Preparation method of arisaema cum bile decoction piece, decoction piece and application

Abstract

The invention relates to a preparation method of a arisaema cum bile decoction piece, the decoction piece and application, and belongs to the technical field of traditional Chinese medicine decoction piece processing. The preparation method comprises the following preparation steps: (1) crushing pachyrhizua angulatus and / or rhizoma arisaematis to obtain medicinal powder; (2) mixing bile and the medicinal powder according to the mass ratio of (4-6): 1, and adding zymophyte for fermentation to obtain fermentation liquor; (3) loading the fermentation liquid into the ox gall bladder, and suspending the fermentation liquid to obtain a fermentation product; and (4) the fermented product is dried at a low temperature and crushed, the arisaema cum bile decoction pieces are obtained, and zymophyte is selected from at least one of proteus mirabilis, vegetable bacillus, Kazakstan saccharomycetes and penicillium bacteria. The decoction piece is prepared by fermenting all pachyrhizua angulatus or adding rhizoma arisaematis and mixed strains, the content of active free cholic acid in the obtained fermentation product is high, and the decoction piece has prevention and treatment effects on various diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of processing Chinese medicine pieces, and particularly relates to a preparation method of Guangdanxing pieces, the pieces and applications. Background Art

[0002] Arisaema ciliata is a must-have medicine and emergency medicine in ancient pharmacies. It is a fermented Chinese herbal medicine with a long history of medicinal use and outstanding clinical efficacy. Arisaema ciliata tastes bitter and slightly spicy, is cool in nature, and enters the heart, liver, and lung meridians. It has the effects of clearing heat and resolving phlegm, calming wind and calming convulsions. It is used to treat cough with phlegm heat, cough with yellow and thick phlegm, phlegm caused by stroke, and epilepsy. It is an important medicine for treating "phlegm" syndrome, including visible phlegm in the lung meridian, invisible phlegm in the heart and liver meridians, and phlegm and blood stasis. The "Chinese Pharmacopoeia" (2020 edition) includes 29 Chinese patent medicines containing Arisaema ciliata, and 131 formulas in the formula preparation standards use Arisaema ciliata. The annual market demand is more than 2,600 tons, making it one of the most widely used Chinese herbal medicines.

[0003] The key points of the ancient method of preparing Nine-Turn Gallbladder Arisaema can be summarized as follows: First, the raw materials, using (the twelfth lunar month) yellow cattle bile, rarely using sheep bile, and not pig bile; using raw Arisaema powder; second, the time, "the twelfth lunar month", "hang it to dry", "a hundred days", and, "Yi Zong Jin Jian": "recollect, do this nine times, the one that has been turned nine times is the best"; third, the brewing place, "avoid the sun", "ventilate", and "dry in the shade"; fourth, the production method, which clearly states "brew", that is, the fermentation method.

[0004] By consulting the "Chinese Herbal Medicine Preparation Specifications" of 21 provinces including Jiangxi and Guizhou, a total of 6 of them contain Arisaema ciliata slices. The preparation processes mainly include fermentation method, mixed steaming method, and replication method; the bile used is from multiple sources, including cattle, sheep, and pig bile; the ratio of Arisaema ciliata powder to bile used is different; there is no clear regulation on the fermentation time, and only Jiangxi Province and Guizhou Province stipulate that the fermentation is about 2 weeks; Jilin, Shanghai, Jiangxi, Hunan and Zhejiang require fermentation conditions to be placed in a warm place or exposed to the sun and night dew, which is completely opposite to the low temperature and cool environment of the ancient preparation method. The regulations on the degree of fermentation are also descriptions of properties, and it is difficult to determine whether the efficacy requirements are met. The "Chinese Pharmacopoeia" (2020 edition) stipulates that Arisaema ciliata is processed by processing Arisaema ciliata powder with cattle, sheep or pig bile, or by fermenting raw Arisaema ciliata powder with cattle, sheep or pig bile. Two completely different preparation methods are stipulated: mixing method and fermentation method.

