A kind of dwarf tea extract and its preparation method and use

Abstract

The present invention discloses a dwarf tea extract and a preparation method and use thereof. Specifically, the dwarf tea extract of the present invention is an ethyl acetate extract of dwarf tea. From the LPS-induced MH-S cell inflammation model and ALI rat model experiments, the anti-inflammatory effects of dwarf tea extracts in different solvents are different. The ethyl acetate extract of dwarf tea has a better anti-inflammatory effect than dwarf tea water extract, ethanol-water extract, n-butanol extract, and petroleum ether extract, and has a better protective effect on organ damage in ALI rats.

Description

Technical Field

[0001] The invention relates to the field of dwarf camellia extract, and in particular to a dwarf camellia extract and a preparation method and application thereof. Background Art

[0002] Acute lung injury (ALI) is an inflammatory syndrome and the early stage of acute respiratory distress syndrome. Its main manifestations include increased pulmonary vascular permeability, tissue fluid infiltration, accumulation of inflammatory cells, and gas exchange dysfunction. Pulmonary and systemic bacterial infections are the main causes of ALI. In addition, inflammation and apoptosis are the main mechanisms of action in the development of ALI. Under the guidance of traditional Chinese medicine theory, the lung is a delicate organ and is easily invaded by pathogens. When warm pathogens attack upward, they become depressed and resolve into phlegm, causing phlegm-heat stagnation and lung failure. The clinical manifestations of ALI diagnosed in traditional Chinese medicine are wheezing, fullness, dizziness, and fever, especially wheezing and fullness. The onset of the disease is mostly caused by real pathogens and is a wind-heat lung heat disease.

[0003] The dried whole herb of the plant Ardisia japonica of the Myrsinaceae family is also known as Ardisia japonica, Pingdimu, and Pearl under the Leaves. It is flat in nature, spicy and slightly bitter in taste, and belongs to the lung and liver meridians. Ardisia japonica has pharmacological effects such as antitussive, expectorant, anti-inflammatory, antiviral, antiasthmatic, diuretic, and antibacterial. "Common Chinese Herbal Medicines in Shanghai" records: "Ardisia japonica has the effects of analgesia, diuresis, spleen strengthening, and hemostasis. It is used to treat night sweats and hemoptysis caused by damp-heat jaundice, hepatitis, acute nephritis, cystitis, and tuberculosis, relieve muscle and bone pain caused by physical fatigue and strain, and can also regulate symptoms such as irregular menstruation and sinusitis." Ardisia japonica is often used to treat chronic bronchitis, tuberculosis, upper respiratory tract inflammation and other diseases. Summary of the invention

[0004] In the previous study, the inventors found that Camellia oleifera has a good anti-inflammatory protective effect on rats with acute lung injury (ALI) model induced by LPS. However, there are differences in the types of main chemical components contained in different extraction parts of traditional Chinese medicine, and there are differences in the strength of their pharmacodynamic effects. Based on the MH-S cell inflammation model and ALI rat model induced by LPS, the present invention studied the anti-inflammatory activity of Camellia oleifera extracts in different solvents (petroleum ether, ethyl acetate, n-butanol, ethanol-water and water), and found that the ethyl acetate extract has high safety and high anti-inflammatory activity.

[0005] Based on this, one of the objects of the present invention is to provide a method for preparing a Camellia sinensis extract.

[0006] The second object of the present invention is to provide a dwarf camellia extract prepared by the above preparation method.

[0007] The third object of the present invention is to provide the use of the above-mentioned Camellia oleifera extract in the preparation of a drug for treating and / or preventing acute lung injury (ALI).

[0008] The technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a Camellia oleifera extract, wherein the Camellia oleifera extract is an ethyl acetate extract of Camellia oleifera, and the preparation method comprises the following steps:

[0010] (1) extracting the dwarf tea with a 60-80% (v / v) ethanol aqueous solution, filtering the obtained ethanol aqueous extract, and concentrating it until there is no alcohol taste, thereby obtaining a dwarf tea extract;

[0011] (2) extracting the Camellia oleifera extract with petroleum ether and ethyl acetate in sequence, concentrating and drying the ethyl acetate extract to obtain the Camellia oleifera ethyl acetate extract.

[0012] In some embodiments, in step (1), the ethanol concentration in the ethanol aqueous solution is 65-75%, v / v, such as 70%, v / v.

[0013] In some embodiments, in step (1), the amount of the ethanol aqueous solution is 5-20 times the amount of the dwarf tea, preferably 10-14 times, for example 12 times.

