Application of three-kernel decoction preparation in protection of lung injury

Abstract

The invention belongs to the field of traditional Chinese medicines, and relates to application of a three-kernel decoction preparation in lung injury protection. The protection effect and mechanism of the three-kernel soup on damp-heat rat lung injury are researched from lung and intestine micro-ecology. The research finds that the protection effect of the three-kernel decoction on the lung injury caused by damp-heat is that the abundance of the phylum firmicalis is reduced, the abundance of the phylum bacteroides is improved, and the abundance of lactobacillus, the abundance of Pseudomonas, the abundance of Brucella Romummiana and the abundance of pseudomonas aeruginosa are changed. Therefore, the three-kernel decoction can treat lung injury caused by damp-heat by adjusting intestinal flora, and has development and application prospects.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine and relates to the application of Sanren decoction preparation in protecting lung injury. The invention studies the protective effect and mechanism of Sanren decoction on damp-heat rat lung injury from the perspective of lung and intestinal microecology. Background Art

[0002] In traditional Chinese medicine theory, dampness and heat belong to the dampness and fire of the "six qi". As early as more than 2,000 years ago, the Yellow Emperor's Classic of Internal Medicine regarded dampness and heat as important pathogenic factors. Dampness and heat are both causes and symptoms in traditional Chinese medicine. With the increasingly fierce social competition, people are under too much work pressure, and they consume too much high-fat and high-sugar food. In addition, environmental pollution leads to bacteria, viruses and other pathogens invading the human body. Especially in the long summer and early autumn, dampness and heat are intertwined, and the number of patients with dampness and heat symptoms is gradually increasing. Ye Tianshi, a famous ancient Chinese medicine expert, pointed out that "the lungs are at the highest position, and evil must be injured first." Lung damage caused by dampness and heat evil invading the lungs is a common clinical disease.

[0003] Lung injury is caused by various factors that damage alveolar epithelial cells and pulmonary capillary endothelial cells, resulting in diffuse interstitial lung and alveolar edema and hypoxic respiratory insufficiency. The pathophysiological characteristics are reduced lung compliance, increased vascular permeability and edema. Clinically, it manifests as pulmonary infiltration, hypoxemia and edema. Its development to a severe stage is called ARDS. In traditional Chinese medicine, lung injury manifests as shortness of breath, dyspnea, dizziness, high fever, dry stool, red tongue, slippery pulse, etc. According to the analysis of clinical manifestations, lung injury belongs to the category of "febrile disease" and "violent asthma" in traditional Chinese medicine, and is mostly caused by new exogenous pathogens such as trauma, evil toxins, six evils, pestilence, etc.

[0004] Sanren Decoction is a classic prescription. It comes from Wu Jutong's "Treatise on Warm Diseases: Upper Jiao" in the Qing Dynasty: "Headache, aversion to cold, heaviness and pain, white tongue without thirst, stringy and thin pulse, pale yellow complexion, chest tightness without hunger, body heat in the afternoon, like yin deficiency, the disease is difficult to cure quickly, and it is called damp-heat. If you sweat, you will be confused and deaf, and even blind and unwilling to speak. If you purge, you will have diarrhea, and if you moisten, the disease will be serious and not cured. The same method is used in the long summer, late autumn and winter, and Sanren Decoction is the main treatment." The full recipe of Sanren Decoction is: 5 qian of almonds, 2 qian of cardamom, 6 qian of coix seeds, 6 qian of talc, 2 qian of light bamboo leaves, 5 qian of ginger pinellia, 2 qian of tongcao, and 2 qian of magnolia bark. In the recipe, almonds are bitter and warm to open the upper Jiao, cardamom seeds are fragrant to promote the middle Jiao, and coix seeds are light and penetrating to benefit the lower Jiao. Pinellia, magnolia bark, talc, tongcao, bamboo leaves, etc. are used to make the clear rise and the turbid fall, and the three Jiaos are resolved.

