Withanolide active component in calyx seu fructus physalis as well as extraction method and application of withanolide active component

Abstract

The invention discloses withanolide active components in calyx seu fructus physalis as well as an extraction method and application thereof, and the extraction method comprises the following steps: crushing the calyx seu fructus physalis, soaking the calyx seu fructus physalis by adopting an ethanol solution with the volume percentage of 0-75% as an extracting agent, and performing reflux extraction to obtain an extracting solution; refining and concentrating the extracting solution to obtain a calyx seu fructus physalis extract; enabling the calyx seu fructus physalis extract to pass through a macroporous adsorption resin column, and carrying out gradient elution by taking an ethanol solution with the volume fraction of 0-100% as an eluent; in the elution process, a high performance liquid chromatography is combined with an ultraviolet detector and an evaporative light detector to analyze the eluent of each gradient so as to determine the elution volume of the ethanol solution of each gradient, and the effluent of the 50%-80% ethanol solution is collected to obtain the withanolide active components. According to the present invention, HPLC, ELSD, in vitro drug activity detection and other methods are combined, and the preparation process with characteristics of high yield, low cost and high safety coefficient is designed to be suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of active components of Lantern Festival, and specifically relates to a withanolide active component in Lantern Festival and an extraction method and application thereof. The withanolide active component can be used in medicines for treating or preventing respiratory diseases. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Respiratory diseases are common diseases with many types, including pneumonia, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute upper respiratory tract infection, pharyngitis, etc. Their high morbidity and mortality rates have brought life and health threats to all mankind. Effective drugs for the treatment of respiratory diseases are urgently needed in clinical practice, and innovative drug research is imperative.

[0004] It is currently believed that inflammatory response, oxidative stress, and immune disorders play an important role in the occurrence and development of most respiratory diseases. When inflammation occurs in the body or locally, the expression level of COX-2 in inflammatory cells can increase to 10-80 times the normal level, causing an increase in the content of PGE2, PGI2, and PGE1 in the inflammatory site, aggravating the inflammatory response and tissue damage. Oxidative stress is an important pathological feature of respiratory diseases. Nrf2, as a key transcription factor that regulates antioxidant stress, plays an important role in inducing the body's antioxidant response. Keap1 binds to Nrf2 and promotes its degradation through the ubiquitin-proteasome pathway, inhibiting Nrf2 and thereby inhibiting the transcription of antioxidant-related genes. Immune disorders also play an important role in the development of respiratory diseases. When allergens are present, T cells proliferate and differentiate into different subtypes such as Th1 and Th2. The cytokines produced by epithelial cells induce Th2 cells to produce IL-5. IL-5 causes eosinophil differentiation, proliferation, airway recruitment and degranulation. Eosinophils are the main effector cells of allergic asthma and aggravate the progression of allergic asthma.

[0005] For common respiratory diseases, Western medicine often uses anti-inflammatory drugs such as glucocorticoids, antibiotics and bronchodilators to achieve therapeutic effects by relieving disease symptoms. Traditional Chinese medicine has a multi-component, multi-target and holistic approach to treating diseases. It has achieved significant therapeutic effects by using traditional Chinese medicine compound prescriptions and their preparations, single herbs and their preparations to treat the above diseases, providing an effective solution for the treatment of the above diseases and a new idea for discovering new therapeutic drugs.

[0006] The dried persistent calyx or the persistent calyx with fruit of Physalis alkekengi L.var.franchetii (Mast.) Makino, a plant of the Solanaceae family. The 2020 edition of the Chinese Pharmacopoeia records that Physalis alkekengi "clears away heat and detoxifies, relieves sore throat and resolves phlegm, and promotes diuresis and relieves stranguria". It can be used for sore throat and hoarseness, cough due to phlegm and heat, and external treatment of pemphigus and eczema. In traditional applications, Physalis alkekengi is used as a decoction for oral administration, but there are problems such as large dosage of raw materials, inconvenience in use, poor taste of the liquid medicine, and poor patient compliance.

[0007] The chemical composition of Lanterna odorata has been shown to contain withanolides (mainly physalis bitters), flavonoids, terpenes, aliphatic compounds and alkaloids. Among them, withanolides and flavonoids may be the main pharmacologically active ingredients for the treatment of respiratory diseases. Withanolide compounds are a class of ergostane-type compounds with 28 carbon atoms, in which the carbon at the 22nd or 23rd position is connected to the carbon at the 26th position to form a side chain of a δ or γ lactone ring. Their special structure and significant activity have attracted attention.

[0008] At present, there are patents related to the preparation process of enriching withanolide components in Lantern Festival, but they all have various shortcomings, such as: (1) the preparation method using commonly used extraction solvents and eluents (ethanol) only collects a specific part of the eluate (less than 1BV) during the macroporous resin separation process, and the yield is extremely low; (2) it is necessary to add catalysts or enzymes, adjust the pH, or use methanol and petroleum ether as eluents, which is costly, has strict conditions and complex processes; (3) the main components of the product are flavonoids such as luteolin, while the withanolide components are small; (4) the content is only determined for a certain monomer component, and the total content of withanolide components is low. Summary of the invention

[0009] In view of the shortcomings of the prior art, the present invention aims to provide an active component of withanolides in Lantern Festival and an extraction method and application thereof.

[0010] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0011] In a first aspect, the present invention provides a method for extracting active components of withanolides from Lantern Festival, comprising the following steps:

[0012] The Chinese lantern is crushed, and an ethanol solution with a volume percentage of 0% to 75% is used as an extractant to soak the Chinese lantern, and then reflux extraction is performed to obtain an extract;

[0013] The extract is refined and concentrated to obtain the Lantern Festival extract;

[0014] Pass the Lantern Festival extract through a macroporous adsorption resin column, and use an ethanol solution with a volume fraction of 0%-100% as an eluent for gradient elution;

[0015] During the elution process, high performance liquid chromatography combined with an ultraviolet detector and an evaporative light detector is used to analyze the eluent of each gradient to determine the elution volume of each gradient ethanol solution, and the effluent of 50%-80% ethanol solution is collected to obtain the active components of withanolides.

[0016] In the disclosed prior art, the detection of withanolide components is carried out by high performance liquid chromatography combined with ultraviolet detector. The inventor found that when using ultraviolet detector alone for detection, the relative content of withanolide components in the detected eluate is too high because the ultraviolet detection method cannot detect components without ultraviolet absorption.

[0017] It was found through experiments that when the eluent is detected by an evaporative light detector, the purity of the obtained withanolide active ingredients can be ensured to be better. Therefore, the present invention uses high performance liquid chromatography combined with an ultraviolet detector and an evaporative light detector for analysis during component detection. The ultraviolet detector can infer its component type by the maximum ultraviolet absorption wavelength of the chromatographic peak, and the evaporative light detector can detect compounds with no or very weak ultraviolet absorption. The combined detection method is more accurate, and based on this, the preparation process is further optimized to remove unknown components without ultraviolet absorption in the active component and improve the purity of the product.

[0018] The obtained withanolide active ingredients include but are not limited to Physalin A, Physalin N, Physalin L, Physalin B, 4,7-Didehydroneophysalin B and its isomers, Physapubescin I and its isomers, etc.

[0019] In some embodiments, the extractant is water.

[0020] In the prior art, the extracting agent for withanolide in Lantern Festival is generally ethanol with a volume fraction of more than 65%. However, the inventors found that when ethanol extraction is used, the extraction efficiency is high, but the selectivity for withanolide is weak, and there are many impurities of non-withanolide active ingredients without UV absorption in the extract. When the extract is further purified, such as by using a macroporous resin column, it is still difficult to obtain withanolide active ingredients with higher purity.

