Traditional Chinese medicine fermentation liquor for treating pediatric acute lung injury as well as preparation method and application of traditional Chinese medicine fermentation liquor

Abstract

The invention provides traditional Chinese medicine fermentation liquor for treating acute lung injury in children and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine. The traditional Chinese medicine aqueous extract is combined with the probiotics, so that the probiotics obtain nutrient substances by utilizing components such as polysaccharides, fibers and proteins in the traditional Chinese medicines, and glycosidase and the like generated by the probiotics can convert macromolecular substances in the traditional Chinese medicines into micromolecular substances which are easier to absorb, so that the toxic and side effects of the traditional Chinese medicines are reduced, and the taste of the traditional Chinese medicines is improved; therefore, the medicine activity is improved. Besides, microorganisms can generate various secondary metabolites in the growth process of the traditional Chinese medicine fermentation liquor, the secondary metabolites comprise a large number of active enzymes, amino acids, organic acids, vitamins, phenols and flavonoid components, and after the secondary metabolites are rapidly absorbed by organisms, the immune function can be adjusted, the disease resistance can be enhanced, and the anti-inflammatory and anti-virus activities of the traditional Chinese medicine fermentation liquor can be greatly improved; the use effect of the traditional Chinese medicine fermentation liquor is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a traditional Chinese medicine fermentation liquid for treating acute lung injury in children, and a preparation method and application thereof. Background Art

[0002] Acute lung injury (ALI) is a clinical syndrome of lung inflammation and increased alveolar-capillary permeability caused by various direct and indirect factors. The pathological manifestation of ALI is diffuse alveolar-capillary membrane damage, leading to acute hypoxic respiratory insufficiency. The pathological process of ALI can develop continuously, manifesting as acute respiratory distress syndrome (ARDS) with more obvious symptoms, eventually causing systemic organ dysfunction or even failure. Pneumonia is the main factor causing acute lung injury in children.

[0003] Xuanfei Huazhuo prescription is a classic prescription in Gansu Province. It has the effects of clearing away the lungs, expelling evil spirits, clearing away heat and removing turbidity. It can be used to treat pneumonia caused by viral infection. Traditional Chinese medicine decoctions have the disadvantages of large dosage, bitter taste, and low utilization of active ingredients in the treatment process. Microbial fermentation of Chinese medicine is based on Chinese medicine processing and modern microbiology. It is a new technology formed by combining traditional processing methods with modern biotechnology such as fermentation engineering. After fermentation, Chinese herbal medicines can fully release the effective ingredients of Chinese medicine, reduce the toxic and side effects of Chinese medicine, improve the taste of Chinese medicine, and thus improve the activity of the medicine. The fermentation broth contains a large amount of active enzymes. After rapid absorption by the body, it can regulate immune function and enhance disease resistance, so that the efficacy of Chinese medicine can be fully exerted. In addition, some components in Chinese medicine can be transformed into new active small molecules after being decomposed by the flora. These active small molecules may further enhance the efficacy of the medicine and show new pharmacological effects. At present, there is no example of combining Xuanfei Huazhuo prescription with microbial fermentation. How to further improve the efficacy of Xuanfei Huazhuo prescription still needs further research. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a Chinese medicine fermentation liquid for treating acute lung injury in children, and a preparation method and application thereof, and to combine the Chinese medicine water extract with microorganisms to prepare a Chinese medicine fermentation liquid with better use effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a traditional Chinese medicine fermentation liquid for treating acute lung injury in children, wherein the traditional Chinese medicine fermentation liquid is obtained by mixed fermentation of a traditional Chinese medicine water extract and Lactobacillus plantarum;

[0007] The Chinese medicine extract is a water extract obtained by decocting the Xuanfei Huazhuo recipe;

[0008] The prescription for purifying the lungs and clearing turbidity comprises the following components in parts by weight: 3-9 parts of honey ephedra, 6-12 parts of patchouli, 6-12 parts of stir-fried atractylodes, 6-12 parts of notopterygium, 3-12 parts of pinellia, 3-9 parts of tangerine peel, 6-12 parts of peucedanum, 6-12 parts of scutellaria, 5-15 parts of forsythia, and 3-9 parts of wine rhubarb.