[0005] Modern pharmacological studies have shown that Sanren Decoction has anti-inflammatory and antioxidant, immune regulation, anti-pathogenic microorganism, endocrine regulation, and anti-diabetic effects. However, there have been no reports on the protective mechanism of Sanren Decoction against lung injury from the perspective of intestinal flora. Summary of the invention

[0006] The purpose of the present invention is to study the protective effect and mechanism of Sanren decoction on damp-heat rat lung injury from the perspective of lung and intestinal microecology, and to apply it as a medicine to protect lung and liver damage.

[0007] To achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0008] First, a model was established to study the role of intestinal flora in lung injury caused by damp heat:

[0009] Lung injury caused by damp heat is related to intestinal flora, and damp heat has obvious changes in intestinal flora, intestinal microbial composition, and species composition at the genus level. Changes in the abundance of Firmicutes, Actinobacteria, Verrucomicrobia, and Bacteroidetes are important causes of damp heat lung injury.

[0010] Secondly, the present invention studies the relationship between Sanren Decoction and lung injury caused by damp-heat from the perspective of intestinal flora.

[0011] The prescription of the Sanren Decoction of the present invention is as follows: each component is measured by weight, 165 parts of bitter almond, 66 parts of cardamom, 198 parts of coix seed, 198 parts of talc, 66 parts of loquat leaf, 165 parts of pinellia, 66 parts of tongcao, and 66 parts of magnolia bark.

[0012] The preparation method is as follows: (1) pressing bitter almonds to extract fatty oil, heating, obtaining bitter almond oil for later use, and the medicinal residue for later use.

[0013] (2) Extract volatile oil from cardamom and magnolia bark separately through supercritical extraction, and keep the residue for later use.

[0014] (3) decoct the residues of cardamom, magnolia bark, bitter almond, coix seed, pinellia tuber, talcum powder, lophatherum gracile, and tongcao with water, combine the decoctions, filter, and concentrate, referred to as concentrate A;

[0015] (4) Allow the concentrated solution A to stand, filter and concentrate, add sodium benzoate, cool, add the bitter almond oil described in step (1) and the volatile oil described in step (2), add water, stir well, and obtain Sanren Decoction oral solution.

[0016] (5) Alternatively, the concentrated solution A is concentrated by spray drying to obtain an extract powder, the bitter almond oil described in step (1) and the volatile oil described in step (2) are encapsulated with cyclodextrin, and the mixture is uniformly mixed with the extract powder to obtain a powder or granules are obtained by granulation.

[0017] The Sanren decoction preparation is preferably in the form of granules, oral liquid or powder.

[0018] The innovation and advantage of the present invention are: establishing a model to study the role of intestinal flora in lung injury caused by damp heat, and finding in the study that intestinal flora disorder is also an important pathological factor and main physiological feature of damp heat, and Sanren decoction reduces the abundance of Firmicutes and increases the abundance of Bacteroidetes, causing changes in the abundance of Lactobacillus, Prevotella, Rombutzia and Pseudomonas aeruginosa, thereby having a protective effect on lung injury caused by damp heat. Therefore, the present invention uses Sanren decoction as a drug for treating lung injury caused by damp heat, which has a good development and application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figures 1 to 15 Capillary electrophoresis of samples from the normal control group, damp-heat group, and fecal microbiota transplantation group;

[0020] Fig.16 The Venn diagram of OTU of fecal microbiota samples in the normal control group, damp-heat group and fecal microbiota transplantation group;

[0021] Fig.17 The relative abundance of species at the phylum level in the normal control group, heat-damp group, and fecal microbiota transplantation group;

[0022] Fig.18 The α diversity dilution curves and levels of the normal control group, damp-heat group, and fecal microbiota transplantation group;

[0023] Fig.19 This is the heat map of β diversity index of normal control group, damp-heat group and fecal microbiota transplantation group;

[0024] Fig. 20 PCoA analysis of normal control group, damp-heat group and fecal microbiota transplantation group;

[0025] Fig.21 NMDS analysis of the normal control group, damp-heat group, and fecal microbiota transplantation group;