[0014] In some embodiments, in step (1), the extraction is performed by adding the dwarf tea to the ethanol aqueous solution and heating to reflux. In some embodiments, heating to reflux may be performed multiple times, for example 2-5 times, each time for 0.5 to 3 hours, preferably 1.5 to 2.5 hours. In some embodiments, cold soaking may be performed before heating to reflux, and the time of cold soaking is 3-24 hours, preferably 8-16 hours.

[0015] In some embodiments, in step (1), the filtrate obtained by filtering the ethanol water extract is concentrated under reduced pressure to 1 / 30-1 / 10, preferably 1 / 25-1 / 15, for example 1 / 20 of the volume of the original ethanol aqueous solution, at which time the ethanol smell is basically gone; the temperature of the reduced pressure concentration is 48-80°C, preferably 50-60°C.

[0016] In some embodiments, in step (2), the volume ratio of petroleum ether to the Camellia dwarfa extract is 1-3:1, preferably 1-2:1; in some embodiments, petroleum ether can be used for multiple extractions, for example 3-8 times, preferably 5 times.

[0017] In some embodiments, in step (2), the volume ratio of ethyl acetate to the dwarf tea extract is 1-3:1, preferably 1-2:1; in some embodiments, ethyl acetate can be used for multiple extractions, for example 3-8 times, preferably 5 times. After the ethyl acetate extracts are combined, they are concentrated in the next step.

[0018] In some embodiments, in step (2), the concentration is preferably concentrated under reduced pressure at a temperature of 48-80°C, preferably 50-60°C.

[0019] In some embodiments, in step (2), the drying is preferably freeze drying, and spray drying or the like may also be used, which is not particularly limited in the present invention.

[0020] In a second aspect, the present invention provides a dwarf camellia extract prepared by the method for preparing the dwarf camellia extract.

[0021] In a third aspect, the present invention provides use of the Camellia oleifera extract in the preparation of a medicament for treating and / or preventing acute lung injury (ALI).

[0022] Beneficial Effects

[0023] From the LPS-induced MH-S cell inflammation model experiment, the extracts of Camellia oleifera in different solvents had different inhibitory effects on the expression of two inflammatory factors, IL-6 and TNF-α, in MH-S cells. The order of inhibitory effects was ethyl acetate extract > Camellia oleifera ethanol-water solution extract > n-butanol extract > petroleum ether extract > water extract, among which the ethyl acetate extract had the best anti-inflammatory effect.

[0024] From the LPS-induced ALI rat model experiment, the ethyl acetate extract has a better anti-inflammatory effect than the ethanol-water extract and water extract of Camellia oleifera, and has a better protective effect on organ damage in ALI rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of screening the survival rate of MH-S cells at different drug concentrations by CCK-8 method in Example 2

[0026] Figure 2 The effects of different extracts of Camellia oleifera on the expression levels of IL-6 (A) and TNF-α (B) in MH-S cells in Example 2 are shown in FIG. Compared with the blank control group: #P<0.05, ##P<0.01; compared with the model group: *P<0.05, **P<0.01.

[0027] Figure 3 The figure is the pathological morphology of lung tissues of rats in each group in Example 3 (HE staining, ×200), wherein A: blank group; B: model group; C: low-dose ethyl acetate extract group; D: medium-dose ethyl acetate extract group; E: high-dose ethyl acetate extract group; F: 70% ethanol extract of Camellia oleifera group; G: Camellia oleifera water extract group; H: dexamethasone group. DETAILED DESCRIPTION

[0028] Materials and sources:

[0029] The medicinal materials of Dalbergia paniculata were purchased from Anhui Shengantang Pharmaceutical Co., Ltd. (origin: Longhui, Hunan, batch number: 210301).

[0030] Lipopolysaccharide (LPS) was purchased from Sigma, USA (lot number: 039M4004V).

[0031] The experimental water was prepared by Milli-QPOD ultrapure water machine. The ethanol, ethyl acetate, petroleum ether and n-butanol used were all analytical grade.

[0032] Example 1. Preparation of Camellia sinensis extract

[0033] (1) Preparation of the dwarf tea water extract: Take 500 g of the dried whole plant of dwarf tea, add 12 times the volume of water, heat and reflux extraction for 3 times, each time for 2 hours, combine the filtrate and concentrate under reduced pressure to 2 g of dwarf tea raw drug / mL to obtain the dwarf tea water extract; freeze-dry the above water extract to obtain a powdered dwarf tea water extract, which is stored at low temperature for later use.