[0021] When the extractant is replaced with water, water has a higher selectivity for the withanolide active ingredients in the lycopersicum erythrorhizome, and the impurity content of the non-ultraviolet-absorbing non-withanolide active ingredients in the extract is much less than the impurity content when ethanol is used as the extractant. When purified by a macroporous resin column, a higher purity withanolide active ingredient can be obtained.

[0022] In some embodiments, the time for soaking the Lantern Festival with the extractant is 1-3 hours. Soaking with the extraction solvent before heating and refluxing is conducive to the precipitation of various effective components and shortens the time of reflux extraction.

[0023] In some embodiments, the temperature of reflux extraction is 50-100° C., and the time of reflux extraction is 1.5-4 h.

[0024] Preferably, the mass of the extractant is 8-25 times the mass of the Lantern Festival.

[0025] In some embodiments, when the extractant is an ethanol solution, the volume concentration of the ethanol solution is 55%-75%.

[0026] In some embodiments, the method for purifying the extract is vacuum filtration.

[0027] In some embodiments, the macroporous adsorption resin in the macroporous adsorption resin column is D101, D201, D301, HPD-100, HPD-400, HPD-BJQH or AB-8.

[0028] Preferably, the macroporous adsorption resin is D101 or HPD-BJQH. Compared with other separation methods, macroporous adsorption resin is more suitable for large-scale industrial production. These two macroporous adsorption resins can separate active components of withanolides with strong activity and high purity.

[0029] Further preferably, the macroporous adsorption resin is D101. The active components separated by D101 macroporous resin have the strongest activity and high yield.

[0030] In some embodiments, when gradient elution is performed, the elution volume of 50%-80% ethanol is 3-6 BV, and the elution flow rate is 1-6 BV / h.

[0031] Preferably, when gradient elution is performed, the effluent with a volume fraction of 55%-70% ethanol is collected.

[0032] More preferably, during gradient elution, the effluent with a volume fraction of 60% ethanol is collected.

[0033] Still further preferably, when gradient elution is performed, the gradient elution program is: elution with ethanol volume fractions of 0%, 25%-35%, 45%-50%, 55%-65% and 65%-70% in sequence, the elution volume of each gradient is 3-6BV, and the elution flow rate is 1-6BV / h.

[0034] More preferably, when gradient elution is performed, the gradient elution program is: eluting with ethanol with volume fractions of 0%, 30%, 50%, 60% and 70% in sequence, the elution volume of each gradient is 3-6BV, and the elution flow rate is 1-6BV / h.

[0035] In a second aspect, the present invention provides an active component of withanolides in Lantern Festival, which is extracted by the extraction method.

[0036] In a third aspect, the present invention provides the use of the withanolide active components in the Lantern Festival in the preparation of a medicine for treating pneumonia, asthma, pharyngitis, pulmonary fibrosis or chronic obstructive pulmonary disease.

[0037] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0038] The innovation of the preparation process of withanolide components in brocade lantern provided by the present invention is:

[0039] The present invention combines HPLC, ELSD and in vitro drug activity detection methods, and designs a high-yield, low-cost and high-safety preparation process (only water and ethanol are used) with the aim of being suitable for industrial production.

[0040] A simple macroporous resin preparation process was adopted, and the composition, biological activity and yield of the target product were used as screening indicators for the first time, and D101 macroporous resin was selected from a variety of macroporous resins.

[0041] Through UPLC-MS / MS analysis of the active components, its composition was further clarified, and it contained a variety of withanolide components: Physalin A, Physalin N, Physalin L, Physalin B, 4,7-Didehydroneophysalin B and its isomers, Physapubescin I and its isomers and other novel components;

[0042] The total withanolide content in the active component exceeds 40% by concentrated sulfuric acid-methanol method. Compared with the currently disclosed preparation methods, the present invention can provide a simple and low-cost preparation process for extracting and preparing high-purity withanolide active components from Lantern Festival.

[0043] There are no one or several withanolide monomer components with strong pharmacological effects that are characteristic, high in content, and have strong pharmacological effects in Lanterna chinensis. Moreover, it is difficult to enrich withanolide components, and there is a lack of efficient and low-cost preparation processes. Therefore, establishing a preparation process for efficiently enriching high-purity withanolide components from Lanterna chinensis is of great significance and value for the development and utilization of Lanterna chinensis and the research of innovative drugs for respiratory diseases.

[0044] At the same time, based on the enrichment and preparation of withanolide components in Lantern Festival, the present invention found that it has a significant therapeutic effect on pulmonary fibrosis, asthma, pharyngitis, and chronic obstructive pulmonary disease, and its mechanism of action is closely related to inhibiting oxidative stress and inflammatory response and regulating immune function.

[0045] The target component prepared by the present invention is rich in withanolide, a special steroid component with strong pharmacological activity (including Physalin A, Physalin N, Physalin L, Physalin B, 4,7-Didehydroneophysalin B and its isomers, Physapubescin I and its isomers and other novel structural components), with a total content of more than 40%. The target component inhibits the expression of inflammation-related proteins such as COX2 and iNOS, inhibits the ubiquitination and degradation of Nrf2 protein, and regulates T cell-mediated immune response, thereby inhibiting inflammatory response and oxidative damage activity and immune regulation activity, and has a significant therapeutic effect on respiratory diseases (including pneumonia, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease and pharyngitis). BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0047] Figure 1 It is a process flow chart for preparing the active components of the brocade lantern in Example 1 of the present invention;

[0048] Figure 2 is a process flow chart for preparing the active components of the brocade lantern in Example 2 of the present invention;

[0049] Figure 3 is a process flow chart for preparing the active components of the brocade lantern in Example 3 of the present invention;

[0050] Figure 4 is a process flow chart for preparing the active components of the brocade lantern in Example 4 of the present invention;

[0051] Figure 5 In Example 2 of the present invention, each type of macroporous resin is used to separate the NO generation inhibition activity and QR induction activity of each gradient component;

[0052] Figure 6 The NO generation inhibition activity and quinone reductase (QR) induction activity of the crude extract provided in Example 2 and the 50-EFP-2 and 70-EFP active components according to Example 5 of the present invention;

[0053] Figure 7 The results of Example 5 of the present invention on the quinone reductase (QR) inducing activity and nitric oxide (NO) generation inhibition activity of the target component of Lantern Festival provided in Examples 1, 3, and 4;

[0054] Figure 8 This is a diagram of Example 6 of the present invention showing that the active component 50-EFP-2 provided in Example 2 inhibits the overexpression of LPS-induced inflammation-related proteins COX2 and iNOS at the cellular level;

[0055] Fig. 9 This is a diagram of Example 6 of the present invention showing that the active component 70-EFP provided in Example 2 inhibits the overexpression of LPS-induced inflammation-related proteins COX2 and iNOS at the cellular level;

[0056] Fig.10 This is a characterization diagram of Example 7 of the present invention, which shows that the active component 50-EFP-2 provided in Example 2 induces Beas-2B cell line to inhibit the ubiquitination degradation of Nrf2 protein, promote the expression of downstream protein NQO1, and exert an antioxidant effect at the cellular level;

[0057] Fig.11 This is a characterization diagram of Example 7 of the present invention, which shows that the active component 70-EFP provided in Example 2 induces Beas-2B cell line to inhibit the ubiquitination degradation of Nrf2 protein, promote the expression of downstream protein NQO1, and exert an antioxidant effect at the cellular level;

[0058] Fig.12 The structures of some compounds of the Withania somnifera class in Example 8 and the corresponding UV absorption spectra;

[0059] Fig.13 is the HPLC chromatogram and component analysis results of the target component W60 provided in Example 1 in Example 9 (except for the red numbers, all are withanolide components);

[0060] Fig.14 The UPLC-MS spectrum of the target component W60 provided in Example 1 in Example 9, wherein A is the ion flow diagram in the negative ion mode; B is the ion flow diagram in the positive ion mode;

[0061] Fig.15 The high performance liquid chromatography spectrum of the target component 50-EFP-2 provided in Example 2 in Example 10;

[0062] Fig.16 The UPLC-MS spectra of the target component 50-EFP-2 provided in Example 2 in Example 10 are shown in Figure 10, where A is the ion flow diagram in the negative ion mode; and B is the ion flow diagram in the positive ion mode.