[0026] Fig. 22 This is a heat map of the differences among the normal control group, damp-heat group, and fecal microbiota transplantation group at the genus level;

[0027] Figures 23 to 37 Capillary electrophoresis of fecal samples from the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0028] Fig.38 This is the Venn diagram of OTU of fecal microbiota samples in the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0029] Fig.39 The relative abundance of species at the phylum level in the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0030] Fig.40 The relative abundance of species at the genus level in the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0031] Fig.41 These are the alpha diversity dilution curves and rank curves for the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0032] Fig.42 This is the heat map of the β diversity index of the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0033] Fig.43 PCoA analysis was performed for the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0034] Fig.44 NMDS analysis was performed for the damp-heat model group, the damp-heat model Sanren decoction administration group, and the damp-heat model Sanren decoction treatment fecal microbiota transplantation group;

[0035] Fig.45 This is a heat map of the differences at the genus level among the damp-heat model group, the damp-heat model Sanren Decoction-administered group, and the damp-heat model Sanren Decoction-treated fecal microbiota transplantation group. DETAILED DESCRIPTION

[0036] In order to better illustrate the present invention, the following embodiments are given:

[0037] Example 1 Preparation of Sanren Decoction Oral Liquid:

[0038] Prescription: Bitter almond 165 g Cardamom 66 g Coix seeds 198 g Talc 198 g

[0039] 66 g of light bamboo leaves, 165 g of pinellia tuber, 66 g of tongcao, 66 g of magnolia bark,

[0040] (1) Press bitter almonds to extract fatty oil, heat to 160°C, and obtain bitter almond oil for later use; the residue is set aside for later use.

[0041] (2) Extract volatile oil from cardamom and magnolia bark separately by supercritical extraction; keep the residues for later use.

[0042] (3) Add water to the residues of cardamom and magnolia bark, bitter almond residue, coix seed, pinellia, talc, lophatherum gracile, and tongcao in a weight ratio of 8:6:6 and decoct three times for 2 hours each time. Combine the decoctions, filter, and concentrate to about 800 mL. Let stand, filter, add 3 g of sodium benzoate, cool, add the above-mentioned bitter almond oil and magnolia bark and cardamom volatile oils, add water to 1000 mL, and stir well to obtain Sanren Decoction oral solution.

[0043] Example 2 Preparation of Sanren Decoction Granules:

[0044] Prescription: 165 g of Armeniacae Semen Amarum, 66 g of Amomi Fructus Rotundus, 198 g of Coicis Semen, 198 g of Talci Pulvis

[0045] 66 g of Lophatheri Herba, 165 g of Pinelliae Rhizoma, 66 g of Tetrapanacis Medulla, 66 g of Magnoliae Officinalis Cortex,

[0046] (1) Press Armeniacae Semen Amarum to obtain fatty oil, heat it to 160 °C to get Armeniacae Semen Amarum oil for standby; reserve its residue for later use.

[0047] (2) Supercritically extract the volatile oils from Amomi Fructus Rotundus and Magnoliae Officinalis Cortex respectively; reserve their residues for later use.

[0048] (3) Decoct the residues of Amomi Fructus Rotundus and Magnoliae Officinalis Cortex, the residue of Armeniacae Semen Amarum together with Coicis Semen, Pinelliae Rhizoma, Talci Pulvis, Lophatheri Herba, and Tetrapanacis Medulla with water according to the weight ratio of 8:6:6 for three times, each time for 2 hours. Combine the decoctions, filter, concentrate, and spray dry to obtain the dry extract powder. After encapsulating the above-mentioned Armeniacae Semen Amarum oil and the volatile oils of Magnoliae Officinalis Cortex and Amomi Fructus Rotundus with cyclodextrin, mix them with the above-mentioned dry extract powder and granulate in one step to obtain the Sanren Decoction granules.

