Fingerprint detection method for shuanghuanglian traditional Chinese medicine composition and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicines, and discloses a fingerprint spectrum detection method of Shuanghuanglian traditional Chinese medicine composition and application thereof. The fingerprint spectrum detection method is as follows: high performance liquid chromatography is used to analyze test sample solution and control sample solution; and a fingerprint spectrum is obtained by processing the color spectrum by using a fingerprint spectrum software; in the liquid chromatography analysis, gradient elution is performed by using a mobile phase, and the mobile phase is as follows: the A phase includes phosphoric acid aqueous solution, and the B phase includes acetonitrile and methanol. The fingerprint spectrum detection method can calibrate at least 22 chromatographic peaks under different detection wavelengths, and identify 10 kinds of effective components such as forsythia ester glycoside E, chlorogenic acid and coffee acid, and simultaneously perform quantitative analysis on multiple components; the fingerprint spectrum detection method can make each batch of medicines have stable and reliable therapeutic effects. The Shuanghuanglian traditional Chinese medicine composition screened by the quality control and having specific active component content characteristics can be used for preparing medicines for reducing susceptibility to influenza.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a fingerprint spectrum detection method for a Shuanghuanglian traditional Chinese medicine composition and application thereof. BACKGROUND

[0002] Influenza, commonly known as flu, is a common acute respiratory infectious disease caused by influenza virus. After the human body is infected with the influenza virus, the early clinical symptoms are mainly upper respiratory tract infection symptoms such as sneezing, runny nose, fever, and some patients may develop viral pneumonia, and then cause acute lung injury, and eventually lead to death. Western medicine often uses antipyretic analgesics to relieve fever, headache and other symptoms caused by influenza, and the commonly used drugs are non-steroidal drugs such as ibuprofen, acetaminophen and naproxen. Although these drugs have a rapid antipyretic effect, they have a large toxic and side effect, and common symptoms include nausea, vomiting, diarrhea, dizziness, lethargy, insomnia, skin rash and the like. Traditional Chinese medicine has a good effect and high safety in the treatment of fever, and has a broad application prospect.

[0003] Influenza belongs to the category of warm disease in traditional Chinese medicine, and is an acute febrile disease caused by exogenous warm pathogen. The patient has a rapid onset, and shows high fever, and severe cases may develop into pneumonia, asthma, and even cause death. Commonly used traditional Chinese medicine compounds include relieving superficies, tonifying qi, and clearing heat and detoxifying. Shuanghuanglian is a well-known traditional Chinese medicine compound for clearing heat, which is composed of three traditional Chinese medicines, namely, honeysuckle (Lonicera japonica Thunb), forsythia (Forsythia suspensa) and scutellaria (Scutellaria baicalensis Georgi). It has the effect of clearing heat and detoxifying, and is often used in the treatment of symptoms such as headache and fever caused by upper respiratory tract infection. It has been on the market for many years and has a definite effect. However, due to factors such as production area, growth period, harvesting period and planting and processing technology, the quality of medicinal materials is highly variable, resulting in unstable chemical components and poor quality consistency of different batches of preparations. Therefore, it is a challenge for the production of Shuanghuanglian traditional Chinese medicine to develop a reasonable limit range for effective components while ensuring efficacy.

[0004] Most of the anti-influenza virus drugs used in clinical practice directly act on the virus to exert therapeutic effect, such as influenza virus M2 ion channel inhibitors (amantadine) and neuraminidase (NA) inhibitors (oseltamivir). However, more and more studies have shown that host factors are an important factor that cannot be ignored in the influence of influenza infection. Studies have found that emotional stress can cause multiple organ damage in the human body, leading to increased susceptibility to various diseases, and the mortality rate significantly increases after influenza infection. Restraint stress load can increase the susceptibility of the body to various diseases, among which the influence on influenza virus infection is more significant. Therefore, it is also of great significance to develop a drug that can reduce the susceptibility of the body to influenza virus. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a fingerprint detection method for Shuanghuanglian traditional Chinese medicine composition and application thereof. The fingerprint detection method for Shuanghuanglian traditional Chinese medicine composition provided by the present application can detect different batches of medicines to ensure the stability of chemical components and the consistency of quality.

[0006] The present application provides a fingerprint detection method for Shuanghuanglian traditional Chinese medicine composition.

[0007] Specifically, the fingerprint detection method for Shuanghuanglian traditional Chinese medicine composition comprises the following steps:

[0008] Preparation of test solution of Shuanghuanglian traditional Chinese medicine composition;

[0009] Preparation of reference solution: linaria esterside E, chlorogenic acid, caffeic acid, linaria esterside I, linaria esterside A, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, baicalin, and forsythia glycoside are dissolved in a solvent to prepare a reference mixture solution;

[0010] High performance liquid chromatography analysis of the test solution and the reference solution to obtain the color spectrum of Shuanghuanglian traditional Chinese medicine composition and the chromatogram of the reference;

[0011] Processing the color spectrum of Shuanghuanglian traditional Chinese medicine composition and the reference by fingerprint software to obtain the fingerprint of Shuanghuanglian traditional Chinese medicine composition;

[0012] In the high performance liquid chromatography analysis, octadecylsilane-bonded silica gel is used as the filler; gradient elution is performed using a mobile phase, and the mobile phase comprises phase A and phase B, wherein phase A comprises 0.05% to 0.25% phosphoric acid aqueous solution, and phase B comprises acetonitrile and methanol.

[0013] Preferably, in the B phase, the volume ratio of acetonitrile to methanol is (8-10):1; further preferably, in the B phase, the volume ratio of acetonitrile to methanol is (8.5-9.5):1.

