Natural product composition as well as preparation method and application thereof

Abstract

The invention provides a natural product composition as well as a preparation method and application thereof, and the natural product composition is prepared by the following steps: extracting rhizoma polygoni cuspidati, radix astragali and herba epimedii granules with water, adding ethanol until the volume fraction of ethanol in the solution is 85%, and drying the supernate. The giant knotweed rhizome, radix astragali and herba epimedii granule extract provided by the invention can effectively relieve asthma allergic airway inflammation and relieve bronchitis.

Description

Technical Field

[0001] The invention relates to a natural product composition and a preparation method and application thereof. Background Art

[0002] Bronchial asthma (BA) is one of the most common respiratory diseases, characterized by recurrent wheezing and shortness of breath with or without chest tightness or cough, accompanied by airway hyperresponsiveness and variable airflow limitation. It is a heterogeneous disease with different clinical manifestations.

[0003] Bronchial asthma is mainly treated with drugs in clinical practice. The use of bronchodilators to reduce airway resistance and relieve airflow limitation is the first-line treatment for bronchial asthma. At the same time, inflammation plays a core role in the formation, development, and deterioration of bronchial asthma. Therefore, anti-inflammatory therapy plays a vital role in alleviating patients' disease burden and stabilizing their condition. Clinically, the most effective anti-inflammatory drug for bronchial asthma is inhaled glucocorticoids. Drugs for the treatment of bronchial asthma are currently divided into control drugs and relieve drugs. Control drugs include glucocorticoids, long-acting β-2 receptor agonists, theophylline, leukotriene modifiers, oral glucocorticoids, anti-IgE antibody biologics, etc. Relief drugs include short-acting β-2 receptor agonists, oral glucocorticoids, theophylline, etc.

[0004] The predecessor of the Polygonum cuspidatum, Astragalus, Epimedium, Morinda officinalis, Radix Rehmanniae, Loquat Leaf, Rhizoma Chuanxiong and Inula flower, Qixian decoction is derived from Bufei decoction and Erxian decoction. It is a modified version of the eight herbal pieces of Polygonum cuspidatum, Astragalus, Epimedium, Morinda officinalis, Radix Rehmanniae, Loquat Leaf, Rhizoma Chuanxiong and Inula flower. It has achieved certain success in the treatment of bronchial asthma. In order to optimize the treatment method and better treat diseases such as bronchial asthma, further research is needed. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect of insufficient therapeutic activity of the prior art and provide a Polygonum cuspidatum and Astragalus membranaceus granule extract which can effectively relieve asthmatic allergic airway inflammation and alleviate bronchitis.

[0006] The present invention solves the above technical problems through the following technical solutions:

[0007] The invention discloses a Polygonum cuspidatum Astragalus sinensis granule extract, which is prepared by the following method, comprising the following steps: extracting the Polygonum cuspidatum Astragalus sinensis granules with water, adding ethanol so that the volume fraction of ethanol in the solution is 85%, and taking the supernatant and drying it.

[0008] In some embodiments, the extract of Polygonum cuspidatum granules comprises the following components by mass fraction: 0.005%-0.05% of magnolia alkaloids, 0.0001%-0.006% of calycosin, 0.02%-0.15% of polydatin, 0.003%-0.022% of rhamnosin-1-O-glucoside, 0.003%-0.032% of icariin A, 0.01%-0.06% of resveratrol, 0.002%-0.011% of calycosin, 0.03%-0.16% of rhamnosin- 8-O-glucoside, 0.001%-0.01% of epimedin B, 0.01%-0.1% of epimedin C, 0.01%-0.06% of icariin, 0.003%-0.013% of baohuoside II, 0.002%-0.014% of quercetin A, 0.01%-0.03% of 2"-O-rhamnoside icariin II, 0.001%-0.004% of baohuoside I and 0.001%-0.008% of rhamnoside; the mass fraction is the percentage of the components in the total mass of the Polygonum cuspidatum and Astragalus sinensis granules extract.

[0009] In some embodiments, the Polygonum cuspidatum, Astragalus membranaceus and Centella asiatica granules include the following raw materials in parts by weight: 15 parts of Polygonum cuspidatum, 15 parts of Astragalus membranaceus, 15 parts of Epimedium, 15 parts of Morinda officinalis, 15 parts of Rehmannia root, 15 parts of Eriobotrya japonica, 7.5 parts of Chuanxiong and 4.5 parts of Inula japonica.

[0010] In some embodiments, the mass ratio of the Polygonum cuspidatum and Astragalus membranaceus granules to the water is 1:6-1:10, for example, 1:8.

[0011] In some embodiments, the daily dosage of the Polygonum cuspidatum and Astragalus membranaceus granules extract is 0.38 g / kg.

[0012] The present invention also discloses a pharmaceutical composition, which comprises the following components in mass fractions: 0.1-2% of magnolamine, 0.01-0.3% of calycosin glucoside, 0.5-8% of polydatin, 0.1-1% of rhamnosin-1-O-glucoside, 0.1-1.5% of icariin A, 0.1-2% of resveratrol, 0.05-0.5% of calycosin glucoside, 1-8% of rhamnosin-8-O-glucoside. glucoside, 0.01-0.5% epimedin B, 0.5-4% epimedin C, 1-3% icariin, 0.1-0.5% baohuoside II, 0.05-0.5% quercetin A, 0.1-1.5% 2"-O-rhamnoside icariin II, 0.01-0.2% baohuoside I, 0.031-0.3% rhamnoside; the mass fraction is the mass percentage of each component in the pharmaceutical composition.