[0049] Example 3 Preparation of the Chinese medicine powder of Sanren Decoction:

[0050] Prescription: 165 g of Armeniacae Semen Amarum, 66 g of Amomi Fructus Rotundus, 198 g of Coicis Semen, 198 g of Talci Pulvis

[0051] 66 g of Lophatheri Herba, 165 g of Pinelliae Rhizoma, 66 g of Tetrapanacis Medulla, 66 g of Magnoliae Officinalis Cortex,

[0052] (1) Press Armeniacae Semen Amarum to obtain fatty oil, heat it to 160 °C to get Armeniacae Semen Amarum oil for standby; reserve its residue for later use.

[0053] (2) Supercritically extract the volatile oils from Amomi Fructus Rotundus and Magnoliae Officinalis Cortex respectively; reserve their residues for later use.

[0054] (3) Decoct the residues of Amomi Fructus Rotundus and Magnoliae Officinalis Cortex, the residue of Armeniacae Semen Amarum together with Coicis Semen, Pinelliae Rhizoma, Talci Pulvis, Lophatheri Herba, and Tetrapanacis Medulla with water according to the weight ratio of 8:6:6 for three times, each time for 2 hours. Combine the decoctions, filter, concentrate, and spray dry to obtain the dry extract powder. After encapsulating the above-mentioned Armeniacae Semen Amarum oil and the volatile oils of Magnoliae Officinalis Cortex and Amomi Fructus Rotundus with cyclodextrin, mix them with the above-mentioned dry extract powder to obtain the Sanren Decoction extract powder.

[0055] Example 4 Role of intestinal flora in lung injury caused by damp-heat

[0056] Experimental subjects:

[0057] 24 6-week-old SPF-grade SD rats, half male and half female, with a body weight of about 180 ± 20 g. They were purchased from the Henan Experimental Animal Center on August 19, 2020. The experimental animal production license number: SCXK(Yu)2017-0001, and the animal quality certificate number: 410975201100048658.

[0058] Experimental instruments:

[0059] Low-temperature high-speed centrifuge (Eppendorf, Germany); high-pressure sterilizer (Gene, USA); refrigerator (Qingdao Haier Co., Ltd.); -80℃ refrigerator (Tokyo Rika Co., Ltd., Japan); GFL-230 oven (Tianjin Laiborui Instrument Equipment Co., Ltd.); artificial climate incubator (Shanghai Shuli Instrument Co., Ltd.);

[0060] Experimental methods:

[0061] Grouping and modeling: 24 SD rats, SPF grade, were randomly divided into three groups according to body weight: normal control group (NC), damp heat group (DH) and fecal microbiota transplantation group (FMT), with 8 rats in each group, half male and half female. The normal control group was fed with ordinary mixed feed in an environment of 23±2℃ and 40%-70% humidity. The damp heat model group was modeled with a composite multi-factor "high-sugar and high-fat feed + high temperature and high humidity environment + Escherichia coli". The rats were fed with high-sugar and high-fat feed for 10 days, and then placed in an artificial climate box at 32±2℃ and relative humidity of 90%-95%, and fed for 6 hours a day for a total of 8 days. Gavage began on the 19th day, and Escherichia coli (ATCC: 25922, concentration: 1×10 9 CFU / mL) 1mL / 100g was given once, and the infection was strengthened once after 24 hours. The animals were removed and placed in a natural environment, and the animals were killed after 2 days. The feces of the rats in the damp-heat model group were collected every day, dissolved in PBS at a ratio of 1:5, vortexed for 3 minutes and then left to stand for 2 minutes. The supernatant was gavaged (1mL / 100g) to the fecal microbiota transplantation group once a day. At the end of the experiment, the rats were anesthetized and intubated to collect alveolar lavage fluid, blood was collected from the abdominal aorta, and tissues such as the lungs, colon, spleen, and tongue were taken for the following analysis and testing.