[0005] To sum up, the preparation of Arisaema consanguineum varies from place to place, and the standards for medicinal pieces are not unified. It has deviated from the traditional production techniques, resulting in large differences in quality and difficulty in ensuring clinical efficacy.

[0006] In response to the problems of unclear processing principles, simple standards, different specifications, and unclear efficacy and mechanism of action, our team conducted a systematic study on Arisaema ciliata. For the first time, the processing principle of Arisaema ciliata was clarified, namely, "the fermentation medium is constructed by Arisaema ciliata powder and ox bile, and under the anaerobic conditions created by the gallbladder skin, bile salt-tolerant specific microorganisms ferment to achieve the biotransformation of bile acids and obtain highly active free bile acids." Based on the traditional production connotation, the Guangdanxing (also known as Jiuzhuandanxing) decoction pieces were innovatively produced. Summary of the invention

[0007] In order to solve the above technical problems, the inventor innovatively discovered that Pueraria lobata is conducive to the generation of active free bile acid, and can replace or partially replace the toxic Chinese medicine Arisaema as a fermentation medium. For this purpose, a method for preparing Guangdanxing (also known as Jiuzhuan Danxing) slices is provided. Through the innovation, Pueraria lobata replaces Arisaema or adds it to Arisaema in a certain proportion, and stirs and ferments it with bile in a fermentation tank first, and then performs suspension fermentation. The relevant parameters in the process are specifically defined, the beneficial bacteria for the conversion of active bile acid are clarified, and a standard standardized process flow is formulated. While inheriting the ancient method of making Arisaema lobata, the inventor innovates Guangdanxing (also known as Jiuzhuan Danxing) based on modern bioengineering technology that conforms to the traditional Arisaema lobata processing connotation. The new production process of Guangdanxing obtained by this method establishes the new standard of Guangdanxing slices, realizes the modern production mode of Guangdanxing, changes the previous backward and simple production mode, and effectively guarantees the product quality, which is of great significance.

[0008] The first aspect of the present invention provides a method for preparing Guangdanxing decoction pieces, comprising the following preparation steps: (1) Grinding Puerariae lobata and / or Arisaema confusa to obtain medicinal powder; (2) Mixing bile and drug powder in a mass ratio of 4-6:1, adding fermentation bacteria for fermentation, and obtaining fermentation liquid; (3) placing the fermentation liquid in a cow gallbladder and suspending it for fermentation to obtain a fermentation product; (4) The fermented product is dried at low temperature, crushed, and pressed into tablets or pellets to obtain Guangdanxing decoction pieces; Wherein the fermentation bacteria is selected from Proteus mirabilis ( Proteus mirabilis strain )、Vegetable Bacillus( Bacillus oleronii ), Saccharomyces cerevisiae ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. ) at least one of the following.

[0009] Preferably, the medicinal powder in step (1) is prepared by crushing Pueraria lobata medicinal materials and Arisaema consanguineum medicinal materials into 80 meshes, and the mass ratio of Arisaema consanguineum medicinal powder to Pueraria lobata medicinal powder is 0-1:0.25-1.

[0010] Preferably, the weight ratio of bile to drug powder in step (2) is 4-6:1.

[0011] Preferably, the amount of fermentation bacteria added in step (2) accounts for 3-6% of the weight of the medicinal powder.

[0012] Preferably, the fermentation temperature in step (2) is 25-28° C., and the fermentation time is 7-15 days.

[0013] Preferably, the temperature of the suspended fermentation in step (3) is lower than 28° C., the relative humidity is lower than 80%, and the fermentation time is about 30 days.

[0014] Preferably, the temperature of low-temperature drying in step (4) is not higher than 80°C, the drying time is 12-24 hours, and the product is crushed into 80-100 mesh fine powder.

[0015] Further preferably, the fine powder is wet granulated and compressed into a cylinder, a pellet or a tablet.

[0016] More preferably, the compressed Guangdanxing decoction pieces are film-coated for easy administration.

[0017] The second aspect of the present invention relates to a Guangdanxing (also known as Jiuzhuandanxing) decoction piece, which is prepared using the above-mentioned preparation method.