[0063] Fig.17 The high performance liquid chromatography spectrum of the target component 70-EFP provided in Example 2 in Example 10;

[0064] Fig.18 The UPLC-MS spectra of the target component 70-EFP provided in Example 2 in Example 10 are shown in Figure 10, where A is the ion flow diagram in the negative ion mode; and B is the ion flow diagram in the positive ion mode.

[0065] Fig.19 This is a diagram showing the effect of the active components 50-EFP-2 and 70-EFP of the Herba Lycopodii in Example 13 on hematoxylin-eosin (H&E) staining of lung tissues of mice with pneumonia induced by LPS, with scale bars of 50 μm in each figure;

[0066] Fig. 20 This is a graph showing the effects of 50-EFP-2 and 70-EFP on anti-inflammatory and antioxidant related proteins (iNOS, COX2, IκB-α, Nrf2, NQO1) in lung tissue of mice with LPS-induced pneumonia in Example 13;

[0067] Fig.21 is a graph showing the effects of 50-EFP-2 and 70-EFP on the contents of SOD, GSH and MDA in the lung tissues of mice with LPS-induced pneumonia in Example 13;

[0068] Fig. 22 This is a diagram showing the effect of the active components 50-EFP-2 and 70-EFP of the Herba Lycopodii in Example 14 on hematoxylin-eosin (H&E) staining of lung tissues of OVA-induced asthmatic mice, with scale bars of 50 μm in each figure;

[0069] Fig.23 This is a graph showing the effects of 50-EFP-2 and 70-EFP on the expression of inflammatory factors (COX2, NF-κB p65, IκB-α) and antioxidant factors (Nrf2, NQO1) in lung tissue of OVA-induced asthmatic mice in Example 14;

[0070] Fig.24 This is a graph showing the effects of 50-EFP-2 and 70-EFP on the amounts of Th1 (A) and Th2 (B) in the lung tissues of OVA-induced asthma model mice in Example 14;

[0071] Fig.25This is a diagram showing the effect of the active ingredient W60 of the Chinese lantern in Example 15 on the hematoxylin-eosin (H&E) staining of the pharyngeal tissue of rats with pharyngitis induced by ammonia water, and the scale bars in the figures are all 100 μm;

[0072] Fig.26 This is a graph showing the effect of the active ingredient W60 of the Chinese lantern on the levels of TNF-α and IL-1β in the serum of rats with pharyngitis in Example 15;

[0073] Fig. 27 This is a graph showing the effect of the active ingredient W60 of the Chinese lantern plant in Example 15 on the Masson staining of lung tissue in mice with bleomycin-induced pulmonary fibrosis, with scale bars of 50 μm in each figure;

[0074] Fig.28 This is a graph showing the effect of the active ingredient W60 of the lycopodiella citrate in Example 15 on fibrosis-related proteins (α-SMA, Vimentin, E-cadherin, Collagen Ⅰ) in the lung tissue of mice with BLM-induced pulmonary fibrosis;

[0075] Fig.29 This is a comparison diagram of the detection of the 60% ethanol and 70% ethanol components of Example 3 by high performance liquid chromatography combined with ultraviolet detector and evaporative light detector;

[0076] Fig.30 This is a comparison spectrum of the detection of the water-extracted active components of Example 1 and the alcohol-extracted active components of Example 4 using an ultraviolet detector and an evaporative light detector in Example 12, respectively. DETAILED DESCRIPTION

[0077] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0078] The present invention describes the anti-inflammatory and anti-oxidative action mechanisms of the withanolide active components provided by the present invention.

[0079] The present invention describes that the anti-inflammatory effect of the withanolide active components provided by the present invention in the LPS-induced RAW 264.7 cell inflammation model is to inhibit the overexpression of inflammation-related proteins COX2 and iNOS, thereby inhibiting the inflammatory process.

[0080] The present invention describes that the antioxidant effect of the withanolide active components provided by the present invention in mouse liver cancer Hepa 1c1c7 cells is achieved by inhibiting the ubiquitination degradation of Nrf2 protein and promoting the expression of downstream protein NQO1, thereby exerting the antioxidant effect.

[0081] The invention also describes the use of the withanolide active component provided by the invention in preparing medicines for respiratory diseases.

[0082] The withanolide active components provided by the present invention can reduce pathological damage in a mouse pneumonia model. The withanolide active components can reduce changes in lung tissue structure, inhibit the infiltration of interstitial inflammatory cells, the increase of inflammatory factors and the decrease of antioxidant factors, and play a protective role in the LPS-induced mouse pneumonia model.

[0083] The withanolide active components provided by the present invention can reduce the pathological damage of lung tissue in the OVA-induced asthma model, reduce the hyperplasia of goblet cells in lung tissue, inhibit Th2 immune response, enhance Th1 immune response to improve allergic airway inflammation, and significantly improve OVA-induced asthma in mice.

[0084] The withanolide active components provided by the present invention can effectively alleviate acute pharyngitis in rats induced by 15% ammonia water. Pathological examination of rats with pharyngitis treated with the withanolide active components of the present invention showed that inflammatory cells in pharyngeal tissues were reduced and systemic symptoms of rats with acute pharyngitis were significantly improved.

[0085] The withanolide active components provided by the present invention can alleviate bleomycin-induced pulmonary fibrosis in mice. The withanolide active components of the present invention can inhibit the production of pulmonary fibrosis-related proteins. Pathological section results show that the withanolide active components can alleviate the problems of a large number of inflammatory cell infiltration, alveolar structure destruction, significant fibroblast proliferation and extracellular matrix deposition in mouse lung tissue, and inhibit pulmonary fibrosis lesions.

[0086] The present invention will be further described below in conjunction with the embodiments.

[0087] Example 1: Preferred preparation process of the target component of the present invention

[0088] 1 kg of Pleurotus eryngii medicinal materials was crushed and soaked in distilled water at room temperature for 2 hours. The amount of extraction solvent used was 15 times the weight of the medicinal materials. The extraction temperature was 85°C and reflux extraction was performed for 3 hours. The extract was refined by vacuum filtration to obtain a refined solution, which was concentrated to obtain the Pleurotus eryngii water extract.

[0089] The water extract of Lantern Festival was passed through D101 macroporous resin, such as Figure 1As shown, separation was performed according to the gradient of 0%, 40%, and 60% ethanol solution. During the elution process, the eluent of each gradient was analyzed by high performance liquid chromatography combined with an ultraviolet detector and an evaporative light detector to determine the elution volume of each concentration of ethanol solution. The elution volume of the 0% ethanol gradient was 3BV, the elution volume of the 40% ethanol gradient was 4BV, and the elution volume of the 60% ethanol gradient was 5BV. The elution flow rate was 1BV / h. The eluent of the 60% ethanol solution was collected, concentrated under reduced pressure, and dried to obtain the active component W60. It was calculated that the yield of the target component obtained from the medicinal material was 2.0%, and the withanolide content thereof exceeded 40% by the concentrated sulfuric acid-methanol method.