[0062] Extraction of fecal microbial DNA from rats: After modeling, rats in each group fasted for 12 hours, and rat feces were collected on a sterile operating table. DNA extraction kits were used to extract fecal microbial DNA from rats in each group. The operation steps are as follows:

[0063] (1) Add 700uL Buffer ATL to the sample and vortex for 5min;

[0064] (2) Incubate at 70°C for 10 min, shake three times during incubation, and centrifuge at 12,000 rpm for 5 min. Take the supernatant and add 100 μL Buffer SN. Mix by shaking and place on ice for 5 min. Centrifuge at 12,000 rpm for 3 min.

[0065] (3) Take 200 μL of supernatant, add 100 μL of Bffer AL and 100 μL of isopropanol, and shake to mix;

[0066] (4) Transfer the mixture to a DNA Mini Column in a Collection Tube and centrifuge at 10,000 g for 1 min.

[0067] (5) Discard the filtrate, add 500 μL Buffer DW1, and centrifuge at 10,000 g for 1 min;

[0068] (6) Discard the filtrate, add 500 μL Buffer DW2, centrifuge at 10,000 g for 1 min, and repeat once;

[0069] (7) Discard the filtrate, centrifuge at 12000g for 5 min, add 100 μL Buffer AE preheated to 70°C, let stand for 3 min, and centrifuge at 12000g for 1 min. The rat fecal microbial DNA was stored at 4°C;

[0070] Bacterial 16S rDNA PCR amplification and detection: Using rat fecal microbial DNA as a template, 338F / 806R was used as a universal primer for polymerase chain reaction (PCR), see Table 1 and Table 2. PCR products were detected by Agilent 5400 fully automatic capillary electrophoresis.

[0071] Upstream primer: 338F, 5'-ACTCCTACGGGAGGCAGCAG-3'

[0072] Downstream primer: 806R, 5'-GGACTACVVGGGTATCTAATC-3'

[0073] Table 1 PCR amplification system

[0074]

[0075] Table 2 Thermal cycle parameters

[0076]

[0077] Intestinal flora sequencing analysis: use The library was constructed using the DNA PCR-Free Sample Preparation Kit. After the library was qualified, it was sequenced using the NovaSeq6000, with technical support provided by Novogene Technology Co., Ltd. The sequencing results were analyzed for species composition, α diversity, β diversity, and species differences.

[0078] The results are as follows:

[0079] High-throughput sequencing results:

[0080] The PCR amplification length was 468 bp, and the PCR product was detected by Agilent 5400 fully automatic capillary electrophoresis. The detection results are shown in Figures 1 to 15 , the target band size of the PCR product is correct, and the total amount meets the needs of more than one library construction, and subsequent library construction can be carried out. The raw data obtained by Illumina NovaSeq sequencing was spliced ​​and quality controlled, and then chimeras were filtered to obtain effective data (Effective Tags) that can be used for subsequent analysis. The statistical results of each sample are shown in Table 3.

[0081] Table 3 Sequencing information of each sample

[0082]

[0083] Species composition analysis:

[0084] The Venn diagram of OTU of fecal bacteria samples in each group is shown in Fig.16 The results showed that although the composition of the intestinal flora OUT of the three groups of rats was similar, damp heat would cause changes in the species composition of the intestinal flora, and the species composition of the intestinal flora of rats would also change to a certain extent after fecal microbiota transplantation.

[0085] The relative abundance of species is shown in the following figure: Fig.17 Compared with the NC group, the abundance of Firmicutes and Actinobacteria in the DH group increased significantly, while Verrucomicrobia and Bacteroidetes decreased significantly; the changes in the flora in the FMT group were similar to those in the DH group. This indicates that the formation of damp-heat lung injury may be related to the increase in the abundance of Firmicutes and Actinobacteria and the decrease in the abundance of Verrucomicrobia and Bacteroidetes.

[0086] Alpha diversity analysis:

[0087] The α diversity analysis index is shown in Table 4. Compared with the NC group, the Shannon, Chao1, and ACE indices in the DH group increased, but there was no significant difference; the Shannon, Chao1, and ACE indices in the FMT group increased (P < 0.01), indicating that the species diversity and abundance in the DH and FMT groups increased.