[0009] Preferably, the Lactobacillus plantarum is one or more of Lactobacillus plantarum LZU-J-Q25, Lactobacillus plantarum LZU-J-Q21, Lactobacillus plantarum LZU-J-QA85, Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-S-ZCJ;

[0010] Lactobacillus plantarum LZU-J-Q25, deposited with GDMCC No: 63278;

[0011] Lactobacillus plantarum LZU-J-Q21, deposit number is GDMCC No: 63277;

[0012] Lactobacillus plantarum LZU-J-QA85, deposit number is GDMCC No: 61192;

[0013] Lactobacillus plantarum LZU-J-TSL6, deposited with GDMCC No: 61242;

[0014] Lactobacillus plantarum LZU-S-ZCJ, the deposit number is GDMCC No: 61402.

[0015] The present invention also provides a method for preparing the traditional Chinese medicine fermentation liquid, comprising the following steps: inoculating probiotics into the traditional Chinese medicine water extract, fermenting at 29-35° C. for 20-28 hours, and then inactivating the probiotics to obtain the traditional Chinese medicine fermentation liquid.

[0016] Preferably, the concentration of live probiotics is 10 6 ~10 10 CFU / mL, and the inoculation volume is 1-5% of the volume of the Chinese medicine water extract.

[0017] Preferably, the inactivation temperature is 85-100°C.

[0018] Preferably, the inactivation time is 15 to 45 minutes.

[0019] The invention provides application of the traditional Chinese medicine fermentation liquid in preparing a medicine for treating acute lung injury.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention improves the use effect of Chinese medicine by combining Chinese medicine water extract with probiotics. During the fermentation process of Chinese medicine fermentation liquid, probiotics use polysaccharides, fibers, proteins and other components in Chinese medicine to obtain nutrients, and the glycosidases produced by probiotics can convert macromolecules in Chinese medicine into small molecules that are easier to absorb, reduce the toxic and side effects of Chinese medicine, improve the taste of Chinese medicine, and thus improve the activity of the medicine. In addition, microorganisms will produce various secondary metabolites during the growth of Chinese medicine fermentation liquid, including a large number of active enzymes, amino acids, organic acids, vitamins, phenols, and flavonoids. After being rapidly absorbed by the body, they can regulate immune function, enhance disease resistance, greatly improve the anti-inflammatory and antiviral activities of Chinese medicine fermentation liquid, and improve the use effect of Chinese medicine fermentation liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the ΔOD600 value of the fermentation broth of different strains;

[0023] Figure 2 is the ΔOD600 value of the fermentation broth of Lactobacillus plantarum LZU-S-ZCJ, Lactobacillus plantarum LZU-J-Q21 and the composite strain;

[0024] Figure 3 is the number of viable bacteria in the fermentation broth under different fermentation temperatures;

[0025] Figure 4 is the number of viable bacteria in the fermentation broth under different inoculation amounts;

[0026] Figure 5 is the number of viable bacteria in the fermentation broth under different fermentation times;

[0027] Figure 6 The pH and total acid content comparison between the Xuanfei Huazhuo Recipe water extract group and the Chinese medicine fermentation liquid group, where S is the Xuanfei Huazhuo Recipe water extract group, and FS is the Chinese medicine fermentation liquid group;

[0028] Figure 7 The figure is a graph showing the changes in the total flavonoid content of the Xuanfei Huazhuo Recipe water extract and the Chinese medicine fermentation broth group, wherein A is a standard curve for total flavonoids, and B is a graph showing the comparison of the total flavonoid content of the Xuanfei Huazhuo Recipe water extract and the Chinese medicine fermentation broth group, wherein S is the Xuanfei Huazhuo Recipe water extract group, and FS is the Chinese medicine fermentation broth group;