[0090] Example 2: Preparation of the target component of the present invention

[0091] 2 kg of Pleurotus eryngii medicinal materials were crushed and soaked at room temperature for 4 hours. The amount of extraction solvent was 12 times the weight of the medicinal materials. The single-factor experiment was used to investigate the effects of extraction ethanol concentration, extraction temperature, extraction time and reflux times. Through the investigation of the above variables, the best process was finally selected: each extraction time was 3 hours, refluxed 2 times, ethanol concentration was 75%, and extraction temperature was 75°C; the extracts were combined and refined by vacuum filtration to obtain refined solution, which was concentrated and dried to obtain Pleurotus eryngii ethanol extract (PCSF).

[0092] The extract of Lantern Festival was passed through AB-8, HPD-BJQH and D101 macroporous resins, such as Figure 2 As shown, separation was performed according to a gradient of 0%, 30%, 40%, 50%, 70%, 80%, 90%, and 100% ethanol solution, with each gradient elution volume being 3 BV and an elution flow rate being 1.5 BV / h. The gradient eluates were collected, concentrated under reduced pressure, and dried to constant weight.

[0093] The anti-inflammatory and antioxidant activities of the components separated by AB-8, HPD-BJQH and D101 macroporous resins were analyzed. Figure 5 As shown in the figure, the 50% ethanol part and the 70% ethanol part of D101 are highly active, the 70% ethanol part of HPD-BJQH is highly active, and the 80% ethanol part of AB-8 is weakly active, so D101 and HPD-BJQH are preferred; because the yield of active components of withanolide separated by D101 macroporous resin is higher than that of HPD-BJQH, D101 macroporous resin is finally preferred. The 50% ethanol part and the 70% ethanol part of D101 were analyzed for components, and both were active components of withanolide (such as Fig.14 , 17 , except the red marked peak, all others are withanolides). The components separated by D101 macroporous resin (50% ethanol and 70% ethanol) were named 50-EFP-2 and 70-EFP, respectively.

[0094] Example 3: Preparation of active components of Lantern Festival

[0095] The alcohol extract of Lantern Festival in Example 2 was passed through D101 macroporous resin, and the process was as follows Figure 3 , separation was performed according to a gradient of 0%, 40%, 50%, 60%, and 70% ethanol solution, with each gradient elution volume being 3BV and an elution flow rate being 1BV / h. The eluates of each gradient ethanol solution were collected, concentrated under reduced pressure, and dried.

[0096] Example 4: Preparation of active components of Lantern Festival

[0097] The alcohol extract of Lantern Festival in Example 2 was passed through D101 macroporous resin, and the process was as follows Figure 4 , separation was performed according to a gradient of 0%, 40%, and 60% ethanol solution, the elution volume of each gradient was 3BV, the elution flow rate was 1BV / h, the eluate of each gradient ethanol solution was collected, concentrated under reduced pressure, and dried.

[0098] Example 5: Evaluation of the anti-inflammatory and antioxidant activities of active ingredients

[0099] Method: The different gradient components provided in Example 2, the alcohol extract of Lantern Festival (PCSF), 50-EFP-2, 70-EFP, and the target components of Examples 1, 3, and 4 were subjected to nitric oxide (NO) generation inhibition assay. RAW264.7 mouse macrophages were inoculated in 96-well plate A. After the cells adhered to the wall, medium containing different concentrations of the test compound and LPS (1 μg / mL) was added and placed in an incubator for incubation for 24 hours. 100 μL of supernatant was added to 96-well plate B, and then 100 μL of Griess reagent (0.1% naphthylethylenediamine, 5% phosphoric acid, 1% sulfonamide) was added and incubated for 15 minutes. The absorbance of the solution was measured at a wavelength of 570 nm using an enzyme reader. The cell survival rate in 96-well plate A was detected using the MTT experiment. The nitrite concentration was calculated using the NaNO2 standard curve. The method for establishing the NaNO2 standard curve is as follows: prepare 0, 3.125μM, 6.25μM, 12.5μM, 25μM, and 50μM NaNO2 (100μL) standard solutions and add them to a 96-well plate, then add Griess reagent, and use an enzyme reader to detect the absorbance.

[0100] The different gradient components provided in Example 2, the alcohol extract of Lantern Festival (PCSF), 50-EFP-2, 70-EFP, and the target components of Examples 1, 3, and 4 were assayed for quinone reductase (QR) induction activity. Hepa 1c1c7 cells (2×104) were inoculated into a 96-well plate, and the original culture medium was removed after the cells adhered to the wall. 200 μL of DMEM culture medium was added to the control group, and 200 μL of DMEM culture medium containing different concentrations of the test drug was added to the drug-treated group. After incubation for 24 hours, the original culture medium was removed, and 30 μL of cell lysate was added to each well. After incubation for 15 minutes, 170 μL of the complete reaction mixture was added to each well. After 4 minutes, the absorbance of the solution was measured at 630 nm.

[0101] The results are as follows Figure 5 As shown, the anti-inflammatory and antioxidant activities of the components separated by AB-8, HPD-BJQH and D101 macroporous resins were analyzed. The 50% ethanol fraction and the 70% ethanol fraction separated by D101 macroporous resin were highly active, the 70% ethanol fraction of HPD-BJQH was highly active, and the 80% fraction of AB-8 had very weak activity.

[0102] The results are as follows Figure 6 As shown, the 75% extract of Psoralea corylifolia (PCSF) provided in Example 2 has anti-inflammatory and antioxidant activities. At the same dosage, the anti-inflammatory and antioxidant activities of the separated 50-EFP-2 and 70-EFP are stronger.

[0103] The results are as follows Figure 7 As shown, the target component provided by Example 1 has strong anti-inflammatory and antioxidant activities; the target component provided by Example 3 has weak anti-inflammatory and antioxidant activities; and the target component provided by Example 4 has strong anti-inflammatory and antioxidant activities. Example 6: Anti-inflammatory mechanism of active components

[0104] Method: The NO activity of the active components 50-EFP-2 and 70-EFP of withanolide obtained in Example 2 was determined. RAW 264.7 cells were inoculated in a 35 mm diameter culture dish and cultured to a density of 70% to 80%. Different concentrations of active components EFP-50-2 and EFP-70 were added and treated with a medium containing LPS (1 μg / mL) for 24 h. The cells were washed twice with PBS, and a cell lysis solution was added to collect the protein.

[0105] The corresponding proteins were detected by immunoblotting. Prepare separation gel and concentrated gel of appropriate concentration; load each group of samples and markers; adjust the voltage of the protein to 93V when passing through the concentrated gel during electrophoresis, and adjust the voltage to 193V when passing through the separation gel; after the electrophoresis, transfer the membrane, and transfer the membrane for about 120min under 36V voltage. After the transfer, 5% skim milk powder was used for blocking, and the membrane was blocked at 30rpm / min for 1h at room temperature, and then washed with PBST solution. After adding the primary antibody, incubate overnight at 4℃, wash 3 times with PBST solution, add the secondary antibody, and incubate at room temperature for 120min. Wash 3 times with PBST, mix ECL chemiluminescent reagent A solution and B solution at 1:1 (v / v), evenly rinse onto the NC membrane, and develop and take pictures in the Bio-Rad ChemiDoc XRS+ system. The bands were processed using ImageJ and Image Lab software.