[0013] In some embodiments, in the pharmaceutical composition, the mass fraction of magnolia alkaloids is 0.21-1.29%, for example, 0.37%, 0.86%, 0.36%, 0.42%, 0.57%, 0.82%, 1.29%, 0.6%, 0.21%, 0.39%, 0.22%, 0.31%, 0.37%, 0.54% or 0.32%.

[0014] In some embodiments, in the pharmaceutical composition, the mass fraction of the calycosin glucoside is 0.01-0.17%, for example, 0.01%, 0.02%, 0.03%, 0.06%, 0.07%, 0.1%, 0.12%, 0.15% or 0.17%.

[0015] In some embodiments, in the drug combination, the mass fraction of polydatin is 0.91-4.64%, for example, 0.91%, 1.1%, 1.41%, 1.86%, 1.88%, 2.28%, 2.53%, 2.58%, 3.11%, 3.13%, 3.22%, 4.08%, 4.56% or 4.64%.

[0016] In some embodiments, in the pharmaceutical composition, the mass fraction of rhamnosyl-1-O-glucoside is 0.16-0.66%, for example, 0.27%, 0.16%, 0.18%, 0.19%, 0.3%, 0.34%, 0.37%, 0.38%, 0.41%, 0.44%, 0.58% or 0.66%.

[0017] In some embodiments, in the pharmaceutical composition, the mass fraction of icariin A is 0.17-1.01%, for example, 0.17%, 0.23%, 0.28%, 0.36%, 0.4%, 0.44%, 0.46%, 0.48%, 0.5%, 0.53%, 0.55%, 0.61%, 0.69%, 0.71% or 1.01%.

[0018] In some embodiments, in the pharmaceutical composition, the mass fraction of resveratrol is 0.38-1.79%, for example, 0.38%, 0.55%, 0.64%, 0.78%, 0.87%, 0.87%, 0.89%, 0.9%, 0.91%, 0.99%, 1.03%, 1.24%, 1.26%, 1.61% or 1.79%.

[0019] In some embodiments, in the pharmaceutical composition, the mass fraction of the calycosin isoflavones is 0.09-0.33%, for example, 0.09%, 0.1%, 0.11%, 0.13%, 0.16%, 0.17%, 0.19%, 0.21%, 0.23%, 0.25% or 0.33%.

[0020] In some embodiments, in the pharmaceutical composition, the mass fraction of rhamnosyl-8-O-glucoside is 1.5-4.84%, for example, 1.5%, 1.72%, 1.82%, 2.08%, 2.22%, 2.22%, 2.35%, 2.76%, 3.03%, 3.04%, 3.37%, 3.49%, 4.35%, 4.62% or 4.84%.

[0021] In some embodiments, in the pharmaceutical composition, the mass fraction of epimedin B is 0.03-0.33%, for example, 0.03%, 0.05%, 0.07%, 0.08%, 0.12%, 0.13%, 0.14%, 0.16%, 0.18%, 0.2%, 0.21%, 0.21%, 0.22%, 0.26% or 0.33%.

[0022] In some embodiments, in the pharmaceutical composition, the mass fraction of epimedin C is 0.76-2.86%, for example, 0.76%, 0.77%, 1.04%, 1.3%, 1.39%, 1.73%, 1.73%, 1.89%, 1.95%, 2.01%, 2.02%, 2.07%, 2.37%, 2.57% or 2.86%.

[0023] In some embodiments, in the pharmaceutical composition, the mass fraction of icariin is 1.33-1.95%, for example 0.33%, 0.47%, 0.72%, 0.76%, 1%, 1.08%, 1.24%, 1.28%, 1.41%, 1.55%, 1.72%, 1.77%, 1.8% or 1.95%.

[0024] In some embodiments, in the pharmaceutical composition, the mass fraction of the Baohuoside II is 0.12-0.38%, for example, 0.12%, 0.14%, 0.16%, 0.17%, 0.19%, 0.23%, 0.24% or 0.38%.

[0025] In some embodiments, in the pharmaceutical composition, the mass fraction of curabioside A is 0.07-0.44%, for example, 0.07%, 0.09%, 0.13%, 0.14%, 0.15%, 0.17%, 0.2%, 0.24%, 0.25%, 0.36% or 0.44%.

[0026] In some embodiments, in the pharmaceutical composition, the mass fraction of the 2"-O-rhamnoside icariin II is 0.42-1.04%, for example, 0.42%, 0.47%, 0.52%, 0.58%, 0.6%, 0.65%, 0.7%, 0.72%, 0.78%, 0.8%, 0.83%, 0.87%, 0.88% or 1.04%.

[0027] In some embodiments, in the pharmaceutical composition, the mass fraction of the Baohuoside I is 0.03-0.12%, for example, 0.03%, 0.05%, 0.07%, 0.09%, 0.1%, 0.11% or 0.12%.

[0028] In some embodiments, in the pharmaceutical composition, the mass fraction of emodin is 0.02-0.24%, for example, 0.03%, 0.09%, 0.07%, 0.12%, 0.22%, 0.06%, 0.02%, 0.1%, 0.08%, 0.24% or 0.14%.

[0029] In some embodiments, the pharmaceutical composition comprises the following active ingredients in mass fractions: 0.21-1.29% of magnolamine, 0.01-0.17% of calycosin glucoside, 0.91-4.64% of polydatin, 0.16-0.66% of rhamnosin-1-O-glucoside, 0.17-1.01% of icariin A, 0.38-1.79% of resveratrol, 0.09-0.33% of calycosin glucoside, 1.5-4.84% of rhamnosin- 8-O-glucoside, 0.03-0.33% of epimedin B, 0.76-2.86% of epimedin C, 1.33-1.95% of icariin, 0.12-0.38% of baohuoside II, 0.07-0.44% of curarioside A, 0.42-1.04% of 2"-O-rhamnoside icariin II, 0.03-0.12% of baohuoside I, and 0.02-0.24% of rhamnoside; the mass fractions are the mass percentages of each component in the pharmaceutical composition.

