Application of extracts from Rhizoma Cibotii in the preparation of drugs for the treatment of acute lung injury

Abstract

The present invention relates to the field of traditional Chinese medicine, and discloses the use of an extract of Rhizoma Cibotii in the preparation of a drug for treating acute lung injury. The present invention obtains an extract of Rhizoma Cibotii with a therapeutic effect on acute lung injury by separating it through a specific extraction method. The present invention further finds that the extract of Rhizoma Cibotii plays an effect of alleviating acute lung injury through the endoplasmic reticulum stress pathway of lung tissue cells, and this discovery can provide an effective preclinical research basis for the clinical application of the extract of Rhizoma Cibotii and the development of new drugs.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to a method for preparing a philadelphus chinensis extract and application of the extract in preparing a drug for treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a disease of pulmonary diffusion dysfunction caused by a variety of pathogenic factors inside and outside the lungs. The main clinical manifestations are hypoxemia, severe edema in the alveoli and lung parenchyma, and dyspnea. In severe cases, it can develop into acute respiratory distress syndrome (ARDS), with a mortality rate of 30-45%. The pathogenesis of ALI is complex. Intrapulmonary factors such as inhalation of harmful gases, tissue fibrosis, destruction of the alveolar capillary barrier / permeability changes, and extrapulmonary factors such as trauma, infection, and shock can all lead to ALI. The most prominent pathophysiological feature of ALI is the destruction of the pulmonary microvascular endothelial barrier and the death of lung epithelial cells.

[0003] Currently, the available treatments for ALI are divided into supportive therapy and drug intervention. The lung protection strategy of mechanical ventilation is currently considered to be the only supportive therapy that can effectively improve survival. Physiologically based drug therapy refers to the use of drugs that affect ventilation, diffusion, or perfusion to relieve the symptoms of ALI. In fact, there is currently no effective drug therapy for ALI that can significantly reduce mortality and improve the quality of life of patients.

[0004] Jiulongteng is a woody vine of the genus Bauhinia in the Leguminosae family. It is also known as Yangti Teng, Wulang Teng, Meihua Rugu Dan, etc. It is distributed in Zhejiang, Jiangxi, Fujian and other provinces and is a common medicine of the She ethnic group. It has the effects of promoting qi and blood circulation, dispelling wind and dampness, and is mainly used to treat rheumatism, bone pain, trauma, etc. In view of the above traditional uses, modern pharmacological studies on Jiulongteng mainly focus on the pharmacological effects of scavenging free radicals, anti-inflammatory, and analgesia in the treatment of trauma and rheumatism. The pharmacological effects of Jiulongteng have no strong correlation and connection with ALI. Therefore, there is no research and literature report on the use of Jiulongteng in the treatment of acute lung injury. Moreover, based only on the traditional uses of Jiulongteng and the results of modern pharmacological studies, it is impossible to determine whether Jiulongteng can be used to treat ALI. On the other hand, the active ingredients in Jiulongteng are numerous and complex, and the specific effects and mechanisms of action of these active ingredients have not yet been fully clarified. Therefore, it is necessary to further determine the specific effects and mechanisms of action of different active ingredients in Jiulongteng in order to better exert its medicinal value. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the use of an extract of Rhizoma Cibotii in the preparation of a drug for the treatment of acute lung injury. The present invention obtains an extract of Rhizoma Cibotii by a specific process, and finds that the extract of Rhizoma Cibotii has a significant therapeutic effect on acute lung injury. The main active ingredient in the extract of Rhizoma Cibotii is PMF1-5 after testing; further, the present invention finds that the extract of Rhizoma Cibotii plays a role in relieving acute lung injury through the endoplasmic reticulum stress pathway of lung tissue cells. This result provides an effective preclinical research basis for the clinical application of Rhizoma Cibotii extract and the development of new drugs.

[0006] The specific technical scheme of the present invention is: First, the present invention provides an application of an extract of Rhizoma Cibotii in the preparation of a drug for treating acute lung injury. The components of the extract of Rhizoma Cibotii include: 5,6,7,3',4'-pentamethoxyflavone (PMF1), 5,6,7,3',4',5'-hexamethoxyflavone (PMF2), 5,7,3',4',5'-pentamethoxyflavone (PMF3), 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavone (PMF4) and 5,7,5'-trimethoxy-3',4'-methylenedioxyflavone (PMF5).

[0007] At present, it is known that Jiulongteng has anti-tumor, antioxidant and anti-hypoxia effects. However, due to the complex chemical composition of Jiulongteng, the types and contents of active ingredients in Jiulongteng extracts obtained by different extraction processes are different. Therefore, there is no Jiulongteng extract with significant therapeutic effect on acute lung injury disclosed in the prior art. The present invention obtains a Jiulongteng extract by a specific process, and it is found that the Jiulongteng extract has a significant therapeutic effect on acute lung injury. After testing, the main active ingredients in the Jiulongteng extract are PMF1-5. In addition, the present invention further finds that the Jiulongteng extract containing the above-mentioned specific active ingredient combination has a synergistic effect on the treatment of acute lung injury compared with a single PMF compound; specifically, the present invention finds through experiments that Jiulongteng extract (PMFs) can alleviate acute lung injury through the endoplasmic reticulum stress pathway of lung tissue cells. This result can provide an effective preclinical research basis for the clinical application of Jiulongteng extract and the development of new drugs.