[0029] Figure 8 The figure is a graph showing the changes in the total polysaccharide content of the Xuanfei Huazhuo Recipe water extract and the Chinese medicine fermentation broth group, wherein A is a standard curve for total polysaccharide, and B is a graph showing the comparison of the total polysaccharide content of the Xuanfei Huazhuo Recipe water extract and the Chinese medicine fermentation broth group, wherein S is the Xuanfei Huazhuo Recipe water extract group, and FS is the Chinese medicine fermentation broth group;

[0030] Fig. 9The results of the measured BALF protein concentration and inflammatory factors are shown in Figure 1, wherein A is the measured interleukin-6 content, B is the measured interleukin-1β content, C is the measured tumor necrosis factor-α content, and D is the measured BALF protein concentration;

[0031] Fig.10 The effects of different experimental groups on the antioxidant in lung tissue, where A is the content of glutathione in lung tissue, B is the content of malondialdehyde in lung tissue, C is the content of macrophage inflammatory protein 2 in lung tissue, D is the content of myeloperoxidase in lung tissue, and E is the content of pro-apoptotic protein BAX in lung tissue;

[0032] Fig.11 The effect diagram of different experimental groups on the pathological changes of lung tissue;

[0033] Fig.12 This is a scoring diagram of the pathological changes in lung tissues in different experimental groups;

[0034] Fig.13 This is a graph showing the effect of different experimental groups on the wet-to-dry ratio of lung tissue.

[0035] Biological Deposit Description

[0036] Lactiplantibacillus plantarum LZU-J-Q25, with the deposit number of GDMCCNo: 63278, was deposited in Guangdong Microbiological Culture Collection Center on March 20, 2023, and the deposit address is 5th Floor, No. 59, No. 100, Xianlie Middle Road, Guangzhou;

[0037] Lactiplantibacillus plantarum LZU-J-Q21, with the deposit number of GDMCCNo: 63277, was deposited in Guangdong Microbiological Culture Collection Center on March 20, 2023, and the deposit address is 5th Floor, No. 59, No. 100, Xianlie Middle Road, Guangzhou;

[0038] Lactobacillus plantarum LZU-J-QA85, with the deposit number of GDMCC No: 61192, was deposited in Guangdong Microbiological Culture Collection Center on September 16, 2020, and the deposit address is 5th Floor, No. 59, No. 100, Xianlie Middle Road, Guangzhou;

[0039] Lactobacillus plantarum LZU-J-TSL6, with the deposit number of GDMCC No: 61242, was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2020, and the deposit address is 5th Floor, No. 59, No. 100, Xianlie Middle Road, Guangzhou;

[0040] Lactobacillus plantarum LZU-S-ZCJ, with the deposit number GDMCCNo: 61402, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 31, 2020. The deposit address is 5th Floor, No. 59, Compound 100, Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0041] The present invention provides a traditional Chinese medicine fermentation liquid for treating acute lung injury in children, wherein the traditional Chinese medicine fermentation liquid is obtained by mixed fermentation of a traditional Chinese medicine water extract and Lactobacillus plantarum;

[0042] The Chinese medicine extract is a water extract obtained by decocting the Xuanfei Huazhuo recipe;

[0043] The Xuanfei Huazhuo recipe comprises the following components in parts by weight:

[0044] 3 to 9 parts of honey ephedra, preferably 4 to 8 parts, more preferably 6 parts;

[0045] Patchouli 6-12 parts, preferably 8-11 parts, more preferably 9 parts;

[0046] 6 to 12 parts of bran-fried Atractylodes lancea, preferably 8 to 11 parts, more preferably 9 parts;

[0047] 6 to 12 parts of Notopterygium wilfordii, preferably 8 to 11 parts, more preferably 9 parts;

[0048] 3 to 12 parts of French Pinellia, preferably 4 to 10 parts, more preferably 6 parts;

[0049] 3 to 9 parts of dried tangerine peel, preferably 4 to 8 parts, more preferably 6 parts;

[0050] Peucedanum 6-12 parts, preferably 8-11 parts, more preferably 9 parts;

[0051] 6 to 12 parts of Scutellaria baicalensis, preferably 8 to 11 parts, more preferably 9 parts;

[0052] 5-15 parts of Forsythia suspensa, preferably 7-12 parts, more preferably 10 parts;

[0053] 3 to 9 parts of wine rhubarb, preferably 4 to 8 parts, more preferably 6 parts.