[0030] In some embodiments, the pharmaceutical composition is prepared by the following method, which comprises the following steps:

[0031] S1, extracting Polygonum cuspidatum and Astragalus sinensis granules with water, adding ethanol, and concentrating the supernatant to obtain a concentrated solution;

[0032] S2. The concentrated liquid is subjected to column chromatography and then dried.

[0033] In some embodiments, the Polygonum cuspidatum, Astragalus membranaceus and Centella asiatica granules include the following raw materials in parts by weight: 15 parts of Polygonum cuspidatum, 15 parts of Astragalus membranaceus, 15 parts of Epimedium, 15 parts of Morinda officinalis, 15 parts of Rehmannia root, 15 parts of Eriobotrya japonica, 7.5 parts of Chuanxiong and 4.5 parts of Inula japonica.

[0034] In some embodiments, in step S1, the volume fraction of ethanol in the solution is 60%-95%, for example, 85%, after the ethanol is added.

[0035] In some embodiments, in step S1, the mass ratio of the Polygonum cuspidatum and Astragalus membranaceus granules to the water is 1:6-1:10, for example, 1:8.

[0036] In some embodiments, in step S1, the mass-to-volume ratio of the Polygonum cuspidatum and Astragalus membranaceus granules and the concentrated solution is 0.3 g / ml-0.6 g / ml, for example, 0.5 g / ml.

[0037] In some embodiments, in step S2, the column chromatography is packed with a macroporous resin, such as AB-8 macroporous resin.

[0038] In some embodiments, in step S2, the volume-to-mass ratio of the resin used in the column chromatography to the Polygonum cuspidatum and Astragalus membranaceus particles in step S1 is 4-8 ml / g, for example, 6 ml / g.

[0039] In some embodiments, in step S2, the column chromatography is eluted with water.

[0040] In some embodiments, in step S2, the column chromatography is eluted with a low concentration ethanol aqueous solution, and the low concentration ethanol aqueous solution is 20%-40% ethanol aqueous solution; for example, 30% ethanol aqueous solution is used for elution; wherein the percentage refers to the ratio of the volume of ethanol in the ethanol aqueous solution to the total volume of the solution.

[0041] In some embodiments, in step S2, the column chromatography is eluted with a medium-concentration ethanol aqueous solution to obtain an eluate, and the medium-concentration ethanol aqueous solution is 50%-70% ethanol aqueous solution, for example, 60% ethanol aqueous solution is used to obtain the eluate.

[0042] In some embodiments, in step S2, the column chromatography is eluted using 3-5 column volumes of water, for example, 4 column volumes of water.

[0043] In some embodiments, in step S2, the column chromatography is eluted using 6-10 column volumes of a low-concentration ethanol aqueous solution, for example, using 8 column volumes of a low-concentration ethanol aqueous solution.

[0044] In some embodiments, in step S2, the column chromatography is eluted with 4-6 column volumes of a medium-concentration ethanol aqueous solution to obtain an eluate, for example, eluted with 5 column volumes of a medium-concentration ethanol aqueous solution to obtain an eluate.

[0045] In some embodiments, in step S2, the drying is to concentrate and evaporate the eluate to dryness to obtain the pharmaceutical composition.

[0046] In some embodiments, in step S2, the column chromatography is eluted with water and low-concentration aqueous ethanol solution in sequence, and then eluted with medium-concentration aqueous ethanol solution, and the liquid eluted with the medium-concentration aqueous ethanol solution is collected to obtain an eluate.

[0047] The present invention also discloses a method for extracting Polygonum cuspidatum and Astragalus sinensis granules, which comprises the following steps:

[0048] S1, extracting Polygonum cuspidatum and Astragalus sinensis granules with water, adding ethanol, and concentrating the supernatant to obtain a concentrated solution;

[0049] S2. The concentrated liquid is subjected to column chromatography and then dried.

[0050] Preferably, the conditions and operations of S1 and S2 are as described in any one of the present invention.

[0051] The present invention also discloses a pharmaceutical preparation, which comprises the Polygonum cuspidatum and Astragalus sinensis granule extract as described in any one of the present invention and / or the pharmaceutical composition as described in any one of the present invention, and pharmaceutical excipients.

[0052] The present invention also discloses the use of a substance X in the preparation of a drug for preventing or treating bronchial asthma, wherein the substance X is one or more of the Polygonum cuspidatum and Astragalus centella asiatica granule extract as described in any one of the present invention, the pharmaceutical composition as described in any one of the present invention, and the pharmaceutical preparation as described in any one of the present invention.

[0053] The present invention also provides a method for detecting the extract of Polygonum cuspidatum and Astragalus sinensis granules, which comprises the following steps: using liquid chromatography to perform gradient elution on the test object; the test object is the extract of Polygonum cuspidatum and Astragalus sinensis granules as described in any one of the present invention or the pharmaceutical composition as described in any one of the present invention;

[0054] In the liquid chromatography method, the chromatographic conditions are as follows: the chromatographic column is Waters Atlantis T3 (5um, 4.6mm*250mm); the flow rate is 0.8-1.2mL / min; the column temperature is 20-40°C; the detection wavelength is 270nm; the mobile phase A is 0.1% formic acid solution, the mobile phase B is acetonitrile, and the elution gradient is as shown in the following table:

[0055]

[0056] In some embodiments, the column temperature is 30°C.