[0106] The results are as follows Figure 8 , 9 As shown, after the cells were treated with active components, EFP-50-2 and EFP-70 could effectively reduce the excessive production of NO induced by LPS and inhibit the overexpression of inflammatory factors such as COX2 and iNOS.

[0107] Example 7: Mechanism of action of active components in inhibiting oxidative damage

[0108] Method: The QR activity of the active components 50-EFP-2 and 70-EFP of the withanolide obtained in Example 2 was determined. Beas-2B cells were inoculated in a 35 mm diameter culture dish and cultured overnight. Different concentrations of active components EFP-50-2 and EFP-70 were cultured for 24 h, washed twice with PBS, and cell lysate was added to collect proteins. The corresponding proteins were then detected by the immunoblotting method of Example 6.

[0109] The results are as follows Fig.10 , 11 As shown, the active components EFP-50-2 and EFP-70 inhibited the ubiquitination degradation of Nrf2 protein and promoted the expression of its downstream NQO1 protein, thereby exerting an antioxidant effect.

[0110] Example 8: Analysis of UV absorption patterns of withanolides

[0111] 14 withanolide monomer components were selected and analyzed by full wavelength scanning using high performance liquid chromatography to detect their ultraviolet absorption wavelengths. The results are shown in Table 1. The maximum absorption wavelengths of withanolide components are mostly between 226-232nm, and there are also ultraviolet absorptions above 310nm or 350nm. Fig.12As shown in the figure, it is speculated that due to the increase in the number of conjugated double bonds in the structure of withanolide, the absorption wavelength red-shifts to around 312nm, 314nm, and 356nm. Therefore, 226-232nm is mainly used as the characteristic wavelength of withanolide compounds, and ultraviolet absorption wavelengths such as 312nm and 356nm are used as auxiliary speculations.

[0112] Table 1

[0113]

[0114] Example 9: Analysis of active ingredient composition

[0115] The active component (W60) of Example 1 was analyzed by high performance liquid chromatography, and the component analysis was performed by comparing the UV absorption law of the withanolide components of Example 8. The results are as follows Fig.13 As shown in Table 2, there are various withanolide components in the active ingredients.

[0116] Table 2

[0117]

[0118] The active component (W60) provided in Example 1 was determined by sulfuric acid-methanol method, and the content of withanolide components in W60 was calculated to be 45%.

[0119] In order to further confirm the chemical composition of W60, UPLC-MS / MS analysis was performed, and the chromatographic conditions were: A was 0.1% formic acid aqueous solution, B was acetonitrile; 0-5min, 2-20% B; 5-50min, 20-45% B; 50-60min, 45-70% B; 60-65min, 70-100% B; flow rate was 0.3mL / min; injection volume was 15μL; column temperature was 35°C. Mass spectrometry conditions: electrospray ion source (ESI), detection adopted positive and negative ion scanning and daughter ion scanning mode, and the full scan m / z range was 120-1500.

[0120] The components were further confirmed by structural speculation based on the mass spectrometry database, fragmentation rules and literature reports. The UPLC-MS / MS fingerprint of W60 is shown in the figure. Fig.14 As shown, the relevant data of each chromatographic peak in the W60 fingerprint are shown in Table 3.

[0121] Table 3

[0122]

[0123] Combined with the fragment ions of the compounds reported in the literature, it was preliminarily speculated that peaks 9 and 11 were Physalin N and Physalin A, peak 10 was 4,7-Didehydroneophysalin B, peak 12 was Physalin B, peaks 13 and 15 were Physalin L and Physalin O, respectively. Based on the molecular weight, it was speculated that peak 22 was 5α-Hydroxy-7-dehydro-25,27-dihydro-7-deoxyneophysalin A, peak 25 was Physapubescin I, peak 30 was Daucosterol, and peaks 1, 20 and 31 were flavonoid compounds Cynaroside, Rhamnazin and Chrysoeriol, respectively.

[0124] According to the compound fragment ions and molecular weight, it is speculated that the compounds corresponding to peaks 2, 6, 7, 8, 14, 16, and 21 in the W60 fingerprint spectrum belong to the physalis bitter compounds. Among them, the peaks 6, 7, 8, 14, 16, and 21 compounds have the same molecular weight and are isomers. Since the structures of the physalis bitter compounds are extremely similar and there are many functional group substitution positions, the specific compound names cannot be finally determined. Peaks 27, 28, and 29 have the same [M+H] + Peak (m / z=589), which are isomers of Physapubescin I, are speculated to be withanolide compounds. Based on the fragment ions and molecular weight, it is speculated that they may be steroidal compounds after highly reduced withanolide. The remaining unidentified compounds are speculated to be mostly physalis bitters based on their UV absorption data and mass spectrometry data.

[0125] The structures of the compounds identified in W60 are shown below:

[0126]

[0127] Example 10: Analysis of active ingredients

[0128] The active components 50-EFP-2 and 70-EFP of Example 2 were analyzed for their components.

[0129] HPLC analysis of active components 50-EFP-2 and 70-EFP. Combined with the UV absorption law of the withanolide components in Example 8, the withanolide components in the active components 50-EFP-2 and 70-EFP were identified. The results are as follows: Fig.15 and Fig.17 (Except those marked in red, all are withanolides).

[0130] In order to further confirm the chemical components in 50-EFP-2 and 70-EFP, UPLC-MS / MS analysis was performed, and the chromatographic conditions were as follows: flow rate of 0.3 mL / min; injection volume of 5 μL; column temperature of 35°C; elution conditions: A was 0.1% formic acid in water, B was acetonitrile; gradient elution: 0-2 min, 2-20% B; 2-10 min, 20-30% B; 10-16 min, 30-33% B; 16-40 min, 33-60% B; 40-45 min, 60-80% B; 45-47 min, 80-100% B; 47-53 min, 100% B. Mass spectrometry conditions: electrospray ionization source (ESI); positive and negative ion detection mode; full scan m / z range of 120-1500.

[0131] A mass spectrometry database of compounds extracted, separated and identified from Lanterna chinensis was established, and the withanolide components were further confirmed based on the fragmentation rules and literature reports. Fig.16 The relevant data of each chromatographic peak are shown in Table 4.

[0132] Table 4

[0133]

[0134] By comparing the fragment ions of the compounds, it was preliminarily inferred that the compounds of peaks 3 and 5 were Physalin N and Physalin A, respectively. Peak 13 was 4,7-Didehydroneophysalin B, and peak 11 was probably 25,27-Dihydro-4,7-dedehydro-7-deoxyneophysalin A, and the molecular weight of peak 11 corresponded to its molecular weight.

[0135] According to the compound fragment ions and molecular weight, it is speculated that the compounds corresponding to peaks 1, 2, 4, 6, 8, 9, and 10 in the 50-EFP-2 fingerprint spectrum belong to the physalis bitter compounds. Peaks 18-22 compounds have the same [M+H] + Peak 25 (m / z = 588), which are isomers of Physapubescin I, are speculated to be withanolide compounds. + The compound with m / z=513 is probably the new steroid compound of Philadelphicalactone A isolated from Lanterna chinensis, which is obtained by reducing the double bond, reducing the two carbonyl groups to hydroxyl groups, and reducing the oxygen-containing ring to two hydroxyl groups, with m / z=512.3349. The remaining unidentified compounds in 50-EFP-2 are mostly physalis bitters, and may also include unknown new withanolide compounds, based on their UV absorption data and mass spectrometry data.

[0136] The UPLC-MS / MS fingerprint of 70-EFP is as follows Fig.18 As shown, the relevant data of each chromatographic peak in the 70-EFP fingerprint are shown in Table 5.