[0034] (2) Preparation of 70% ethanol extract of Camellia sinensis: Take 500 g of the whole plant of Camellia sinensis dried, add 12 times the volume of 70% ethanol, cold soak for 12 h, heat reflux extraction 3 times, each time for 2 h, filter, combine the filtrates and recover ethanol under reduced pressure until there is no alcohol taste, and concentrate under reduced pressure to 2 g Camellia sinensis crude drug / mL to obtain 70% ethanol extract of Camellia sinensis; concentrate the above 70% ethanol extract of Camellia sinensis under reduced pressure to an appropriate amount (about 300 mL), freeze-dry, and obtain a powdered 70% ethanol extract of Camellia sinensis.

[0035] (3) Preparation of petroleum ether, ethyl acetate and n-butanol extracts of Camellia davidiana:

[0036] Take 2500g of dried dwarf tea, add 12 times the amount of 70% ethanol, cold soak for 12h, heat reflux 3 times, 2h each time, filter, combine the 3 extracts, let stand for 12h, recover ethanol until there is no alcohol taste, and concentrate under reduced pressure to an appropriate amount (about 1500mL). The obtained concentrated solution is extracted 5 times with three organic solvents of petroleum ether, ethyl acetate, and n-butanol in order of polarity from low to high with a volume of 1:1.5 (concentrated solution: petroleum ether / ethyl acetate / n-butanol), and the extracts of the same solvent are combined, the solvent is recovered under reduced pressure, concentrated under reduced pressure, and freeze-dried to finally obtain different powdered dwarf tea extracts: dwarf tea petroleum ether extract, dwarf tea ethyl acetate extract, and dwarf tea n-butanol extract, which are stored at low temperature for use. The powder extraction rates of the above three dwarf tea extracts are 1.06% (dwarf tea petroleum ether extract), 1.83% (dwarf tea ethyl acetate extract), and 2.87% (dwarf tea n-butanol extract), respectively.

[0037] Example 2 In vitro cell activity test of Camellia sinensis extract

[0038] 1. Preparation of stock solutions of different extracts of Camellia oleifera

[0039] Accurately weigh 20 mg of Camellia oleifera water extract, Camellia oleifera 70% ethanol extract, Camellia oleifera petroleum ether extract, Camellia oleifera ethyl acetate extract, and Camellia oleifera n-butanol extract, and add 400 μL DMSO to prepare 50 mg / mL stock solutions for use.

[0040] Preparation of working solution: The above-mentioned Camellia sinensis extract stock solution was serially diluted with RPMI-1640 medium. The specific concentrations are shown in Table 1.

[0041] 2. Cell Culture

[0042] Mouse alveolar macrophages (MH-S cells, purchased from Wuhan Pronocell Life Science Co., Ltd.) were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO 2 The cells were cultured in a saturated humidity cell culture incubator. When the cell confluence reached 80%-90%, the cells were subcultured and the experiments were performed in the logarithmic growth phase.

[0043] 3. CCK-8 method to screen the drug concentration of MH-S cells

[0044] Take MH-S cells in the logarithmic growth phase and adjust the cell suspension density to 2.5×10 6 / mL, 100 μL per well was inoculated into 96-well culture plates, and cultured in the incubator for 24 hours. After the cells were well attached to the wall, the original culture medium was discarded and the cells were randomly divided into blank control group, different concentrations of dwarf tea water extract administration group, different concentrations of dwarf tea 70% ethanol extract administration group, different concentrations of petroleum ether extract administration group, different concentrations of ethyl acetate extract administration group, and different concentrations of n-butanol extract administration group. The concentration design of the administration group is shown in Table 1. The blank control group was added with culture medium containing 10% fetal bovine serum, and the other administration groups were added with relative concentrations of drugs. Six replicates were set for each experimental group. After administration, the cells were incubated at 37°C and 5% CO 2 The cells were cultured in a saturated humidity cell culture incubator for 24 hours. After 24 hours, CCK-8 reagent (batch number: GK100011, Shanghai Hongye Biotechnology Co., Ltd.) solution was added and cultured in the incubator for 3 hours. The absorbance of each group was measured at 450 nm using an ELISA reader to calculate the cell survival rate of each group.