[0057] In some embodiments, the flow rate is 1 mL / min.

[0058] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0059] The raw materials of the kit used in the present invention are all commercially available.

[0060] The positive and progressive effect of the present invention is that it provides a Polygonum cuspidatum and Astragalus membranaceus granule extract and a pharmaceutical composition which can effectively relieve asthmatic allergic airway inflammation and alleviate bronchitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 HPLC spectrum of small molecule compound components of Polygonum cuspidatum and Astragalus sinensis granules extract

[0062] Figure 2 This is the triple quadrupole mass spectrum of the small molecule compound components of Polygonum cuspidatum and Astragalus sinensis granules extract

[0063] Figure 3 HE staining and Sirius red staining of mouse lung tissue (200x) DETAILED DESCRIPTION

[0064] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0065] Instruments and reagents

[0066] Freeze dryer L-200 (BUCHI, USA); Hei-VAP Value rotary evaporator (Heidolph, Germany); MS204TS electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); automatic ice maker (Focusun, Germany); H2500R high-speed desktop refrigerated centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); VX-Ⅱ multi-tube vortex oscillator (Beijing Tajin Technology Co., Ltd.); electric constant temperature blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); Waters AcquityTM ultra-high performance liquid chromatography system (Waters, USA), equipped with binary pump, autosampler and PDA detector; Waters Xevo G2 single quadrupole time-of-flight mass spectrometer equipped with electrospray ionization source (Waters, USA); SB-5200DTD ultrasonic cleaner (240W, 53KHz, Ningbo Xinzhi Biotechnology Co., Ltd.); Merck Milli-Q ultrapure water machine (Millipore, USA); microplate reader (Thermo Multiskan MK3); oscillator (QILINBEIERQB8002); vortex mixer (QILINBEIER KB3); centrifuge (QILINBEIER LX200); three-dimensional cryo-grinder (wonbio); desktop high-speed refrigerated microcentrifuge (DragonLab); vortex mixer (Thermo); handheld centrifuge (Thermo); fluorescence quantitative PCR instrument (Bio-rad); PCR instrument (Thermo); dry constant temperature metal bath (Thermo); ultra-micro spectrophotometer (Thermo); film sealer (intelligent) (Thermo); PCR reaction plate (Axygen); fluorescence quantitative PCR plate sealing film (Axygen); upright white light camera microscope (Nikon, Japan).

[0067] Polygonum cuspidatum and Astragalus sinensis granules (specification: 12g per bag, batch number: 2210801), Shanghai Baolong Pharmaceutical Co., Ltd. Among them, by mass fraction, 12.5% ​​are excipients and 87.5% are active ingredients of traditional Chinese medicine.

[0068] Acetonitrile, chromatographic grade, was purchased from Fisher, USA; sodium formate and leucine-enkephalin, mass spectrometry grade, were purchased from Sigma, USA; methanol, chromatographic grade, was purchased from Shanghai Titan Technology Co., Ltd.; anhydrous formic acid was purchased from Sinopharm Chemical Reagent Co., Ltd.; and water was ultrapure water.

[0069] Ingredient testing method:

[0070] Take an appropriate amount of sample, accurately weigh 10 mg, place in a 10 mL volumetric flask, add 9 mL of methanol, ultrasonically (250 W, 40 kHz) for 30 minutes, let it cool to room temperature, add methanol to the scale line, shake well, filter with a 0.22 μm microporous filter membrane, and perform component detection.

[0071] HPLC chromatographic conditions: chromatographic column is Waters Atlantis T3 (5um, 4.6mm*250mm); flow rate is 1mL / min; column temperature is 30℃; detection wavelength is 270nm; mobile phase is 0.1% formic acid solution (A)-acetonitrile (B), elution gradient: (0-5min (90-90% A), 5-10min (90-80% A), 10-20min (80-80% A), 20-45min (80-75% A), 45-65min (75-70% A), 65-70min (70-70% A), 70-75min (75-65% A), 75-85min (65-40% A), 85-90 (40-30% A), 90-95 (30-90% A), 95-100 (90-90% A)). Inject 10uL of both the test solution and the reference solution.

[0072] Triple quadrupole conditions:

[0073] Chromatographic conditions: instrument: Shimadzu HPLC liquid chromatography system; chromatographic column: Waters ACQUITY UPLCTM BEH C18 (150 mm×2.1 mm, 1.8 μm); column temperature: 30°C; sample chamber temperature: 10°C; flow rate: 0.35 ml / min; injection volume: 5 μl; maximum pressure: 120 MPa; mobile phase: A: 0.1% formic acid in water, B: acetonitrile; gradient elution: 0-5 min (20% B); 5-9 min (20%-40% B); 9-15 min (40%-41% B); 15-16.5 min (41-60% B); 16.5-16.6 min (60%-20% B); 16.6-18 min.

[0074] Mass spectrometry conditions: Instrument: AB SCIEX 4500QTRAP MS, MSE mode; Ion source: ESI; Mode: The multiple reaction monitoring (MRM); Protective gas: nitrogen, pressure: 50 psi, temperature is 500°C; Auxiliary gas: nitrogen, pressure: 50 psi; Electron spray voltage: 4.5 KV (negative electrode), 5.5 KV (positive electrode).