[0014] Preferably, the A phase further comprises heptane sulfonate with a molar concentration of 0.0025-0.01 mol / L; further preferably, the A phase further comprises heptane sulfonate with a molar concentration of 0.003-0.008 mol / L. For example, sodium heptane sulfonate. The composition of the sample mobile phase is optimized and selected with the peak relative fingerprint information index TQ as the optimization objective function. The more the number of peaks, the better the homogenization, and the larger the index TQ value. Research has found that the addition of heptane sulfonate not only improves the TQ value, but also, in combination with the analysis of chromatogram peak type and fingerprint peak separation degree, the addition of ionic reagent (heptane sulfonate) is beneficial to the separation of fingerprint peaks.

[0015] Preferably, in the A phase, the mass concentration of the aqueous phosphoric acid solution is 0.1% to 0.25%; further preferably, in the A phase, the mass concentration of the aqueous phosphoric acid solution is 0.15% to 0.25%.

[0016] Preferably, the process of the gradient elution is as follows:

[0017] 0-5 min, the volume percentage of the B phase in the mobile phase is increased from 4% to 6% to 8% to 10%;

[0018] 5-10 min, the volume percentage of the B phase in the mobile phase is increased from 8% to 10% to 11% to 12%;

[0019] 10-12 min, the volume percentage of the B phase in the mobile phase is increased from 11% to 12% to 14% to 16%;

[0020] 12-20 min, the volume percentage of the B phase in the mobile phase is increased from 14% to 16% to 18% to 20%;

[0021] 20-25 min, the volume percentage of the B phase in the mobile phase is increased from 18% to 20% to 21% to 22%;

[0022] 25-30 min, the volume percentage of the B phase in the mobile phase is increased from 21% to 22% to 24% to 27%;

[0023] 30-45 min, the volume percentage of the B phase in the mobile phase is increased from 24% to 27% to 30% to 34%;

[0024] 45-50 min, the volume percentage of the B phase in the mobile phase is increased from 30% to 34% to 43% to 48%;

[0025] 50-55 min, the volume percentage of the B phase in the mobile phase is decreased from 43% to 48% to 4% to 6%.

[0026] Further preferably, the process of the gradient elution is as follows:

[0027] 0-5 min, the volume percentage of the B phase in the mobile phase is increased from 6% to 10%;

[0028] 5-10 min, the volume percentage of the B phase in the mobile phase is increased from 6% to 10%;

[0029] 10-12 min, the volume percentage of the B phase in the mobile phase is increased from 12% to 15%;

[0030] 12-20 min, the volume percentage of the B phase in the mobile phase is increased from 15% to 20%;

[0031] 20-25 min, the volume percentage of phase B in the mobile phase is increased from 20% to 21%;

[0032] 25-30 min, the volume percentage of phase B in the mobile phase is increased from 21% to 25%;

[0033] 30-45 min, the volume percentage of phase B in the mobile phase is increased from 25% to 32%;

[0034] 45-50 min, the volume percentage of phase B in the mobile phase is increased from 32% to 45%;

[0035] 50-55 min, the volume percentage of phase B in the mobile phase is decreased from 45% to 6%.

[0036] It is found that the gradient elution condition can effectively improve the fingerprint peak shape and the fingerprint information index TQ value is larger.

[0037] In the high performance liquid chromatography analysis, the detection wavelength is 220 nm, 326 nm.

[0038] Preferably, in the control sample mixed solution, the concentration of chlorogenic acid is 110-140 μg / mL, the concentration of isochlorogenic acid C is 40-50 μg / mL, the concentration of forsythoside I is 70-90 μg / mL, the concentration of caffeic acid is 5-20 μg / mL, the concentration of forsythoside A is 240-360 μg / mL, the concentration of forsythoside is 30-50 μg / mL, and the concentration of forsythoside E is 50-80 μg / mL; further preferably, in the control sample mixed solution, the concentration of chlorogenic acid is 115-130 μg / mL, the concentration of isochlorogenic acid C is 45-50 μg / mL, the concentration of forsythoside I is 75-90 μg / mL, the concentration of caffeic acid is 5-15 μg / mL, the concentration of forsythoside A is 250-350 μg / mL, the concentration of forsythoside is 30-45 μg / mL, and the concentration of forsythoside E is 56-75 μg / mL.

[0039] The application further provides a Shuanghuanglian traditional Chinese medicine composition, which is subjected to quality control according to the above fingerprint detection method, and contains active ingredients obtained by extraction and purification of Scutellaria baicalensis Georgi, Lonicera japonica and Forsythia suspensa; in terms of mass percentage, the content of baicalin in the active ingredients is greater than 8.68‰, the content of chlorogenic acid is greater than 0.46‰, the content of forsythoside is greater than 0.26‰, the content of forsythoside E is greater than 0.39‰, the content of caffeic acid is greater than 0.03‰, the content of forsythoside I is greater than 0.32‰, the content of forsythoside A is greater than 1.24‰, and the content of isochlorogenic acid C is greater than 0.21‰.

[0040] Preferably, the content of baicalin in the active ingredient is 10.85-11.97 ‰, the content of chlorogenic acid is 0.58-0.90 ‰, the content of forsythoside is 0.33-0.37 ‰, the content of forsythoside E is 0.49-0.66 ‰, the content of caffeic acid is 0.04-0.06 ‰, the content of forsythoside I is 0.40-0.63 ‰, the content of forsythoside A is 1.54-2.13 ‰, and the content of isochlorogenic acid C is 0.26-0.36 ‰.

