CN117653681A - Bupleurum chinense L ethanol extract, preparation method thereof and application thereof in treating pulmonary fibrosis - Google Patents
Bupleurum chinense L ethanol extract, preparation method thereof and application thereof in treating pulmonary fibrosis Download PDFInfo
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- CN117653681A CN117653681A CN202311716970.7A CN202311716970A CN117653681A CN 117653681 A CN117653681 A CN 117653681A CN 202311716970 A CN202311716970 A CN 202311716970A CN 117653681 A CN117653681 A CN 117653681A
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- bupleurum
- glucosyl
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- ethanol
- bupleurum chinense
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Abstract
The invention provides a preparation method of a bupleurum chinense ethanol extract, which comprises the following steps: step one, drying whole herb of bupleurum, cutting into segments, crushing Cheng Zhushe bupleurum powder, adding petroleum ether with 15-20 times of density, heating, refluxing and degreasing; and step two, adding 75% ethanol with the density of 10-15 times, heating and refluxing in water bath for extraction for 3 times, filtering, combining the filtrates, concentrating by a rotary evaporation evaporator, recovering the ethanol, rotary evaporating until the ethanol recovery is finished, and drying the residual water in an oven. The preparation cost is low, and the obtained bupleurum chinense ethanol extract can be used for treating pulmonary fibrosis and has good curative effect.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, and in particular relates to a preparation method of a bupleurum chinense ethanol extract and application of the bupleurum chinense ethanol extract in treating pulmonary fibrosis.
Background
Pulmonary fibrosis (pulmonary fibrosis, PF) is a fibrotic interstitial lung disease (interstitial lung disease, ILD) that is chronic, progressive and has a strong destructive effect on lung tissue. Clinically, it is mainly manifested by progressive dyspnea with a concomitant decline in lung function, ultimately leading to permanent deterioration and death of lung function. Pathologically, in the early stages of PF, lung tissue mainly exhibits inflammatory cell infiltration, edema and congestion, and then converts into alveolar epithelial cell injury and excessive repair, and lung tissue structure destruction, abnormal proliferation and differentiation of fibroblasts into myofibroblasts, resulting in massive deposition of extracellular matrix (extracellular matrix, ECM) within lung tissue, ultimately leading to fibrous occlusion of lung tissue and even "honeycomb" lung.
Pulmonary fibrosis is two of secondary pulmonary fibrosis and idiopathic pulmonary fibrosis (idiopathic pulmonary fibrosis, IPF), where idiopathic pulmonary fibrosis fails to find its cause, is most common clinically, patient appetite is reduced, wasting, cardiopulmonary function declines, and most IPF patients eventually die from respiratory failure. IPF develops after more than 50 years of age, with increased incidence with age, with males being higher than females. IPF is a fatal pulmonary disease with varying natural courses of disease, and data on it show that IPF patients have a median survival of 3-5 years from diagnosis to death, with a survival rate of only 30% in 5 years, and are known as "neoplastic-like disease" because of their higher mortality than tumors. In addition, pulmonary fibrosis caused by secondary factors, including environmental pollution, drugs and diseases, such as those caused by 2019 coronavirus (covd-19), connective tissue diseases, tuberculosis, etc.
At present, the pathogenesis of pulmonary fibrosis is not completely clear, and specific medicines and treatment means are not clinically available. Pirfenidone and nidanib are approved by FDA for use, but only slow down the decline of lung function, and are expensive, and most households are not burdened. There is no method for stopping or reversing the decline of lung function other than lung transplantation, but this method has limited application due to the small number of donors and the like. Therefore, it has been a hot spot and difficult problem of current researches to determine the pathogenesis of PF and search for new therapeutic means. The traditional Chinese medicine is used as a traditional medical treasury, and has the characteristics of multi-component, multi-target and multi-way treatment and potential anti-pulmonary fibrosis effect, so that the search for the medicine for preventing and treating PF is of great interest. In recent years, with the continuous and deep research on PF, the search for PF treatment by traditional Chinese medicine has been increasing, and there are a considerable amount of traditional Chinese medicine compounds, and the traditional Chinese medicine raw materials and extracts and the traditional Chinese medicine compounds show the prevention and treatment effects on PF in the research.
Therefore, the invention obtains the bupleurum chinense ethanol extract by a reflux extraction method and verifies the application in treating pulmonary fibrosis.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a preparation method of a bupleurum chinense ethanol extract and application thereof in treating pulmonary fibrosis. The anti-pulmonary fibrosis activity in the TGF-beta 1 induced A549 cell is good, the E-cadherin mRNA content can be up-regulated, and the N-cadherin, vimentin, alpha-SMA, fibrauretin and COL1A1 mRNA content can be down-regulated; the anti-pulmonary fibrosis activity is good in the bleomycin-induced SD rat, and the GSH and SOD content can be up-regulated, and the Hyp, MDA, TGF-beta 1, TNF-alpha and IL-1 beta content can be down-regulated.
The invention provides the following technical scheme:
a preparation method of a bupleurum chinense ethanol extract comprises the following steps:
step one, drying whole herb of the bupleurum chinense, cutting into segments, crushing Cheng Zhushe bupleurum chinense powder, adding petroleum ether with 15-20 times of density, heating, refluxing and degreasing, repeating degreasing for 2-3 times until filtrate has no obvious dark green color, volatilizing residual petroleum ether until the bupleurum chinense powder is completely dried;
and step two, adding 75% ethanol with the density of 10-15 times into the bupleurum powder obtained in the step one, heating and refluxing in water bath for 3 times, filtering, merging filtrate, concentrating by a rotary evaporation evaporator, recovering ethanol, rotary evaporating until the ethanol recovery is finished, drying residual water by an oven, and vacuum drying to obtain the bupleurum ethanol extract.
An application of bupleurum chinense L ethanol extract in preparing medicines for resisting pulmonary fibrosis is provided.
Further, the bupleurum chinense ethanol extract comprises a compound of 3-O-beta-D-glucosyl- (1-6) - [ -alpha-L-rhamnosyl- (1-3) ] -beta-D-glucosyl-11, 13 (18) -diene- (16 beta, 29) -dihydroxyl-28-O-beta-D-glucosyl-oleanane; the compound 3-O- β -L-rhamnosyl- (1→2) - [ β -D-xylosyl- (1→3) ] - β -D-glucosyl-11,13 (18) -diene- (16 β,23, 29) -trihydroxy-28-O- β -D-glucosyl-oleanane; the compound 3-O-rhamnosyl- (1→4) - [ rhamnose- (1→3) ] -glucosyl- (1→6) - [ glucosyl- (1→2) ] -glucosyl-11,13 (18) -diene-16-hydroxy-28-O-glucosyl-oleanane; compound Luo Tonggan V; the compound anemonin III; the compound saikosaponin Q; compound synucleoside B; the compound saikosaponin C; the compound saikosaponin H; the compound rutin; the compound saikosaponin D.
