the serum of rats with acute pharyngitis.

[0025] Figure 6 The figure shows the effect of QAFE on the relative expression levels of TNF-α and IL-1β mRNA in the pharyngeal tissue of rats with acute pharyngitis. In the figure, A shows the relative expression level of TNF-α mRNA in the pharynx of rats with acute pharyngitis, and B shows the relative expression level of IL-1β mRNA in the pharynx of rats with acute pharyngitis.

[0026] Figure 7 The effect of QAFE on PI3K-Akt pathway-related proteins in pharyngeal tissue of rats with acute pharyngitis. In the figure, A is a protein blot, B and C are the relative quantitative results of p-PI3K / PI3K and p-Akt / Akt protein expressions, respectively;

[0027] Figure 8 The effect of QAFE on the cough latency and cough frequency induced by ammonia in mice. In the figure, A is the cough latency and B is the cough frequency;

[0028] Fig. 9 The effect of QAFE on the cough latency and cough frequency induced by citrate in guinea pigs. In the figure, A is the cough latency and B is the cough frequency.

[0029] Fig.10 The effect of QAFE on oxidative stress-related indicators in BALF of coughing guinea pigs. In the figure, A is SOD activity, and B is GSH content;

[0030] Fig.11 The effect of QAFE on the levels of serum TNF-α, IL-1β, and SP in coughing mice. In the figure, A is the level of serum TNF-α in coughing mice, B is the level of serum IL-1β in coughing mice, and C is the level of serum SP in coughing mice;

[0031] Fig.12 The effect of QAFE on the relative expression levels of TNF-α and IL-6 mRNA in lung tissues of coughing mice. In the figure, A is the relative expression level of TNF-α mRNA in lung tissues of coughing mice, and B is the relative expression level of IL-6 mRNA in lung tissues of coughing mice;

[0032] Fig.13The effect of QAFE on the expression of p-STAT3, IL-17A, and TRPV1 proteins in lung tissues of coughing mice. In the figure, A is Western blot, and B and D are the quantitative results of p-STAT3 / STAT3, IL-17A, and TRPV1 proteins, respectively;

[0033] Fig.14 This is the HPLC chromatogram for the identification of Quzhi Fructus Extract. In the figure, A is the chromatogram of the reference substance, B is the chromatogram of the test substance, and the peak components from left to right are naringin, naringin, hesperidin, and neohesperidin.

[0034] Figure 1-Figure 13 middle, # P<0.05, ## P<0.01vs blank control group; * P<0.05, ** P<0.01vs model control group. DETAILED DESCRIPTION

[0035] The invention provides application of Quzhizi extract in preparing medicine for treating acute pharyngitis and cough and a preparation method of the Quzhizi extract.

[0036] In the present invention, it is preferred that the Quzhi Fructus extract treats acute pharyngitis by inhibiting the PI3K-Akt signaling pathway; it is further preferred that the Quzhi Fructus extract can reduce the number of leukocytes and neutrophils in whole blood; increase the SOD activity in serum, reduce the MDA content in serum, increase the GSH content in serum; reduce the levels of TNF-α, IL-6, and IL-1β in serum; and reduce the mRNA expression levels of TNF-α and IL-1β in pharyngeal tissue.

[0037] In the present invention, it is preferred that the Quzhi Fructus extract treats cough by regulating JAK-STAT, IL-17A signaling pathways and TRPV1 protein; it is further preferred that the Quzhi Fructus extract can reduce the number of coughs; increase SOD activity and GSH content in BALF; reduce serum TNF-α, IL-1β, SP levels; reduce the mRNA expression levels of TNF-α and IL-6 in lung tissue; increase the protein expression levels of p-STAT3 / STAT3 and IL-17A in lung tissue and down-regulate the protein expression level of TRPV1 in lung tissue.

[0038] The preparation process of the Quzhi Fructus extract of the present invention comprises the following steps: (1) extracting the Quzhi Fructus slices with slightly boiling hot water under reflux, filtering, and combining the filtrate; (2) concentrating the filtrate under reduced pressure, vacuum drying, and pulverizing to obtain the Quzhi Fructus extract.

[0039] In the present invention, the amount of water added in the water reflux extraction in step (1) is preferably 8-12 times the amount of the Quzhi Fructus decoction pieces, more preferably 10 times; the number of extractions is preferably 1-3 times, the extraction time is 0.5-1.5h, more preferably 3 times, each extraction is 1.5h. The temperature of the reduced pressure concentration in step (2) is preferably 60-80°C, more preferably 70°C; the temperature of the vacuum drying is preferably 50-70°C, more preferably 60°C.

[0040] The contents of naringin, naringin, hesperidin and neohesperidin in the Quzhi Fructus extract prepared by the invention are 12.46, 126.70, 6.39 and 82.02 mg / g respectively; preferably, the contents of each component are controlled within ±30% during pharmaceutical preparation, that is, the content of naringin is 8.7-16.2 mg / g, the content of naringin is 88.7-164.7 mg / g, the content of hesperidin is 4.5-8.3 mg / g and the content of neohesperidin is 57.4-106.6 mg / g.

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] Example 1

[0043] The therapeutic effect and mechanism of Qufuzhi Fructus Extract (QAFE) on acute pharyngitis

[0044] 1. Experimental Animals

[0045] Thirty-six specific pathogen-free SD rats, all male, weighing 180-220 g, were housed in the barrier system animal room of the Safety Evaluation Research Center of Hangzhou Medical College. No more than 5 animals of the same sex were housed in each cage, with free access to water and food, room temperature of 20.0-26.0℃, relative humidity of 40%-70%, central air conditioning and centralized ventilation ≥15 times / hour, and lighting of 12h light and 12h dark.