[0041] Preferably, the similarity of the fingerprint spectrum of the Shuanghuanglian traditional Chinese medicine composition to the control fingerprint spectrum is greater than 0.9.

[0042] Preferably, the dosage form of the Shuanghuanglian traditional Chinese medicine composition is one of extracts, decoctions, soft capsules, pills, oral liquids, tinctures, syrup agents, suppositories, gels, and sprays.

[0043] The application also provides the use of the Shuanghuanglian traditional Chinese medicine composition.

[0044] Specifically, the Shuanghuanglian traditional Chinese medicine composition is used in the preparation of a medicine for reducing the susceptibility to influenza.

[0045] It is found that the Shuanghuanglian traditional Chinese medicine composition can significantly reduce the susceptibility of mice to influenza virus, effectively restore the response of the RIG1-MAVS signal pathway of influenza virus H1N1 infection additional restraint stress inhibition, and restore the expression of IFN-β in blood and lungs by using the above fingerprint spectrum detection method for quality control of the Shuanghuanglian traditional Chinese medicine composition and controlling the content of baicalin in the active ingredient to be greater than 8.68 ‰, the content of chlorogenic acid to be greater than 0.46 ‰, the content of forsythoside to be greater than 0.26 ‰, the content of forsythoside E to be greater than 0.39 ‰, the content of caffeic acid to be greater than 0.03 ‰, the content of forsythoside I to be greater than 0.32 ‰, the content of forsythoside A to be greater than 1.24 ‰, and the content of isochlorogenic acid C to be greater than 0.21 ‰.

[0046] The application also provides a medicine for reducing the susceptibility to influenza, which comprises the Shuanghuanglian traditional Chinese medicine composition.

[0047] Compared with the prior art, the application has the following beneficial effects:

[0048] (1) The fingerprint detection method provided by the present application can mark at least 22 chromatographic peaks under different detection wavelengths, and identify 10 kinds of effective components such as forsythiaside E, chlorogenic acid, caffeic acid, forsythiaside I, forsythiaside A, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, baicalin and forsythoside, and simultaneously quantitative analysis of multiple components. The fingerprint detection method has the advantages of comprehensive detection components, simple operation, stable and reliable method; the fingerprint detection method can be used for controlling the internal quality of Shuanghuanglian traditional Chinese medicine composition, so that each batch of drugs has stable and reliable therapeutic effect.

[0049] (2) The Shuanghuanglian traditional Chinese medicine composition screened by the quality control method of the present application has specific active ingredient content characteristics, can significantly reduce the susceptibility of mice to influenza virus, and can be used for preparing a drug for reducing the susceptibility to influenza. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Fingerprint of forty batches of Shuanghuanglian traditional Chinese medicine composition;

[0051] Figure 2 Fingerprint of reference substance;

[0052] Figure 3 Influence diagram of Shuanghuanglian traditional Chinese medicine composition on morbidity and survival rate of restraint stress H1N1 infected mice;

[0053] Figure 4 Influence diagram of Shuanghuanglian traditional Chinese medicine composition on lung index, spleen index and lung injury of restraint stress H1N1 infected mice;

[0054] Figure 5 Influence diagram of Shuanghuanglian traditional Chinese medicine composition on lung tissue pathological changes of restraint stress H1N1 infected mice;

[0055] Figure 6 Influence diagram of Shuanghuanglian traditional Chinese medicine composition on viral protein NP and gene NP of restraint stress H1N1 infected mice;

[0056] Figure 7 Influence diagram of Shuanghuanglian traditional Chinese medicine composition on inflammatory factors of restraint stress H1N1 infected mice;

[0057] Figure 8 Influence diagram of Shuanghuanglian traditional Chinese medicine composition on antiviral protein ALOX15 of restraint stress H1N1 infected mice;

[0058] Figure 9 Influence diagram of Shuanghuanglian traditional Chinese medicine composition on response to RIG1-MAVS antiviral innate immune signaling pathway. DETAILED DESCRIPTION

[0059] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are listed for illustration. It should be pointed out that the following examples do not constitute a limitation on the scope of protection required by the present application.

[0060] The raw materials, reagents or devices used in the following examples, if not specifically stated, can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0061] Example 1: Preparation of Shuanghuanglian traditional Chinese medicine composition

[0062] The preparation method is described in Chinese Pharmacopoeia 2020 edition. Specifically, take 375 parts by weight of Huangqin and decoct three times, the first time for 2 hours, and the second and third times for 1 hour each. Combine the decoction, filter, concentrate the filtrate, and at 80°C, add an appropriate amount of 2 mol / L hydrochloric acid solution to adjust the pH value to 1.0-2.0, and keep warm for 1 hour. Stand for 12 hours, filter, add 6-8 times the amount of water to the precipitate, adjust the pH value to 7.0 with 40% sodium hydroxide solution, and then add an equal volume of ethanol. Stir to dissolve, filter, adjust the pH value of the filtrate to 2.0 with 2 mol / L hydrochloric acid solution, keep warm at 60°C for 30 minutes, stand for 12 hours, filter, wash the precipitate with ethanol until the pH value is 7.0, and recover the ethanol for future use. Take 375 parts by weight of Jinyinhua and 750 parts by weight of Lianqiao, warm soak in water for 30 minutes, then decoct twice, each time for 1.5 hours. Combine the decoction, filter, and concentrate the filtrate to a clear paste with a relative density of 1.20-1.25 (70-80°C). Slowly add ethanol when the temperature cools to 40°C to make the alcohol content reach 75%, stir thoroughly, stand for 12 hours, filter the supernatant, add an appropriate amount of 75% ethanol to the residue, stir evenly, stand for 12 hours, filter, combine the ethanol solutions, recover the ethanol until there is no alcohol taste, add the Huangqin extract described above, and add an appropriate amount of water to adjust the pH value to 7.0 with 40% sodium hydroxide solution. Stir well, store in a cool place (4-8°C) for 72 hours, filter, add 300 parts by weight of sucrose to the filtrate, stir to dissolve, add an appropriate amount of essence, adjust the pH value to 7.0, add water to make 1000 volumes, stir well, stand for 12 hours, filter, fill, sterilize, and you get it. Volume parts / weight parts correspond to ml / g.