Further, the bupleurum chinense ethanol extract is applied to the preparation of medicines for inhibiting the proliferation of pulmonary fibrosis cells.
Furthermore, the bupleurum chinense ethanol extract is applied to the preparation of the lung fibrosis cell antioxidant drugs.
Furthermore, the bupleurum chinense ethanol extract is applied to the preparation of medicines for inhibiting inflammatory factors TGF-beta 1, TNF-alpha and IL-1 beta by pulmonary fibrosis.
By adopting the technical scheme, the invention has the following beneficial effects:
1. the invention provides a preparation method of a bupleurum chinense ethanol extract, which comprises the steps of obtaining the bupleurum chinense ethanol extract, carrying out HPLC-MS qualitative analysis, and determining the contained compound components.
2. Experiments prove that the bupleurum chinense ethanol extract prepared by the invention has good treatment effect on A549 cell fibrosis and SD rat pulmonary fibrosis, thereby showing good anti-pulmonary fibrosis activity.
3. The effect of the bupleurum chinense ethanol extract for treating pulmonary fibrosis is obviously better than that of the positive drug pirfenidone, and the bupleurum chinense ethanol extract has wide popularization and application values.
Drawings
FIG. 1 is a graph showing the results of drug toxicity of bupleurum chinense ethanol extract on A549 cells;
FIG. 2 is a graph showing the effect of bupleurum extract on pulmonary fibrosis cell proliferation;
FIG. 3 is a graph showing the effect of bupleurum extract on the N-cadhererin mRNA content of pulmonary fibrosis cells;
FIG. 4 is a graph showing the effect of bupleurum extract on E-cadhererin mRNA content of pulmonary fibrosis cells;
FIG. 5 is a graph showing the effect of bupleurum extract on the level of Vimentin mRNA in pulmonary fibrosis cells;
FIG. 6 is a graph showing the effect of bupleurum extract on the α -SMAmRNA content of pulmonary fibrosis cells;
FIG. 7 is a graph showing the effect of bupleurum extract on the mRNA level of fibroblastin in lung fibrosis cells;
FIG. 8 is a graph showing the effect of bupleurum extract on COL1A1 mRNA content of pulmonary fibrosis cells;
FIG. 9 is a graph showing the effect of bupleurum extract on the body weight of PF rats;
FIG. 10 is a graph showing the effect of bupleurum extract on the lung index of PF rats;
FIG. 11 is a graph showing the effect of bupleurum extract on the hydroxyproline content of PF rats;
FIG. 12 is a graph showing the effect of bupleurum extract on PF rat lung tissue on HE staining;
FIG. 13 is a graph showing the result of Masson staining of the effect of bupleurum extract on PF rat lung tissue
FIG. 14 is a graph showing the result of dyeing on sirius gifuensis with ethanol extract of Bupleurum scorzonerifolium
FIG. 15 is a graph showing the effect of bupleurum extract on the GSH content of PF rat lung;
FIG. 16 is a graph showing the effect of bupleurum extract on MDA content in lung tissue of PF rats;
FIG. 17 is a graph showing the effect of the alcoholic extract of Bupleurum scorzonerifolium on the SOD content of lung tissue of PF rats;
FIG. 18 is a graph showing the effect of bupleurum extract on the serum TGF-beta 1 content of PF rats;
FIG. 19 is a graph showing the effect of bupleurum extract on the serum TNF- α content of PF rats;
FIG. 20 is a graph showing the effect of bupleurum extract on serum IL-1. Beta. Content of PF rats;
FIG. 21 is a block diagram of the compound 3-O-beta-D-glucosyl- (1.fwdarw.6) - [ -alpha-L-rhamnosyl- (1.fwdarw.3) ] -beta-D-glucosyl-11, 13 (18) -diene- (16. Beta., 29) -Dihydroxy-28-O-beta. -D-glucosyl-oleanane (3-O-beta. -D-glucosyl- (1.fwdarw.6) - [ -alpha-L-rhamnose- (1.fwdarw.3) -beta. -D-glucosyl-11,13 (18) -diene- (16. Beta., 29) -Dihydroxy-28-O-beta. -D-glucosyl-olennane);
FIG. 22 is a mass spectrum of the compound 3-O- β -D-glucosyl- (1→6) - [ - α -L-rhamnosyl- (1→3) ] - β -D-glucosyl-11,13 (18) -diene- (16β, 29) -Dihydroxy-28-O- β -D-glucosyl-oleanane (3-O- β -D-glucosyl- (1→6) - [ - α -L-rhamnose- (1→3) - β -D-glucosyl-11,13 (18) -diene- (16β, 29) -Dihydroxy-28-O- β -D-glucosyl-olennane);
FIG. 23 is a block diagram of the compound 3-O- β -L-rhamnosyl- (1→2) - [ β -D-xylosyl- (1→3) ] - β -D-glucosyl-11,13 (18) -diene- (16 β,23, 29) -Trihydroxy-28-O- β -D-glucosyl-oleanane (3-O- β -L-rhamnose- (1→2) - [ β -D-xylose- (1→3) - β -D-glucosyl-11,13 (18) -diene- (16 β,23, 29) -Trihydroxy-28-O- β -D-glucosyl-olerane;
FIG. 24 is a mass spectrum of the compound 3-O- β -L-rhamnosyl- (1→2) - [ β -D-xylosyl- (1→3) ] - β -D-glucosyl-11,13 (18) -diene- (16 β,23, 29) -Trihydroxy-28-O- β -D-glucosyl-oleanane (3-O- β -L-rhamnose- (1→2) - [ β -D-xylose- (1→3) - β -D-glucosyl-11,13 (18) -diene- (16 β,23, 29) -Trihydroxy-28-O- β -D-glucosyl-olennane);
FIG. 25 is a block diagram of the compound 3-O-rhamnosyl- (1.fwdarw.4) - [ rhamnose- (1.fwdarw.3) ] -glucosyl- (1.fwdarw.6) - [ glucosyl- (1.fwdarw.2) ] -glucosyl-11,13 (18) -diene-16-hydroxy-28-O-glucosyl-oleanane (3-O-rhamnose- (1.fwdarw.4) - [ rhamnose- (1.fwdarw.3) ] -glucosyl- (1.fwdarw.6) - [ glucosyl- (1.fwdarw.2) ] -glucosyl-11,13 (18) -dien-16-hydroxy-28-O-glucosyl-olernane;
FIG. 26 is a mass spectrum of the compound 3-O-rhamnosyl- (1.fwdarw.4) - [ rhamnosyl- (1.fwdarw.3) ] -glucosyl- (1.fwdarw.6) - [ glucosyl- (1.fwdarw.2) ] -glucosyl-11,13 (18) -diene-16-hydroxy-28-O-glucosyl-oleanane (3-O-rhamnose- (1.fwdarw.4) - [ rhamnose- (1.fwdarw.3) ] -glucosyl- (1.fwdarw.6) - [ glucosyl- (1.fwdarw.2) ] -glucosyl-11,13 (18) -dien-16-hydroxy-28-O-glucosyl-olernane);
FIG. 27 is a block diagram of compound Luo Tonggan V (Rotundioside V);
fig. 28 is a mass spectrum of compound Luo Tonggan V (Rotundioside V);
FIG. 29 is a block diagram of the compound Clinopodium polycephalum saponin III (Clinoposaponin III);
FIG. 30 is a mass spectrum of the compound Clinopodium polycephalum saponin III (Clinoposaponin III);
FIG. 31 is a block diagram of the compound saikosaponin Q (Saikosaponin Q);
FIG. 32 is a mass spectrum of the compound saikosaponin Q (Saikosaponin Q);
FIG. 33 is a block diagram of compound zygotic saponin B (Comastomasaponin B);
FIG. 34 is a mass spectrum of compound zygotic saponin B (Comastomasaponin B);
FIG. 35 is a block diagram of the compound saikosaponin C (Saikosaponin C);
FIG. 36 is a mass spectrum of the compound saikosaponin C (Saikosaponin C);
FIG. 37 is a block diagram of the compound saikosaponin H (Saikosaponin H);
FIG. 38 is a mass spectrum of the compound saikosaponin H (Saikosaponin H);
FIG. 39 is a structural diagram of Rutin (Rutin);
FIG. 40 is a mass spectrum of Rutin (Rutin);
FIG. 41 is a block diagram of the compound saikosaponin D (Saikosaponin D);
FIG. 42 is a mass spectrum of the compound saikosaponin D (Saikosaponin D);
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the drawings and detailed description are only intended to illustrate the invention and are not intended to limit the invention.