[0054] In the present invention, the Lactobacillus plantarum is preferably one or more of Lactobacillus plantarum LZU-J-Q25, Lactobacillus plantarum LZU-J-Q21, Lactobacillus plantarum LZU-J-QA85, Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-S-ZCJ, further preferably LZU-S-ZCJ, LZU-J-Q21, LZU-S-ZCJ and LZU-J-Q21 (v:v, 1:1), LZU-S-ZCJ and LZU-J-Q21 (v:v, 3:1), LZU-S-ZCJ and LZU-J-Q21 (v:v, 1:3), and further preferably Lactobacillus plantarum LZU-J-Q21.

[0055] The present invention also provides a method for preparing the traditional Chinese medicine fermentation liquid, comprising the following steps: inoculating probiotics into the traditional Chinese medicine water extract, fermenting at 29-35° C. for 20-28 hours, and then inactivating the probiotics to obtain the traditional Chinese medicine fermentation liquid.

[0056] In the present invention, the fermentation temperature is preferably 29 to 35°C, more preferably 30 to 34°C, more preferably 33°C, and the fermentation time is preferably 20 to 28 hours, more preferably 22 to 26 hours, and even more preferably 24 hours. The concentration of live probiotics is preferably 10 6 ~10 10 CFU / mL, more preferably 10 7 ~10 9 CFU / mL, more preferably 10 8 CFU / mL; the inoculation volume is preferably 1-5% of the volume of the Chinese medicine water extract, more preferably 1.5-3%, and more preferably 2%; in the present invention, the inactivation temperature is preferably 85-100°C, more preferably 87-95°C, and more preferably 90°C. The inactivation time is preferably 15-45min, more preferably 20-40min, and more preferably 30min.

[0057] The invention provides application of the traditional Chinese medicine fermentation liquid in preparing a medicine for treating acute lung injury.

[0058] The preparation method of the Xuanfei Huazhuo prescription water extract is preferably:

[0059] Take 6g of honey ephedra, 9g of bran-fried atractylodes, 9g of Notopterygium wilfordii, 6g of French pinellia, 6g of dried orange peel, 9g of peucedanum, 9g of scutellaria, 10g of forsythia, and 3g of wine rhubarb, add 6.5 times the weight of water, soak for 0.5h, decoct for 0.5h, and add 9g of patchouli 10 minutes before turning off the heat. After boiling, sterilize the Chinese medicine liquid, collect the filtrate through a filter, sterilize and fill it, and obtain the water extract of Xuanfei Huazhuo prescription.

[0060] ELISA kit source description:

[0061] Interleukin 6 kit (Cat. No. F2163-A), interleukin 1β kit (Cat. No. F2040-A), tumor necrosis factor α kit (Cat. No. F2132-A), glutathione kit (Cat. No. F2799-A), malondialdehyde kit (Cat. No. F9264-A), macrophage inflammatory protein 2 kit (Cat. No. F2152-A), myeloperoxidase kit (Cat. No. F2338-A), pro-apoptotic protein BAX kit (Cat. No. F9178-A), all of the above kits are from Fancovi. BCA protein concentration kit (Cat. No. A045-4-2) is from Nanjing Jiancheng Bioengineering Institute.

[0062] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] Example 1 Confirmation of fermentation strains and fermentation conditions

[0064] (1.1) Screening of fermentation strains

[0065] Take 9 10mL test tubes, add 9.8mL of Xuanfei Huazhuo Decoction water extract, and 8 CFU / mL, 2% water extract volume inoculation volume were inoculated into plant lactobacillus LZU-J-Q25, LZU-J-QA85, LZU-J-Q21, LZU-J-TSL6 and LZU-S-ZCJ, placed in a constant temperature incubator, cultured at 37°C, sampled every 12 hours, zeroed with Xuanfei Huazhuo Fang water extract, and measured the ΔOD600 value of the fermentation liquid. Results are shown in Figure 1 .