[0046] 2. Test methods

[0047] 2.1 Preparation of Qufuzhi Fructus Extract (QAFE)

[0048] The origin of Quzhike used in the experiment is Qingshigedi, Quzhou, Zhejiang. It is processed into Quzhike slices by Changshan Tiandao Chinese Medicine Pieces Co., Ltd., and Quzhike extract is prepared by Zhejiang Kangenbei Pharmaceutical Co., Ltd. The extraction method is as follows: 150kg of Quzhike slices are added to ten times the volume of water, soaked for 2 hours, and then slightly boiled and refluxed for extraction 3 times, each time for 1.5 hours. The extracts are combined, vacuum dried, collected, and crushed to obtain dry paste powder. Properties: brown dry powder, slightly fragrant, bitter taste; Storage conditions: dry, room temperature.

[0049] 2.2 Animal grouping, modeling and drug administration

[0050] 2.2.1 Drug preparation

[0051] 10% ammonia water (V / V) preparation: Add ultrapure water to 120 mL of 25%-28% ammonia water to make up to 300 mL to prepare ammonia water with a concentration of about 10%.

[0052] Preparation of QAFE solution: The prepared QAFE was dissolved in ultrapure water to a final concentration of 44, 88, and 176 mg / mL.

[0053] 2.2.2 Animal grouping and dosing

[0054] Thirty-six male SD rats were randomly divided into 6 groups: blank control group, model control group, low, medium and high doses of QAFE (0.44 g / kg, 0.88 g / kg, 1.76 g / kg), and positive control group (Shuanghuanglian oral liquid, 20 mL / kg), with 6 rats in each group. As shown in Table 1.

[0055] Table 1 Experimental groups and drug administration of rats

[0056]

[0057] 2.2.3 Modeling and drug administration

[0058] Use a laryngeal sprayer to spray 10% ammonia water into the rat pharynx (spread area is about 150mm 2 ), twice a day, once in the morning and once in the afternoon, 3 sprays each time (0.2-0.4mL), for 3 consecutive days.

[0059] Drug administration began the next day after the modeling was completed. The three QAFE dose groups were gavaged with corresponding doses of QAFE, the blank control and model control groups were gavaged with the same volume of normal saline, and the positive control group was gavaged with corresponding doses of Shuanghuanglian oral liquid for 5 consecutive days, twice a day, once in the morning and once in the afternoon.

[0060] 2.3 General status observation and scoring

[0061] During the experimental period, the rats' food intake, activity, hair around the mouth and nose, scratching, pharyngeal mucosal status and secretions were observed every day. One hour after the last administration, the same person scored the hair loss around the mouth and nose, oral secretions, coughing and wheezing, and activity of each group of animals. The scoring criteria are shown in Table 2.

[0062] Table 2 General behavior scoring criteria for rats with acute pharyngitis

[0063] score Hair loss around the mouth and nose Amount of oral secretions Cough and asthma Activity 0 none normal none normal 1 Mild shedding Mild drooling Mild cough Mild decline 2 Moderate shedding Moderate drooling Moderate cough and wheezing Moderate decline 3 Severe shedding Severe drooling Severe cough and asthma Severe decline

[0064] 2.4 Specimen Collection

[0065] After the last administration, all animals were fasted overnight, but not water. 12 hours later, rats were intraperitoneally injected with 1% sodium pentobarbital (0.1 mL / 100 g). Blood was collected from the abdominal aorta, and 500 μL of whole blood was placed in an anticoagulant tube for white blood cell and neutrophil counting. The remaining whole blood was allowed to stand at room temperature for 30 minutes, centrifuged at 4°C, 3000 r / min for 15 minutes to separate serum, and the blood was stored at -80°C for subsequent testing. Use scissors to cut open the corners of the rat's mouth to fully expose the palate and pharynx. Use scissors to gently cut the pharyngeal mucosa and submucosal tissue, which is about 10 mm × 5 mm × 1 mm in size. Take the pharyngeal mucosa and its submucosal tissue of the same size close to the hard palate end and place it in 10% formaldehyde for fixation. Pathological histological analysis was performed later, and the rest was stored at -80°C.

[0066] 2.5 Pharyngeal histopathological examination

[0067] (1) Fixed

[0068] The pharyngeal tissue was immersed in 10% formaldehyde for more than 24 h.

[0069] (2) Dehydration

[0070] Take appropriate sized pharyngeal tissues from each group and lay the cut surface flat in an embedding box. Dehydrate in 75% ethanol for 4h → dehydrate in 85% ethanol for 2h → dehydrate in 95% ethanol for the third time for 2h → 95% ethanol for 1h → 100% ethanol for 1h → dehydrate in 100% ethanol for 1h, and complete the above steps to achieve gradient dehydration. Place the dehydrated pharyngeal tissues in xylene I and II for transparent immersion for 40min, and finally place them in paraffin I and paraffin II for 1h and 1.5h respectively.

[0071] (3) Embedding

[0072] The processed tissues were embedded in paraffin using an embedding machine and the wax blocks were stored at 4°C.

[0073] (4) Slicing

[0074] Wax slices with a thickness of approximately 4 μm were cut using a microtome.

[0075] (5) Baking sheet

[0076] Unfold the wax slice and place it on a glass slide, bake it on a slide baker for 15 minutes, and then bake the slices in a 60℃ constant temperature box for 4-5 hours.