[0063] Example 2: Eight-component quantitative detection of Shuanghuanglian traditional Chinese medicine composition

[0064] 1. Instruments and reagents

[0065] Agilent 1100 liquid chromatograph; Agilent OpenLAB CDS Chemstation (Edition C.01.07) network workstation (Agilent Technologies); Sarturius-BS110S analytical balance (Beijing Sartorius Balance Co., Ltd.); ES-E120D electronic analytical balance (Tianjin Deantest Sensing Technology Co., Ltd.); Shuanghuanglian traditional Chinese medicine composition prepared in Example 1; eight component reference substances: forsythiaside E, forsythin, chlorogenic acid, isochlorogenic acid C, baicalin, caffeic acid, forsythiaside I, and forsythiaside A.

[0066] 2. Preparation of reference substance solution

[0067] An appropriate amount of chlorogenic acid reference substance, isochlorogenic acid C reference substance, forsythiaside I reference substance, caffeic acid reference substance, forsythiaside A reference substance, forsythin reference substance, and forsythiaside E reference substance were precisely weighed and dissolved in methanol to prepare a mixed reference substance solution containing 122 μg of chlorogenic acid, 48 μg of isochlorogenic acid C, 81 μg of forsythiaside I, 7 μg of caffeic acid, 311 μg of forsythiaside A, 37 μg of forsythin, and 69 μg of forsythiaside E per 1 mL, and then the mixture was shaken to obtain the solution.

[0068] 3. Preparation of test substance solution

[0069] 2.5 mL of Shuanghuanglian traditional Chinese medicine composition prepared in Example 1 was precisely pipetted into a 20 mL volumetric flask, diluted to the mark, filtered through a 0.45 μm filter membrane, and then obtained.

[0070] 4. Chromatographic conditions

[0071] Condition 1: octadecylsilane-bonded silica gel as the filler; 0.2% phosphoric acid-water solution (containing 0.005 mol / L sodium heptanesulfonate) as mobile phase A, acetonitrile-methanol (9:1) solution as mobile phase B, gradient elution, gradient elution program: 0-5 min, 6%→10% B; 5-10 min, 10%→12% B; 10-12 min, 12%→15% B; 12-20 min, 15%→20% B; 20-25 min, 20%→21% B; 25-30 min, 21%→25% B, 30-45 min, 25%→32% B, 45-50 min, 32%→45% B; 50-55 min, 45%→6% B, detection wavelength: 220 nm, 326 nm (seven components including forsythiaside E);

[0072] Condition 2: octadecylsilane-bonded silica gel as the filler; methanol-water-glacial acetic acid (50:50:1) as the mobile phase; detection wavelength: 274 nm (baicalin).

[0073] 5. Determination

[0074] Accurately pipette 5 μl of the mixed control solution and the sample solution into the liquid chromatograph, and record the chromatogram. The similarity degree should be greater than 0.9. The peak area and retention time of the control fingerprint are shown in Table 2.

[0075] The eight-component content of forty batches of Shuanghuanglian traditional Chinese medicine composition was detected according to the above method.

[0076] Table 1 Eight-component content of forty batches of Shuanghuanglian traditional Chinese medicine composition (‰)

[0077]

[0078]

[0079] Example 3: Fingerprint construction

[0080] 1. Preparation of the control solution, accurately take chlorogenic acid control, isochlorogenic acid A control, isochlorogenic acid B control, isochlorogenic acid C control, forsythia ester glycoside I control, coffee acid control, forsythia ester glycoside A control, forsythia glycoside control, forsythia ester glycoside E control, and accurately weigh, add methanol to prepare 1 mL of mixed control solution containing 122 μg of chlorogenic acid, 200 μg of isochlorogenic acid A, 200 μg of isochlorogenic acid B, 48 μg of isochlorogenic acid C, 81 μg of forsythia ester glycoside I, 7 μg of coffee acid, 311 μg of forsythia ester glycoside A, 37 μg of forsythia glycoside, and 69 μg of forsythia ester glycoside E, shake well, and obtain.

[0081] 2. Preparation of the sample solution, the same as in Example 2.

[0082] 3. Chromatographic conditions. The same as in Example 2, chromatographic condition 1, detection wavelength 220 nm.

[0083] 4. Determination, accurately pipette 5 μl of the mixed control solution and the sample solution into the liquid chromatograph, and record the chromatogram. The sample fingerprint ( Figure 1 ) and the control fingerprint ( Figure 2 ), with not less than 22 fingerprint peaks as indicated in the control fingerprint diagram as the reference correction peak, calculated by the calculation software, the similarity degree should be greater than 0.9. The peak area and retention time of the control fingerprint are shown in Table 2.