[0008] Preferably, the mass ratio of 5,6,7,3',4'-pentamethoxyflavone (PMF1), 5,6,7,3',4',5'-hexamethoxyflavone (PMF2), 5,7,3',4',5'-pentamethoxyflavone (PMF3), 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavone (PMF4) and 5,7,5'-trimethoxy-3',4'-methylenedioxyflavone (PMF5) in the Jiulongteng extract is (2-3):(35-40):(38-42):(14-18):(2-4).

[0009] Secondly, the present invention provides a method for preparing the safflower extract, which comprises the following steps: 1) The decoction pieces of the Herba Cynoglossi are crushed and extracted with methanol, and then concentrated to obtain an extract 1. This step mainly obtains the total extract of the Herba Cynoglossi, and the extract 1 mainly contains plant pigments, polysaccharides, water-soluble phenolic acids, catechins, flavonoid glycosides, flavonoids, steroids, terpenes and other impurities.

[0010] 2) Mix extract 1 and diatomaceous earth, load the sample onto a glass column, elute with petroleum ether and ethyl acetate in turn, collect the ethyl acetate eluate, and concentrate to obtain extract 2. In this step, petroleum ether is first used to remove low-polarity fat-soluble impurities in the total extract, and then ethyl acetate is used to elute the extract 2, which mainly contains catechins, flavonoid glycosides, flavonoids, steroids, and terpenes. Polysaccharides and water-soluble phenolic acids are left on the diatomaceous earth for removal.

[0011] 3) Mix extract 2 with silica gel, load it onto a silica gel chromatography column, and elute it with a gradient of petroleum ether-ethyl acetate with decreasing volume ratio of petroleum ether, collect the eluate, and concentrate it to obtain extract 3. This step mainly utilizes the polarity difference of the forward chromatography, and uses a high ratio of petroleum ether-ethyl acetate to elute and remove the steroids and terpenoids with low polarity first, and then elutes with petroleum ether-ethyl acetate with appropriate polarity to obtain extract 3, which mainly contains catechins, flavonoid glycosides, and flavonoids.

[0012] 4) Load extract 3 onto an ODS-C18 column, elute with methanol-water at different volume ratios, collect the eluate, and concentrate to obtain extract 4. This step mainly utilizes the difference in elution power of reverse chromatography, and uses a low ratio of methanol-water to elute and remove hydrophilic catechins and most flavonoid glycosides, and then uses methanol-water with an appropriate elution power to obtain extract 4 containing flavonoids and a small amount of flavonoid glycosides.

[0013] 5) HPLC separation of extract 4 to obtain the extract of Jiulongteng. This step mainly utilizes the separation ability of preparative high performance liquid chromatography to further remove a small amount of flavonoid glycosides and other types of flavonoid impurities before and after the polymethoxyflavonoid chromatographic peak to obtain five polymethoxyflavonoid effective parts PMFs with high purity.

[0014] Preferably, in 1), the mass ratio of the Herba Artemisiae Rhizoma decoction pieces to methanol is 1:(5-15).

[0015] Preferably, in 1), the extraction is firstly soaking in methanol for 10-15h, then ultrasonically extracting at least twice at 40-60°C, combining the extracts and concentrating to obtain extract 1.

[0016] Preferably, in 2), the extract 1 is freeze-dried and then mixed with 8-12 weight percent diatomaceous earth and dried.

[0017] Preferably, in 2), the mixture is first eluted with petroleum ether for 3-7 times of column volume, and then eluted with ethyl acetate for 3-7 times of column volume.

[0018] Preferably, in 3), during the gradient elution, the volume proportion of petroleum ether in petroleum ether-ethyl acetate is 35-45%, 25-35%, 15-25% and 5-15% respectively; and the column volume of each elution is 2-6 times.

[0019] Preferably, in 3), the mesh size of the silica gel is 50-200 meshes.

[0020] Preferably, in 3), the mass ratio of the extract 2 to silica gel is 1:(1.5-2.5).

[0021] Preferably, in 3), the mesh size of the silica gel chromatography column is 100-300 meshes.

[0022] Preferably, in 4), elution is performed with methanol-water having a methanol volume content of 45-55%, 25-35% and 35-45% in sequence; the column volume of each elution is 3-7 times.

[0023] Preferably, in 5), the extract 4 is dissolved in methanol and injected into a preparative high performance liquid chromatography with methanol-water as the mobile phase, and the eluate is collected and concentrated to obtain the Herba Codonopsis pilosulae extract.