Example 1
Degreasing of bupleurum chinense
The whole herb of bupleurum chinense is dried, cut into segments and crushed Cheng Zhushe bupleurum chinense powder. Taking 100 bamboo leaf bupleurum powder, adding 1500ml petroleum ether according to the density of 1:15 (weight/volume), fully mixing uniformly, placing in a 2L round bottom flask, heating, refluxing and degreasing for 1h at 65 ℃, carrying out suction filtration to remove filtrate, repeating degreasing for 2-3 times until the filtrate after suction filtration has no obvious dark green color, and placing the degreased bamboo leaf bupleurum powder in a fume hood to volatilize residual petroleum ether until the bamboo leaf bupleurum powder is completely dried.
Preparation of bupleurum chinense ethanol extract
Taking the defatted bupleurum powder, adding 1000ml of 75% ethanol according to the density of 1:10 (weight/volume), fully mixing, placing the solution into a 2L round bottom flask, installing a heating reflux device, heating and reflux extracting for 3 times in a micro boiling water bath at 85 ℃ for 1h each time, filtering and merging the filtrates for 3 times. Standing the filtrate in a 4 deg. refrigerator for 12 hr, and suction filtering again to remove tannin. Concentrating the extractive solution with rotary evaporator, recovering ethanol, rotary evaporating until the extractive solution is about 100ml, pouring out, oven drying the residual water, vacuum drying, weighing, and storing. 23.59g of bupleurum chinense ethanol extract is obtained, and the extraction rate is 23.59%.
Example 2
HPLC-MS analysis of the alcoholic extract of Bupleurum scorzonerifolium
Dissolving small amount of the obtained bupleuri radix ethanol extract in 75% methanol solution at concentration of 10mg/ml, filtering with 0.22 μm microporous membrane, and performing HPLC-MS analysis.
Chromatographic conditions: thermo Scientific Ultimate 3000 UHPLC (Massachusetts, USA), with DGP-3600SDN dual ternary pumps, WPS-3000SL Analytical autosampler, TCC-3000RS column incubator and DAD-3000 detector, equipped with Chromeleon workstation software.
Chromatographic column: ACQUITY UPLC BEH C18 chromatographic column (1.7 μm, 2.1X100 mm)
Pre-column: vanGuardTM BEH C18.7 μm
Column temperature: 45 DEG C
Sample injection volume: 3 mu L
Flow rate: 0.6mL/min
Mobile phase: a (H2O, 0.1% formic acid); b (ACN), gradient elution. Specific gradient elution conditions were as follows:
mass spectrometry conditions:
the results are shown in FIGS. 21 and 22, and the compound is 3-O-beta-D-glucosyl- (1.fwdarw.6) - [ -alpha-L-rhamnosyl- (1.fwdarw.3) ] -beta-D-glucosyl-11, 13 (18) -diene- (16. Beta., 29) -Dihydroxy-28-O-beta-D-glucosyl-oleanane (3-O-beta-D-glucosyl- (1.fwdarw.6) - [ -alpha-L-rhamno-1.fwdarw.3) -beta-D-glucosyl-11, 13 (18) -diene- (16. Beta., 29) -Dihydroxy-28-O-beta-D-glucosyl-olernane). The results are shown in FIGS. 23 and 24, and the compound is 3-O-beta-L-rhamnosyl- (1.fwdarw.2) - [ beta-D-xylosyl- (1.fwdarw.3) ] -beta-D-glucosyl-11, 13 (18) -diene- (16. Beta., 23, 29) -Trihydroxy-28-O-beta-D-glucosyl-oleanane (3-O-beta-L-rhamnose- (1.fwdarw.2) - [ beta-D-xylose- (1.fwdarw.3) -beta-D-glucosyl-11, 13 (18) -diene- (16. Beta., 23, 29) -Trihydroxy-28-O-beta-D-glucosyl-olernane). The results are shown in FIGS. 25 and 26, and the compound is 3-O-rhamnosyl- (1.fwdarw.4) - [ rhamnosyl- (1.fwdarw.3) ] -glucosyl- (1.fwdarw.6) - [ glucosyl- (1.fwdarw.2) ] -glucosyl-11,13 (18) -diene-16-hydroxy-28-O-glucosyl-oleanane (3-O-rhamnose- (1.fwdarw.4) - [ rhamnose- (1.fwdarw.3) ] -glucosyl- (1.fwdarw.6) - [ glucosyl- (1.fwdarw.2) ] -glucosyl-11,13 (18) -dien-16-hydroxy-28-O-glucosyl-olernane). The results are shown in fig. 27, 28, compound Luo Tonggan V (Rotundioside V). The results are shown in fig. 29, 30, where the compound is anemonin III (Clinoposaponin III). The results are shown in fig. 31, 32, the compound is saikosaponin Q (Saikosaponin Q). The results are shown in FIGS. 33 and 34, where the compound is zygosaponin B (Comastomasaponin B). The results are shown in fig. 35, 36, the compound is saikosaponin C (Saikosaponin C). The results are shown in fig. 37 and 38, and the compound is saikosaponin H (Saikosaponin H). As a result, as shown in FIGS. 39 and 40, the compound was Rutin. The results are shown in fig. 41 and 42, and the compound is saikosaponin D (Saikosaponin D).