[0084] Table 2 Peak area and retention time of the control fingerprint

[0085]

[0086] Example 4: Efficacy test

[0087] Based on the eight-component content range determined in Example 2 and the control fingerprint information determined in Example 3, the Shuanghuanglian traditional Chinese medicine composition with the content characteristics of active ingredients and the similarity meeting the conditions is selected for efficacy evaluation. Specifically, the content of active ingredients of the Shuanghuanglian traditional Chinese medicine composition meets: baicalin 10.85-11.97‰, chlorogenic acid 0.58-0.90‰, forsythoside 0.33-0.37‰, forsythoside E 0.49-0.66‰, caffeic acid 0.04-0.06‰, forsythoside I 0.40-0.63‰, forsythoside A 1.54-2.13‰, and isochlorogenic acid C 0.26-0.36‰. And the similarity of the fingerprint spectrum compared with the control fingerprint spectrum is greater than 0.9.

[0088] Based on the lung heat model built by emotional stress plus influenza virus infection, the heat-clearing efficacy of the Shuanghuanglian traditional Chinese medicine composition is evaluated. The emotional stress mouse model is selected to simulate the emotional stress state, and the lung heat model with increased susceptibility to influenza virus is successfully established, and the heat-clearing efficacy of the above Shuanghuanglian traditional Chinese medicine composition is evaluated.

[0089] 1. Experimental materials

[0090] The Shuanghuanglian traditional Chinese medicine composition of the present application is self-made by Henan Fuxin Pharmaceutical Co., Ltd., water, and oseltamivir (Oseltamivir).

[0091] 4-week-old male SPF Kunming mice were purchased from the Experimental Animal Center of Southern Medical University, license number SCXK (Yue) 2021-0041, and were raised and dosed under the conditions of alternating day and night (12h / 12h) and constant temperature (23±2)℃, constant humidity (50%±10%).

[0092] 2. Animal grouping and dosing

[0093] Normal control group (Control), virus control group (H1N1), virus + restraint stress model group (H1N1+Stress), virus + restraint stress + western medicine positive drug oseltamivir (Oseltamivir), virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition low-dose administration group (SHL-L), virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition medium-dose administration group (SHL-M), virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition high-dose administration group (SHL-H), 10 in each group. The specific method steps are as follows:

[0094] (1) From three days before infection with virus, normal control group (Control), virus control group (H1N1) and virus + restraint stress model group (H1N1+Stress) were given water with the same volume and concentration. The virus + restraint stress + western medicine positive drug Oseltamivir, virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition low-dose administration group (SHL-L), virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition medium-dose administration group (SHL-M), virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition high-dose administration group (SHL-H) were given Oseltamivir (30.75 mg / kg / d), low-dose Shuanghuanglian traditional Chinese medicine composition (36.9 g crude drug / kg / d), medium-dose Shuanghuanglian traditional Chinese medicine composition (73.8 g crude drug / kg / d), high-dose Shuanghuanglian traditional Chinese medicine composition (147.8 g crude drug / kg / d), respectively. On the second day after administration, the mice were restrained for 18 h (from 18:00 in the afternoon to 12:00 the next morning), and the restraint device for the mice was a well-ventilated 50 mL sharp-bottomed polypropylene plastic centrifuge tube. At the same time, the food and water of the non-stressed control mice were deprived. After restraint, on the third day of recovery, the mice were lightly anesthetized with ether and infected with 2xLD50 of H1N1 (A / FM / 1 / 47) influenza virus liquid (35 μL per mouse) by nasal instillation, and the control group was given an equal amount of PBS solution by nasal instillation.

[0095] (2) H1N1 infection of mice: 2xLD50 of H1N1 (A / FM / 1 / 47) influenza virus liquid (35 μL).

[0096] (3) Detection index: From the first day after the mice were infected with influenza virus, the body weight of the mice was measured every day at a fixed time, and the symptoms of the mice in each group were observed and recorded (when the mice showed a decrease in appetite, a decrease in body weight of more than 1 g compared with the previous day, and symptoms such as changes in piloerection, kyphosis or breathing pattern, etc., they were determined to be sick), the time of onset, the time of death, and continuous observation for 21 days.

[0097] 3. Data analysis

[0098] Statistical analysis of data was performed using Graphpad 8.0 software, and data were expressed as Mean ± SD. Significant differences between two groups were compared by Student’s t test, and significant differences between multiple groups were compared by one-way ANOVA. P<0.05 indicated a significant difference.

[0099] 4. Results

[0100] The influenza virus additional restraint stress mouse pneumonia model was established. In the usual observation, it was found that the mice began to show symptoms of viral infection on the first day of viral infection, such as listlessness, weakness in limbs, decreased appetite, hunched back, respiratory changes, ruffled fur, and slow movement. The symptoms of viral infection were most obvious on the sixth day of viral infection. The health status of the mice was observed to verify the heat-clearing effect of the composition. The incidence of the mice was evaluated based on the changes in body weight and activity of the mice, and the results are shown in Table 1. Figure 3 Figure 3 A and B are the incidence of mice, Figure 3 C and D are the survival rate of mice for 21 days. From Figure 3 A and B, it can be seen that the incidence of mice in the virus + restraint stress model group (H1N1 + Stress) reached the highest value on the fourth day after influenza virus infection, and the three different dose groups can delay and reduce the incidence of mice. From Figure 3 C and D, it can be seen that the survival rate of the virus control group (H1N1) is 60%, the survival rate of the virus + restraint stress model group (H1N1 + Stress) is 20%, the survival rate of the positive drug oseltamivir group (Oseltamivir) is 90%, the survival rate of the low-dose drug group (SHL-L) is 60%, the survival rate of the medium-dose drug group (SHL-M) is 70%, and the survival rate of the high-dose drug group (SHL-H) is 90%; compared with the model group (H1N1 + Stress), the three different dose groups can significantly improve the survival rate of H1N1 additional restraint stress mice.