[0077] (6) HE staining

[0078] The sections were placed in xylene I and xylene II for 5 minutes each for dewaxing; in anhydrous ethanol solution I, anhydrous ethanol solution II, 95% ethanol solution, 90% ethanol solution for 3 minutes each, and 80% ethanol solution for 1 minute, and the above steps were completed for xylene dewaxing; rinsed with running water for 10 seconds to wash away the excess ethanol solution on the sections, immersed in hematoxylin staining solution for 10 minutes for nuclear staining, rinsed with running water for 5 seconds to wash away the excess unbound dye on the sections, differentiated with differentiation solution for 1 second to remove excess dye on the cells, rinsed with running water for 7 minutes, removed the differentiation solution and the removed dye, and terminated the differentiation; immersed in eosin staining solution for 5 minutes for contrast staining; 80% ethanol solution for 30 seconds, 90% ethanol solution, 95% ethanol solution for 1 minute each, anhydrous ethanol I and anhydrous ethanol II for 3 minutes each, and the above steps were completed for dehydration; xylene I, xylene II, and xylene III for 3 minutes each, and the above steps were completed for transparent treatment.

[0079] 2.6 Determination of the number of inflammatory cells in whole blood

[0080] The whole blood was placed in ADVIA2120 blood cell analyzer for white blood cell and neutrophil counts.

[0081] 2.7 Determination of serum TNF-α and IL-1β levels

[0082] According to the instructions of the ELISA kit, the levels of TNF-α and IL-1β in the serum of each group were detected.

[0083] 2.8 Detection of SOD, MDA and GSH levels in serum

[0084] According to the kit instructions, the levels of SOD, MDA and GSH in the serum of each group were detected.

[0085] 2.9 RT-qPCR

[0086] (1) RNA extraction from pharyngeal tissue

[0087] The frozen pharyngeal tissue was taken out, about 30 mg was weighed and placed in a new enzyme-free 1.5 mL EP tube, 1 mL RNAiso-plus was added, and the mixture was placed on ice for homogenization. After the homogenization was completed, it was allowed to stand for 5 minutes and centrifuged at 4°C and 12000g for 5 minutes. The supernatant was transferred to a new enzyme-free 1.5 mL EP tube, 200 μL chloroform was added, and the mixture was shaken by hand. After standing at room temperature for 5 minutes, it was centrifuged at 4°C and 12000g for 15 minutes. The supernatant was transferred to a new enzyme-free 1.5 mL EP tube, the same volume of isopropanol as the supernatant was added, the mixture was shaken by hand, and the mixture was allowed to stand at room temperature for 10 minutes and centrifuged at 4°C and 12000g for 10 minutes. The liquid in the tube was discarded, and the precipitate at the bottom of the tube was washed with 75% ethanol prepared with DEPC water. This was repeated twice. After centrifugation, the supernatant was discarded, dried, and 20 μL DEPC water was added to dissolve the precipitate to obtain the pharyngeal total RNA solution, which was stored at -80°C for later use.

[0088] (2) Reverse transcription

[0089] Take the total RNA out of the -80℃ freezer, thaw it on ice, and then quantify it using Nanodrop One. Then add the corresponding volume of DEPC water to the total RNA sample to adjust the concentration to 500ng / μL. Take out the relevant reagents required for reverse transcription, thaw them on ice, and add the corresponding reagents in turn to a new 200μL enzyme-free EP tube according to the ratio described in Table 3.

[0090] Table 3 Reverse transcription reagent ratio

[0091] Reagents volume 5×PrimeScript RT Master Mix 2μL Total RNA 1μL <![CDATA[RNase Free dH2O]]> 7μL

[0092] Mix the above reagents evenly, centrifuge the liquid in the tube to the bottom, put it into the PCR instrument, set the program to 37℃15min→85℃5s→4℃ cycle, and when the first two steps are completed and the reaction system is cooled to 4℃, the reverse transcription is completed and the cDNA sample is obtained.

[0093] (3) qPCR

[0094] Add the specific primers of the target gene and cDNA samples to the eight-tube strip, then add the corresponding volume of TBGreen Premix Ex Taq, ROX reference Dye and DEPC water, mix well and amplify in a PCR instrument. -ΔΔCt The relative expression of mRNA in each group was calculated by the method. The reaction system reagents are shown in Table 4, and the primer sequences of related genes are shown in Table 5. The amplification steps are as follows: 95℃10min, 1 cycle for pre-denaturation; 95℃15s for 35 cycles for denaturation; 60℃1min for annealing / extension.

[0095] Table 4 qPCR reaction system

[0096] Reagents volume TB Green Premix Ex TaqII(Tli RNaseH Plus)(2×) 10μL PCR Forward Primer (10 μM) 0.8μL PCR Reverse Primer (10μM) 0.8μL ROX reference dye (50×) 0.4μL cDNA Solution 2μL <![CDATA[RNase H2O]]> 6μL Total 20μl

[0097] Table 5 Related gene primer sequences (Rat)

[0098]

[0099] 2.10 Western blot analysis

[0100] (1) Tissue protein extraction

[0101] Take out the tissue sample from the -80℃ freezer, weigh 50mg and place it in a 1.5mL EP tube, cut the tissue into small pieces with scissors on ice, add 600μL of the prepared lysis buffer (RIPA: phosphatase inhibitor: PMSF = 97:2:1) to the tube, and fully homogenize on ice. Centrifuge at 12000g for 10min at 4℃, take the supernatant to a new 1.5mL EP tube, determine the protein concentration according to the Bradford protein concentration determination kit, add 5× Loading Buffer to the sample at a ratio of protein sample: Loading Buffer = 4:1, adjust the protein concentration to 5μg / μL, place in a metal bath at 100℃ for 10min, cool at room temperature, and store at -20℃.