Example 3
Toxicity of bupleurum chinense ethanol extract in A549 cells
Human lung adenocarcinoma a549 cells have alveolar type II epithelial cell characteristics, and are capable of undergoing epithelial mesenchymal transition from hexagonal to spindle-shaped under the action of TGF- β1, and thus can be used for studying in vitro lung fibrosis models.
A549 cells were grown to log phase at 5×10 3 After cells were allowed to adhere to 96-well plates for 24 hours at 100. Mu.L/well, the cells were treated with 0, 10, 20, 40, 80, 160, 320, 500, 1000. Mu.g/ml of bupleurum chinense ethanol extract, 5 duplicate wells were set for each concentration, and incubated for 24 hours.
2. Cell viability was determined using the CCK-8 method (CCK-8 assay; solarbio), the original medium was discarded, and CCK-8 was added to each well with medium in a ratio of 1:9 and 5 compound wells added with 100 mu l of the mixed solution are set as blank control groups, and after incubation for 1-4 hours in an incubator, the absorbance at 450nm is measured by using an enzyme-labeled instrument.
3. Cell viability was proportional to absorbance per well, and the mean ± standard deviation (mean ± standard) of cell viability was calculated for each group of at least three replicate wells.
The cell viability was able to represent the toxic effect of the bupleurum alcoholic extract on a549 cells, and the results are shown in fig. 1, and it can be seen that the cell viability was not statistically different from that of the control group at less than 160 μg/ml, indicating that the bupleurum alcoholic extract did not exhibit significant cytotoxicity in this concentration range.
Example 4
The ethanol extract of Bupleurum scorzonerifolium has effect in inhibiting proliferation of lung cell
A549 cells were grown to log phase at 3×10 3 After cells were allowed to adhere to a 96-well plate for 24 hours with 5 wells left as negative controls, the cells were allowed to stand for 24 hours in an incubator with a medium containing TGF-. Beta.1 at a concentration of 10 ng/ml.
2. Microscopic observation a549 cells were activated, changing from hexagonal to elongated spindle shape. Cells were treated with ethanol extracts of bupleurum chinense at concentrations of 20, 30, 40, 60, 80, 120, 160 μg/ml, 5 duplicate wells were set for each group, 5 wells were left as positive controls, and placed in an incubator for 24 hours of culture.
3. Cell viability was determined using the CCK-8 method (CCK-8 assay; solarbio), the original medium was discarded, and CCK-8 was added to each well with medium in a ratio of 1:9 and 5 compound wells added with 100 mu l of the mixed solution are set as blank control groups, and after incubation for 1-4 hours in an incubator, the absorbance at 450nm is measured by using an enzyme-labeled instrument.
4. Cell viability was proportional to the absorbance of each well. Mean ± standard deviation (mean ± standard) of cell viability was calculated for each group of at least three wells.
The results are shown in FIG. 2, and it can be seen that the viability of A549 cells was measured in the presence of TGF-. Beta.1 to simulate a pro-fibrotic environment. TGF- β1 significantly promoted proliferation of a549 cells compared to the control group. The bupleurum chinense ethanol extract can obviously inhibit the proliferation of A549 cells induced by TGF-beta 1 at the concentration of 30-160 mug/ml which does not show cytotoxicity to A549 cells, and the inhibition rate is concentration-dependent. Therefore, the bupleurum chinense ethanol extract provided by the invention can effectively inhibit the proliferation of lung fibrotic cells.
Example 5
Determination of intracellular N-cadherin, E-cadherin, vimentin, alpha-SMA, fibratectin, COL1A1 mRNA content. During PF, lung epithelial cells can react to microenvironment signals and transform into mesenchymal cells, such as fibroblasts and myofibroblasts, through a process called epithelial-mesenchymal-mesenchymal transition (EMT), which are direct contributors to organ fibrosis. During EMT, intrinsic epithelial markers such as E-cadherin (E-cadherin) are lost and mesenchymal features such as N-cadherin (N-cadherin), vimentin (vimentin) and alpha-smooth muscle actin (alpha-SMA) are obtained. Mesenchymal cells, particularly myofibroblasts, can secrete large amounts of ECM such as collagen type I (COL 1 A1), laminin (Laminin), fibronectin (Fibronectin), and α -SMA upon stimulation with cytokines such as transforming growth factor- β (TGF- β), angiotensin II (angiotensin II), and interleukin 6 (IL-6). Thus, drugs directed to these events may promote the development of PF therapies.
1. Extraction of RNA
1) A549 cells were 1.5x10 per well 5 Culturing in 6-well plate for 24 hr, adding TGF-beta 1 to activate, keeping 3 holes as negative control, adding bupleuri radix ethanol extract at 60, 90, 120 μg/ml concentration after 24 hr, and keeping 3 holes as positive control.
2) The six-well plate after 24 hours of medicated culture was removed and the medium was aspirated. 1ml of pre-chilled PBS was added to each well and washed 3 times, and the PBS was aspirated. 1ml Trizol was added to each well, and the mixture was gently blown repeatedly with a gun head to uniformly distribute the reagent on the cell surface, and the mixture was sucked into a 1.5ml RNase-free EP tube to allow the cells to be sufficiently lysed, and allowed to stand at room temperature for 5-10min.
3) 200 μl of chloroform was added to the EP tube in sequence, and mixed with vigorous shaking for 30s to allow the aqueous phase and the organic phase to be in full contact, and allowed to stand at room temperature for 3-5min. Centrifugation at 12,000g for 15min at 4℃was seen as three layers, and RNA was transferred in the upper aqueous phase to another new RNase-free EP tube (approximately 400. Mu.L).
4) Adding an equal volume of precooled isopropanol, gently and fully mixing (reversing an EP tube for 6-8 times) and standing for 10min at room temperature to increase RNA precipitation; centrifuging at 4deg.C for 10min at 12,000g, discarding the waste liquid at the upper layer of EP tube, observing a small amount of white RNA precipitation tube bottom, and collecting RNA precipitate.
5) Washing twice with 1ml of absolute ethanol (4 ℃ C., 7500g for 5 min), opening an EP pipe orifice, pouring out the supernatant, and air-drying RNA in an ultra clean bench (room temperature);
6) An appropriate amount of DEPC water (at least 15. Mu.L) was added depending on the amount of precipitate to dissolve the precipitate. And (3) measuring the concentration and purity of the extracted RNA by using an ultra-micro spectrophotometer, calculating the purity according to the ratio of A260/A280 nm, and placing a qualified sample in a refrigerator at the temperature of-80 ℃ for standby, wherein the purity of the sample is qualified in the range of 1.8-2.0.