[0101] (II) Based on the lung heat model built by adding emotional stress and influenza virus infection, the heat-clearing mechanism of Shuanghuanglian traditional Chinese medicine composition was evaluated

[0102] 1. Experimental materials

[0103] The Shuanghuanglian traditional Chinese medicine composition of the present application is self-made by Henan Fuxin Pharmaceutical Co., Ltd., water, and oseltamivir. 4-week-old male SPF Kunming mice were purchased from the Experimental Animal Center of Southern Medical University, license number SCXK (Yue) 2021-0041, and were raised and administered under the conditions of alternating day and night (12h / 12h) and constant temperature (23±2)℃, constant humidity (50%±10%).

[0104] 2. Experimental method

[0105] 2.1 Animal grouping and administration

[0106] ​Control, virus control (H1N1), virus + restraint stress model (H1N1 + Stress), virus + restraint stress + western medicine positive drug Oseltamivir, virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition low-dose administration group (SHL-L), virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition medium-dose administration group (SHL-M), virus + restraint stress + Shuanghuanglian traditional Chinese medicine composition high-dose administration group (SHL-H), 6 in each group. The specific method steps are as follows:

[0107] Virus infection: same as above

[0108] (2) H1N1 infection of mice: 2xLD50 of H1N1 (A / FM / 1 / 47) influenza virus liquid (35 μL).

[0109] (3) Detection index: On the 4th day after the mice were infected with influenza virus, the lung tissue of the mice was taken by thoracotomy. HE staining was used to detect the pathological changes of the brain tissue of the virus-infected mice. q-RCR was used to detect the expression of H1N1 virus gene NP and lipoxygenase ALOX15 in the lung tissue of the mice. Western blot was used to detect the expression of H1N1 virus protein NP and lipoxygenase ALOX15 in the lung tissue of the mice.

[0110] 2.2 Data analysis of Western blot experiment for detecting the expression of virus protein NP

[0111] (1) Preparation of protein sample: for tissue samples, add protein lysis solution at a ratio of 1:10 (g / mL), add 2 steel balls per tube, and homogenize with a full-automatic tissue homogenizer for 2 min. For cell samples, after PBS rinsing, centrifuge at 1500 rpm for 5 min, discard the supernatant, collect the cells and add an appropriate amount of protein lysis solution. After ultrasonic crushing, the samples are placed on ice for lysis for 30 min, and during this period, vortex for 30 s every 5 min to fully lyse the samples. Then, centrifuge the samples at 4°C, 12000 rpm, for 10 min, and collect the supernatant. After determining the protein concentration with a kit, add 1 / 4 volume of 5x loading buffer to the protein solution, heat denaturation at 100°C for 10 min, and store at -80°C or use for electrophoresis analysis.

[0112] (2) SDS-PAGE electrophoresis: load 30-50 μg of total protein. When compressing the gel of the base layer, use a voltage of 60 V, and electrophorese for 20-30 min; when separating the gel of the lower layer, use a voltage of 120 V, and electrophorese for 90-120 min. After electrophoresis, perform the membrane transfer operation at a constant current of 300 mA for 60-90 min.

[0113] (3) Antibody incubation: Block the protein band with 5% skim milk at room temperature for 1-2 hours to remove non-specific binding. Then, place the band to be detected in the corresponding primary antibody dilution buffer and dilute according to the dilution ratio on the antibody instructions: NP (1:1000) and GAPDH (1:3000). Incubate overnight at 4°C. The next day, wash 5 times with TBST for 8 minutes each time. Then, incubate the band with the corresponding HRP-labeled secondary antibody dilution buffer (1:5000) at room temperature for 2 hours. Finally, wash with TBST to remove unbound secondary antibody.

[0114] (4) ECL chemiluminescence detection: Prepare ECL working solution by mixing solution A and solution B in a 1:1 ratio, immerse the band completely in the working solution, and expose it in a chemiluminescence imager to detect protein expression.

[0115] 2.3 Western blot analysis to detect the expression level of lipoxygenase ALOX15 in lung tissue

[0116] The experimental method is the same as in 2.2.

[0117] 3. Data Analysis

[0118] Statistical analysis was performed on the data using Graphpad 8.0 software, and the data are expressed as Mean ± SD. Significant differences between two groups were compared using Student's t-test, and significant differences among multiple groups were analyzed using one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0119] 4. Results

[0120] 4.1 Lung index, spleen index, and lung tissue pathological changes in mice

[0121] The experimental procedure is as follows Figure 4 As shown in Figure A, on the 4th day after infection with the virus, the mice were weighed and euthanized by exsanguination. The lungs and spleen were then removed via thoracotomy. The organs were washed twice with 0.9% saline solution, then blotted dry with filter paper. They were weighed using an analytical balance, and the lesions in the lung tissue were observed visually. The spleen and lung indices were then calculated using the following formulas: Spleen index = Spleen weight / Body weight (mg / g); Lung index = Lung weight / Body weight (mg / g). The organ indices showed that compared to the virus control group (H1N1), the lung index of the model group (H1N1+Stress) mice was significantly increased, while the spleen index showed no significant change. This indicates that the model group (H1N1+Stress) mice exhibited severe pulmonary edema, and the three different dosage groups effectively improved this pathological symptom. Figure 4 (B, C). In an experiment evaluating indicators related to influenza virus pneumonia in mice, lung morphology examination showed that the three different dosage groups effectively improved the symptoms of pulmonary congestion in mice.Figure 4 (D). Results of HE staining of transverse lung tissue sections are as follows: Figure 5 As shown, by Figure 5 It can be seen that the normal control group (Control) showed no inflammatory cell infiltration; compared with the virus control group (H1N1), the model group (H1N1+Stress) showed severe alveolar hemorrhage and inflammatory infiltration, and severe tissue damage. The three different dose groups could effectively improve the pathological damage of the lungs of mice subjected to influenza virus H1N1 plus restraint stress.