[0024] Preferably, the drug is in the form of an oral preparation, an injection preparation, an inhalation aerosol, or a nasal drop.

[0025] Preferably, the medicine comprises the extract of Rhizoma Cibotii as a medicinal ingredient, and pharmaceutically acceptable excipients.

[0026] In a second aspect, the present invention provides a method for reducing in vitro lung tissue cell damage using the above-mentioned Rhizoma Cibotii extract, wherein the lung tissue cell damage is caused by LPS activating the endoplasmic reticulum stress signaling pathway.

[0027] The present invention has found through research that LPS can activate different endoplasmic reticulum stress signaling pathways in the lungs, activating IRE1α, PERK and ATF6 respectively. The nephrolepis extract obtained by the specific extraction method of the present invention plays a negative regulatory role in the endoplasmic reticulum stress activated by LPS. Specifically, after the lung tissue cells were treated with the nephrolepis extract, the expression of endoplasmic reticulum stress signals in the lung tissue cells was significantly reduced, and the phosphorylation level of eIF2α in the lung tissue cells was reduced. The nephrolepis extract blocked the apoptosis signaling pathways of JNK, CHOP and TRAF2 to reduce lung tissue cell damage. The nephrolepis extract also directly reduced the expression of p-eIF2α in lung tissue, which means that the nephrolepis extract plays a role in controlling endoplasmic reticulum stress.

[0028] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention obtains a Herba Corydalis extract by a specific extraction process, and it is found that the Herba Corydalis extract has a significant therapeutic effect on acute lung injury. The main active ingredient in the Herba Corydalis extract is PMF1-5.

[0029] (2) The present invention discovered that the extract of Rhizoma Corydalis can alleviate acute lung injury through the endoplasmic reticulum stress pathway of lung tissue cells. This result provides an effective preclinical research basis for the clinical application of Rhizoma Corydalis extract and the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 :A is the HPLC-QTOF-MS total ion chromatogram of extract 2 (a) and PMFs (b); BF is the total ion chromatogram of PMF 1-5 1 H-NMR spectrum and high-resolution mass spectrometry identification results. G is the HPLC-DAD chromatogram of PMF 1-5 standard (a) and PMFs sample (b).

[0031] Figure 2 : Effects of extract 1, extract 2, and PMFs on the expression level of IL-6 inflammatory factor in LPS-induced RAW264.7 cell inflammation model.

[0032] Figure 3 : Effects of extract 1, extract 2, and PMFs on the expression level of IL-1β inflammatory factor in LPS-induced RAW264.7 cell inflammation model.

[0033] Figure 4:Effects of PMF1-5 and PMFs on the expression level of IL-6 inflammatory factor in LPS-induced RAW264.7 cell inflammation model.

[0034] Figure 5 :Effects of PMF1-5 and PMFs on the expression level of IL-1β inflammatory factor in LPS-induced RAW264.7 cell inflammation model.

[0035] Figure 6 :Protective effect of PMFs on acute lung injury induced by LPS in mice. A is the experimental schedule of the study design. B Experimental results of body weight change (n = 12); C Experimental results of lung weight / body weight ratio (n = 6); D Experimental results of lung wet-to-dry weight ratio (W / D) (n = 6); E H&E staining to detect histological changes in mouse lung tissue, scale bar = 200 μm. Data are expressed as mean ± SD, *p < 0.05, **p < 0.01.

[0036] Figure 7 :Study on the protective mechanism of PMFs against LPS-induced acute lung injury in mice. AD shows the effects of PMFs on the expression of IL-6, IL-1β, TNF-α and IFN-γ in mouse lung tissue; E shows the Western blot analysis of Ikkα, IκB, p-IκB, NF-κB, NF-κB, p-NF-κB and GAPDH protein expression; F shows the quantitative analysis of fp-IκB / IκB, p-NF-κB / NF-κB and Ikkα expression. Data are expressed as mean ± SD; n = 3, *p < 0.05, **p < 0.01.

[0037] Figure 8 :Regulatory effect of PMFs on oxidative stress in LPS-induced acute lung injury in mice. A is a micrograph of bronchoalveolar lavage fluid (BALF), scale bar = 20 μm; B is the total cell count in BALF; C is the neutrophil count in BALF; D is the macrophage count in BALF; E is the level of GSH-Px in BALF; F is the level of CAT in BALF; G is the level of MDA in BALF; H is the level of HYP in BALF; I is the quantitative analysis of the proportion of DHE-positive cells; J is the DHE staining picture of lung tissue. Scale bar = 50 μm. Data are expressed as mean ± SD; n = 3, *p < 0.05, **p < 0.01.