Reverse transcription of RNA into cDNA
The reverse transcription kit used was the Solaro company general RT-PCR kit (M-MLV), cat# RP1100. Reverse transcription was performed according to the kit instructions.
Sample RNA and reagents were placed in 300. Mu.L EP tube of RNase-free according to system one configuration. And then cooling the mixture on ice for 2min at 70 ℃ for 5min, and adding the second system after collecting the reaction liquid by short centrifugation.
After simple centrifugation and mixing, the reaction was stopped after 60min at 42℃and 5min at 95℃and 300. Mu.L of EP tube was placed on ice or stored at-20 ℃.
3.RT-qPCR
The Takara kit TB was used for the real-time quantitative polymerase chain reaction (quantitative real-time PCR, qPCR)Premix Ex Taq TM (Tli RNaseH Plus), cat No. RR420A. The experiments were performed according to the following system configuration system (TB Green 5. Mu.L; upstream primer 0.5. Mu.L; downstream primer 0.5. Mu.L; DEPC water 3. Mu.L; cDNA sample 1. Mu.L) and then 40 cycles were performed, each cycle comprising three steps. The first step is denaturation at 95℃for 10 seconds, which causes the DNA to unwind into single strands. The second step is annealing at 55℃for 30 seconds, and the primer can bind to the target sequence. The last step is an extension at 98℃for 5 seconds, the primer extending onto the target sequence and generating a new DNA strand. After 40 cycles were completed, the reaction was completed, data was obtained, and calculation was performed.
TGF-beta 1 has strong fibrillation promoting effect and can induce EMT. This is a phenomenon in which intrinsic epithelial markers such as E-cadherein are lost to gain mesenchymal characteristics such as N-cadherin, vimentin and alpha-SMA. When EMT occurs, the morphology of a549 cells changes from hexagonal to elongated spindle shape. In this experiment, A549 cells were spindle-shaped after 24h of TGF- β1 treatment. When the concentration of the bupleurum chinense ethanol extract is 60, 90 or 120 mug/ml, the mesenchymal morphology is obviously weakened compared with the TGF-beta 1 induction group. To further verify the EMT inhibition effect of bupleurum chinense ethanol extract from molecular level, qPCR was used to detect EMT related markers. The results are shown in FIGS. 3,4,5 and 6, and after 24h of culture, TGF-beta 1-induced E-cadherein gene expression was down-regulated in A549 cells, while N-cadherin, vimentin and alpha-SMA gene expression was up-regulated. Compared with the model group, the ethanol extract of the bupleurum chinense has increased E-cadhererin expression and down-regulated N-cadherin, vimentin and alpha-SMA gene expression at 60, 90 or 120 mug/ml. These results indicate that the bupleurum chinense ethanol extract inhibits TGF- β1 induced EMT of a549 cells.
Cells with mesenchymal characteristics, such as fibroblasts and myofibroblasts, secrete extracellular matrix proteins such as COL1A1, fibrinectin and Laminin. ECM excessive deposition is a significant cause of organ fibrosis. In order to investigate the mechanism of promoting ECM degradation by the bupleurum chinense ethanol extract, the gene expression levels of ECM key markers, namely fibrauretin and COL1A1, were detected by adopting a qPCR method. The results are shown in FIGS. 7,8, where fibrinectin and COL1A1 were significantly up-regulated at the gene level in TGF-. Beta.1 induced A549 cells. After 24 hours of treatment of the bupleurum chinense ethanol extract, the levels of fibrauretin and COL1A1 were reduced. These results indicate that bupleurum chinense ethanol extract stimulates TGF- β1-induced ECM degradation of a549 cells.
In conclusion, the bupleurum chinense ethanol extract has good activity of resisting pulmonary fibrosis, and can be applied as a medicine for treating pulmonary fibrosis.
Example 6
Influence of bupleurum chinense ethanol extract on body weight, lung index and hydroxyproline content of PF rats
Establishment, grouping and administration of PF animal models
1) 36 male SPF SD rats, 5-6 weeks old, body weight (180+ -20 g), were randomly divided into a blank control group, a model group, a bupleurum group, and a positive drug pirfenidone group, 9 animals each after one week of adaptive feeding.
2) Establishment of rat PF model: isoflurane anesthetized rats were supine fixed on a rat plate, the laryngoscope against the tongue root, the glottis exposed, the rats were non-invasively intubated with an atomising syringe, the needle of the atomising syringe was pierced into the trachea when the glottis opened, bleomycin solution (5 mg/kg) was injected, and the blank control group was injected with an equal amount of physiological saline. The rat board is rapidly swayed left and right for 1-2min after the injection of the medicine, the bleomycin is uniformly distributed in the lung, and the rat is in right lateral recumbent position after the injection is finished, and waits for natural awakening.
3) The next day of molding, 100mg/kg of the bupleurum alcohol extract is administrated by the bupleurum group in a daily gastric lavage mode, 50mg/kg of the pirfenidone capsule is administrated by the pirfenidone group in a daily gastric lavage mode, and the normal saline with the same amount is administrated by the blank control group and the model group in a daily gastric lavage mode, and the continuous gastric lavage is carried out for 28 days.
2. Weighing of rat body weight
The day of modeling, the weight of each group of rats was weighed, and the rats were weighed once every 7 days later and recorded until the rats were sacrificed.
3. Rat sacrifice and specimen collection
Rats were sacrificed 4 weeks after gavage and specimens were collected by isoflurane anesthesia.
1) Serum was collected: the chest cavity is opened, the apex of the heart is sampled to 3-4ml, the mixture is stood for 24 hours at 4 ℃,3500r/min at 4 ℃, and serum is taken after centrifugation for 10min and is split-packed and stored in a refrigerator at-80 ℃.
2) Taking lung tissue: taking out the rat lung tissue, cleaning the rat lung tissue by using normal saline, removing other adhered tissues, sucking the water on the lung tissue by using filter paper, and weighing the rat lung tissue. The upper leaf of the right lung is fixed in paraformaldehyde solution for HE, masson and sirius red staining, and the rest tissue is placed in a refrigerator at-80 ℃ for standby.
4. Rat lung index assay
After the rat is sacrificed, the lung tissue is taken out, washed clean by normal saline, the water is absorbed, and the weight of the rat lung tissue is weighed and compared with the weight of the rat. Rat lung index (%) =rat lung tissue weight (g)/rat body weight (g) ×100%
5. Rat lung tissue hydroxyproline content determination
Hydroxyproline content was measured using a hydroxyproline (Hyp) assay kit (alkaline hydrolysis) from the institute of bioengineering, nanjing, cat No. a030-2-1, which was operated according to the description.