[0045] Cell survival rate = absorbance value of drug administration group / absorbance value of blank control group × 100%

[0046] Table 1. Concentration design of drug administration groups

[0047]

[0048]

[0049] 4. Expression levels of IL-6 and TNF-α in MH-S cells

[0050] The concentration design of MH-S cell administration based on CCK-8 method screening is shown in Table 2. MH-S cells in the logarithmic growth phase were digested and the density of the cell suspension was adjusted to 1×10 5 1 mL was inoculated into each well of a 12-well plate and incubated at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator with saturated humidity for 24 hours. After the cells adhered to the wall and grew, they were randomly divided into a blank control group, an LPS group (model group), an LPS+water extract group, an LPS+70% ethanol extract group, an LPS+petroleum ether extract group, an LPS+ethyl acetate extract group and an LPS+n-butanol extract group. The blank control group was added with a culture medium containing 10% fetal bovine serum, and the model group and the LPS+drug group were added with 100 ng / mL of LPS to induce the construction of an MH-S cell inflammation model. The cells were cultured for 24 hours after drug addition, and the cell supernatant was used to detect IL-6 and TNF-α inflammatory factors.

[0051] Table 2. Optimal dosing concentration design for each dosing group

[0052]

[0053] 5 Data processing

[0054] SPSS 26.0 software was used to process and analyze the data, and GraphPad Prism 7.0 software was used to draw statistical graphs.

[0055] 6. Experimental Results

[0056] 6.1 CCK-8 method screening MH-S cell survival rate results at different drug concentrations

[0057] The viability of MH-S cells was detected by CCK-8 method, and the effects of water extract, 70% ethanol extract, petroleum ether extract, ethyl acetate extract and n-butanol extract of Camellia oleifera on the growth and survival rate of MH-S cells were evaluated. Figure 1 .

[0058] The results showed that, in the concentration range of 0-25μg / mL of petroleum ether extract and 0-50μg / mL of n-butanol extract, the survival rate of MH-S cells was above 85%, and higher administration concentrations of petroleum ether extract and n-butanol extract had a certain effect on the growth of MH-S cells; in the concentration range of 0-6.25μg / mL of ethyl acetate extract, 0-200μg / mL of water extract, and 0-100μg / mL of 70% ethanol extract, the survival rate of MH-S cells was above 90%, indicating that the above concentration ranges of dwarf tea water extract, 70% ethanol extract, and ethyl acetate extract will not affect the normal growth of MH-S cells and have low cytotoxicity.

[0059] 6.2 Effects of Camellia sinensis and its different extracts on the expression of IL-6 and TNF-α in MH-S cells

[0060] Figure 2 Table 3 shows the effects of different extracts of Camellia oleifera on the expression levels of IL-6 (A) and TNF-α (B) in MH-S cells.

[0061] Table 3 Effects of different extracts of Camellia oleifera on the expression levels of IL-6 and TNF-α in MH-S cells

[0062]

[0063] Note: Compared with the blank group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, *** P<0.01.

[0064] Depend on Figure 2 As shown in Table 3, the expression levels of IL-6 and TNF-α in the MH-S cells of the model group were significantly higher than those in the blank control group, and the difference was statistically significant (P < 0.01), indicating that the LPS-induced inflammatory cell model was successfully established. The four concentrations of petroleum ether extract, ethyl acetate extract and n-butanol extract all had different degrees of inhibitory effects on the release of IL-6 and TNF-α in MH-S cells; the four concentrations of 70% ethanol extract had an inhibitory effect on the release of IL-6 in MH-S cells, and the three high concentrations had an inhibitory effect on the release of TNF-α; the water extract had no significant inhibitory effect on the release of IL-6 and TNF-α.

[0065] The study further introduces the parameter P 0 The anti-inflammatory activities of four extracts of Camellia oleifera were comprehensively evaluated.

[0066] Introducing parameter P 0 ,definition

[0067] P 1 is the P value of the one-way ANOVA between each drug group and the model group. The smaller the value, the greater the difference between each drug group and the model group. 2 is the P value of the one-way ANOVA between each drug-treated group and the blank control group. The larger the value, the closer the inhibition of inflammatory factor release in each drug-treated group is to that in the blank group. The expression of IL-6 and TNF-α inflammatory factors has the same importance, so the direct addition method is used for comprehensive scoring. The P value of each group is calculated. 0 The values ​​and comprehensive score results are shown in Table 4.

[0068] Table 4 Comprehensive scoring results of IL-6 and TNF-α expression levels of water extract, 70% ethanol extract, petroleum ether extract, ethyl acetate extract and n-butanol extract of Camellia ternata

[0069]

[0070] Note: The P1 value in each group of the water part is "-" because the water extract has no inhibitory effect on the expression of IL-6 and TNF-α, causing the expression of IL-6 and TNF-α in each water extract administration group to show an upward trend.