[0088] Table 4 α diversity index

[0089]

[0090]

[0091] Rarefaction Curve and Rank Abundance Curve Fig.18As shown in the figure, the rarefaction curve and the hierarchical aggregation curve tend to be flat, and the Good's coverage is above 0.998 (Table 4). The analysis results show that the sequencing data has reached saturation and can include the vast majority of species of rat intestinal flora.

[0092] Beta diversity analysis:

[0093] The β diversity index heat map results are shown in Fig.19 The numbers in the squares in the figure are the dissimilarity coefficients between the samples. The smaller the dissimilarity coefficients, the smaller the difference in species diversity between the two samples. The results show that there are large differences in species diversity between the DH group and the SR group, and between the DH group and the DFS group.

[0094] PCoA analysis Fig. 20 The closer the sample distance is, the more similar the species composition structure is. It can be seen from the figure that the sample communities in the same group have high similarity and tend to gather together, while the sample communities in different groups are very different and will be far apart. In the PCoA analysis, the contribution value of the PC1 axis to the sample difference is 52.91%, and the contribution value of the PC2 axis to the sample difference is 17.81%. The results show that the species composition of each group of samples is different and has significant differences.

[0095] The results of NMDS analysis are shown in Fig.21 Each point in the figure represents a sample, and the distance between points represents the degree of difference. Samples in the same group are represented by the same color. Stress = 0.107, which is less than 0.2, indicating that NMDS can accurately reflect the degree of difference between samples. The results show that the composition of intestinal flora in each group of samples is significantly different.

[0096] Species difference analysis:

[0097] Species differences analysis at the genus level Fig. 22The top 10 species with the largest abundance were Firmicutes, Bacteroidota, Actinobacteriota, Verrucomicrobiota, Unidentified bacteria, Proteobacteria, Cyanobacteria, DesuLfobacterota, Deferribacteres, and Fusobacteriota. The top three dominant phyla and abundances in the three groups of samples were slightly different. The dominant phyla in the NC group were Firmicutes (46.67%), Bacteroidetes (43.42%), and Verrucomicrobia (4.00%); the dominant phyla in the DH group were Firmicutes (53.33%), Bacteroidetes (34.83%), and Actinobacteria (6.5%); the dominant phyla in the FMT group were Firmicutes (51.08%), Bacteroidetes (37.92%), and Actinobacteria (3.33%). Compared with the NC group, the abundance of Firmicutes and Actinobacteria in the DH group increased significantly, and the abundance of Verrucomicrobia and Bacteroidetes decreased significantly; the changes in the flora in the FMT group were similar to those in the DH group. This indicates that the formation of damp-heat lung injury may be related to the increased abundance of Firmicutes and Actinobacteria and the decreased abundance of Verrucomicrobia and Bacteroidetes.

[0098] In summary, the results of intestinal flora sequencing showed that the number of unique OTUs in the DH group and the FMT group increased compared with the NC group, indicating that damp heat had a significant effect on intestinal flora. Changes in the abundance of Firmicutes, Actinobacteria, Verrucomicrobia, and Bacteroidetes may be an important cause of damp heat lung injury. In addition, the results of β diversity index, PCoA, and NMDS showed that the intestinal microbial composition of rats in each group was significantly different. Species difference analysis showed that the species composition of the NC group and the DH group, and the NC group and the FMT group was different at the genus level.

[0099] Example 5: Study on the protective effect of Sanren decoction on lung injury in rats with damp-heat based on microbiome

[0100] Experimental subjects: Same as Example 4.

[0101] Experimental materials: Same as Example 4.

[0102] Experimental equipment: Same as Example 4.