[0075] Mass spectrometry parameters

[0076]

[0077] Example 1

[0078] 1. Preparation of Polygonum cuspidatum and Astragalus sinensis granules extract

[0079] 48 g of Polygonum cuspidatum and Astragalus membranaceus granules (batch number: 2210801) were added with 400 mL of water and 2.67 L of anhydrous ethanol until the ethanol concentration reached 85%. The mixture was allowed to stand for 24 h, and the supernatant was taken. The mixture was dried under reduced pressure until there was no alcohol taste, and freeze-dried to obtain the Polygonum cuspidatum and Astragalus membranaceus granules extract with a yield of 53.87% (the yield was calculated based on the non-excipient part of the Polygonum cuspidatum and Astragalus membranaceus granules as the raw material mass).

[0080] The contents of various components in the Polygonum cuspidatum and Astragalus sinensis granules extract are as follows:

[0081]

[0082]

[0083] 2. Preparation of small molecule components of Polygonum cuspidatum and Astragalus sinensis granules extract

[0084] 48g of Polygonum cuspidatum and Astragalus membranaceus granules (batch number: 2210801) were added with 400mL of water and 2.67L of anhydrous ethanol until the ethanol concentration reached 85%. The mixture was allowed to stand for 24h, and the supernatant was concentrated to 100mL, and purified by column chromatography. The chromatography column was 40mm*300mm, and was loaded with 300mL of AB-8 macroporous resin. The elution was performed with water (4 column volumes), 30% ethanol (8 column volumes), 60% ethanol (5 column volumes), and 90% ethanol (10 column volumes), respectively. The eluate obtained by eluting with 60% ethanol was concentrated and evaporated to dryness to obtain the small molecule component group of the Polygonum cuspidatum and Astragalus membranaceus granules extract. After mass spectrometry characterization and comparison with the standard, the HPLC spectrum of the components of the Polygonum cuspidatum and Astragalus membranaceus granules extract was obtained ( Figure 1 ) and triple quadrupole spectra ( Figure 2 ).

[0085] The content of active ingredients in the small molecule component group of Polygonum cuspidatum and Astragalus sinensis granules extract is as follows

[0086]

[0087] Example 2 The improving effect of the extract of Polygonum cuspidatum and Astragalus sinensis granules on OVA-induced asthma model mice

[0088] Thirty-two healthy female SPF BALB / c mice, 4 to 6 weeks old, with an average body weight of (20±2) g, were purchased from Shanghai Slake Laboratory Animal Co., Ltd., with a production license of SCXK (Shanghai) 2017-0005 and a use license of SYXK (Shanghai) 2019-0027. They were kept in the animal room of Shanghai Institute of Pharmaceutical Industry, approved by the Animal Experiment Ethics Committee of Shanghai Institute of Pharmaceutical Industry, with a use license of SYXK (Shanghai) 2019-0027. Before the experiment, the animals were adaptively fed in the breeding room for 7 days (humidity 50±10%; temperature 25±2℃; 12h day and night cycle), with free access to food and drinking water.

[0089] 32 mice were randomly divided into 4 groups. The mice were adaptively fed for 1 week and then divided into control group, model group, granule group (KL group) and extract group (TQW group), with 8 mice in each group. On the 1st and 14th day after adaptive feeding, 200 μL of OVA sensitization solution was intraperitoneally injected for sensitization; the mice in the model group, KL group and TQW group were challenged with 50 μL of OVA provocation solution drops in the nose on the 14th, 25th, 26th and 27th days, respectively. The control group mice followed the above method, but normal saline was used instead of OVA during sensitization and challenge. The mice were continuously gavaged from the 28th day. The granule group was given Polygonum cuspidatum granules, and the extract group was given Polygonum cuspidatum granules extract, once a day, and the dose was 8 g kg according to the KL group. -l ·d -1 , XFZ group 3.8 g·kg -l ·d -1 The mice in the model group and the control group were given an equal amount of 0.9% NaCl solution instead of drug administration. On the 42nd day, the mice were killed after blood sampling, and the alveolar lavage fluid (BALF) and lungs were obtained and various indicators were tested.

[0090] 3.1 HE staining and picrosirius red staining

[0091] Part of the lung tissue was taken for HE section staining and picrosirius red section staining. The tissue was fixed in paraformaldehyde solution. After fixation for 24 hours, conventional paraffin sections and H&E and picrosirius red staining were performed, and the images were observed under a microscope and collected.

[0092] From the changes in lung tissue pathology Figure 3 ), the control group mice had no inflammatory cell infiltration in the lung tissue bronchi, no airway thickening, and no obvious airway mucus secretion. The model group mice had obvious inflammatory infiltration in the lung tissue, obvious airway thickening, and significantly increased airway mucus secretion. The inflammatory infiltration of the lung tissue of the KL group and the TQW group mice was alleviated compared with the asthma group, the airway was slightly thickened, and the airway mucus secretion was significantly reduced.

[0093] 3.2 Elisa detection of cytokine expression in serum and BALF

[0094] Cytokines in serum and BALF were measured using Elisa kits. Please refer to the instructions for specific operations. ELISA results are shown in the following table.

[0095]

[0096] Compared with the control group, the Th2-related IL-4 and IL-5 in the serum of the mice in the model group were significantly increased, the Th1-related TNF-α in the BALF was significantly increased, the Th2-related IL-13 was increased, and the Th1-related IFN-γ, Treg-related IL-10 and TGF-β were significantly decreased; compared with the model group, the Th2-related IL-4 and IL-5 in the serum of the mice in the KL group and TQW group were significantly decreased, the Th1-related TNF-α in the BALF was significantly decreased, and the Th1-related IFN-γ, Treg-related IL-10 and TGF-β were significantly increased, indicating that the extract of Polygonum cuspidatum and Astragalus membranaceus granules has an improving effect on OVA-induced asthma in mice, and the effect is better than that of Polygonum cuspidatum and Astragalus membranaceus granules, and its mechanism of action is related to Th1 / Th2 imbalance.