[0071] The results showed that the water extract, 70% ethanol extract, petroleum ether extract, ethyl acetate extract and n-butanol extract of Camellia oleifera had different degrees of inhibitory effects on the release of IL-6 and TNF-α in the LPS-induced MH-S cell inflammation model. The order of inhibitory effects was ethyl acetate extract > 70% ethanol extract > n-butanol extract > petroleum ether extract > water extract. Therefore, the anti-inflammatory effect of the ethyl acetate extract of Camellia oleifera was better than that of the 70% ethanol extract, n-butanol extract, petroleum ether extract and water extract of Camellia oleifera.

[0072] Example 3 In vivo activity test of Camellia sinensis extract

[0073] 1. Preparation of working solutions of different extracts of Camellia sinensis

[0074] Camellia sinensis water extract solution, 70% ethanol extract solution and ethyl acetate extract solution: Add 0.5% CMC-Na solution to the Camellia sinensis water extract, 70% ethanol extract or ethyl acetate extract prepared in Example 1 to make a suspension equivalent to 2g Camellia sinensis crude drug / mL to obtain the working solution of the corresponding extract.

[0075] Dexamethasone: Grind dexamethasone tablets into fine powder, accurately weigh an appropriate amount, and add normal saline to prepare a suspension equivalent to 23 μg dexamethasone / mL, i.e., dexamethasone solution.

[0076] 2. Experimental Animal Grouping and Dosage

[0077] The rats were fed adaptively for 7 days. They were fed with sterilized feed and free to drink sterilized water. The purification and water and electricity operating systems were checked regularly and the environment was kept quiet. A total of 64 male rats were randomly divided into 8 groups: blank group, model group, low-dose ethyl acetate extract group (5g dwarf tea herb / kg / d), medium-dose ethyl acetate extract group (10g dwarf tea herb / kg / d), high-dose ethyl acetate extract group (20g dwarf tea herb / kg / d), 70% ethanol extract of dwarf tea group (20g dwarf tea herb / kg / d), dwarf tea water extract group (20g dwarf tea herb / kg / d) and dexamethasone group (2.3×10 -4 g / kg / d). The low, medium and high doses of ethyl acetate extract, the 70% ethanol extract group of Camellia oleracea, the Camellia oleracea water extract group and the dexamethasone administration group were intragastrically administered with corresponding doses, and the blank group and the model group were intragastrically administered with the same dose of distilled water for 7 consecutive days.

[0078] 3. Preparation of ALI Rat Model

[0079] One hour after the last administration, rats were anesthetized by intraperitoneal injection of 20% urethane. ALI rat model was prepared by tracheal instillation of lipopolysaccharide (LPS, 2 mg / kg) in the model group, low-, medium-, and high-dose groups of ethyl acetate extract of Camellia oleracea, 70% ethanol extract group of Camellia oleracea, Camellia oleracea water extract group, and dexamethasone group, and normal saline was instilled in the blank group. After the rats woke up naturally, the rats in each group were placed in metabolic cages and fasted for 24 hours without water or food.

[0080] 4. Sample Collection

[0081] Serum samples: Blood was collected from the abdominal aorta. One part of the blood was collected using a lithium heparin negative pressure tube for the detection of routine blood pharmacodynamic indicators (white blood cell count (WBC), neutrophil ratio (NEU%), lymphocyte ratio (LYM%), monocyte ratio (MONO%)); the other part was placed in a negative pressure tube without sodium heparin to collect serum, placed for 2 hours, centrifuged at 4°C for 15 minutes, and the speed was 3000r / min. The supernatant was used for the detection of IL-6, IL-10, CRP and TNF-α inflammatory factor levels.

[0082] Lung tissue: The rat trachea and whole lungs were removed by thoracotomy, and the left lung alveolar lavage fluid (BALF) was extracted after ligating the right main bronchus for the detection of IL-6, IL-10, CRP and TNF-α expression levels; the left lung was perfused with 4% paraformaldehyde for 30 min and directly fixed in 4% paraformaldehyde for pathological examination; the right upper lobe of the lung, spleen and thymus were washed in physiological saline, the surface moisture was absorbed by filter paper, and the weight was used for the detection of lung dry-to-wet weight ratio and organ indicators.