[0103] Experimental methods:

[0104] 24 SD rats, SPF grade, were randomly divided into three groups according to body weight: damp-heat model group (DH), damp-heat model Sanren decoction administration group (SR) and damp-heat model Sanren decoction treatment fecal microbiota transplantation group (DFS), 8 rats in each group, half male and half female. Damp-heat model group: after the damp-heat model was established, physiological saline (1mL / 100g) was administered once a day, and the animals were killed after 7 days. Damp-heat model Sanren decoction administration group: 12h after the damp-heat model was established, Sanren decoction of the present invention (0.93mL / 100g) was administered once a day, and the animals were killed after 7 days. Damp-heat model Sanren decoction treatment fecal microbiota transplantation group: 24h after the damp-heat model was established, the collected fecal supernatant of the Sanren decoction group rats was administered once a day, and the animals were killed after 7 days.

[0105] Extraction of rat feces microbial DNA: Same as Example 4.

[0106] Bacterial 16S rDNA PCR amplification and detection: Same as Example 4.

[0107] Intestinal flora sequencing analysis: same as Example 4.

[0108] The results are as follows:

[0109] The PCR amplification length was 468 bp, and the PCR product was detected by Agilent 5400 fully automatic capillary electrophoresis. The detection results are shown in Figures 23 to 37 , the target band size of the PCR product is correct, and the total amount meets the needs of more than one library construction, and subsequent library construction can be carried out. The raw data obtained by Illumina NovaSeq sequencing was spliced ​​and quality controlled, and then chimeras were filtered to obtain effective data (Effective Tags) that can be used for subsequent analysis. The statistical results of each sample are shown in Table 5.

[0110] Table 5 Sequencing information of each sample

[0111]

[0112] Species composition analysis:

[0113] The Venn diagram of OTU of fecal bacteria samples in each group is shown in Fig.38 The results showed that the number of unique OTUs in the SR and DFS groups was significantly reduced compared with that in the DH group, indicating that Sanren decoction may exert its therapeutic effect on damp-heat lung injury by changing the intestinal flora structure of rats.

[0114] At the phylum level, the relative abundance of species is shown in the following table: Fig.39The dominant bacterial phyla in the three groups of samples were Firmicutes and Bacteroidetes. Compared with the DH group, the proportion of Firmicutes in the SR group decreased (P<0.05) and the proportion of Bacteroidetes increased (P<0.05). This shows that Sanren Decoction may reduce the abundance of Firmicutes and increase the abundance of Bacteroidetes, thereby alleviating the inflammatory response and thus playing a protective role against lung injury caused by damp-heat.

[0115] At the genus level, the relative abundance of species is shown in the following table. Fig.40 , respectively Lactobacillus, Prevotella, Romboutsia, FaecalibacuLum, Ruminococcus, Turicibacter, Alloprevotella, Clostridium sensu stricto 1, Prevotellaceae NK3B31 group, and Prevotellaceae Ga6A1 group. The dominant bacterial genera in the DH group were Lactobacillus (9.07%), Rombutzia (9.06%), and Pseudomonas aeruginosa (6.14%); the dominant bacterial genera in the SR group were Lactobacillus (29.72%) and Prevotella (20.34%); the dominant bacterial genera in the DFS group were Lactobacillus (36.06%) and Prevotella (5.67%); the relative abundance of Lactobacillus and Prevotella in the SR group was higher than that in the DH group, while the relative abundance of Rombutzia and Pseudomonas aeruginosa was lower than that in the DH group. This indicates that Sanren Decoction may reduce the inflammatory response by increasing the relative abundance of Lactobacillus and Prevotella, reducing the relative abundance of Rombutzia and Pseudomonas aeruginosa, and thus play a protective role against lung injury caused by damp-heat.

[0116] Alpha diversity analysis:

[0117] The α diversity analysis index is shown in Table 6. Compared with the DH group, the Shannon and Simpson indices of the SR and DFS groups were smaller (P<0.01), indicating that the species diversity of the SR and DFS groups was reduced; compared with the DH group, the Chao1 and ACE indices of the SR group were reduced (P<0.05), indicating that the abundance of the SR group was reduced.