[0102] (2) Preparation of electrophoresis gel

[0103] Follow Yazyme Omni-Easy TM One-step PAGE gel rapid preparation kit instructions for preparing 1.00 mm electrophoresis gels of different concentrations.

[0104] (3) Preparation of electrophoresis buffer, transfer buffer, TBST, and blocking buffer

[0105] Add ultrapure water to make up to 1L of Tris 3.03g, glycine 14.4g, and SDS 1g, mix until the powder is dissolved to obtain the electrophoresis solution; add water to make up to 1L of Tris 3.03g, glycine 14.4g, and methanol 200mL, mix until the solid is dissolved to obtain the transfer solution; take 1 packet of TBS powder, add 1mL of Tween, and add ultrapure water to make up to 1L to obtain TBST; weigh an appropriate amount of BSA, add TBST to make a 3% BSA solution, which is the blocking solution.

[0106] (4) SDS-PAGE electrophoresis

[0107] Install the prepared electrophoresis gel into the electrophoresis tank, pour the newly configured electrophoresis solution into it, add 4μL Maker and the corresponding amount of protein sample to the wells in sequence, the amount of protein sample loaded is about 20μg per well, turn on the power after loading, and perform electrophoresis under a constant voltage of 80V. When the protein band moves to the lower layer of gel, change the constant voltage to 120V until the protein band moves to the bottom of the lower layer of gel.

[0108] (5) Transfer

[0109] Cut out a PDVF membrane of appropriate size and soak it in methanol for 30 seconds for activation; soak the transfer clips, sponges, and filter paper with transfer solution, spread them flat, place the PDVF membrane on the filter paper, then take out the SDS-PAGE gel and spread it flat on the membrane, remove bubbles, and clamp it in the order of sponge-filter paper-PDVF membrane-SDS-PAGE gel-filter paper-sponge to make a flat sandwich structure. Put the clips in the transfer tank, put the transfer tank in an ice water bath, set the transfer conditions to a constant current of 300mA, and set the transfer time according to the molecular weight.

[0110] (6) Closed

[0111] After the transfer, the PVDF membrane was taken out and placed in the blocking solution and shaken at room temperature for 1 hour.

[0112] (7) Primary antibody incubation

[0113] After blocking, the PVDF membrane was removed and washed three times with TBST, each time for 5 min. After washing, it was placed in the prepared primary antibody (antibody: primary antibody diluent = 1:1000) and incubated at 4°C overnight.

[0114] (8) Secondary antibody incubation

[0115] After the primary antibody incubation was completed, the PDVF membrane was removed and washed three times with TBST, each time for 5 minutes. After washing, it was placed in the corresponding secondary antibody (secondary antibody: TBST = 3000:1) and incubated at 37°C for 1.5 hours.

[0116] (9) Development

[0117] After the secondary antibody incubation is completed, the PDVF membrane is removed and washed with TBST for 3 times, 5 minutes each time. After washing, mix the A and B solutions in the BeyoECLStar ultra-sensitive ECL chemiluminescence kit in a 1:1 ratio to prepare an appropriate amount of developer, drop an appropriate amount of developer onto the PDVF membrane, and use a gel imager to take pictures and analyze.

[0118] 3. Data statistics and analysis

[0119] Statistical analysis was performed using Excel and / or Graphpad prism 9 software packages, and the data were expressed as Mean ± SEM. Each data was tested for normal distribution. If it was in accordance with normal distribution (P>0.05), one-way analysis of variance was performed. If it was not in accordance with normal distribution (P≤0.05), non-parametric test was performed. Compared with the model group, P<0.05 was considered to be statistically significant. When comparing the two groups, an independent sample t test was performed, and P<0.05 was considered to be statistically significant.

[0120] 4. Test results

[0121] 4.1 Effect of QAFE on the general condition of rats with acute pharyngitis

[0122] During the experiment, the blank control group had a good mental state, agile movements, normal food and water intake, shiny white hair, normal scratching frequency, and no oral secretions. Three days after modeling, the rats in the model group were listless, slow in movement, with messy hair, reduced food intake and water intake, increased scratching, increased secretions around the mouth and nose, and salivation, indicating that the acute pharyngitis model was successfully established. The specific scoring results of the general state of rats with acute pharyngitis are shown in Figure 1 The scoring results showed that compared with the model control group, the QAFE medium and high dose groups and the Shuanghuanglian oral liquid group could significantly reduce the hair loss around the mouth and nose and the amount of oral secretions (P < 0.05 or P < 0.01), and the QAFE high dose group and the Shuanghuanglian oral liquid group could significantly reduce the scores of indicators such as cough and asthma and activity ability (P < 0.05 or P < 0.01). These results indicate that QAFE administration can effectively relieve the symptoms of acute pharyngitis.

[0123] 4.2 Effect of QAFE on pharyngeal injury in rats with acute pharyngitis

[0124] The results of the macroscopic observation of the pharynx are as follows Figure 2 As shown in A, compared with the normal group, the model group had red and swollen pharynx, dark red mucosal surface, bleeding on the mucosal surface (blue arrow), and ulcers on the hard palate (red circle). After treatment, compared with the model group, the pharynx of the Shuanghuanglian oral liquid group, QAFE medium and high dose groups improved to a greater extent, the redness and swelling basically disappeared, the mucosal gloss and wetness improved, and the pharyngeal morphology was basically close to the normal group; the QAFE low dose group also improved to a certain extent. Pharyngitis in the QAFE medium and high dose groups improved significantly, while the QAFE low dose group did not improve significantly.