[0097] Compared with the control group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01;

[0098] Example 4 Effects of small molecule compound components on H2O2-induced apoptosis of 16HBE cells

[0099] Reagents and instruments

[0100] Cell Counting Kit-8 (BS350E, China Biosharp Company); RPMI1640 culture medium (GP21040040120, Wuhan Sewell Company); Gibco trypsin digestion solution (2661762, Thermo Fisher Scientific Company, USA); DMSO (133900, Shanghai Experimental Reagent Co., Ltd.); H2O2 (102564192, Sigma Company, USA). Fetal bovine serum (504062722, Shanghai Shusheng Biotechnology Co., Ltd.).

[0101] Electronic balance (ALC-210.3, Sartorius, Germany); clean bench (SW-CJ-2FD, Suzhou Purification Equipment Co., Ltd.); carbon dioxide incubator, refrigerated centrifuge, full-wavelength multifunctional microplate reader (371, Micro2R, Varioskan Flash, Thermo Corporation, USA); low-speed centrifuge (TDZ5-WS, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); cell counter (Celldrop 1, DeNovix, USA).

[0102] Experimental methods:

[0103] Cell culture and grouping: 16HBE cells were cultured in a cell culture dish using RPMI 1640 medium containing 10% serum at 37°C and 5% CO2. When the cell fusion reached 80%, they were digested with 0.5% trypsin and grouped. The inoculation concentration was 10,000 cells / well, and 0.1 ml was added to each well.

[0104] Grouping and administration: According to the type of drug to be investigated, the patients were divided into (1) control group; (2) H2O2 group; (3) H2O2 + Polygonum cuspidatum and Astragalus membranaceus granules group; (4) H2O2 + small molecule component group of Polygonum cuspidatum and Astragalus membranaceus granules extract.

[0105] Incubation time: Observe the cell status and activity 24 hours after administration according to experimental needs.

[0106] Indicator observation:

[0107] The morphology of 16HBE cells was observed by inverted optical microscope after 24 hours of exposure to H2O2 and test drugs.

[0108] After the cell culture was completed, 10 μL of CCK-8 detection reagent was added to each well, incubated at 37°C in the dark for 4 h (appropriate adjustment is required), and the absorbance value (A) at 450 nm of each well was detected by an ELISA reader, and the cell survival rate was calculated according to the formula.

[0109] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0110] Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0111] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution);

[0112] Ac: absorbance of control well (containing cells, culture medium, CCK-8 solution, but no drug);

[0113] Ab: absorbance of blank well (containing culture medium and CCK-8 solution, but not cells or drugs);

[0114] Experimental results:

[0115] 3.1 Inhibitory effects of Polygonum cuspidatum and Astragalus sinensis granules and small molecule components of Polygonum cuspidatum and Astragalus sinensis granules extract on normal 16HBE cells

[0116] As shown in the following table, different concentrations of Polygonum cuspidatum and Astragalus membranaceus granules and small molecule compound component groups had no significant growth inhibitory effect on normal 16HBE cells when the maximum concentration (1 mg / mL) was reached, and even had some cell proliferation promoting effects, indicating that Polygonum cuspidatum and Astragalus membranaceus granules and small molecule compound component groups had no obvious 16HBE cell toxicity.

[0117] Inhibitory effects of different concentrations of Polygonum cuspidatum and Xiandihua granules and small molecule compound components on normal 16HBE cells

[0118]

[0119] The modeling concentration of H2O2 was set at 40μmol / mL, and the administration time was 24h. The inhibitory effects of Polygonum cuspidatum and Astragalus membranaceus granules and the small molecule components of Polygonum cuspidatum and Astragalus membranaceus granules extract on H2O2-induced 16HBE cell apoptosis were tested. The results showed that the small molecule components of Polygonum cuspidatum and Astragalus membranaceus granules extract had a significant inhibitory effect.

[0120]

[0121]

[0122] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A Polygonum cuspidatum and Astragalus membranaceus granule extract, characterized in that: The preparation method is as follows, which comprises the following steps: after extracting the Polygonum cuspidatum and Astragalus sinensis granules with water, ethanol is added to make the volume fraction of ethanol in the solution be 85%, and the supernatant is taken and dried.

2. The Polygonum cuspidatum and Astragalus membranaceus granule extract according to claim 1, characterized in that It meets one or more of the following conditions: (1) The extract of Polygonum cuspidatum granules comprises the following components in mass fractions: 0.005%-0.05% of magnoline, 0.0001%-0.006% of calycosin, 0.02%-0.15% of polydatin, 0.003%-0.022% of rhamnosin-1-O-glucoside, 0.003%-0.032% of icariin A, 0.01%-0.06% of resveratrol, 0.002%-0.011% of calycosin, 0.03%-0.16% of rhamnosin-8-O-glucoside. -glucoside, 0.001%-0.01% epimedin B, 0.01%-0.1% epimedin C, 0.01%-0.06% icariin, 0.003%-0.013% baohuoside II, 0.002%-0.014% arbutin A, 0.01%-0.03% 2"-O-rhamnoside icariin II, 0.001%-0.004% baohuoside I and 0.001%-0.008% rhamnoside; the mass fraction is the percentage of the component in the total mass of the Polygonum cuspidatum granules extract; (2) The Polygonum cuspidatum, Astragalus, Epimedium, Morinda officinalis, Rehmannia root, Loquat leaf, 7.5 parts Chuanxiong and 4.5 parts Inula flower; (3) The mass ratio of the Polygonum cuspidatum Astragalus Cercidiphyllum Granules to the water is 1:6-1:10, preferably 1:8; (4) The daily dosage of the Polygonum cuspidatum and Astragalus sinensis granules extract is 0.38 g / kg; and (5) The extract of Polygonum cuspidatum and Astragalus sinensis granules is used to prepare a drug for preventing or treating bronchial asthma.