[0122] 4.2 Detection of influenza virus replication in the lungs

[0123] In this experiment, RNA was extracted from mouse lung tissue on day 4 after viral infection, and changes in viral gene NP levels were detected by q-PCR. The results are as follows: Figure 6 As shown in Figure B, compared with the virus control group (H1N1), the transcriptional expression of viral gene NPs in the model group (H1N1+Stress) was significantly increased; compared with the model group (H1N1+Stress), the transcriptional expression of NPs in the three different dose groups and the positive control drug oseltamivir was decreased. Simultaneously, the expression of H1N1 protein NPs was detected by Western blot analysis of proteins extracted from mouse lung tissue. NPs can reflect the replication status of H1N1 virus in tissues, and the results are shown below. Figure 6 As shown in Figure A. From Figure 6 As shown in Figure A, compared with the virus control group (H1N1), the expression of viral protein NP in the lung tissue of the model group (H1N1+Stress) was significantly upregulated; compared with the model group (H1N1+Stress), the expression of NP was downregulated in the three different dose groups and the positive control drug oseltamivir. Figure 6 The expression of influenza virus protein NP in the lung tissue of mice in each group was detected by immunohistochemistry. The results showed that the expression and distribution of NP protein in the lung tissue of mice in the three different doses of Shuanghuanglian oral liquid were significantly less than those in the model group (H1N1+Stress).

[0124] The above experimental results indicate that restraint stress increases viral replication in the lung tissue of H1N1-infected mice, and administration of the composition can effectively reduce the expression of viral genes and proteins in mouse lung tissue and inhibit viral replication.

[0125] 4.3 Effects on the expression of inflammatory factors in the lungs of mice under restraint stress from H1N1 influenza virus infection load

[0126] The changes in inflammatory factors TNF-α, IL-6, and IL-1β in lung tissue of each group were detected by q-PCR, and the results are as follows: Figure 7 As shown. Figure 7Figures A, B, and C are graphs showing the effects of the test drugs on the expression of inflammatory factors TNF-a, IL-6, and IL-1b, respectively. Figure 7 It can be seen that the TNF-a, IL-6, and IL-1b in the normal control group (Control) were at very low levels. After infection with the influenza virus H1N1, the expression of TNF-a, IL-6, and IL-1b increased. Compared with the virus control group (H1N1), the levels of TNF-a, IL-6, and IL-1b in the model group (H1N1+Stress) increased more obviously. The three different dose groups and the western medicine positive drug Oseltamivir (Oseltamivir) could effectively reduce the expression of inflammatory factors and alleviate the inflammatory response in the lung tissue of the influenza virus infection restraint stressed mice.

[0127] 4.4 Effects on lipoxygenase ALOX15

[0128] Previous studies have shown that the expression level changes significantly in the emotional stress mouse model and that ALOX15 has a positive regulatory effect on the RIG1-MAVS antiviral signaling pathway. Studies have also reported that the catalytic product of ALOX15, protectin D1 (PD1), significantly inhibits the nuclear export of influenza virus RNA and improves the survival rate and pathological changes of severe influenza mice, which suggests the importance of ALOX15 in the antiviral response. In this experiment, on the fourth day after the mice were infected with the virus, the protein of the mouse lung tissue was extracted, and the changes in the protein level of lipoxygenase ALOX15 were detected by Western Blot. The results are shown in Figure 8 Compared with the normal control group (Control), the expression of ALOX15 protein in the virus control group (H1N1) increased significantly. Compared with the virus control group (H1N1), the expression of ALOX15 in the model group (H1N1+Stress) decreased significantly, while the three different dose groups and the western medicine positive drug Oseltamivir (OSTW) could effectively increase the expression of ALOX15. At the same time, the RNA in the mouse lung tissue was extracted to detect the changes in the transcription level of ALOX15 by q-PCR. The results are shown in Figure 8 Compared with the normal control group (Control), the expression of ALOX15 protein in the virus control group (H1N1) increased significantly. Compared with the virus control group (H1N1), the expression of ALOX15 in the model group (H1N1+Stress) decreased significantly, while the three different dose groups and the western medicine positive drug Oseltamivir (OSTW) could effectively increase the expression of ALOX15. At the same time, the RNA in the mouse lung tissue was extracted to detect the changes in the transcription level of ALOX15 by q-PCR. The results are shown in

[0129] 4.5 Restoring the response to RIG1-MAVS antiviral innate immune signaling pathway

[0130] According to the foregoing experimental results, it is known that the expression of ALOX15 in the lung of the mouse infected with the influenza virus H1N1 is inhibited by additional restraint stress, and the Shuanghuanglian traditional Chinese medicine composition provided by the application can effectively increase the expression of ALOX15. The inhibition of the expression of ALOX15 can weaken the positive regulation of the RIG1-MAVS antiviral signaling pathway. The experimental results are shown in Figure 9 Figure 9 Fig. 1A is a result graph of detecting the RIG1-MAVS signaling pathway by Western Blot, and it can be known from Figure 9 Fig. 1A that the low-dose Shuanghuanglian traditional Chinese medicine composition can effectively restore the response of the RIG1-MAVS signaling pathway inhibited by the influenza virus H1N1 infection and additional restraint stress. The expression amount of IFN-β in the lung and serum is detected by Elisa, wherein Figure 9 Fig. 1B is the expression amount of IFN-β in the lung, Figure 9 Fig. 1C is the expression amount of IFN-β in the serum. It can be known from Figure 9 Figs. 1B and 1C that the three different dose administration groups and the western medicine positive drug Oseltamivir can restore the expression of IFN-β in the blood and the lung.