[0118] Table 6 α diversity index

[0119]

[0120] Rarefaction Curve and Rank Abundance Curve Fig.41 As shown, the three groups of rarefaction curves and hierarchical aggregation curves tend to be flat, and the Good's coverage is above 0.998 (Table 6). The analysis results show that the sequencing data has reached saturation and can include the vast majority of species of rat intestinal flora.

[0121] Beta diversity analysis:

[0122] The β diversity index heat map results are shown in Fig.42 The results showed that there were significant differences in species diversity between the DH group and the SR group, and between the DH group and the DFS group.

[0123] PCoA analysis Fig.43 In the PCoA analysis, the contribution of PC1 axis to sample differences was 57.27%, and the contribution of PC2 axis to sample differences was 22.11%. The results showed that the species composition of each group of samples was different and had significant differences (P<0.05).

[0124] The results of NMDS analysis are shown in Fig.44 Stress = 0.117, which is less than 0.2, indicating that NMDS can accurately reflect the degree of difference between samples. The results show that the composition of intestinal flora in each group of samples is significantly different.

[0125] Species difference analysis:

[0126] Species differences analysis at the genus level Fig.45 There were 7 genera with significant differences in abundance between the DH group and the SR group (P<0.05), and 9 genera with extremely significant differences (P<0.01); there were 6 genera with significant differences in abundance between the DH group and the DFS group (P<0.05), and 8 genera with extremely significant differences (P<0.01); there were 3 genera with significant differences in abundance between the SR group and the DFS group (P<0.05).

[0127] In summary, the results of intestinal flora sequencing showed that the number of unique OTUs in the SR group and the DFS group was significantly reduced compared with the DH group, indicating that Sanren Decoction had a significant change in the intestinal flora. At the phylum level, Sanren Decoction could significantly reduce the abundance of Firmicutes and increase the abundance of Bacteroidetes. At the genus level, the changes in the abundance of Lactobacillus, Prevotella, Rombutzia, and Pseudomonas aeruginosa may be an important reason for the therapeutic effect of Sanren Decoction. In addition, the results showed that there were significant differences in the intestinal microbial composition of rats in each group. Species difference analysis showed that there were 15 species with significant differences in species abundance at the genus level between the DH group and the SR group.

Claims

1. The pharmaceutical application of Sanren decoction preparation is characterized in that: The drug is used to prepare a drug for preventing or treating lung injury; the prescription of the Sanren Decoction is as follows: each component is calculated by weight: 165 parts of bitter almond, 66 parts of cardamom, 198 parts of coix seed, 198 parts of talc, 66 parts of lophatherum gracile, 165 parts of pinellia tuber, 66 parts of tongcao, and 66 parts of magnolia bark; The preparation method is as follows: (1) pressing bitter almonds to extract fatty oil, heating, and obtaining bitter almond oil for later use; and the medicinal residue for later use; (2) Extract volatile oil from cardamom and magnolia bark by supercritical extraction, and keep the residue for later use; (3) Decoction the residues of cardamom, magnolia bark, bitter almond, coix seeds, pinellia tuber, talcum powder, lophatherum gracile, and tongcao with water, combine the decoctions, filter, and concentrate, referred to as concentrated solution A; (4) allowing the concentrated solution A to stand, filtering and concentrating, adding sodium benzoate, cooling, adding the bitter almond oil described in step (1) and the volatile oil described in step (2), adding water, stirring, and obtaining Sanren Decoction oral solution; (5) Alternatively, the concentrated solution A is concentrated by spray drying to obtain an extract powder, the bitter almond oil described in step (1) and the volatile oil described in step (2) are encapsulated with cyclodextrin, and the mixture is mixed evenly with the extract powder to obtain a powder or the Sanren Decoction granules are obtained by granulation.

2. The pharmaceutical application of the Sanren decoction preparation according to claim 1, characterized in that: The invention is used for preparing medicine for preventing or treating lung damage and respiratory diseases caused by damp heat.

3. The pharmaceutical application of the Sanren decoction preparation according to claim 1 or 2, characterized in that: The Sanren decoction preparation is selected from granules, oral liquid or powder.

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