3. A pharmaceutical composition, characterized in that The invention comprises the following components in mass fractions: 0.1-2% of magnolia alkaloids, 0.01-0.3% of calycosin glucoside, 0.5-8% of polydatin, 0.1-1% of rhamnosin-1-O-glucoside, 0.1-1.5% of icariin A, 0.1-2% of resveratrol, 0.05-0.5% of calycosin glucoside, 1-8% of rhamnosin-8-O-glucoside, 0.01 -0.5% of epimedin B, 0.5-4% of epimedin C, 1-3% of icariin, 0.1-0.5% of baohuoside II, 0.05-0.5% of curarioside A, 0.1-1.5% of 2"-O-rhamnoside icariin II, 0.01-0.2% of baohuoside I, and 0.031-0.3% of rhamnoside; the mass fractions are the mass percentages of each component in the pharmaceutical composition.

4. The pharmaceutical composition according to claim 3, characterized in that It meets one or more of the following conditions: (1) The mass fraction of the magnolia alkaloids is 0.21-1.29%; (2) The mass fraction of the calycosin isoflavone glucoside is 0.01-0.17%; (3) The mass fraction of polydatin is 0.91-4.64%; (4) The mass fraction of rheumatoid artemisia selengensis-1-O-glucoside is 0.16-0.66%; (5) The mass fraction of icariin A is 0.17-1.01%; (6) The mass fraction of resveratrol is 0.38-1.79%; (7) The mass fraction of the calycosin isoflavone is 0.09-0.33%; (8) The mass fraction of rheumatoid artemisia selengensis-8-O-glucoside is 1.5-4.84%; (9) The mass fraction of epimedin B is 0.03-0.33%; (10) The mass fraction of epimedin C is 0.76-2.86%; (11) The mass fraction of icariin is 1.33-1.95%; (12) The mass fraction of the Baohuoside II is 0.12-0.38%; (13) The mass fraction of the scutellarin A is 0.07-0.44%; (14) The mass fraction of the 2″-O-rhamnoside icariin II is 0.42-1.04%; (15) The mass fraction of the Baohuoside I is 0.03-0.12%; and (16) The mass fraction of the rhein is 0.02-0.24%.

5. The pharmaceutical composition according to claim 4, characterized in that It meets one or more of the following conditions: (1) The mass fraction of the magnoline is 0.37%, 0.86%, 0.36%, 0.42%, 0.57%, 0.82%, 1.29%, 0.6%, 0.21%, 0.39%, 0.22%, 0.31%, 0.37%, 0.54% or 0.32%; (2) The mass fraction of the calycosin glucoside is 0.01%, 0.02%, 0.03%, 0.06%, 0.07%, 0.1%, 0.12%, 0.15% or 0.17%; (3) the mass fraction of polydatin is 0.91%, 1.1%, 1.41%, 1.86%, 1.88%, 2.28%, 2.53%, 2.58%, 3.11%, 3.13%, 3.22%, 4.08%, 4.56% or 4.64%; (4) the mass fraction of rheumatoid artemisia selengensis-1-O-glucoside is 0.27%, 0.16%, 0.18%, 0.19%, 0.3%, 0.34%, 0.37%, 0.38%, 0.41%, 0.44%, 0.58% or 0.66%; (5) The mass fraction of icariin A is 0.17%, 0.23%, 0.28%, 0.36%, 0.4%, 0.44%, 0.46%, 0.48%, 0.5%, 0.53%, 0.55%, 0.61%, 0.69%, 0.71% or 1.01%; (6) The mass fraction of resveratrol is 0.38%, 0.55%, 0.64%, 0.78%, 0.87%, 0.87%, 0.89%, 0.9%, 0.91%, 0.99%, 1.03%, 1.24%, 1.26%, 1.61% or 1.79%; (7) The mass fraction of the calycosin isoflavones is 0.09%, 0.1%, 0.11%, 0.13%, 0.16%, 0.17%, 0.19%, 0.21%, 0.23%, 0.25% or 0.33%; (8) The mass fraction of rheumatoid artemisia selengensis-8-O-glucoside is 1.5%, 1.72%, 1.82%, 2.08%, 2.22%, 2.22%, 2.35%, 2.76%, 3.03%, 3.04%, 3.37%, 3.49%, 4.35%, 4.62% or 4.84%; (9) The mass fraction of epimedin B is 0.03%, 0.05%, 0.07%, 0.08%, 0.12%, 0.13%, 0.14%, 0.16%, 0.18%, 0.2%, 0.21%, 0.21%, 0.22%, 0.26% or 0.33%; (10) The mass fraction of epimedin C is 0.76%, 0.77%, 1.04%, 1.3%, 1.39%, 1.73%, 1.73%, 1.89%, 1.95%, 2.01%, 2.02%, 2.07%, 2.37%, 2.57% or 2.86%; (11) The mass fraction of icariin is 0.33%, 0.47%, 0.72%, 0.76%, 1%, 1.08%, 1.24%, 1.28%, 1.41%, 1.55%, 1.72%, 1.77%, 1.8% or 1.95%; (12) The mass fraction of the Baohuoside II is 0.12%, 0.14%, 0.16%, 0.17%, 0.19%, 0.23%, 0.24% or 0.38%; (13) The mass fraction of curabioside A is 0.07%, 0.09%, 0.13%, 0.14%, 0.15%, 0.17%, 0.2%, 0.24%, 0.25%, 0.36% or 0.44%; (14) The mass fraction of the 2″-O-rhamnoside icariin II is 0.42%, 0.47%, 0.52%, 0.58%, 0.6%, 0.65%, 0.7%, 0.72%, 0.78%, 0.8%, 0.83%, 0.87%, 0.88% or 1.04%; (15) The mass fraction of the baohuoside I is 0.03%, 0.05%, 0.07%, 0.09%, 0.1%, 0.11% or 0.12%; and (16) The mass fraction of the rhein is 0.03%, 0.09%, 0.07%, 0.12%, 0.22%, 0.06%, 0.02%, 0.1%, 0.08%, 0.24% or 0.14%.