[0066] The results showed that when the fermentation time reached 36h, the fermentation effects of Lactobacillus plantarum LZU-S-ZCJ and Lactobacillus plantarum LZU-J-Q21 were the best, and these two strains were selected to explore the ratio of fermentation strains.

[0067] Take 6 10ml test tubes, add 9.8ml of Xuanfei Huazhuo Decoction water extract, and 8 CFU / mL, 2% volume inoculation volume was inoculated into LZU-S-ZCJ, LZU-J-Q21, LZU-S-ZCJ and LZU-J-Q21 (v:v, 1:1), LZU-S-ZCJ and LZU-J-Q21 (v:v, 3:1), LZU-S-ZCJ and LZU-J-Q21 (v:v, 1:3), placed in a constant temperature incubator, cultured at 37°C, sampled every 12 hours, zeroed with Xuanfei Huazhuo Fang water extract, and measured the ΔOD600 value of the fermentation liquid. For details, see Figure 2 .

[0068] The results showed that when the fermentation time reached 36h, the fermentation effect of Lactobacillus plantarum LZU-J-Q21 was the best, and LZU-J-Q21 was selected for optimization of subsequent fermentation process.

[0069] (1.2) Determination of fermentation temperature

[0070] Take 30mL of Xuanfei Huazhuo Decoction water extract and add 10 8 CFU / mL, 2% water extract volume inoculation volume inoculated with Lactobacillus plantarum LZU-J-Q21, fermented at 29℃, 31℃, 33℃, 35℃, 37℃ for 24h, and plated to calculate the number of viable bacteria in the fermentation liquid to determine the optimal fermentation temperature. Figure 3 .

[0071] The results showed that when the fermentation temperature was higher than 33°C, the number of live bacteria in the fermentation liquid began to decrease, and the fermentation temperature was determined to be 33°C.

[0072] (1.3) Determination of the inoculum quantity

[0073] Take 30mL of Xuanfei Huazhuo Decoction water extract and add 10 8 CFU / mL, 1%, 2%, 3%, 4%, 5% of the volume of the water extract was inoculated into Lactobacillus plantarum LZU-J-Q21, fermented at 37°C for 24h, and the number of live bacteria in the fermentation liquid was calculated on the plate to determine the optimal inoculation amount. Figure 4 .

[0074] The results showed that when the inoculum amount was higher than 2%, the number of live bacteria in the fermentation broth began to decrease, so the inoculum amount was determined to be 2%.

[0075] (1.4) Determination of fermentation time

[0076] Take 30mL of Xuanfei Huazhuo Decoction water extract and add 10 8 CFU / mL, 2% volume inoculation volume was inoculated into Lactobacillus plantarum LZU-J-Q21, and fermented at 37°C for 12h, 18h, 24h, 30h, and 36h respectively. The number of viable bacteria in the fermentation liquid was calculated by plating the plate to determine the optimal fermentation time. Figure 5 .

[0077] The results showed that when the fermentation time reached 24 hours, the number of live bacteria in the fermentation liquid no longer increased, and the fermentation time was determined to be 24 hours.

[0078] In summary, the fermentation conditions were determined as follows: fermentation time 24h, fermentation temperature 33°C, inoculation amount 2%, and inactivation at 90°C for 30min after fermentation.

[0079] Example 2 Differences in the physicochemical properties of the Xuanfei Huazhuo Recipe water extract and the Chinese medicine fermentation broth

[0080] (2.1) Changes in pH and total acidity of the water extract of Xuanfei Huazhuo recipe and the fermented Chinese medicine broth

[0081] During its growth, Lactobacillus plantarum will produce certain organic acids, such as lactic acid, which will reduce the pH of the fermentation liquid and increase the total acid content of the fermentation, which are two important indicators for evaluating the degree of fermentation. Therefore, this experiment uses a pH meter and measures data at different fermentation times according to the national standard for the determination of total acid in food (GB 12456-2021). For details of the results, see Figure 6 .