[0038] Fig. 9:Effects of PMFs on ER stress in LPS-induced acute lung injury in mice. A, B are Western blot analyses of ER stress-related proteins, including ATF4, PERK, p-PERK, eIF2α, eIF2α-eIF2α, CHOP, IRE-1α, p-IRE-1α, TRAF2, ATF6, JNK, p-JNK, and GAPDH; C, D are quantitative analyses of the expression of ATF4, CHOP, TRAF2, and ATF6 and p-PERK / PERK, p-eIF2α / eIF2α, p-IRE-1α / IRE-1α, and p-JNK / JNK; E is immunohistochemistry of GRP78 antibody in lung tissue, scale bar = 50 μm. Data are expressed as mean ± SD; n = 3, *p < 0.05, **p < 0.01.

[0039] Fig.10 :Effects of PMFs on apoptosis of lung cells in LPS-induced acute lung injury in mice. A is TUNEL staining of the lung, green dots are TUNEL-positive cells, scale bar = 20 μm; B is quantitative analysis of the proportion of TUNEL-positive cells; C is Western blot analysis of apoptosis-related proteins Caspase 3, Cleaved-Caspase 3, Bcl-2, BAX and GAPDH; D is quantitative analysis of the relative expression of Bcl-2 and BAX and the expression of Cleaved-Caspase 3 / Caspase 3. Data are expressed as mean ± SD; n = 3, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with the embodiments.

[0041] Overall embodiment First, the present invention provides an application of an extract of Rhizoma Cibotii in the preparation of a drug for treating acute lung injury. The components of the extract of Rhizoma Cibotii include: 5,6,7,3',4'-pentamethoxyflavone (PMF1), 5,6,7,3',4',5'-hexamethoxyflavone (PMF2), 5,7,3',4',5'-pentamethoxyflavone (PMF3), 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavone (PMF4) and 5,7,5'-trimethoxy-3',4'-methylenedioxyflavone (PMF5).

[0042] Preferably, the mass ratio of 5,6,7,3',4'-pentamethoxyflavone (PMF1), 5,6,7,3',4',5'-hexamethoxyflavone (PMF2), 5,7,3',4',5'-pentamethoxyflavone (PMF3), 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavone (PMF4) and 5,7,5'-trimethoxy-3',4'-methylenedioxyflavone (PMF5) in the Jiulongteng extract is (2-3):(35-40):(38-42):(14-18):(2-4).

[0043] Secondly, the present invention provides a method for preparing the safflower extract, which comprises the following steps: 1) The pieces of Rhizoma Cibotii were crushed and extracted with methanol, and then concentrated to obtain extract 1.

[0044] In some preferred implementation cases, in 1), the Herba Artemisiae Rhizoma decoction pieces are crushed and then passed through a No. 2 sieve.

[0045] In some preferred implementation cases, in 1), the mass ratio of the Herba Artemisiae Rhizoma decoction pieces to methanol is 1:(5-15).

[0046] In some preferred implementation cases, in 1), the extraction is firstly soaking in methanol for 10-15h, then ultrasonically extracting at least twice at 40-60°C, combining the extracts and concentrating to obtain extract 1.

[0047] In some more preferred implementation cases, in 1), the ultrasonic extraction is performed 4-6 times; and the concentration is performed under reduced pressure at a temperature of 40-60°C.

[0048] 2) Mix extract 1 and diatomaceous earth, load the mixture onto a glass column, elute with petroleum ether and ethyl acetate in turn, collect the eluate, and concentrate to obtain extract 2.

[0049] In some preferred implementation cases, in 2), the extract 1 is freeze-dried and then mixed with 8-12 weight percent diatomaceous earth and dried.

[0050] In some preferred implementation cases, in 2), petroleum ether is first used for elution for 3-7 times of column volume, and then ethyl acetate is used for elution for 3-7 times of column volume.

[0051] In some more preferred implementation cases, in 2), the freeze-drying time is 40-60 hours; the drying is reduced pressure drying at a temperature of 40-60°C for 20-30 hours; and the concentration is reduced pressure concentration at a temperature of 40-60°C.

[0052] 3) Mix extract 2 and silica gel, load the mixture onto a silica gel chromatography column, and elute with a gradient of petroleum ether-ethyl acetate with a decreasing volume ratio of petroleum ether. Collect the eluate and concentrate it to obtain extract 3.

[0053] In some preferred implementation cases, in 3), during the gradient elution, the volume proportion of petroleum ether in petroleum ether-ethyl acetate is 35-45%, 25-35%, 15-25% and 5-15% respectively; the column volume of each elution is 2-6 times.

[0054] In some preferred implementation cases, in 3), the mesh size of the silica gel is 50-200 meshes.

[0055] In some preferred implementation cases, in 3), the mass ratio of the extract 2 to silica gel is 1:(1.5-2.5).

[0056] In some preferred implementation cases, in 3), the mesh size of the silica gel chromatographic column is 100-300 meshes.

[0057] In some preferred implementation cases, in 3), the concentration is concentrated under reduced pressure at a temperature of 40-60°C.