[0131] The above-described embodiments only express several embodiments of the application, the description is more specific and detailed, but it cannot be understood as the limitation of the patent scope of the application. It should be noted that for ordinary skilled in the art, without departing from the concept of the application, several modifications and improvements can be made, which belong to the protection scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.​

Claims

1. A method for detecting a fingerprint of a Shuanghuanglian traditional Chinese medicine composition, characterized in that, Includes the following steps: Preparation of the test solution for the Shuanghuanglian herbal composition: Take 375 parts by weight of Scutellaria baicalensis, decoct three times with water, the first time for 2 hours, the second and third times for 1 hour each. Combine the decoctions, filter, concentrate the filtrate, and adjust the pH to 1.0–2.0 with an appropriate amount of 2 mol / L hydrochloric acid solution at 80℃. Keep warm for 1 hour, let stand for 12 hours, filter, add 6–8 times the amount of water to the precipitate, adjust the pH to 7.0 with 40% sodium hydroxide solution, add an equal volume of ethanol, stir to dissolve, filter, adjust the pH to 2.0 with 2 mol / L hydrochloric acid solution, keep warm at 60℃ for 30 minutes, let stand for 12 hours, filter, wash the precipitate with ethanol until the pH is 7.0, and recover the ethanol for later use; Take 375 parts by weight of Lonicera japonica and 750 parts by weight of Forsythia suspensa, soak in water for 30 minutes, decoct twice, 1.5 hours each time, combine the decoctions, filter, concentrate the filtrate to a relative density of 1 at 70–80℃. The extract, weighing 0.20–1.25, was cooled to 40°C. Ethanol was slowly added to achieve an alcohol content of 75%. The mixture was stirred thoroughly and allowed to stand for 12 hours. The supernatant was filtered off. An appropriate amount of 75% ethanol was added to the residue, stirred well, and allowed to stand for 12 hours. The residue was filtered again, and the ethanol solutions were combined. Ethanol was recovered until no alcohol odor remained. The above-mentioned Scutellaria baicalensis extract was added, along with an appropriate amount of water. The pH was adjusted to 7.0 with 40% sodium hydroxide solution. The mixture was stirred well and refrigerated at 4–8°C for 72 hours. The residue was filtered, and the filtrate was added… Add 300 parts by weight of sucrose, stir to dissolve, then add an appropriate amount of flavoring, adjust the pH to 7.0, add water to make 1000 parts by volume, stir well and let stand for 12 hours, filter, fill and sterilize to obtain the Shuanghuanglian traditional Chinese medicine composition; the volume parts / weight parts correspond to ml / g; weigh 2.5 mL of the Shuanghuanglian traditional Chinese medicine composition, place it in a 20 mL volumetric flask, make up to volume, filter through a 0.45 μm filter membrane to obtain the test solution of the Shuanghuanglian traditional Chinese medicine composition; Preparation of reference solution: Take forsythoside E, chlorogenic acid, caffeic acid, forsythoside I, forsythoside A, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, baicalin, and forsythoside, dissolve them in a solvent to prepare a mixed solution of reference solution; The test solution and the reference solution were analyzed by high performance liquid chromatography to obtain the chromatogram of the Shuanghuanglian traditional Chinese medicine composition and the chromatogram of the reference. The chromatograms of the Shuanghuanglian traditional Chinese medicine composition and the reference standard were processed using fingerprinting software to obtain the fingerprint spectrum of the Shuanghuanglian traditional Chinese medicine composition. In the high-performance liquid chromatography analysis, octadecylsilane-bonded silica gel was used as the packing material; Gradient elution is performed using a mobile phase, wherein the mobile phase comprises: phase A, which includes a 0.05%–0.25% aqueous phosphoric acid solution and a heptane sulfonate with a molar concentration of 0.0025–0.01 mol / L; and phase B, which includes acetonitrile and methanol, wherein the volume ratio of acetonitrile to methanol is (8–10):

1. The gradient elution process is as follows: Within 0–5 minutes, the volume percentage of phase B in the mobile phase increased from 4%–6% to 8%–10%. Over 5–10 minutes, the volume percentage of phase B in the mobile phase increased from 8%–10% to 11%–12%. 10-12 min, the volume percentage of the B phase in the mobile phase is increased from 11-12% to 14-16%; 12-20 min, the volume percentage of the B phase in the mobile phase is increased from 14-16% to 18-20%; 20-25 min, the volume percentage of the B phase in the mobile phase is increased from 18-20% to 21-22%; 25-30 min, the volume percentage of the B phase in the mobile phase is increased from 21-22% to 24-27%; 30-45 min, the volume percentage of the B phase in the mobile phase is increased from 24-27% to 30-34%; 45-50 min, the volume percentage of the B phase in the mobile phase is increased from 30-34% to 43-48%; 50-55 min, the volume percentage of the B phase in the mobile phase is decreased from 43-48% to 4-6%; In the high performance liquid chromatography analysis, the detection wavelength is 220 nm, 326 nm.

2. The method according to claim 1, wherein The molar concentration of the heptane sulfonate in the A phase is 0.003-0.008 mol / L. The molar concentration of the heptane sulfonate in the A phase is 0.003-0.008 mol / L.

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