[0082] The results showed that compared with the Xuanfei Huazhuo recipe water extract group, the pH of the Chinese medicine fermentation broth group was significantly decreased and the total acid content was significantly increased, indicating that Lactobacillus plantarum LZU-J-Q21 can grow in the Xuanfei Huazhuo recipe water extract and produce metabolites.

[0083] (2.2) Changes in total polysaccharides and total flavonoids in the water extract of Xuanfei Huazhuo recipe and the fermented liquid of traditional Chinese medicine

[0084] Polysaccharides and flavonoids are important pharmacological substances in Xuanfei Huazhuo Fang, but some macromolecular substances have low oral availability. Microbial fermentation can convert macromolecular substances into small molecular substances, improve bioavailability and thus improve effectiveness. The phenol-sulfuric acid method was used to determine the content of total polysaccharides in the water extract of Xuanfei Huazhuo Fang and the fermentation broth of traditional Chinese medicine, and the AlCl3 method was used to determine the difference in the content of total flavonoids and total polysaccharides in the water extract of Xuanfei Huazhuo Fang and the fermentation broth of traditional Chinese medicine. The results are detailed in Figure 7 and Figure 8 .

[0085] The results showed that compared with the Xuanfei Huazhuo Decoction water extract group, the total flavonoids in the Chinese medicine fermentation liquid group were reduced ( Figure 7 Figure B in the figure shows that the flavonoid components in the Xuanfei Huazhuo formula were decomposed into other components by Lactobacillus plantarum. The total polysaccharide content in the Xuanfei Huazhuo formula water extract group and the traditional Chinese medicine fermentation liquid group did not change significantly ( Figure 8 Figure B), indicating that LZU-J-Q21 may produce extracellular polysaccharides during its growth.

[0086] Example 3 Study on the effect of Chinese herbal fermentation liquid on improving acute lung injury in children

[0087] (3.1) This study was conducted in accordance with the Gansu Provincial Laboratory Animal Management Standards and the Chinese Laboratory Animal Management Standards. All experimental procedures and techniques were reviewed and approved by the Animal Ethics Committee of Lanzhou University. Eighty Balb / c mice (4 weeks old, weighing 13 g to 15 g), half male and half female, were obtained from Lanzhou Veterinary Research Institute (Lanzhou, China) and housed in an animal facility that meets specific pathogen-free (SPF) requirements (room temperature 20-22 °C, humidity 45-55%, 12 h light / dark cycle) with unrestricted access to standard laboratory diet and water. After one week of adaptive feeding, the mice were randomly divided into 8 groups, including the negative control group (called "Group C"), the model group (called "Group M"), the positive control dexamethasone group (called "Group D"), the Xuanfei Huazhuo recipe water extract group (called "Group S"), the low, medium and high doses of Chinese medicine fermentation liquid groups (abbreviated as "FL", "FM" and "FH" groups, respectively), and the Lactobacillus plantarum LZU-J-Q21 group (called "Q21 group"), each group consisting of 10 mice, the medium dose group of fermentation Xuanfei Huazhuo was the clinical dose, the low dose group was 0.5 times the clinical dose, and the high dose group was 2 times the clinical dose.

[0088] (3.2) Acute lung injury model

[0089] Except for group C, mice in other groups were nasally instilled with LPS solution at a concentration of 12 mg / mL, with 10 μL dripped into the left and right nostrils of each mouse. After 24 hours of modeling, mice in each group were gavaged for 7 days. Mice in groups C and M were gavaged with 0.4 mL of normal saline every day, mice in group D were gavaged with 0.4 mL of dexamethasone acetate solution (concentration of 0.23 mg / kg) every day, mice in group S were gavaged with 0.4 mL of Xuanfei Huazhuo Fang water extract (concentration of 4.4 g / kg) every day, mice in group FL were gavaged with 0.4 mL of low-dose Chinese medicine fermentation liquid (concentration of 2.2 g / kg) every day, mice in group FM were gavaged with 0.4 mL of medium-dose Chinese medicine fermentation liquid (concentration of 4.4 g / kg) every day, mice in group FH were gavaged with 0.4 mL of low-dose Chinese medicine fermentation liquid (concentration of 8.8 g / kg) every day, and mice in group Q21 were gavaged with 0.4 mL of 1.46×10 10 cfu / mL (the number of viable bacteria is the same as that of medium-dose Chinese medicine fermentation liquid). The mice in each group were killed 7 days after intragastric administration.