6. The pharmaceutical composition according to claim 3, characterized in that It is prepared by the following method: It includes the following steps: S1, extracting Polygonum cuspidatum and Astragalus sinensis granules with water, adding ethanol, and concentrating the supernatant to obtain a concentrated solution; S2, the concentrated solution is subjected to column chromatography and dried; Preferably, the preparation method satisfies one or more of the following conditions: (1) The Polygonum cuspidatum, Astragalus, Epimedium, Morinda officinalis, Rehmannia root, Loquat leaf, 7.5 parts Chuanxiong and 4.5 parts Inula flower; (2) In step S1, the volume fraction of ethanol in the solution is 60%-95%, for example 85% after the ethanol is added; (3) In step S1, the mass ratio of the Polygonum cuspidatum Astragalus Cercidiphyllum granules to the water is 1:6-1:10, for example, 1:8; (4) In step S1, the mass-to-volume ratio of the Polygonum cuspidatum and Astragalus sinensis granules to the concentrated solution is 0.3 g / ml-0.6 g / ml, for example, 0.5 g / ml; (5) In step S2, the column chromatography is packed with a macroporous resin, such as AB-8 macroporous resin; (6) In step S2, the volume-to-mass ratio of the resin used in the column chromatography to the Polygonum cuspidatum and Astragalus sinensis granules in step S1 is 4-8 ml / g, for example, 6 ml / g; (7) In step S2, the column chromatography is eluted with water; (8) In step S2, the column chromatography is eluted with a low concentration ethanol aqueous solution, wherein the low concentration ethanol aqueous solution is 20%-40% ethanol aqueous solution; for example, 30% ethanol aqueous solution is used for elution; wherein the percentage refers to the ratio of the volume of ethanol in the ethanol aqueous solution to the total volume of the solution; (9) In step S2, the column chromatography is eluted with a medium-concentration ethanol aqueous solution to obtain an eluent, wherein the medium-concentration ethanol aqueous solution is a 50%-70% ethanol aqueous solution, for example, a 60% ethanol aqueous solution is used to obtain an eluent; (10) In step S2, the column chromatography is eluted using 3-5 column volumes of water, for example, 4 column volumes of water; (11) In step S2, the column chromatography is eluted using 6-10 column volumes of a low-concentration ethanol aqueous solution, for example, using 8 column volumes of a low-concentration ethanol aqueous solution; (12) In step S2, the column chromatography is eluted with 4-6 column volumes of a medium-concentration ethanol aqueous solution to obtain an eluate, for example, eluted with 5 column volumes of a medium-concentration ethanol aqueous solution to obtain an eluate; and (13) In step S2, the drying is to concentrate and evaporate the eluate to dryness to obtain the pharmaceutical composition; More preferably, in step S2, the column chromatography is eluted with water and low concentration ethanol aqueous solution in sequence, and then eluted with medium concentration ethanol aqueous solution, and the liquid eluted with the final concentration ethanol aqueous solution is collected to obtain the eluate.

7. A method for extracting Polygonum cuspidatum and Astragalus sinensis granules, characterized in that: It includes the following steps: S1, extracting Polygonum cuspidatum and Astragalus sinensis granules with water, adding ethanol, and concentrating the supernatant to obtain a concentrated solution; S2, the concentrated solution is subjected to column chromatography and dried; Preferably, the conditions and operations of S1 and S2 are as described in claim 6.

8. A pharmaceutical preparation, characterized in that The invention comprises the Polygonum cuspidatum and Astragalus sinensis granule extract according to any one of claims 1 to 2 and / or the pharmaceutical composition according to any one of claims 3 to 6, and pharmaceutical excipients.

9. Use of a substance X in the preparation of a drug for preventing or treating bronchial asthma, wherein the substance X is one or more of the Polygonum cuspidatum and Astragalus centella asiatica granule extract according to any one of claims 1 to 2, the pharmaceutical composition according to any one of claims 3 to 6, and the pharmaceutical preparation according to claim 8.

10. A method for detecting an extract of Polygonum cuspidatum and Astragalus sinensis granules, characterized in that: It includes the following steps: The analyte is subjected to gradient elution by liquid chromatography; the analyte is the Polygonum cuspidatum and Astragalus sinensis granule extract according to any one of claims 1 to 2 or the pharmaceutical composition according to any one of claims 3 to 6; In the liquid chromatography method, the chromatographic conditions are as follows: the chromatographic column is Waters Atlantis T3 (5um, 4.6mm*250mm); flow rate is 0.8-1.2mL / min, for example 1mL / min; column temperature is 20-40℃; The detection wavelength was 270 nm; mobile phase A was 0.1% formic acid solution, mobile phase B was acetonitrile, and the elution gradient was as follows: Preferably, the chromatographic conditions meet one or more of the following conditions: (1) The column temperature is 30°C; (2) The flow rate is 1 mL / min.

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