[0137] Table 5

[0138]

[0139] It is speculated that peaks 5 and 8 in the 70-EFP fingerprint are compounds Physalin B and Physalin M, respectively. The peak 1 compound has the same molecular weight as the peaks 18-22 compounds in 50-EFP-2. They are isomers and are also speculated to be withanolide compounds. The peak 6 compound has the same molecular weight as Alkekengilin A and Alkekengilin B in Lanterna chinensis. The peak 6 compound is their isomers and is speculated to be a physalis bitter compound.

[0140] The molecular weights of peaks 10-24 were not found in the mass spectrometry database of the established compound of the Chinese lantern. The characteristics of peaks 14-16 and 19-24 are that they have no or weak UV absorption. By consulting the literature and the book "Study on Withanolides" edited by Professor Yang Bingyou, it is speculated that they may be new steroidal compounds obtained after highly reduced Withanolides.

[0141] In summary, qualitative analysis showed that 70-EFP contained physalis bitter compounds and a variety of unknown new withanolide compounds.

[0142] The structures of the compounds identified in 50-EFP-2 and 70-EFP are shown below:

[0143]

[0144] Embodiment 11

[0145] Analysis of the active components of Psoralea corylifolia, illustrating the use of ELSD detector to determine the target components.

[0146] Each gradient component of Example 3 was analyzed by high performance liquid chromatography combined with ultraviolet detector and evaporative light detector. Fig.29As shown, the result of the ultraviolet detector shows that the result similarity of the 60% ethanol elution component and the 70% ethanol elution component is extremely high, and the main chromatographic peaks are all withanolides. However, the evaporative light detector can detect that the two are significantly different, and the 70% ethanol elution component contains multiple components without ultraviolet absorption, and the relative content is relatively large. Thus, the 60% ethanol elution component is determined as the target component of the present invention, and the preparation process optimization is promoted, the withanolide component is verified by the ultraviolet detector, and the unknown components without ultraviolet absorption are excluded by the evaporative light detector, ensuring that the same parts of the 60% ethanol component and the 70% ethanol component are collected together, excluding multiple components without ultraviolet absorption in the 70% ethanol component, and improving its yield and purity.

[0147] Example 12

[0148] The analysis of the active components of P. quinata showed that the purity of withanolide in the separated product was higher when water extraction was determined using ELSD detector than when alcohol extraction was used.

[0149] The active components of Example 1 (water extraction, 60% ethanol elution portion obtained by macroporous resin column chromatography) and the active components of Example 4 (alcohol extraction, 60% ethanol elution portion obtained by macroporous resin column chromatography) were subjected to high performance liquid chromatography analysis combined with an ultraviolet detector and an evaporative light detector for detection and analysis, and the ultraviolet absorption law of the withanolide components of Example 8 was compared for component analysis.

[0150] like Fig.30 As shown, the active components of Example 4, after detection by the ultraviolet detector, have main chromatographic peaks that are all withanolides, but the difference between the evaporative light detection spectrum and the ultraviolet detection spectrum is large, and the evaporative light detector detects multiple components without ultraviolet absorption. The active components of Example 1, the evaporative light detection spectrum is highly consistent with the ultraviolet detection spectrum, indicating that the components detected by the ultraviolet detector can represent the entire composition of the active components, that is, the main components of the components are all withanolides. In summary, on the basis of the conventional HPLC-UV detector detection method, combined with the evaporative light detector, it can be determined that the water extract of Lantern Festival is easier to separate into high-purity withanolide components than the alcohol extract of Lantern Festival.

[0151] Example 13: Establishment and administration of in vivo mouse pneumonia model

[0152] (1) Experimental methods

[0153] The active components of the withanolide 50-EFP-2 and 70-EF provided in Example 2 were verified for in vivo activity. Male C57BL / 6N mice (6-8 weeks, 20-22g, purchased from Beijing Weitong Lihua Company) were used in this study to complete the experiment. The mice were raised in a growth environment with a 12h day and night cycle, ensuring free drinking water and regular feeding. All in vivo experiments were in accordance with the regulations of the Ethics Committee of the School of Pharmacy of Shandong University. Adaptive feeding for one week.

[0154] Grouping: The mice were randomly divided into 5 groups for treatment: blank control group, LPS induced group (10 mg / kg), dexamethasone (Dex) group (1 mg / kg), 50-EFP-2 low-dose group (10 mg / kg), 50-EFP-2 high-dose group (20 mg / kg), 70-EFP low-dose group (10 mg / kg), and 70-EFP high-dose group (20 mg / kg).

[0155] Preparation of drugs: Prepare 5% ethanol (dissolved in saline) solution of positive control drug (Dex) and drug to be tested. LPS is prepared with saline and prepared immediately before use. To prevent clogging of the pulmonary atomization administration device, it is dissolved by ultrasound and filtered with a microporous filter membrane before atomization administration.

[0156] Modeling and administration: The specific experimental operation of the LPS-induced pneumonia modeling process in mice is as follows: grab the mouse and ensure that the mouse head is fixed and the body is in an upright position, insert the lung nebulizer into the mouse trachea to evenly atomize LPS to the lungs on both sides of the mouse. At this time, it can be observed that the tip of the mouse's nose is slightly moist or there are small bubbles coming out, which means that LPS atomization is successful. At the same time, keep the mouse upright for 1 minute. Before LPS atomization, gavage is first used for protection, and the positive control drug (Dex) and the blank control group (5% ethanol) are gavaged in the same manner and volume. The drug is administered every day, and LPS is atomized once every two days. 24 hours after the last LPS atomization, the mice were killed by cervical dislocation and lung tissues were collected.

[0157] Histopathological evaluation: Hematoxylin-eosin (H&E) staining, the upper lobe of the right lung of the mouse was cleaned in normal saline and placed in 3mL of 4% paraformaldehyde for 24 hours, and incubated at room temperature with shaking. The lung tissue was cut from the middle of the largest area, the paraformaldehyde was rinsed with double distilled water, dehydrated and embedded in paraffin. After the wax block cooled, it was marked, the surface was trimmed and cut into slices with a thickness of 3-4μm. Dewaxed in water paraffin, stained with hematoxylin and eosin (H&E), then dried, sealed, and dried again. The image was observed and photographed in the bright field of the BX53+DP73 microscope system.

[0158] Detection of inflammatory factor levels in lung tissue: Take the weighed lung tissue in a 1.5 mL centrifuge tube, add Sample buffer (without bromophenol blue) at a ratio of 1:10, grind and crush the homogenate with an electric grinder until there is no obvious tissue block, let it stand in an ice bath for 30 minutes, centrifuge (4°C, 3500 rpm / min, 15 minutes), take the supernatant, heat it in a 100°C heater for 10 minutes, and ultrasonically crush it for 36 seconds. According to the immunoblot detection method of Example 6, the level of related proteins in the lung tissue homogenate was detected.

[0159] Contents of SOD, GSH and MDA in lung tissue: weighed lung tissue was placed in a 1.5 mL centrifuge tube, and physiological saline was added at a ratio of 1:10 and homogenized. The experimental process was completed according to the instructions of the kit to determine the contents of SOD, GSH and MDA in lung tissue.

[0160] Statistical analysis: Graphpad Prism 8.0 software was used for data statistical processing, and one-way ANOVA (analysis of variance) was used to analyze the significance of data differences.

[0161] (2) Experimental results

[0162] like Fig.19 As shown, LPS induced thickening of alveolar septa, infiltration of interstitial inflammatory cells in lung tissue and proliferation of goblet cells in tracheal wall. Active components 50-EFP-2 and 70-EFP could alleviate LPS-induced pathological damage of mouse lung tissue to varying degrees.