[0090] (3.3) Biochemical index detection

[0091] (3.3.1) BALF protein concentration and inflammatory factor determination

[0092] The right lung of the mouse was ligated, and the left lung was lavaged with 0.5 mL of pre-cooled PBS. The bronchoalveolar lavage fluid (BALF) of the mouse was collected, and the content of IL-6 (interleukin 6), IL-1β (interleukin 1β), and TNF-α (tumor necrosis factor α) was determined using Elisa kits, and the protein concentration was determined using BCA kits. The results are shown in Table 9.

[0093] The results are shown in the figure. Compared with the model group, the IL-6 ( Fig. 9 A in), IL-1β ( Fig. 9 B), TNF-α ( Fig. 9 C), protein concentration ( Fig. 9 The D) in the above two groups showed a significant decrease. Compared with the Xuanfei Huazhuo Recipe water extract group, the IL-6, IL-1β, TNF-α, and protein concentrations of each Chinese medicine fermentation liquid group decreased more significantly. However, there was no significant difference between the Q21 group and the model group. This indicates that the Xuanfei Huazhuo Recipe water extract can inhibit the production of inflammatory factors after fermentation with Lactobacillus plantarum, thereby improving the anti-inflammatory effect.

[0094] (3.3.2) Determination of antioxidant indexes in lung tissue

[0095] The lower and posterior lobes of the right lung of mice were homogenized and ground, and the supernatant was collected after centrifugation. The contents of GSH (glutathione), MDA (malondialdehyde), MIP-2 (macrophage inflammatory protein 2), MPO (myeloperoxidase) and pro-apoptotic protein BAX were determined using Elisa kits. Fig.10 .

[0096] The results are as follows Fig.10 As shown in the figure, compared with the model group, the oxidative stress index GSH ( Fig.10 A in), MDA( Fig.10 B in), MPO( Fig.10 At the same time, compared with the model group, the pro-inflammatory factor MIP-2 ( Fig.10 C) and the pro-apoptotic protein BAX ( Fig.10 The content of D in the Q21 group also decreased, and the downward trend was more obvious than that in the water extract group of the Xuanfei Huazhuo recipe. In the Q21 group, there was no significant difference in various indicators compared with the model group. This shows that the Xuanfei Huazhuo recipe can inhibit oxidative damage to the lungs after fermentation with plant lactobacillus, and at the same time inhibit the production of pro-inflammatory factors and pro-apoptotic proteins.

[0097] (3.4) Lung tissue pathology sections

[0098] The middle lobe of the right lung of the mouse was removed and rinsed with pre-cooled 4°C PBS buffer. The lung tissue was fixed in 10% neutral formalin solution and placed at room temperature for 24 hours. It was then dehydrated with ethanol, transparentized with xylene, and embedded in conventional paraffin wax, cut into 4 μm slices, and processed for anti-dewaxing, scooping, baking, etc. It was stained with hematoxylin and eosin, and then dehydrated, transparentized, and sealed. The pathological changes of lung tissue in different experimental groups were observed under an ordinary optical microscope and the lung tissue injury score was calculated. The results are detailed in Fig.11 and Fig.12 .