Firstly, carrying out sample pretreatment on lung tissues, accurately weighing 30-100mg of wet weight of the lung tissues, putting the lung tissues into a test tube, accurately adding 1mL of hydrolysate, and uniformly mixing. Covering, hydrolyzing at 95deg.C or boiling water bath for 20min (mixing for 10min for more complete hydrolysis).
Adding 10 mu L of indicator into each tube after each tube is cooled by running water, and shaking uniformly;
accurately adding 1ml of pH-regulating first solution into each tube, and uniformly mixing (the solution should be red at this time);
the pH-adjusted solution B was aspirated with a 200. Mu.L sample applicator, and mixed well after each drop until the color of the indicator in the solution turned yellow-green. At this time, the pH value is about 6.0-6.8;
then adding double distilled water to 10mL, and uniformly mixing;
adding appropriate amount of activated carbon (about 20-30mg, based on supernatant, centrifuging, clarifying, and colorless) into 3-4mL diluted hydrolysate, mixing, centrifuging at 3500r/min for 10min, and carefully taking 1mL supernatant for detection.
And then carrying out detection analysis according to the instruction of the kit.
Finally, uniformly mixing, carrying out water bath at 60 ℃ for 15min, centrifuging at 3500r/min for 10min after cooling, taking the supernatant at the wavelength of 550nm, measuring the absorbance value of each tube, and then calculating the Hyp content according to the following formula.
Hydroxyproline content (μg/mg tissue) = [ (assay-blank)/(standard-blank) ]. Times.5 μg/mL× (10 mL +.W)
W: tissue mass, mg.
As shown in FIG. 9, the weight of the rats in the other groups was significantly reduced (P < 0.05) on days 0-7, and there was no significant difference in the weight change between the rats in the model group and the drug groups (P > 0.05) as compared with the control group; starting on day 7, rats in each group gradually increased in weight, and model groups showed a slower rate of weight increase (P < 0.05) on days 7-14 compared to the control group; there was no significant difference in net increase in body weight (P > 0.05) between groups of rats at days 14-21, 21-28. In the lung fibrosis model, the collagen of lung tissues is increased, the lung index is increased, and the content of the hydroxyproline in the lung is increased. As shown in FIG. 10, the lung index of rats in the model group was increased (P < 0.05) compared to the control group, and the lung index of rats in the Bupleurum polycephalum (P < 0.05) compared to the model group was decreased. As shown in FIG. 11, the lung hydroxyproline content of rats in the model group was increased (P < 0.05) compared to the control group, and the lung hydroxyproline content of rats in the bupleurum group was decreased (P < 0.05) compared to the model group. The results show that the bupleurum chinense ethanol extract can treat pulmonary fibrosis, and the treatment effect is better than that of the positive drug pirfenidone.
Example 7
Effect of bupleurum chinense ethanol extract on PF rat lung tissue slice staining
1. Paraffin section
Lung tissue was fixed in 4% paraformaldehyde and then removed, and paraffin sections were prepared as follows.
Dehydrating: rat lung tissue was sequentially passed through ethanol solutions of different concentrations: 50%, 70%, 85%, 95% ethanol solution for 90min and 100% ethanol solution for 2h.
And (3) transparency: embedding cassettes containing rat lung tissue in ethanol and xylene at 1:1 in xylene for 120min after 60 min.
Penetration: transparent lung tissue was treated with xylene and paraffin at 1:1 in the mixed solution for 90min and then in paraffin for 120min.
And (3) paraffin embedding: and taking out the treated tissue from the tissue embedding box, and embedding the tissue in a metal embedding frame by using paraffin.
Slicing: the paraffin-embedded specimens were removed and serially sectioned along the sagittal plane to a thickness of 5 μm. The lower end of the wax sheet belt is gently supported by the left hand holding writing brush, the upper end of the wax sheet belt is clamped by the right hand holding forceps, and the wax sheet belt is placed into the water box of the tablet display instrument in the front direction.
And (5) spreading: spreading in warm water at 50 ℃, after the slice is fully spread and flattened, vertically inserting the slide glass coated with polylysine into water to lean on the slice, immediately lifting the slide glass vertically, and correcting the slice position.
Baking slices: the slide glass is put into a 60 ℃ oven for baking for 2 hours, and then is put into a 37 ℃ oven for fixing for 12 hours.
Dewaxing: the xylene I and the xylene II are respectively soaked for 10min, and the absolute ethyl alcohol I and the absolute ethyl alcohol II are respectively soaked for 5min,95% ethyl alcohol for 5min,85% ethyl alcohol for 5min and 75% ethyl alcohol for 5min, and are washed by running water.
HE staining
The cut sections were dewaxed conventionally to water and HE stained as follows. Hematoxylin solution dyeing for 8min, distilled water washing for 1-2 times, 1% hydrochloric acid alcohol differentiation for 5s, distilled water washing for 1-2 times, eosin solution dyeing for 1min, distilled water washing for 1-2 times, 85% ethanol dehydration for 10s,95% ethanol dehydration for 10s, absolute ethanol I dehydration for 30s, absolute ethanol II dehydration for 30s, transparent for 5min x 2 times in xylene solution, neutral resin sealing, airing, and observing rat lung histopathological changes under an optical microscope.
Masson staining
The cut sections were dewaxed to water and Masson stained as follows. The prepared Weibert iron hematoxylin staining solution is used for 10min, the acid ethanol differentiation solution is differentiated for 5-15s, the water washing is performed, the Masson blue staining solution is reversely blue for 3-5min, the water washing is performed, the distilled water washing is performed for 1min, the ponceau red fuchsin staining solution is used for 5-10min, the weak acid working solution is used for 1min, the phosphomolybdic acid solution is used for 1-2min, the weak acid working solution is used for 1min, the aniline blue staining solution is used for 1-2min, the weak acid working solution is used for 1min,95% ethanol is rapidly dehydrated, the absolute ethanol is dehydrated for 3 times, the xylene is transparent for 3 times, the neutral gum is sealed for 1-2min, and the lung tissue morphology change is observed after airing.
4. Dyeing of sirius scarlet
The cut sections were dewaxed to water and stained with sirius scarlet as follows. Adding into the saturated picric acid sirius scarlet staining solution, staining for 8min, and taking out. Rinsing with absolute ethanol for several minutes, taking out, placing under a microscope for observation, and rinsing until the color is proper under the microscope for observation. The slices are transferred into a 60 ℃ oven for baking until the slices are dried, placed into dimethylbenzene for transparency for 5min, and are sealed by neutral resin.