[0018] Preferably, the content of active free bile acid in the Guangdanxing decoction piece is ≥6.5%, and the content of deoxycholic acid is ≥2%.

[0019] The third aspect of the present invention relates to the use of the Guangdanxing (also known as Jiuzhuandanxing) decoction pieces prepared by the above-mentioned preparation method in the preparation of drugs for clearing heat phlegm in the lung meridian, respiratory tract infection, sore throat, phlegm-heat cough, lung injury, high fever convulsions, clearing phlegm and blood stasis in the blood, protecting the liver and lowering blood lipids, promoting metabolism, protecting nerves, preventing and treating liver injury, stroke, dizziness, Parkinson's disease, epilepsy, etc.

[0020] Preferably, the Guangdanxing (also known as Jiuzhuandanxing) decoction pieces can be used directly, or can be used as a raw material for preparing other compound preparations.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The preparation method provided by the present invention covers the connotation target of preparing nine-turn gallbladder arisaema by the traditional ancient nine-turn process (9 fermentations), and establishes the content determination standard with free bile acid and deoxycholic acid as the quality control index; (2) The fermentation is carried out in a process-controllable fermentation tank to investigate the influence of raw material ratio, fermentation temperature and fermentation time on the generation of free bile acid and deoxycholic acid; (3) The innovative screening of kudzu vine can be used as both a starch-rich culture medium and a promoter of active free bile acid generation, which can partially or completely replace the toxic arisaema medicinal material, making the medicine safer; (4) The innovative screening of fermentation strains that are beneficial to the generation of active bile acid; (5) The present invention combines the use of the traditional hanging gallbladder drying method for drying, and the prepared new medicinal slice "Guangdanxing" has the same content as the traditional nine-turn gallbladder arisaema by the ancient method of 9 fermentations, and the preparation time is shorter than that of the ancient method, the conversion rate of the target product is high, the process is controllable, and the product quality is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Pathological sections of lung tissues of lung injury treated with Guangdanxing (HE staining, 200×); Figure 2 :Effects of Guangdanxing on the levels of TNF-α, IL-6, IL-1β and IL-18 in lung tissues of mice with lung injury; Figure 3 :Effects of Guangdanxing on the pathological morphology of liver tissue in NASH mice (HE, 200×); Figure 4 :Effects of Guangdanxing on serum IL-1β, TNF-α and brain substantia nigra IL-1β, TNF-α, COX-2 and iNOS levels in MPTP Parkinson's mouse model; Figure 5 :Effects of guandanxin on the relative expression of PKA, GPX4 and FTH1 in the substantia nigra of MPTP Parkinson's disease mouse model. DETAILED DESCRIPTION

[0023] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0024] In the following examples and experimental examples, the raw materials are: bile from bovine animals Bos niaTaurus domestic Fresh bile of Gmelin; Pueraria lobata is a plant of the Leguminosae family Pueraria thomsonii The dried root of Benth. Arisaema erubescens(Wall.) Schott, Arisaema heterophyllum Arisaema heterophyllum Bl.、Arisaema edulis Arisaema amurense Maxim. or Tiger Palm Arisaema ( Brushes cut into pieces Schott . ) of dried tubers. The fermentation bacteria are selected from Proteus mirabilis ( Miraculous Proteus strain )、Vegetable Bacillus( Bacillus oleronii ), Saccharomyces cerevisiae ( Kazakhstan servazzii ) and Penicillium ( Penicillium sp. ) are all commercially available strains.

[0025] Take the dried Pueraria lobata and Arisaema consanguineum medicinal materials and grind them into 80-mesh fine powder for later use.

[0026] Experimental Example 1: According to the traditional process, the nine-time fermented choledoch radix arisaema is prepared. The preparation steps are as follows: Fresh raw ox bile and raw arisaema are added as materials, with the ratio of raw arisaema to bile being 1:1. The traditional fermentation conditions are as follows: the temperature is 19-28°C (varies day and night, seasonally), ventilation is allowed, and the feeding time is in the twelfth lunar month (i.e. the eleventh lunar month). The first fermentation is carried out, and the fermentation end point is considered after the fermentation becomes dry. The fermentation time for one turn is calculated to be 28-32 days. The fermentation is taken out and crushed, and then fermented again with new bile. A total of 9 fermentations are performed to obtain the product.