[0163] like Fig. 20 As shown, the active components 50-EFP-2 and 70-EFP can reverse the upregulation of inflammatory-related proteins such as COX2 induced by LPS. In addition, the effects of the active components 50-EFP-2 and 70-EFP on the levels of the antioxidant factor Nrf2 and its downstream NQO1 protein were analyzed. The active components 50-EFP-2 and 70-EFP inhibited the ubiquitination degradation of Nrf2 protein in mouse lung tissue and promoted the expression of its downstream NQO1 protein.

[0164] like Fig.21 As shown, the active components 50-EFP-2 and 70-EFP protected lung tissue from oxidative damage by reducing MDA and increasing the expression of GSH and SOD.

[0165] Example 14: Establishment and administration of in vivo mouse asthma model

[0166] (1) Experimental methods

[0167] The in vivo activity of the active components of Withanolide 50-EFP-2 and 70-EFP provided in Example 2 was verified. This study used 48 female Balb / c mice (7-8 weeks, 20-22g, purchased from Beijing Weitong Lihua Company). The mice were raised in the same environment as the pneumonia model mice. Adaptive feeding for 7 days. All in vivo experiments were in accordance with the regulations of the Ethics Committee of the School of Pharmacy of Shandong University. Adaptive feeding for one week.

[0168] Animal grouping: The animals were randomly divided into 6 groups for treatment: dexamethasone (Dex) group (1 mg / kg), blank control group, OVA-induced group, 50-EFP-2 low-dose group (10 mg / kg), 50-EFP-2 high-dose group (20 mg / kg), 70-EFP low-dose group (10 mg / kg), 70-EFP high-dose group (20 mg / kg), and Polygonum multiflorum 75% ethanol extract (PCSF) group (20 mg / kg).

[0169] Modeling and administration: On days 0, 7, and 14, 200 μL of sensitizer was injected intraperitoneally for sensitization. Days 22-28 were the OVA aerosol challenge stage. On days 15-28, mice were gavaged for protection for 3 hours and then aerosolized with the stimulator for 30 minutes. The positive control drug (Dex) and the blank control group (5% ethanol) were gavaged in the same way. Mice were killed by cervical dislocation 24 hours after the last challenge (day 29), and lung tissues were collected for detection.

[0170] Histopathological evaluation: Lung tissue was processed using the method of Example 13.

[0171] Detection of inflammatory factor levels in lung tissue: The method of Example 13 was used.

[0172] Proportion of immune cells in tissues: Cut the lung tissue of mice into pieces, digest it with collagenase working solution, grind it and filter it into a centrifuge tube to obtain a single cell suspension; the single cell suspension was lysed with red blood cell lysis solution, and then cell stimulating factors were added. Stimulation culture was carried out in culture medium for 4 hours, and cells were scraped with a cell spatula and collected into a flow tube, serum was added, and the tube was blocked at room temperature for 30 minutes. FITC anti-mouse CD4 and Zombie Aqua were labeled for it. TM Fixable Viability Kit antibody; Next, incubate with the nuclear transfection solution prepared according to the instructions, incubate at 4°C in the dark for 30 minutes; Finally, label with Brilliant Violet421 TM anti-mouse IFN-γ and APC anti-mouse IL-4 antibodies.

[0173] Statistical analysis: Graphpad Prism 8.0 software was used for data statistical processing, and one-way ANOVA (analysis of variance) was used to analyze the significance of data differences.

[0174] (2) Experimental results

[0175] like Fig. 22 As shown, no abnormalities were found in the blank control group, while the lung sections of mice in the OVA-induced group showed inflammatory cell infiltration in the alveoli and proliferation of goblet cells around blood vessels. The administration of active components 50-EFP-2 and 70-EFP significantly alleviated the pathological features and the accumulation of inflammatory cells in the lungs.

[0176] like Fig.23 As shown in the figure, OVA can induce the upregulation of COX2 and NF-κB proteins and promote the degradation of IκB-α. The active components 50-EFP-2 and 70-EFP can reverse the ubiquitination degradation of Nrf2 protein and the expression of its downstream NQO1 induced by OVA, exerting an antioxidant effect.

[0177] The results are as follows Fig.24 As shown, the proportion of Th1 cells in the lung tissue of mice induced by OVA decreased, while the proportion of Th2 cells increased. After treatment with the active components 50-EFP-2 and 70-EFP, the proportion of Th1 cells increased, while the proportion of Th2 cells decreased, regulating the immune response and alleviating OVA-induced asthma in mice.

[0178] Example 15: Establishment and administration of rat pharyngitis model in vivo

[0179] (1) Experimental methods

[0180] The active ingredient W60 provided in Example 1 was verified for in vivo activity. This study used 48 SD rats (180-200 g, purchased from Beijing Weitong Lihua Company). The rats were raised in the same environment as the pneumonia model mice. All in vivo experiments were in accordance with the regulations of the Ethics Committee of the School of Pharmacy of Shandong University. Adaptive feeding for one week.

[0181] Animal grouping: The animals were randomly divided into 6 groups for treatment: amoxicillin group (0.3 g / kg), blank control group, ammonia stimulation group, W60 low-dose group (10 mg / kg), W60 high-dose group (20 mg / kg), and extract group (20 mg / kg).

[0182] Modeling and drug administration: Except for the blank control group, the rats in the other groups were sprayed with 15% ammonia water into the pharynx of the rats using a laryngeal sprayer twice a day, once in the morning and once in the afternoon, 3 times each time, for 3 consecutive days. Drug administration began on the 4th day, once a day, by gavage, for 5 days. The weight of the rats was measured every day, and their behavior and pharyngeal state were observed, and their orbital blood was collected.

[0183] Histopathological evaluation: Hematoxylin-eosin (H&E) staining was the same as in Example 13.

[0184] Collection of orbital blood and ELISA test: Before killing the rats, orbital blood was collected, left to stand at room temperature for 3 hours, and then centrifuged (3000 rpm / min, 20 min). The supernatant was collected as serum and stored at -80°C until use. The serum was then subjected to ELISA analysis of IL-1 and TNF-α.

[0185] Statistical analysis: Graphpad Prism 8.0 software was used for data statistical processing, and one-way ANOVA (analysis of variance) was used to analyze the significance of data differences.

[0186] (2) Experimental results

[0187] like Fig.25 Observation of staining results showed that the pharyngeal tissue of the blank control group was clearly layered, the mucosal epithelium did not proliferate, the inflammatory cells in the lamina propria were rare, and the submucosal glandular structure was normal; the pharyngeal tissue of the model group was irregularly layered, the mucosal epithelium was desquamated or keratinized, the epithelium was slightly thickened, a large number of inflammatory cells infiltrated in the lamina propria, and there was an increase in glands; the changes in the pharyngeal tissue structure of the amoxicillin (20mg / kg) group and the W60-H (40mg / kg) group were significantly alleviated compared with the model group, the desquamation of the mucosal epithelium was repaired, the infiltration of inflammatory cells was reduced, the number of glands was reduced, and the epithelial thickening phenomenon of the W60-H (40mg / kg) group was improved. W60 can reduce inflammatory cell infiltration to a certain extent and promote the repair of mucosa and glands. W60 has a certain therapeutic effect on pharyngitis in rats.

[0188] like Fig.26 As shown in the results, the levels of IL-1 and TNF-α in the serum of rats with pharyngitis were significantly increased, and the levels of IL-1 and TNF-α in the serum of rats with pharyngitis were significantly reduced after treatment with the active component of Lanterna chinensis (W60), Lanterna chinensis extract and the positive control drug amoxicillin capsule. This indicates that W60 can reduce the levels of inflammatory factors in the pharyngeal tissue of rats with pharyngitis.