[0125] HE staining results Figure 2As shown in B, the pharyngeal tissue structure of rats in the blank control group was clear, the epithelial layer was smooth, and no squamous epithelial hyperplasia was observed; no inflammatory cell infiltration or congestion was observed in the submucosal tissue. The submucosal gland secretion was normal, and the linear gland structure was normal; the model control group had squamous epithelial hyperplasia (yellow arrow), submucosal inflammatory cell infiltration (green arrow), reduced submucosal gland secretion, and glandular epithelial degeneration (red arrow); pharyngitis was significantly improved in the Shuanghuanglian oral group, QAFE medium and high dose groups, but not significantly improved in the QAFE low dose group.

[0126] 4.3 Effect of QAFE on the number of leukocytes and neutrophils in whole blood of rats with acute pharyngitis

[0127] like Figure 3 As shown in A, compared with the blank control group, the number of white blood cells in the blood of rats in the model control group was significantly increased (P < 0.05); compared with the model group, the number of white blood cells in the low-, medium-, and high-dose QAFE groups and the Shuanghuanglian oral liquid group was significantly reduced (P < 0.01 or P < 0.05).

[0128] like Figure 3 As shown in B, compared with the blank control group, the number of neutrophils in the blood of rats in the model control group was significantly higher than that in the blank control group (P < 0.05); after treatment with low, medium and high doses of QAFE and Shuanghuanglian oral liquid, the number of neutrophils was significantly reduced (P < 0.01 or P < 0.05).

[0129] 4.4 Effect of QAFE on serum SOD, MDA and GSH in rats with acute pharyngitis

[0130] like Figure 4 As shown in A, compared with the blank control group, the serum SOD activity of rats in the model group was significantly decreased (P < 0.01); compared with the model group, the SOD activity of the low, medium and high dose groups of QAFE was significantly increased (P < 0.05), and although the SOD activity of the Shuanghuanglian oral liquid group showed an increasing trend, it was not statistically significant (P > 0.05).

[0131] like Figure 4 As shown in B, compared with the blank control group, the serum MDA content of rats in the model group was significantly increased (P < 0.05); compared with the model group, the MDA content in the QAFE medium-dose, high-dose group and Shuanghuanglian oral liquid group was significantly decreased (P < 0.05), and although the MDA content in the QAFE low-dose group showed a decreasing trend, it was not statistically significant (P > 0.05).

[0132] like Figure 4As shown in Figure C, compared with the blank control group, the serum GSH content of rats in the model group was significantly decreased (P < 0.05); compared with the model group, the GSH content in the high-dose QAFE group was significantly increased (P < 0.01), and although the GSH content in the low-dose QAFE group, the medium-dose QAFE group and the Shuanghuanglian oral liquid group showed an upward trend, it was not statistically significant (P > 0.05).

[0133] The above results indicate that QAFE can reduce the oxidative stress level in the serum of rats with acute pharyngitis.

[0134] 4.5 Effect of QAFE on the levels of inflammatory factors in the serum of rats with acute pharyngitis

[0135] like Figure 5 As shown in A, compared with the blank control group, the serum TNF-α level of rats in the model group was significantly increased (P < 0.05); compared with the model group, the TNF-α levels of the low-, medium-, and high-dose QAFE groups and the Shuanghuanglian oral liquid group were significantly decreased (P < 0.05).

[0136] like Figure 5 As shown in B, compared with the blank control group, the serum IL-6 level of rats in the model group was significantly increased (P < 0.01); compared with the model group, the IL-6 levels of the QAFE medium and high dose groups and the Shuanghuanglian oral liquid group were significantly decreased (P < 0.05), and although the IL-6 level in the QAFE low dose group showed a downward trend, it was not statistically significant.

[0137] like Figure 5 As shown in Figure C, compared with the blank control group, the serum IL-1β level of rats in the model group was significantly increased (P < 0.05); compared with the model group, the IL-1β level of the QAFE high-dose group was significantly decreased (P < 0.05), and although the IL-1β level of the QAFE low-dose, medium-dose groups and Shuanghuanglian oral liquid group showed a downward trend, it was not statistically significant.

[0138] 4.6 Effect of QAFE on TNF-α and IL-1β mRNA levels in pharyngeal tissues of rats with acute pharyngitis

[0139] like Figure 6 As shown in A, compared with the blank control group, the mRNA expression level of TNF-α in the pharyngeal tissue of rats in the model group was significantly increased (P < 0.01), and the mRNA expression level of TNF-α in the QAFE medium and high dose groups and Shuanghuanglian oral liquid group was significantly decreased, which was statistically significant (P < 0.01). Although the mRNA expression level of TNF-α in the QAFE low dose group showed a downward trend, it was not statistically significant.

[0140] like Figure 6As shown in Figure B, compared with the blank control group, the mRNA expression level of IL-1β in the pharyngeal tissue of rats in the model group was significantly increased (P < 0.05), and the mRNA expression level of IL-1β in the high-dose QAFE group and Shuanghuanglian oral liquid group was significantly decreased, which was statistically significant (P < 0.01 or P < 0.05), while the mRNA expression level of TNF-α in the low- and medium-dose QAFE groups showed a downward trend, but it was not statistically significant.

[0141] The above results indicate that QAFE can play a protective role by alleviating the inflammatory response of pharyngeal tissue in rats with acute pharyngitis.