[0058] 4) Load extract 3 onto an ODS-C18 column, elute with methanol-water in different volume ratios, collect the eluate, and concentrate to obtain extract 4.

[0059] In some preferred implementation cases, in 4), elution is performed with methanol-water having a methanol volume content of 45-55%, 25-35% and 35-45% in sequence; the column volume of each elution is 3-7 times.

[0060] In some preferred implementation cases, in 4), the concentration is concentrated under reduced pressure at a temperature of 40-60°C.

[0061] 5) The extract 4 was separated by HPLC to obtain the Herba Cibotii Herba Extract.

[0062] In some preferred implementation cases, extract 4 is dissolved in methanol and injected into preparative high performance liquid chromatography, with methanol-water as the mobile phase, and the eluate is collected and concentrated to obtain the phylloxera extract.

[0063] In some more preferred implementation cases, the volume ratio of methanol to water in methanol-water is 40:60-55:45.

[0064] In some more preferred implementation cases, a chromatographic column Daisogel C18 10 μm 100A 20 mm×250 mm is used.

[0065] In some preferred implementation cases, the drug is in the form of an oral preparation, an injection preparation, an inhalation aerosol, or a nasal drop; more preferably, the oral preparation is specifically a tablet, a capsule, or a granule; the injection preparation is specifically an injection solution, a sterile powder for injection, or a concentrated solution for injection, which can be administered by intramuscular injection, intravenous injection, or intravenous drip.

[0066] Preferably, the medicine comprises the extract of Rhizoma Cibotii as a medicinal ingredient, and pharmaceutically acceptable excipients.

[0067] Finally, the present invention provides a method for reducing in vitro lung tissue cell damage using the above-mentioned Jiulongteng extract, wherein the damage to the lung tissue cells is caused by LPS activating the endoplasmic reticulum stress signaling pathway. The present invention has found through research that LPS can activate different endoplasmic reticulum stress signaling pathways in the lungs, activating IRE1α, PERK and ATF6 respectively. The Jiulongteng extract obtained by the specific extraction method of the present invention plays a negative regulatory role in endoplasmic reticulum stress activated by LPS. Specifically, after the Jiulongteng extract treats lung tissue cells, the expression of endoplasmic reticulum stress signals in lung tissue cells is significantly reduced, and the phosphorylation level of eIF2α in lung tissue cells is reduced. Jiulongteng extract blocks the apoptosis signaling pathways of JNK, CHOP and TRAF2 to reduce lung tissue cell damage. Jiulongteng extract also directly reduces the expression of p-eIF2α in lung tissue, which means that Jiulongteng extract plays a role in controlling endoplasmic reticulum stress.

[0068] Example 1 (I) Preparation of Prunus mume extracts (PMFs): 1.5 kg of Bauhinia championii Benth. pieces (collected in Fujian, identified as Bauhinia championii Benth. by Associate Professor Chen Jianzhong of Fujian University of Traditional Chinese Medicine) were crushed and passed through a No. 2 sieve, soaked in 10 times the mass of methanol for 12 h, ultrasonically extracted at 50°C for 5 times, the extracts were combined, and concentrated under reduced pressure at 50°C to obtain Extract 1.

[0069] Extract 1 was freeze-dried for 48 h, mixed with 10 times the mass of diatomaceous earth, dried under reduced pressure at 50°C for 24 h, loaded into a glass column, eluted with petroleum ether for 5 times the column volume, and then eluted with ethyl acetate for 5 times the column volume, the ethyl acetate eluate was collected, and concentrated under reduced pressure at 50°C to obtain extract 2.

[0070] Extract 2 was mixed with 2 times the mass of 100 mesh silica gel, loaded onto a 200 mesh silica gel column, eluted with petroleum ether-ethyl acetate (volume ratio 4:6) for 4 column volumes, then gradient eluted with petroleum ether-ethyl acetate (volume ratio 3:7 - 1:9), the eluate in the petroleum ether-ethyl acetate (volume ratio 1:9) section was collected, and concentrated under reduced pressure at 50°C to obtain extract 3.

[0071] Extract 3 was loaded onto an ODS-C18 column and eluted with methanol-water (volume ratio of 1:1) for 5 column volumes, then with methanol-water (volume ratio of 3:7) for 4 column volumes, and then with methanol-water (volume ratio of 4:6) for 5 column volumes. The methanol-water (volume ratio of 4:6) eluate was tracked and detected by HPLC-QTOF-MS. The eluate was collected and concentrated under reduced pressure at 50°C to obtain extract 4.

[0072] Extract 4 was dissolved in methanol and injected into a preparative HPLC (chromatographic column: Daisogel C18 10μm100A 20mm×250mm), with methanol-water (volume ratio 48:52) as the mobile phase, the peak was eluted at 254nm, the eluate was collected, and concentrated under reduced pressure at 50°C to obtain the Prunella Vulgaris extract (PMFs).