The results are shown in FIG. 12, and the HE staining results show that the control rats have normal alveolar structure, intact alveolar epithelial cells, almost no obvious lung blebs formed, and no obvious fibrosis of the lung interstitium. The model group can be used for treating pulmonary alveolus structural disorder, obvious pulmonary alveolus formation, inflammatory cell infiltration and obvious pulmonary interstitial fibrosis. Compared with the model group, the alveolus structures of the bupleurum group and the pirfenidone group are recovered, and the fibrosis is obviously reduced. As shown in fig. 13, the Masson staining results showed that the control group had a clear alveolar structure with less collagen deposition between alveolar spaces and few blue stained areas. The model group has almost no normal alveolar structure, obvious collagen deposition among alveoli and more whole blue-stained areas. The bupleurum group and pirfenidone group recovered to different degrees compared to the model group, with blue collagen fibers only appearing at the alveolar edges. These results indicate that the bupleurum chinense ethanol extract can effectively treat pulmonary fibrosis. As shown in fig. 14, the result of staining sirius scarlet showed that the lung tissue of the control group was yellow in color as a whole, and only a part of the vicinity of the cavity was pale red, indicating that collagen in the tissue was small. The model group can see an orange-red background of the whole lung tissue, indicating the presence of collagen deposition in all alveolar spaces. Compared with the model group, the red collagen fibers of the bupleurum group and the pirfenidone group are greatly reduced, and only a small amount of red collagen fibers appear at the alveoli edge.
Example 8
Influence of bupleurum chinense ethanol extract on PF rat oxidative stress index GSH, MDA, SOD content
1.GSH
The GSH content of rat lung tissue was measured using a reduced Glutathione (GSH) assay kit (microplate method) from Nanjing institute of biological engineering, cat No. A006-2-1, which was operated according to the specification.
Firstly, carrying out sample pretreatment on lung tissues, accurately weighing the weight of the tissues, and according to the weight (g): after adding reagent one in a volume (ml) =1:9 ratio, mechanical homogenization was performed, centrifugation was performed at 3500r/min for 10 minutes, and the supernatant was used as a color reaction. And then carrying out detection analysis according to the instruction of the kit.
Mix well plate by gently shaking, stand for 5 minutes, at 405nm, and measure absorbance value of each well by enzyme label instrument. The GSH content was then calculated according to the following formula.
GSH content in tissue (μmol/g tissue) = [ (assay-blank)/(standard-blank)]×20μmol/L×V Sample assembly ÷W
V Sample assembly : the volume of the first reagent L added when the sample is homogenized with the first reagent;
w: sample mass, g.
2.MDA
MDA content of rat lung tissue was measured using a Malondialdehyde (MDA) assay kit (TBA method) from Nanjing institute of biological engineering, cat No. A003-1-2, which was operated according to the instructions.
Firstly, carrying out sample pretreatment on lung tissues, accurately weighing the weight of the tissues, and according to the weight (g): after adding 9 times of physiological saline in a volume (ml) =1:9 ratio, homogenizing on ice, centrifuging at 2500r/min for 10 minutes, and taking a supernatant to be measured. After the completion of the first, second and third reagent arrangements according to the kit instructions, the solutions were prepared in the following two systems by taking centrifuge tubes. And (3) injection: after the system I is prepared, shaking a few centrifuge tubes, uniformly mixing, and adding the system II.
System one
System II
After the preparation, a small hole is punched on the centrifuge tube, a cover is covered tightly, the vibrator is vibrated uniformly, water bath is carried out at 95 ℃ for 40 minutes, flowing water is taken out for cooling, and then the centrifuge tube is centrifuged for 10 minutes at 3500 r/min. The supernatant was read with a microplate reader at 532nm and 200. Mu.L. The MDA content was then calculated according to the following formula.
(measurement tube-control tube)/(standard tube-blank tube). Times.10 nmol/ml/(concentration of sample to be measured = MDA content in tissue (nmol/mgprot)
3.SOD
The SOD content of rat lung tissue was measured using total superoxide dismutase (T-SOD) assay kit (WST-1 method) from Nanjing institute of biological engineering, cat# A001-3-2, operated according to the specification.
Firstly, carrying out sample pretreatment on lung tissues, accurately weighing the weight of the tissues, and according to the weight (g): after adding 9 times of physiological saline in a volume (ml) =1:9 ratio, homogenizing on ice, centrifuging at 2500r/min for 10 minutes, and taking a supernatant to be measured. And then carrying out detection analysis according to the instruction of the kit.
All liquids were mixed and incubated at 37℃for 20min and read at 450nm using an enzyme-labeled instrument. The activity of SOD was then calculated according to the following formula:
1) [ (control-control blank) - (assay-assay blank) ] ≡ (control-control blank) ×100% = SOD inhibition (%)
2) SOD inhibition rate ≡50% ++dilution ≡concentration of sample protein to be tested (mgprot/ml) =sod activity (U/mgprot)
As shown in FIG. 15, the lung tissue GSH content of rats in the model group was decreased (P < 0.05) compared with the control group, and the lung tissue GSH content of rats in the bupleurum and pirfenidone groups was increased (P < 0.05) compared with the model group, indicating that bupleurum and pirfenidone can restore the antioxidant capacity of rats with pulmonary fibrosis. As shown in fig. 16, the MDA content of the rat lung tissue of the model group is increased (P < 0.05) compared with the control group, and the MDA content of the rat lung tissue of the bupleurum chinense is decreased (P < 0.05) compared with the model group, which indicates that the bupleurum chinense can reduce the degree of attack of free radical on the body cells of the rat with pulmonary fibrosis. As shown in FIG. 17, the SOD content of the rat lung tissue of the model group was decreased (P < 0.05) compared with the control group, and the SOD content of the rat lung tissue of the bupleurum chinense group was increased to be equal to the control group (P < 0.05) compared with the model group, which indicates that the bupleurum chinense can restore the oxidation/antioxidation balance of the rat with pulmonary fibrosis. These results indicate that the bupleurum chinense ethanol extract can treat pulmonary fibrosis through antioxidation.
Example 9
Effect of Bupleurum scorzonerifolium ethanol extract on the content of PF rat inflammatory factors TGF-beta 1, TNF-alpha, IL-1 beta
1.TGF-β1
The TGF-beta 1 content of Rat serum was measured using the Rat TGF-beta 1ELISA KIT from Shanghai old biotechnology Co., ltd, cat# YX-E21175, according to the instructions.
First, the ELISA kits all contained the following: the kit comprises a microporous enzyme label plate (12 holes multiplied by 8 strips) coated with antigen or antibody, detection antibody-HRP, sample diluent, chromogenic liquid A, chromogenic liquid B, stop solution, 20 multiplied by washing buffer solution, standard substance, sealing plate film and self-sealing bag. The ELISA kit standard concentration gradient of TGF-beta 1 is: 0. 125, 250, 500, 1000, 2000pg/mL.
Next, the sample treatment portion, rat serum, was centrifuged at 3000r/min for 10 minutes, and the supernatant was taken for measurement.
Finally, the whole operation steps mainly comprise 6 steps.