[0142] 4.7 Effect of QAFE on PI3K-Akt Signaling Pathway

[0143] like Figure 7 As shown in the figure, the phosphorylation levels of PI3K and Akt proteins in the pharyngeal tissue of rats in the model group were significantly increased, indicating that the PI3K-Akt signaling pathway in the pharyngeal tissue of rats was activated after ammonia modeling; compared with the model group, the phosphorylation levels of PI3K and Akt proteins in the QAFE dose group were significantly reduced (P < 0.05 or P < 0.01). The above results indicate that QAFE can inhibit the PI3K-Akt signaling pathway to reduce inflammation in the pharynx of rats, thereby treating acute pharyngitis.

[0144] Example 2

[0145] Antitussive effect and mechanism of extract from Citrus aurantium

[0146] 1. Experimental Animals

[0147] 60 C57 BL / 6 mice, SPF grade, half male and half female, with a body weight of 22±2g.

[0148] 70 C57 BL / 6 mice, SPF grade, half male and half female, with a body weight of 22±2g.

[0149] 60 Hartley guinea pigs, ordinary grade, half male and half female, body weight 275±25g.

[0150] All animals were given free access to food and water, a 12-h day-night rhythm, room temperature of 22±2°C, and humidity of 55±15%.

[0151] 2. Test methods

[0152] 2.1 Preparation of Qufuzhi Fructus Extract (QAFE)

[0153] Same as Example 1.

[0154] 2.2 Animal grouping, modeling and drug administration

[0155] 2.2.1 Drug preparation

[0156] QAFE solution: Weigh an appropriate amount of QAFE and add pure water to prepare solutions with concentrations of 60, 75, 120, 150, 240, and 300 mg / mL, respectively, for intragastric administration.

[0157] Positive control aminophylline: Weigh an appropriate amount of aminophylline and add pure water to prepare solutions with concentrations of 6.4 and 8 mg / mL, respectively, for intragastric administration.

[0158] Positive control Pediatric Kechuanling Granules: Weigh an appropriate amount of Pediatric Kechuanling Granules, add pure water to prepare solutions with concentrations of 25 and 31 mg / mL, respectively, for intragastric administration.

[0159] 2.2.2 Animal grouping and dosing

[0160] 2.2.2.1 Mouse cough suppression experiment

[0161] Place C57 BL / 6 mice in the recording box of the EMKA animal lung function monitoring system, open the Medlab software, and start recording after the animals have adapted. Use the instrument's built-in sprayer to atomize 25% ammonia water for 5 seconds and record for 5 minutes. Mice that cough 10-50 times are selected for subsequent experiments.

[0162] 60 mice that met the requirements were selected, half male and half female, and randomly divided into 6 groups according to body weight, 10 in each group. The experimental animals were divided into model group (Model, pure water was given by gavage), QAFE low-dose group (0.6g / kg), QAFE medium-dose group (1.2g / kg), QAFE high-dose group (2.4g / kg), aminophylline group (0.064g / kg), and pediatric cough and asthma group (0.25g / kg). Gavage was administered twice a day for 5 consecutive times. One hour after the last administration, the mice were placed in the recording box of the EMKA animal lung function monitoring system, and the Medlab software was opened. After the animals were adapted, the recording began. The 25% ammonia water was atomized for 5s through the instrument's own sprayer, and the recording was performed for 5min. The cough signals of the mice within 5min were collected using the Medlab biological signal acquisition system, and the cough latency and cough frequency were recorded.

[0163] 2.2.2.2 Guinea pig cough test

[0164] Place the Hartley guinea pig in the recording box of the EMKA animal lung function monitoring system, open the Medlab software, and start recording after the animal has adapted. Use the instrument's built-in nebulizer to atomize 17.5% citric acid for 40 seconds, and record the cough latency and the number of coughs within 5 minutes. Screen guinea pigs that cough 10-35 times for subsequent experiments.

[0165] 60 guinea pigs that met the requirements were selected, half male and half female, and randomly divided into 6 groups according to body weight, with 10 animals in each group. The experimental animals were divided into model group (gavage with pure water), QAFE low-dose group (0.375g / kg), QAFE medium-dose group (0.75g / kg), QAFE high-dose group (1.5g / kg), aminophylline group (0.04g / kg), and pediatric Kechuanling group (0.155g / kg). Gavage was administered twice a day for 5 consecutive times. One hour after the last administration, the guinea pigs were placed in the recording box of the EMKA animal lung function monitoring system, and the Medlab software was opened. After the animals adapted, the recording began. The 17.5% citric acid was atomized for 40s through the instrument's own sprayer, and the cough signals of the guinea pigs within 5 minutes were collected using the Medlab biological signal acquisition system, and the cough latency and cough frequency were recorded.

[0166] 2.3 Study on cough suppressant mechanism

[0167] Another 70 C57 BL / 6 mice were used to explore the antitussive mechanism of QAFE. Among them, 10 mice without coughing were used as blank control group, and 60 mice that met the above requirements were randomly divided into 6 groups, namely model group, QAFE low, medium and high dose groups, aminophylline group and pediatric cough and asthenia group, with 10 animals in each group. Mice in the blank control group were gavaged with the same volume of pure water, twice a day, for 5 consecutive times, and the dosing schedule of the other groups was the same as above.

[0168] 2.4 Specimen Collection

[0169] After the last administration, all guinea pigs were fasted overnight, but not water. After the coughing experiment, 1% sodium pentobarbital (0.1 mL / 100 g) was injected into the guinea pigs intraperitoneally, and the abdominal aorta was bled for euthanasia. The guinea pigs were placed in a supine position after being killed, and their limbs were fixed. First, the chest cavity was cut open, the ribs on both sides were separated, and the right lung hilum was ligated with silk thread. The right lung tissue was taken and frozen at -80°C. The skin of the neck was cut open, the tissue was peeled off layer by layer, the trachea was exposed, an incision was made above the trachea, and the hose was inserted into the trachea from the incision, and it was tied with silk thread to fix it. 1 mL of normal saline was drawn with a syringe and poured into the lungs to rinse back and forth to ensure that the amount of recovered liquid was greater than 0.8 mL. The collected BALF was collected in a sterile EP tube and frozen at -80°C for later use.