[0073] HPLC-DAD-Q-TOF-MS analysis of PMFs and extract 2 Figure 1 A. The analysis results showed that PMFs consisted of five polymethoxyflavones: 5,6,7,3',4'-pentamethoxyflavone (PMF1), 5,6,7,3',4',5'-hexamethoxyflavone (PMF2), 5,7,3',4',5'-pentamethoxyflavone (PMF3), 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavone (PMF4), and 5,7,5'-trimethoxy-3',4'-methylenedioxyflavone (PMF5). The NMR spectra, chemical structures, and HPLC chromatograms are shown in Figure 2. Figure 1 As shown in BG.

[0074] In summary, 1.5 kg of Jiulongteng slices were used to obtain 248.8 g of extract 1 (yield of about 16.59%), 93.6 g of extract 2 (yield of about 6.24%), and 15.4 g of Jiulongteng extract (PMFs) (yield of about 1.03%). The contents of each component in Jiulongteng extract (PMFs) are as follows: PMF1 = 2.34 g / 100g, PMF2 = 37.74 g / 100g, PMF3 = 40.25 g / 100g, PMF4 = 16.13 g / 100g, PMF5 = 2.74 g / 100g.

[0075] (II) Evaluation of the anti-inflammatory effects of extracts, PMFS and PMF1-5 in vitro (1) Evaluation of the anti-inflammatory effects of extract 1, extract 2, and PMFs in vitro LPS was used to induce the RAW264.7 cell inflammation model, and the levels of IL-6 and IL-1β inflammatory factors in the cells were detected to examine the anti-inflammatory effects of extract 1, extract 2, and PMFs. Experimental groups: blank control group; 1 μg / mL LPS (model control group); positive control group (dexamethasone, DXMS, 1 μM); extract 1 treatment group (50, 10, 2μg / mL); extract 2 treatment group (50, 10, 2μg / mL); PMFs treatment group (50, 10, 2μg / mL). After the positive control group and treatment group were treated with the corresponding drugs for 2 hours, LPS was added to the model group, positive control group, and treatment group. After LPS induction for 24 hours, the cell supernatant was collected and the level of inflammatory factors was detected using an ELISA kit.

[0076] The experimental results showed that different extracts of Jiulongteng can significantly downregulate the expression levels of inflammatory factors IL-6 and IL-1β, and are dose-dependent. The anti-inflammatory effects of the extracts obtained by step-by-step purification are PMFs>Extract 2>Extract 1 from strong to weak. Among them, PMFs has the strongest anti-inflammatory effect. The above results show that PMFs prepared by the method of the present invention is the effective part of Jiulongteng to exert its anti-inflammatory effect.

[0077] (2) Evaluation of the anti-inflammatory effects of PMF1-5 and PMFS in vitro LPS was used to induce the RAW264.7 cell inflammation model, and the levels of IL-6 and IL-1β inflammatory factors in the cells were detected to examine the anti-inflammatory effects of PMF1-5 and PMFs. Experimental groups: blank control group; 1 μg / mL LPS (model control group); positive control group (dexamethasone, DXMS, 1 μM); PMF1 treatment group (50, 10, 2μg / mL); PMF2 treatment group (50, 10, 2μg / mL); PMF3 treatment group (50, 10, 2μg / mL); PMF4 treatment group (50, 10, 2μg / mL); PMF5 treatment group (50, 10, 2μg / mL); PMFs treatment group (50, 10, 2μg / mL). After the positive control group and treatment group were treated with the corresponding drugs for 2 hours, LPS was added to the model group, positive control group and treatment group. After LPS induction for 24 hours, the cell supernatant was collected and the levels of inflammatory factors were detected using ELISA kits.

[0078] The experimental results showed that PMF1-5 and PMFS could significantly downregulate the expression levels of inflammatory factors IL-6 and IL-1β in a dose-dependent manner. The anti-inflammatory effects of each single compound of PMF1-5 were weaker than those of PMFS. The results showed that the anti-inflammatory effect of PMFs came from the synergistic effect of the five compounds PMF1-5, and the anti-inflammatory effect of PMFs was significantly better than that of each compound alone.

[0079] 3. Evaluation of the anti-inflammatory effect of PMFs in vivo C57BL / 6J mice were randomly divided into 6 groups: blank control, model control group (LPS, 20 μg / mouse), positive control group (dexamethasone), and PMFs treatment group (900, 300, and 100 mg / kg), with 10 mice in each group. After oral gavage for 2 days, mice in the model group and treatment group were induced to have inflammation by LPS nasal drops. Four hours after induction, the positive control group and treatment group were given corresponding drug treatments, and each index was detected 24 hours after LPS induction.