1) The required strips were removed from the bag at a storage temperature of 4 c, allowed to equilibrate for 20 minutes at room temperature, the remaining strips sealed in the bag and returned to the refrigerator at 4 c for storage.
2) In order to generate a standard curve and determine the concentration of a sample to be measured, a standard well and a sample well need to be provided. In the standard wells, 50 μl of standard at different concentrations needs to be added in order to establish the standard curve. In the sample well, 10. Mu.L of the sample to be measured is added first, and 40. Mu.L of the sample diluent is added to dilute the concentration of the sample to an appropriate range. Blank wells do not need to be added.
3) In both standard wells and sample wells, 100 μl of HRP-labeled detection antibody was added to each well. Such antibodies are used to detect the presence of the antigen of interest. A closing membrane is used to seal the reaction wells to prevent evaporation and cross-contamination. Incubation was performed in an incubator at 37℃for 60min to allow the antibodies to bind to the target antigen in the sample. This process is important because the subsequent detection step can only be performed when the antibody binds to the antigen of interest.
4) The liquid in the wells is discarded, the residual liquid can be drained by being filled with filter paper, then each well is filled with 1X of washing liquid, the washing liquid is allowed to stand in the well for 1min, then the washing liquid is thrown off, and the water-absorbing paper is used for draining, and the process needs to be repeated 5 times. The purpose of this step is to remove unbound antibodies and non-specific binding substances to reduce the occurrence of false positives and to improve the specificity and accuracy of the detection.
5) 50. Mu.L of each of the color-developing solution A and the color-developing solution B was added to each well, and incubated at 37℃for 15 minutes in the absence of light. The color solution A and the color solution B are enzyme reaction substrates for detecting the binding of the antibody to the target antigen. After the antibodies interact with the target antigen, they will produce a fluorescent or chromogenic reaction under the action of HRP. The time and temperature conditions of this step are such that the reactants have sufficient time to react and avoid interference of external light with the reaction.
6) After 50. Mu.L of the stop solution was added to each well, the enzyme reaction of substrates A and B was stopped by the stop solution to stop the fluorescence or color reaction (blue color immediately changed to yellow color), and then the absorbance value of each well was measured at a wavelength of 450nm using an microplate reader. The results of the measurement may reflect the concentration of the antigen of interest in each sample. The absorbance values can be converted to the concentration of the antigen of interest by a standard curve. And (5) ending the detection.
2.TNF-α
The TNF-alpha content of Rat serum was measured using the Rat TNF-alpha ELISA KIT from Shanghai old biotechnology Co., ltd, cat# YX-201406R, which was operated according to the instructions.
The ELISA kit standard concentration gradient of TNF-alpha is: 0. 20, 40, 80, 160, 320pg/mL.
The specific method of operation is the same as TGF-. Beta.1 and will not be described in detail herein.
3.IL-1β
The IL-1β ELISA KIT from Shanghai old Biotechnology Co was used to measure the IL-1β content of Rat serum, product number YX-091201R, which was operated according to the instructions.
The ELISA kit standard concentration gradient of IL-1 beta is: 0. 2.5, 5, 10, 20, 40pg/mL.
The specific method of operation is the same as TGF-. Beta.1 and will not be described in detail herein.
As a result, as shown in FIG. 18, it can be seen that the serum TGF-. Beta.1 content of rats in the model group was increased (P < 0.05) compared to the control group, and the serum TGF-. Beta.1 content of rats in the bupleurum and pirfenidone groups was decreased to be close to that in the normal group (P < 0.05) compared to the model group. As shown in FIG. 19, it can be seen that the serum TNF- α content of rats in the model group was increased (P < 0.05) compared to the control group, and that the serum TNF- α content of rats in the bupleurum group and pirfenidone group was decreased (P < 0.05) compared to the model group. As shown in FIG. 20, it can be seen that the serum IL-1. Beta. Content of rats in the model group was increased (P < 0.05) compared to the control group, and the serum IL-1. Beta. Content of rats in the bupleurum group and pirfenidone group was decreased (P < 0.05) compared to the model group. These results indicate that the bupleurum chinense ethanol extract can inhibit inflammatory factors TGF-beta 1, TNF-alpha and IL-1 beta, and has good anti-pulmonary fibrosis activity. Thus can be applied as a medicine for treating pulmonary fibrosis.
The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (6)
1. The preparation method of the bupleurum chinense ethanol extract is characterized by comprising the following steps:
step one, drying whole herb of the bupleurum chinense, cutting into segments, crushing Cheng Zhushe bupleurum chinense powder, adding petroleum ether with 15-20 times of density, heating, refluxing and degreasing, repeating degreasing for 2-3 times until filtrate has no obvious dark green color, volatilizing residual petroleum ether until the bupleurum chinense powder is completely dried;
and step two, adding 75% ethanol with the density of 10-15 times into the bupleurum powder obtained in the step one, heating and refluxing in water bath for 3 times, filtering, merging filtrate, concentrating by a rotary evaporation evaporator, recovering ethanol, rotary evaporating until the ethanol recovery is finished, drying residual water by an oven, and vacuum drying to obtain the bupleurum ethanol extract.
2. An application of bupleurum chinense L ethanol extract in preparing medicines for resisting pulmonary fibrosis is provided.
3. The use according to claim 2, wherein the bupleurum chinense alcohol extract comprises the compound 3-O- β -D-glucosyl- (1→6) - [ - α -L-rhamnosyl- (1→3) ] - β -D-glucosyl-11,13 (18) -diene- (16 β, 29) -dihydroxy-28-O- β -D-glucosyl-oleanane; the compound 3-O- β -L-rhamnosyl- (1→2) - [ β -D-xylosyl- (1→3) ] - β -D-glucosyl-11,13 (18) -diene- (16 β,23, 29) -trihydroxy-28-O- β -D-glucosyl-oleanane; the compound 3-O-rhamnosyl- (1→4) - [ rhamnose- (1→3) ] -glucosyl- (1→6) - [ glucosyl- (1→2) ] -glucosyl-11,13 (18) -diene-16-hydroxy-28-O-glucosyl-oleanane; compound Luo Tonggan V; the compound anemonin III; the compound saikosaponin Q; compound synucleoside B; the compound saikosaponin C; the compound saikosaponin H; the compound rutin; the compound saikosaponin D.
4. The use according to claim 2, wherein the bupleurum chinense extract is used for the preparation of a medicament for inhibiting proliferation of pulmonary fibrotic cells.
5. The use according to claim 2, wherein the bupleurum chinense extract is used for the preparation of a medicament for the oxidation of pulmonary fibrosis cells.
6. The use according to claim 2, wherein the bupleurum chinense alcohol extract is used for preparing medicines for inhibiting inflammatory factors TGF-beta 1, tnf-alpha and IL-1 beta in pulmonary fibrosis.
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