[0170] 30 minutes after the modeling, all mice in the cough mechanism study were eyeballed and blood was collected. The blood was placed in a sterile EP tube and allowed to stand at room temperature for 30 minutes. The blood was centrifuged at 4°C and 3000r / min for 15 minutes to separate the serum. The blood was stored at -80°C for subsequent testing. After blood collection, the mice were euthanized by cervical dislocation. The mice were placed in a supine position after being killed, their limbs were fixed, the chest cavity was cut open, the ribs on both sides were separated, the lung tissue was exposed, and the lung tissue was placed in a 1.5mL EP tube and frozen at -80°C.

[0171] 2.5 Determination of SOD and GSH levels in serum

[0172] According to the kit instructions, the levels of SOD and GSH in the bronchoalveolar lavage fluid of guinea pigs in each group were detected.

[0173] 2.6 Determination of serum TNF-α, IL-1β, and SP levels

[0174] According to the kit instructions, the levels of TNF-α, substance P (SP), and IL-1β in the serum of each group of mice were detected.

[0175] 2.7 RT-qPCR

[0176] The steps are the same as those in Example 1. The primer sequences are shown in Table 6.

[0177] Table 6 Related gene primer sequences (Mouse)

[0178]

[0179] 2.8 Western blot analysis

[0180] The expression levels of STAT3, p-STAT3, IL-17A and TRPV1 proteins in mouse lung tissue were detected using the same method as in Example 1.

[0181] 3. Data statistics and analysis

[0182] Statistical analysis was performed using Excel and / or Graphpad prism 9 software packages, and the data were expressed as Mean ± SEM. Each data was tested for normal distribution. If it was in accordance with normal distribution (P>0.05), one-way analysis of variance was performed. If it was not in accordance with normal distribution (P≤0.05), non-parametric test was performed. Compared with the model group, P<0.05 was considered to be statistically significant. When comparing the two groups, an independent sample t test was performed, and P<0.05 was considered to be statistically significant.

[0183] 4. Test results

[0184] 4.1 Antitussive effect of QAFE on mouse cough model

[0185] As shown in Table 7 and Figure 8As shown in the results, compared with the model control group, the number of coughs within 5 minutes in the high-dose QAFE group, theophylline group, and the Kechuanling granule group was significantly reduced (P < 0.01), and the inhibition rates were 28.65%, 39.89%, and 48.03%, respectively; the low-dose and medium-dose QAFE groups had no significant effect on the number of coughs (P > 0.05), and the inhibition rates were -12.36% and 6.46%, respectively. Compared with the model control group, there was no difference in the cough latency among the groups (P > 0.05).

[0186] Table 7 Effects of QAFE on ammonia-induced cough model in mice

[0187]

[0188] In the table, a: inhibition rate is the inhibition rate compared with the Model group; * P<0.05, ** P<0.01vs model control group.

[0189] 4.2 Antitussive effect of QAFE on guinea pig cough model

[0190] As shown in Table 8 and Fig. 9 As shown in the results, compared with the model control group, the number of coughs within 5 minutes in the QAFE medium-dose, high-dose and Xiaoer Kechuanling granule groups was significantly reduced (P < 0.01 or P < 0.05), and the inhibition rates were 42.67%, 60.00% and 42.22%, respectively; the number of coughs within 5 minutes in the QAFE low-dose group and aminophylline group showed a decreasing trend, but there was no statistical difference (P > 0.05), and the inhibition rates were 25.33% and 31.85%, respectively. Compared with the model control group, there was no difference in the cough latency among the groups (P > 0.05).

[0191] Table 8 Effect of QAFE on citrate-induced cough model in guinea pigs

[0192]

[0193]

[0194] In the table, a: 4 mice died during the administration of aminophylline in the control group; b: The inhibition rate is the inhibition rate compared with the number of coughs in the Model group; * P<0.05, ** P<0.01vs model control group.

[0195] 4.3 Effect of QAFE on SOD and GSH in BALF of coughing guinea pigs

[0196] like Fig.10As shown in the figure, compared with the model group, the SOD activity of the high-dose QAFE group was significantly increased (P < 0.05), and the SOD of the low- and medium-dose QAFE groups, the aminophylline group, and the pediatric Kechuanling group showed an upward trend, but there was no statistical significance (P > 0.05). Compared with the model group, the GSH content of the high-dose QAFE group and the aminophylline group was significantly increased (P < 0.05), and the GSH of the low- and medium-dose QAFE groups and the pediatric Kechuanling group showed an upward trend, but there was no statistical difference (P > 0.05).

[0197] 4.4 Effect of QAFE on serum TNF-α, IL-1β, and SP levels in cough mice

[0198] like Fig.11 As shown in A, compared with the blank group, the serum TNF-α level in the model group was significantly increased (P < 0.05), and the serum TNF-α levels in the high-dose QAFE group, theophylline group and the pediatric Kechuanling group were significantly decreased compared with the model group (P < 0.05).

[0199] like Fig.11 As shown in B, compared with the blank group, the serum IL-1β level in the model group was significantly increased (P < 0.01), and the serum IL-1β level in the QAFE medium and high dose groups, aminophylline group and pediatric Kechuanling group was significantly decreased compared with the model group (P < 0.01).