[0080] (1) Protective effect of PMFs on LPS-induced acute lung injury in mice Three doses of PMFs were used to treat LPS-induced acute lung injury in mice, with dexamethasone as a positive control. The results showed that PMFs significantly alleviated the weight loss of model mice and significantly inhibited LPS-induced lung injury. By detecting the lung wet-to-dry weight ratio (W / D) and lung weight / body weight ratio, the model mice had obvious pulmonary edema, while the PMFs group effectively alleviated pulmonary edema, and the effect of PMFs in the high-dose group on relieving pulmonary edema was similar to that of dexamethasone ( Figure 6 C and Figure 6 D). Histological examination showed that LPS caused severe granulocyte infiltration in lung tissue, destroyed alveolar structure, and led to extensive lung tissue damage. The PMFs group could reduce LPS-induced inflammatory cell infiltration and lung tissue damage in a dose-dependent manner ( Figure 6 E).

[0081] (2) Effects of PMFs on LPS-induced lung inflammation We determined the expression levels of pro-inflammatory factors in lung tissue. The results showed that LPS increased the mRNA levels of IL-6, IL-1β, TNF-α, and IFN-γ. After treating mice with PMFs, the mRNA levels of pro-inflammatory factors in lung tissue were significantly reduced ( Figure 7 AD). Western blot analysis showed that LPS induced the release of proinflammatory factors by activating the NF-κB signaling pathway, while PMFs reduced the expression of IKKα, decreased the phosphorylation of IκB, blocked the activation of the NF-κB signaling pathway, and inhibited the inflammatory signaling pathway in the lung tissue of mice ( Figure 7 E and 7F). High doses of PMFs can achieve similar effects as dexamethasone in reducing inflammatory responses in the lungs of mice.

[0082] (3) Regulatory effect of PMFs on oxidative stress in the lung When examining BALF, we found that LPS increased the permeability of lung tissue, resulting in the presence of a large number of cells in BALF. By sorting and counting cells in BALF, we found that LPS induced the accumulation of neutrophils and macrophages in BALF, while dexamethasone significantly reduced the accumulation of inflammatory cells in BALF. Similarly, PMFs were able to reduce the increase of inflammatory cells in BALF induced by LPS ( Figure 8 AD). LPS-induced inflammatory cell accumulation in BALF can also promote oxidative stress. Figure 8 As shown in EH, LPS reduced the levels of GSH-Px and CAT in BALF and increased the levels of MDA and HYP. After PMFs treatment, the levels of GSH-Px and CAT were significantly restored, while the levels of MDA and HYP in BALF were significantly reduced. DHE staining of lung tissue showed that PMFs significantly inhibited the increase of ROS levels in lung tissue induced by LPS ( Figure 8 I and 8J).

[0083] (4) PMFs restore LPS-induced pulmonary endoplasmic reticulum stress The present invention further studied the levels of ER stress-related proteins in lung tissue. Western blot analysis showed that LPS activated different ER stress signaling pathways in the lungs, activating IRE1α, PERK, and ATF6 respectively. After PMFs were treated in mice, the expression of ER stress signals in the lungs of mice was significantly reduced, and the phosphorylation level of eIF2α in the lungs of mice was reduced. PMFs blocked the apoptosis signaling pathways of JNK, CHOP, and TRAF2 to reduce lung cell damage ( Fig. 9 AD). PMFs also directly reduced the expression of p-eIF2α in lung tissue ( Fig. 9 E and 9F), implying that PMFs play a role in controlling ER stress.

[0084] (5) Effects of PMFs on LPS-induced lung cell apoptosis The present invention further detects the progression of lung cell apoptosis after LPS induction. It is obvious from the TUNEL results that LPS induces lung cell apoptosis by damaging intracellular DNA, exposing the 3'-OH end, and producing positive terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) results. PMFs play an important role in protecting lung cells from LPS-induced apoptosis ( Fig.10 A and 10B). PMFs treatment significantly reduced the number of apoptotic cells in the lung tissue of mice, reduced the expression of pro-apoptotic proteins caspase 3 and BAX, effectively restored the expression level of Bcl-2, and reduced LPS-induced apoptosis ( Fig.10 C and 10D).

[0085] 4. Conclusion LPS induces the release of proinflammatory factors, leading to the activation of pulmonary inflammatory signals, producing pneumonia characterized by inflammatory leukocyte infiltration, and ultimately triggering apoptosis of alveolar cells or pulmonary interstitial cells, inducing pulmonary dysfunction. The research results of the present invention show that on the second day after LPS induction, a large number of inflammatory cells infiltrated the mouse lungs, the expression level of inflammatory factors increased, the inflammatory signaling pathway was activated, and the high oxidative stress signal environment penetrated from the lung tissue to the trachea / bronchioles. The present invention discovered and separated the effective part of polymethoxyflavonoids PMFs from Jiulongteng, and the main chemical substances were 5,6,7,3',4'-pentamethoxyflavonoids, 5,6,7,3',4',5'-hexamethoxyflavonoids, 5,7,3',4',5'-pentamethoxyflavonoids, 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavonoids, and 5,7,5'-tetramethoxy-3',4'-methylenedioxyflavonoids. PMFs could effectively alleviate LPS-induced ALI, significantly reduce the lung inflammation area in mice, and restore lung tissue damage in a dose-dependent manner.