[0083] 5. Detection indicators

[0084] 5.1 Lung wet-to-dry weight ratio and organ index

[0085] The right upper lobe of the lung of each group of rats was removed and weighed to obtain the wet weight (W), then placed in an oven at 60°C for 48 hours and weighed to obtain the dry weight (D). The degree of edema of the lung tissue was evaluated by calculating the W / D ratio. The thymus and spleen of each group of rats were taken and weighed, and the rat organ index was calculated, and the calculation results were statistically analyzed.

[0086] 5.2 Lung function

[0087] 24 hours after the modeling, the whole body volume scanning detection system (wBP) was used to non-invasively detect the lung function parameters of rats in each group, including airway stenosis index (Penh), maximum expiratory flow rate (PEFb), mid-expiratory flow rate (EF50) and apnea at 37° (PAU).

[0088] 5.3 Routine blood test

[0089] Blood was collected from the abdominal aorta for routine blood tests, and the white blood cell count (WBC), neutrophil ratio (NEU%), lymphocyte percentage (LYM%) and monocyte percentage (MONO%) were calculated.

[0090] 5.4 Determination of IL-10, IL-6, CRP and TNF-α levels in serum and BALF

[0091] The expressions of IL-10, IL-6, CRP and TNF-α in serum and BALF were determined by enzyme-linked immunosorbent assay (ELISA).

[0092] 5.5 Lung histopathological examination

[0093] The left lung was fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, sliced ​​at 4 μm, and stained with HE. The pathological changes of the lung tissues of the rats in each group were observed under a microscope.

[0094] 6 Data processing

[0095] Data were processed using SPSS Statistics 26.0 statistical analysis software. One-way ANOVA was used for inter-group comparisons. The least significant difference method (LSD) was used for those who met the variance homogeneity test, and the Dunnett's T3 method was used for those who did not meet the variance homogeneity test. The results are expressed as mean ± standard deviation. The test level is α=0.05.

[0096] 7. Experimental Results

[0097] 7.1 Changes in lung wet-to-dry weight ratio and organ index

[0098] The results are shown in Table 5. Compared with the blank group, the thymus and spleen indexes of ALI rats were significantly decreased (P < 0.01); compared with the model group, the thymus index and spleen index of rats in the medium and high dose groups of ethyl acetate extract, 70% ethanol extract group of dwarf tea and dexamethasone group were significantly increased (P < 0.01); compared with the blank group, the lung wet-to-dry weight ratio of ALI rats was significantly increased (P < 0.01), indicating that the model animals had severe pulmonary edema; compared with the model group, the lung wet-to-dry weight ratio of the medium and high dose groups of ethyl acetate extract, 70% ethanol extract group of dwarf tea, water extract group of dwarf tea and dexamethasone group was significantly decreased (P < 0.05, 0.01), and with the increase of the dose of ethyl acetate extract, the lung wet-to-dry weight was significantly decreased; and compared with the water extract group of dwarf tea, the lung wet-to-dry weight ratio of the high dose group of ethyl acetate extract was significantly decreased, and the thymus index and spleen index were significantly increased (P < 0.01). The results showed that the ethyl acetate extract had a certain protective effect on organ damage in ALI rats in a dose-dependent manner.

[0099] Table 5. Effects of ethyl acetate extract of Camellia oleifera on lung wet-to-dry weight ratio and organ index in ALI rats

[0100]

[0101]

[0102] Note: Compared with the blank group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, ** P<0.01; compared with the dwarf tea water extract group: ▲ P<0.05, ▲▲ P<0.01.

[0103] 7.2 Changes in lung function

[0104] The results are shown in Table 6. Compared with the blank group, the EF50, PEF, PAU and Penh in the model group were significantly increased (P < 0.05, 0.01); compared with the model group, the PEF in the medium and high dose groups of ethyl acetate extract, the water extract group of dwarf tea, the 70% ethanol extract group and the dexamethasone group was significantly reduced (P < 0.05, 0.01), and the EF50, PAU and Penh in the low, medium and high dose groups of ethyl acetate extract and the dexamethasone group were significantly reduced (P < 0.05, 0.01); compared with the water extract group of dwarf tea, the medium and high dose groups of ethyl acetate extract of dwarf tea could significantly inhibit the increase of EF50, PEF, PAU and Penh (P < 0.05, 0.01). The above results show that ethyl acetate extract can intervene in the degree of bronchoconstriction and airway resistance of ALI model rats induced by LPS, and significantly improve the lung function of ALI model rats.

[0105] Table 6. Effects of ethyl acetate extract of Camellia oleifera on lung function in ALI model rats

[0106]

[0107] Note: Compared with the blank group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, ** P<0.01; compared with the dwarf tea water extract group: ▲ P<0.05, ▲▲ P<0.01.