[0099] The results are as follows Fig.11 As shown in the figure, under 200x eyepiece observation, the alveolar structure of mice in the normal group was intact, and no large number of inflammatory cells were found in the pulmonary interstitium and alveoli. The alveolar structure of mice in the model group was significantly damaged, and a large number of red blood cells and inflammatory cells gathered in the alveolar cavity. There was no obvious damage to the alveolar tissue in the dexamethasone group and the low and medium dose groups of Chinese medicine fermentation liquid, and a small number of inflammatory cells infiltrated into the alveoli. The alveolar structure of mice in the high dose group of Chinese medicine fermentation liquid was intact, a small number of inflammatory cells infiltrated into the alveoli, and the alveolar wall thickened. The alveolar wall of mice in the Xuanfei Huazhuo Decoction water extract group showed more obvious thickening, and inflammatory cells infiltrated into the alveoli. The alveoli of mice in the Lactobacillus plantarum Q21 group were significantly damaged, with a small number of blood cells and inflammatory cells infiltrating, and the alveolar wall was significantly thickened.

[0100] (3.5) Lung tissue wet-to-dry ratio

[0101] The right upper lobe of the mouse lung was weighed for wet weight, dried in a constant temperature oven at 40°C to constant weight, and the dry weight was weighed to calculate the wet-to-dry ratio of the lung tissue.

[0102] The results are as follows Fig.13 As shown, compared with the model group, the lung wet-to-dry ratio of the dexamethasone group and the low and medium dose groups of the Chinese medicine fermentation liquid showed a very significant decrease, the lung wet-to-dry ratio of the Xuanfei Huazhuo formula water extract group and the high dose group of the Chinese medicine fermentation liquid showed a significant decrease, and the lung wet-to-dry ratio of the Q21 group did not show a significant difference from the model group. The larger the lung wet-to-dry ratio, the more severe the pulmonary edema of the mouse. The results show that the Chinese medicine fermentation liquid can effectively inhibit the pulmonary edema caused by the inflammatory response in mice.

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicinal fermentation liquid for treating acute lung injury in children, characterized in that: The Chinese medicine fermentation liquid is obtained by mixed fermentation of the Chinese medicine water extract and Lactobacillus plantarum; The Chinese medicine extract is a water extract obtained by decocting the Xuanfei Huazhuo recipe; The prescription for purifying the lungs and clearing turbidity comprises the following components in parts by weight: 3-9 parts of honey ephedra, 6-12 parts of patchouli, 6-12 parts of stir-fried atractylodes, 6-12 parts of notopterygium, 3-12 parts of pinellia, 3-9 parts of tangerine peel, 6-12 parts of peucedanum, 6-12 parts of scutellaria, 5-15 parts of forsythia, and 3-9 parts of wine rhubarb.

2. The Chinese medicine fermentation broth according to claim 1, characterized in that: The Lactobacillus plantarum is one or more of Lactobacillus plantarum LZU-J-Q25, Lactobacillus plantarum LZU-J-Q21, Lactobacillus plantarum LZU-J-QA85, Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-S-ZCJ; Lactobacillus plantarum LZU-J-Q25, classified as Lactiplantibacillus plantarum, with a deposit number of GDMCC No: 63278; Lactobacillus plantarum LZU-J-Q21, classified as Lactiplantibacillus plantarum, with a deposit number of GDMCC No: 63277; Lactobacillus plantarum LZU-J-QA85, classified as Lactobacillus plantarum, with a deposit number of GDMCC No: 61192; Lactobacillus plantarum LZU-J-TSL6, classified as Lactobacillus plantarum, with a deposit number of GDMCC No: 61242; Lactobacillus plantarum LZU-S-ZCJ, classified and named Lactobacillus plantarum, has a preservation number of GDMCC No: 61402.

3. The method for preparing the Chinese medicine fermentation broth according to claim 1 or 2, characterized in that: The following steps are involved: The probiotics are inoculated into the Chinese medicine water extract, fermented at 29-35°C for 20-28 hours and then inactivated.

4. The preparation method according to claim 3, characterized in that: The concentration of live probiotics is 10 6 ~10 10 CFU / mL, and the inoculation volume is 1-5% of the volume of the Chinese medicine water extract.

5. The preparation method according to claim 3, characterized in that: The inactivation temperature is 85-100℃.

6. The preparation method according to claim 3, characterized in that: The inactivation time is 15 to 45 minutes.

7. Use of the Chinese medicinal fermentation liquid according to claim 1 or 2 in the preparation of a medicine for treating acute lung injury.

Images