[0086] The results of the present invention show that LPS activates P-ERK, P-eIF2α, increases the expression of ATF4 and CHOP, and induces endoplasmic reticulum stress. In addition, LPS increases the phosphorylation level of IRE1α, guides IRE1α to bind to TRAF2, and recruits the NF-κB kinase inhibitor IKKα to activate the NF-κB signaling pathway to induce an inflammatory response. The LPS-activated IRE1α signaling pathway mediates the JNK pathway and leads to cell apoptosis, which is also one of the main mechanisms of LPS-induced ALI. PMFs play a negative regulatory role in LPS-activated endoplasmic reticulum stress. PMFs can alleviate ALI by regulating endoplasmic reticulum stress, which is related to PMFs regulating endoplasmic reticulum stress targets to reduce lung cell apoptosis, which has been verified in mouse lung tissue studies.

[0087] In summary, the present invention discovered and isolated the effective part PMFs of Jiulongteng for treating acute lung injury. Animal experiments confirmed that the anti-inflammatory, antioxidant and anti-apoptotic effects of PMFs can alleviate LPS-induced acute lung injury. Further studies have shown that PMFs alleviate acute lung injury through the endoplasmic reticulum stress pathway of lung tissue cells. These results provide an effective preclinical research basis for the clinical application of PMFs and the development of new drugs.

[0088] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0089] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. The use of the extract of Rhizoma Cibotii in the preparation of a drug for treating acute lung injury, characterized in that: The extract of the safflower contains the following active compounds: 5,6,7,3',4'-pentamethoxyflavone, 5,6,7,3',4',5'-hexamethoxyflavone, 5,7,3',4',5'-pentamethoxyflavone, 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavone and 5,7,5'-trimethoxy-3',4'-methylenedioxyflavone.

2. The use according to claim 1, characterized in that: The mass ratios of 5,6,7,3',4'-pentamethoxyflavone, 5,6,7,3',4',5'-hexamethoxyflavone, 5,7,3',4',5'-pentamethoxyflavone, 5,6,7,5'-tetramethoxy-3',4'-methylenedioxyflavone and 5,7,5'-trimethoxy-3',4'-methylenedioxyflavone in the vine extract are (2-3):(35-40):(38-42):(14-18):(2-4).

3. The use according to claim 1, characterized in that: The preparation method of the strychnos nux vomica extract comprises the following steps: 1) The decoction pieces of Rhizoma Cibotii were crushed and then extracted with methanol to obtain extract 1; 2) Mix the extract 1 with diatomaceous earth, and elute with petroleum ether and ethyl acetate successively to obtain the extract 2; 3) Mix extract 2 with silica gel, and elute with a gradient of petroleum ether-ethyl acetate with a decreasing volume ratio of petroleum ether to obtain extract 3; 4) Extract 3 was eluted with methanol-water in different volume ratios to obtain extract 4; 5) The extract 4 was separated by HPLC to obtain the Herba Cibotii Herba Extract.

4. The use according to claim 3, characterized in that: 1) In The mass ratio of the spatholobiaceae slices to methanol is 1:(5-15); The extraction is performed by first soaking in methanol for 10-15 hours, then ultrasonically extracting at least twice at 40-60° C., combining the extracts and concentrating them to obtain extract 1.

5. The use according to claim 3, characterized in that: 2) In The extract 1 is freeze-dried and mixed with 8-12 weight of diatomaceous earth, and dried; First, elute with petroleum ether for 3-7 column volumes, and then elute with ethyl acetate for 3-7 column volumes.

6. The use according to claim 3, characterized in that: 3), during the gradient elution, the volume proportion of petroleum ether in petroleum ether-ethyl acetate is 35-45%, 25-35%, 15-25% and 5-15% respectively; the column volume of each elution is 2-6 times.

7. The use according to claim 3, characterized in that: 3) In The mesh number of the silica gel is 50-200 mesh; The mass ratio of the extract 2 to the silica gel is 1:(1.5-2.5); The mesh number of the silica gel chromatographic column is 100-300 meshes.

8. The use according to claim 3, characterized in that: 4) In The mixture was eluted with methanol-water having a methanol volume content of 45-55%, 25-35% and 35-45% in sequence; The column volume for each elution was 3-7 times.

9. The use according to claim 3, characterized in that: 5), dissolving extract 4 in methanol, injecting the sample into preparative high performance liquid chromatography, using methanol-water as mobile phase, collecting the eluate, and concentrating it to obtain the extract of Rhizoma Cibotii.

10. A method for reducing in vitro lung tissue cell damage using an extract of Rhizoma Cynoglossi, characterized in that: The damage to the lung tissue cells is caused by LPS activating the endoplasmic reticulum stress signaling pathway; The Exigua oleracea extract is the Exigua oleracea extract in the application described in any one of claims 1-9.

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