[0108] 7.3 Changes in blood routine

[0109] The results are shown in Table 7. Compared with the blank group, the peripheral blood WBC, NEU% and MONO% of the model group increased significantly, and LYM% decreased significantly (P < 0.05, 0.01); compared with the model group, the WBC and MONO% of the high-dose ethyl acetate extract group decreased significantly (P < 0.05), the NEU% of the low, medium and high-dose ethyl acetate extract groups, the 70% ethanol extract group of the dwarf tea and the dwarf tea water extract group decreased significantly, and the LYM% increased significantly (P < 0.01), and the high-dose ethyl acetate extract group had a significantly better effect of reducing NEU% and increasing LYM% than the dwarf tea water extract group (P < 0.05, 0.01). Compared with the dwarf tea water extract group, the high-dose ethyl acetate extract group of the dwarf tea could significantly inhibit the increase of NEU% and MONO% content (P < 0.05, 0.01), and extremely significantly promote the release of LYM% (P < 0.01).

[0110] Table 7. Effects of ethyl acetate extract of Camellia oleifera on blood routine test in ALI model rats

[0111]

[0112] Note: Compared with the blank group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, ** P<0.01; compared with the dwarf tea water extract group: ▲ P<0.05, ▲▲ P<0.01.

[0113] 7.4 Expression levels of TNF-α, IL-6, and IL-10 in serum and bronchoalveolar lavage fluid

[0114] As shown in Table 8, compared with the blank group, the levels of TNF-α and IL-6 in the serum of the model group were significantly increased, and the levels of IL-10 were significantly decreased (P < 0.01); compared with the model group, the levels of TNF-α in the serum of the medium and high doses of ethyl acetate extract groups and the 70% ethanol extract of Camellia sinensis group were significantly decreased, the level of IL-6 in the serum of the high dose of ethyl acetate extract group was significantly decreased, and the level of IL-10 was significantly increased (P < 0.05, 0.01); compared with the Camellia sinensis water extract group, the levels of TNF-α in the medium and high doses of ethyl acetate extract groups were significantly decreased (P < 0.01), the level of IL-10 was significantly increased (P < 0.01), and the level of IL-6 in the high dose of ethyl acetate extract group was significantly decreased (P < 0.05).

[0115] Table 8. Effects of ethyl acetate extract of Camellia oleifera on serum TNF-α, IL-6 and IL-10 levels in ALI model rats

[0116]

[0117]

[0118] Note: Compared with the blank group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, ** P<0.01; compared with the dwarf tea water extract group: ▲ P<0.05, ▲▲ P<0.01.

[0119] As shown in Table 9, compared with the blank group, the CRP and TNF-α levels in BALF of the model group were significantly increased, the IL-10 level was significantly decreased (P < 0.01), and the IL-6 level had an increasing trend, but there was no significant difference; compared with the model group, the TNF-α level in BALF of the medium and high dose groups of ethyl acetate extract, the 70% ethanol extract group of dwarf tea, the water extract group of dwarf tea and the dexamethasone group was significantly decreased, while IL-10 was significantly increased (P < 0.05, 0.01), and the IL-6 level in BALF of the high dose group of ethyl acetate extract was significantly decreased (P < 0.05). Compared with the water extract group of dwarf tea, the TNF-α level in BALF of the medium and high dose groups of ethyl acetate extract was significantly decreased (P < 0.05, 0.01), and the IL-6 and IL-10 levels were significantly decreased (P < 0.05). In summary, the effect of ethyl acetate extract is more obvious than that of water extract group, and with the increase of dose, the regulatory effect of ethyl acetate extract on inflammatory factors in ALI model rats is enhanced, showing a certain dose dependence.

[0120] Table 9. Effects of ethyl acetate extract of Camellia oleifera on the levels of CRP, TNF-α, IL-6 and IL-10 in BALF of ALI model rats

[0121]

[0122] Note: Compared with the blank group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, ** P<0.01; compared with the dwarf tea water extract group: ▲ P<0.05, ▲▲ P<0.01.

[0123] 7.5 Pathological changes of lung tissue in ALI rats

[0124] The pathological morphology of lung tissue of rats in each group (HE staining, ×200) is shown in Figure 3 .