[0200] like Fig.11 As shown in C, compared with the blank group, the serum SP level in the model group was significantly increased (P < 0.01), and the serum SP level in the QAFE medium and high dose groups, aminophylline group and pediatric Kechuanling group was significantly decreased compared with the model group (P < 0.01 or P < 0.05).

[0201] 4.5 Effect of QAFE on TNF-α and IL-6 mRNA levels in lung tissue of coughing mice

[0202] like Fig.12 As shown in the data, compared with the blank group, the mRNA expression levels of TNF-α and IL-6 in lung tissue of the model group were significantly increased (P < 0.01). Compared with the model group, the mRNA expression levels of TNF-α and IL-6 in the QAFE medium and high dose groups, aminophylline group, and Xiaoer Kechuanling group were significantly decreased (P < 0.05 or P < 0.01).

[0203] 4.6 Effect of QAFE on p-STAT3, IL-17A, and TRPV1 protein expressions in lung tissues of coughing mice

[0204] like Fig.13As shown in AC, compared with the blank group, p-STAT3 / STAT3 and IL-17A in the model group were significantly upregulated. Compared with the model group, p-STAT3 / STAT3 and IL-17A in the high-dose QAFE group, aminophylline group, and Xiaoer Kechuanling group were significantly downregulated.

[0205] like Fig.13 As shown in D, compared with the blank group, TRPV1 in the model group was significantly upregulated, and compared with the model group, TRPV1 levels in the QAFE high-dose group, aminophylline group, and Xiaoer Kechuanling granule group were significantly downregulated.

[0206] The above results suggest that QAFE exerts its antitussive effect by regulating JAK-STAT, IL-17A signaling pathway and TRPV1 protein.

[0207] Example 3

[0208] Quantification of main components of Qufuzhi Fructus Extract (QAFE)

[0209] Quantitative chromatographic conditions: Agilent Extend C18 (5 μm, 4.6×250 nm) was used as the chromatographic column, acetonitrile-0.1% formic acid water (18:82, v / v) was used as the mobile phase for isocratic elution; the flow rate was 1.0 mL / min; the detection wavelength was 330 nm; the column temperature was 30°C; and the injection volume was 10 μL.

[0210] Preparation of reference solution: Take appropriate amount of naringin, naringin, hesperidin, and neohesperidin reference substances, accurately weigh them, add 50% methanol to dissolve them, and prepare mixed reference solutions of about 60, 150, 120, and 120 μg / mL. Preparation of test solution: Take 0.1g of the extract of Citrus aurantium in Example 1, accurately weigh it, put it in a 25mL volumetric flask, add appropriate amount of 50% methanol, ultrasonicate for 20 minutes, cool to room temperature, add 50% methanol to the scale, shake well, centrifuge, and take the supernatant.

[0211] The sample volume extracted was 52.95 kg, with a yield of 35.3%. The identification results of QAFE are shown in Fig.14 The results of content determination showed that the contents of naringin, naringin, hesperidin and neohesperidin were 12.46, 126.70, 6.39 and 82.02 mg / g, respectively. Therefore, the extract of Quzhi Fructus is used as the QAFE quality control standard when preparing medicines for the treatment of acute pharyngitis and cough.

[0212] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of Quzhi Fructus Extract in the preparation of medicines for the treatment of acute pharyngitis.

2. The use according to claim 1, characterized in that: The Quzhi Fructus extract treats acute pharyngitis by inhibiting the PI3K-Akt signaling pathway.

3. The use according to claim 1, characterized in that: The extract of Quzhi Fructus can reduce the number of leukocytes and neutrophils; increase SOD activity, reduce MDA content, increase GSH content; reduce TNF-α, IL-6, IL-1β levels; and reduce the mRNA expression levels of TNF-α and IL-1β.

4. Application of Quzhi Fructus Extract in the preparation of medicine for treating cough.

5. The use according to claim 4, characterized in that: The Quzhi Fructus extract treats cough by regulating JAK-STAT, IL-17A signaling pathway and TRPV1 protein.

6. The use according to claim 4, characterized in that: The Quzhi Fructus extract can reduce the number of coughs; increase SOD activity and GSH content; reduce TNF-α, IL-1β, and SP levels; reduce the mRNA expression levels of TNF-α and IL-6; increase the protein expression levels of p-STAT3 / STAT3 and IL-17A and down-regulate the protein expression level of TRPV1.

7. The use according to any one of claims 1 to 6, characterized in that: The preparation process of the Fructus Aurantii Immaturus extract comprises the following steps: (1) Extract the dried pieces of Quzhi Fructus with reflux extraction using slightly boiling hot water, filter, and combine the filtrate; (2) The filtrate is concentrated under reduced pressure, dried in vacuum, and crushed to obtain the Fructus Aurantii Immaturus extract.

8. The use according to claim 7, characterized in that: In the step (1), the amount of water added in the water reflux extraction is 8-12 times the amount of the Quzhi Fructus decoction pieces, the number of extractions is 1-3 times, and the extraction time is 0.5-1.5h.

9. The use according to claim 7, characterized in that: The temperature for reduced pressure concentration in step (2) is 60-80°C, and the temperature for vacuum drying is 50-70°C.

10. The use according to claim 7, characterized in that: In the Quzhi Fructus extract, the content of naringin is 8.7-16.2 mg / g, the content of naringin is 88.7-164.7 mg / g, the content of hesperidin is 4.5-8.3 mg / g, and the content of neohesperidin is 57.4-106.6 mg / g.

Images