[0189] Example 16: Establishment and administration of an in vivo mouse pulmonary fibrosis model

[0190] (1) Experimental methods

[0191] The active component W60 provided in Example 1 was verified for in vivo activity. This study used 36 male C57BL / 6N mice (6-8 weeks, 20-22g, purchased from Beijing Weitong Lihua Company) to complete the experiment. The mice were raised in the same environment as the pneumonia model mice. All in vivo experiments were in accordance with the regulations of the Ethics Committee of the School of Pharmacy of Shandong University. Adaptive feeding for one week.

[0192] Animal grouping: The animals were randomly divided into 6 groups for treatment: pirfenidone group (1 mg / kg), blank control group, bleomycin induced group, W60 low-dose group (60 mg / kg), W60 high-dose group (120 mg / kg), and extract group (60 mg / kg).

[0193] Modeling and administration: The blank control group was injected with 0.1 mL of saline intratracheally, and the model group was injected with bleomycin (6 mg / kg) dissolved in saline intratracheally, 0.1 mL per group. After injection, the mice were quickly rotated upside down to mix the bleomycin injection or saline in the lung tissue. After the operation, the mice were kept warm and woke up and fed and watered normally. On the second day after the operation, the drug was administered by gavage for 3 weeks, and the mice were killed by cervical dislocation, and the lung tissue was collected for detection.

[0194] Histopathological evaluation: Masson staining: lung tissue was dewaxed and rehydrated, stained with Weigert's iron hematoxylin, differentiated with acetic acid ethanol differentiation solution, washed with water, bluing with Masson blueing solution, washed with water, washed with deionized water, stained with Ponceau fuchsin staining solution, washed with 0.15% acetic acid working solution, differentiated with phosphomolybdic acid solution, observed under a microscope, terminated differentiation, washed with 0.15% acetic acid working solution, stained with aniline blue staining solution, washed with 0.2% acetic acid working solution, rapidly dehydrated with 95% alcohol twice, dehydrated with 100% ethanol three times, transparentized with xylene twice, sealed with neutral gum and coverslip, and then Masson staining was performed.

[0195] Detection of proteins related to pulmonary fibrosis: The method of Example 13 was used.

[0196] Statistical analysis: Graphpad Prism 8.0 software was used for data statistical processing, and one-way ANOVA (analysis of variance) was used to analyze the significance of data differences.

[0197] (2) Experimental results

[0198] like Fig. 27 As shown in the figure, the tissue structure of the blank control group was intact, the alveolar cavity was clear, there was only a small amount of blue collagen deposition, and the alveolar septum was not edematous or widened. The lung tissue of the mice in the bleomycin-induced group had inflammatory cell infiltration. Compared with the bleomycin-induced group, the mice in the W60-administered group and the extract group had less inflammatory cell infiltration in the lung tissue and less damage to the alveolar structure. The effect of W60 was significantly better than that of the extract group. This shows that W60 can reduce the fibrotic lesions of the lung tissue in mice.

[0199] like Fig.28As shown in the figure, in the model group, the expression of fibrosis-related proteins (CollagenⅠ, Vimentin, α-SMA) was significantly higher than that in the blank group, while in the pirfenidone group and the active component of Lantern Festival (W60) administration group, the expression of fibrosis-related proteins was significantly reduced; in the model group, the protein expression of epithelial cell marker E-cadherin was significantly reduced, while in the pirfenidone group and the active component of Lantern Festival (W60) administration group, the protein expression of E-cadherin was significantly increased. This shows that W60 can reduce the extracellular matrix deposition in mice with pulmonary fibrosis.

[0200] Example 17: Preparation of tablets

[0201] 30 g of active component W60 (provided in Example 1) is added with 40 g of corn starch and 20 g of dextrin, and the mixture is thoroughly mixed to prepare granules. The granules are dried and granulated, and an appropriate amount of talcum powder is added and tablets are obtained.

[0202] Example 18: Preparation of capsules

[0203] 30 g of active component W60 (provided in Example 1) is added with 30 g of starch, 15 g of dextrin and 10 g of microcrystalline cellulose, and the mixture is thoroughly mixed to prepare granules. The granules are dried and granulated, and an appropriate amount of magnesium stearate is added to the granules. The granules are then filled into capsules to obtain the product.

[0204] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting active components of withanolides from Lantern Festival, characterized in that: The steps include: The Chinese lantern is crushed, and an ethanol solution with a volume percentage of 0% to 75% is used as an extractant to soak the Chinese lantern, and then reflux extraction is performed to obtain an extract; The extract is refined and concentrated to obtain the Lantern Festival extract; Pass the Lantern Festival extract through a macroporous adsorption resin column, and use an ethanol solution with a volume fraction of 0%-100% as an eluent for gradient elution; During the elution process, high performance liquid chromatography combined with an ultraviolet detector and an evaporative light detector is used to analyze the eluent of each gradient to determine the elution volume of each gradient ethanol solution, and the effluent of 50%-80% ethanol solution is collected to obtain the active components of withanolides.

2. The method for extracting active components of withanolides from Lantern Festival according to claim 1, characterized in that: The extractant is water.

3. The method for extracting active components of withanolides from Lantern Festival according to claim 1, characterized in that: The time for soaking the Lantern Festival with the extractant is 1-3 hours; Preferably, the reflux extraction temperature is 50-100°C, and the reflux extraction time is 1.5-4h; Preferably, the mass of the extractant is 8-25 times the mass of the Lantern Festival.

4. The method for extracting withanolide active components from Lantern Festival according to claim 1, characterized in that: When the extractant is an ethanol solution, the volume concentration of the ethanol solution is 55%-75%.

5. The method for extracting withanolide active components from Lantern Festival according to claim 1, characterized in that: The method for purifying the extract is vacuum filtration.

6. The method for extracting withanolide active components from Lantern Festival according to claim 1, characterized in that: The macroporous adsorption resin in the macroporous adsorption resin column is D101, D201, D301, HPD-100, HPD-400, HPD-BJQH or AB-8.

7. The method for extracting withanolide active components from Lantern Festival according to claim 1, characterized in that: The macroporous adsorption resin is D101 or HPD-BJQH; preferably, the macroporous adsorption resin is D101.

8. The method for extracting withanolide active components from Lantern Festival according to claim 1, characterized in that: When gradient elution is performed, the elution volume of 50%-80% ethanol is 3-6BV, and the elution flow rate is 1-6BV / h; Preferably, when gradient elution is performed, the effluent having a volume fraction of 55%-70% ethanol is collected; Preferably, when gradient elution is performed, the effluent having a volume fraction of 60% ethanol is collected; Preferably, when gradient elution is performed, the gradient elution program is: eluting with ethanol with volume fractions of 0%, 25%-35%, 45%-50%, 55%-65% and 65%-70% in sequence, the elution volume of each gradient is 3-6BV, and the elution flow rate is 1-6BV / h; Preferably, when gradient elution is performed, the gradient elution program is: eluting with ethanol with volume fractions of 0%, 30%, 50%, 60% and 70% in sequence, the elution volume of each gradient is 3-6BV, and the elution flow rate is 1-6BV / h.

9. An active ingredient of withanolides in Lantern Festival, characterized in that: The extract is obtained by extraction using any one of the extraction methods described in claims 1-8.

10. Use of the withanolide active ingredients in the Lantern Festival of Claim 9 in the preparation of a medicine for treating pneumonia, asthma, pharyngitis, pulmonary fibrosis or chronic obstructive pulmonary disease.

Images