[0125] like Figure 3As shown, compared with the blank group, a large number of inflammatory cells infiltrated in the alveolar cavity of ALI rats, the alveolar wall thickened, the lung damage was more serious, and the range of lung damage was larger. Compared with the model group, the lung damage of rats in the dexamethasone group and the medium and high dose groups of ethyl acetate extract of dwarf tea was greatly improved, indicating that dexamethasone, medium and high dose groups of ethyl acetate extract had a certain protective effect on LPS-induced ALI; in addition, the degree of lung damage in rats in the high dose group of ethyl acetate extract was significantly weaker than that in the water extract group of dwarf tea and the 70% ethanol extract group of dwarf tea, indicating that the anti-inflammatory effect of the high dose group of ethyl acetate extract was better than that of the water extract group of dwarf tea and the 70% ethanol extract group of dwarf tea.

Claims

1. Use of the ethyl acetate extract of Camellia oleifera in the preparation of a medicament for treating and / or preventing acute lung injury, wherein: The preparation method of the ethyl acetate extract of Camellia oleifera comprises the following steps: (1) extracting the dwarf tea with a 60-80% v / v ethanol aqueous solution, wherein the amount of the ethanol aqueous solution is 5-20 times the amount of the dwarf tea, and the extraction is performed as follows: adding the dwarf tea to the ethanol aqueous solution, heating and refluxing for 2-5 times, each time for 0.5-3 hours, and cold soaking the dwarf tea before heating and refluxing, and the cold soaking time is 3-24 hours; filtering the obtained ethanol aqueous extract, and concentrating it until there is no alcohol taste, to obtain a dwarf tea extract; (2) extracting the Camellia oleifera extract with petroleum ether and ethyl acetate in sequence, wherein the volume ratio of petroleum ether to the Camellia oleifera extract is 1-3:1, and the petroleum ether is used for extraction 3-8 times; the volume ratio of ethyl acetate to the Camellia oleifera extract is 1-3:1, and the ethyl acetate is used for extraction 3-8 times; concentrating and drying the ethyl acetate extract to obtain the Camellia oleifera ethyl acetate extract.

2. The use according to claim 1, characterized in that: In step (1), the ethanol concentration in the ethanol aqueous solution is 65-75%, v / v.

3. The use according to claim 2, characterized in that: The ethanol concentration in the ethanol aqueous solution is 70%, v / v.

4. The use according to claim 1, characterized in that: In step (1), the amount of the ethanol aqueous solution is 10-14 times the amount of the dwarf tea.

5. The use according to claim 4, characterized in that: The amount of the ethanol aqueous solution is 12 times that of the dwarf tea.

6. The use according to claim 1, characterized in that: In step (1): the extraction is carried out as follows: add the dwarf camellia sinensis to the ethanol aqueous solution and heat under reflux for 2-5 times, each time for 1.5-2.5 hours.

7. The use according to claim 1, characterized in that: In step (1): the cold soaking treatment time is 8-16 hours.

8. The use according to claim 1, characterized in that: In step (1): the filtrate obtained by filtering the ethanol water extract is concentrated under reduced pressure to 1 / 30-1 / 10 of the volume of the original ethanol water solution; the temperature of the reduced pressure concentration is 48-80°C.

9. The use according to claim 8, characterized in that: The filtrate obtained by filtering the ethanol water extract is concentrated under reduced pressure to 1 / 25-1 / 15 of the volume of the original ethanol aqueous solution.

10. The use according to claim 9, characterized in that: The filtrate obtained by filtering the ethanol water extract is concentrated under reduced pressure to 1 / 20 of the volume of the original ethanol aqueous solution.

11. The use according to claim 8, characterized in that: The temperature of the reduced pressure concentration is 50-60°C.

12. The use according to claim 1, characterized in that: In step (2), the volume ratio of petroleum ether to the Camellia oleifera extract is 1-2:

1.

13. The use according to claim 1, characterized in that: In step (2): extraction was performed 5 times using petroleum ether.

14. The use according to claim 1, characterized in that: In step (2), the volume ratio of ethyl acetate to the Camellia oleifera extract is 1-2:

1.

15. The use according to claim 1, characterized in that: In step (2): extraction was performed 5 times using ethyl acetate.

16. The use according to claim 1, characterized in that: In step (2): after combining the ethyl acetate extracts, proceed to the next step of concentration.

17. The use according to claim 1, characterized in that: In step (2), the concentration is carried out under reduced pressure at a temperature of 48-80°C.

18. The use according to claim 17, characterized in that The temperature is 50-60℃.

19. The use according to claim 1, characterized in that: In step (2): drying is selected from freeze drying and spray drying.

Images