[0027] According to the capacity of the gallbladder, the ratio of bile to raw materials was adjusted, the average amount of bile was calculated, and a total of 15 batches were prepared. According to the literature, the weight ratio of raw arisaema to bile after 9 fermentations was finally determined to be 1:4-6.

[0028] The chemical study of Arisaema ciliata after 9 fermentations was conducted, and the content of the main active markers free bile acid, deoxycholic acid and low-activity taurocholic acid was determined. The determination method is as follows: (1) Test sample solution: Take an appropriate amount of the test sample, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 70% methanol to make a 10 mg / mL solution, weigh it, ultrasonicate it for 30 min, let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, centrifuge it (speed: 8000 rpm, time: 10 min), filter it, and take the filtrate.

[0029] (2) Reference substance solution: Take an appropriate amount of reference substance, weigh it accurately, and add methanol to make up to volume in a 1 ml volumetric flask to make a solution containing 2 mg per 1 ml.

[0030] (3) Detection method Chromatographic column: Waters Symmetry C 18 (4.6×250 mm, 5 μm); mobile phase: acetonitrile (A)-0.2% acetic acid water (B), gradient elution; flow rate: 1.0 mL·min -1 ; Column temperature: 30°C. Evaporative light detector carrier gas flow rate: 2.2 mL / min; Drift tube temperature: 55°C. Chromatographic elution conditions are shown in Table 1, and test results are shown in Table 2.

[0031] Table 1: Gradient elution program

[0032] Table 2: Contents of related substances in 15 batches of 9 fermented cholanthus

[0033] The results of the traditional method of determining the nine-turn gallbladder arisaema showed that the average content of taurocholic acid was 48.02%, which is a conjugated bile acid with low activity; the average content of bile acid was 3.47%, and the average content of deoxycholic acid was 1.29%.

[0034] According to the traditional process, the nine-time fermented Arisaema consanguineum is prepared by the ancient method to determine that bile acid and deoxycholic acid are the active bile acid components converted by fermentation. The present invention uses the content of bile acid and deoxycholic acid as the standard for screening process conditions.

[0035] Experimental Example 2: Optimal Ratio of Puerariae lobata and Arisaema consanguineum Take fresh ox bile and 80 mesh kudzu powder and arisaema powder, mix them in the ratio of 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, whole kudzu powder to obtain mixed powders of different weight ratios; mix them evenly in the ratio of bile: powder by weight of 4:1, add 4% of the weight of the powder Proteus mirabilis ( Miraculous Proteus strain )、Vegetable Bacillus( Bacillus oleronii ), Saccharomyces cerevisiae ( Kazakhstan servazzii ) and Penicillium ( Penicillium sp. ) (the four bacteria were composed of a mass ratio of 1:1:1:1), stirred and fermented for 15 days at 25°C, with a stirring rate of 150 rpm. Samples were taken on the 15th day of fermentation to determine the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of bound taurocholic acid in the fermentation broth. The determination method was the same as that of Experimental Example 1, and the results are shown in Table 3.

[0036] Table 3: Contents of related substances in fermented products with different Pueraria lobata-Arisaema mass ratios

[0037] The results showed that Pueraria lobata could significantly increase the production of two free bile acids, active deoxycholic acid and cholic acid. The mass ratio of Arisaema consanguineum powder to Pueraria lobata powder was 0-1:0.25-1, and both were well converted. Arisaema consanguineum had the effect of resolving phlegm and dispersing nodules. As a fermentation substrate, Arisaema consanguineum could enhance the effect of "resolving phlegm and dispersing nodules".

[0038] Experimental Example 3: Investigation of the ratio of bile-powder raw materials Fresh ox bile and kudzu vine root powder and arisaema powder crushed into 80 mesh were mixed evenly at a bile: powder (kudzu vine root: arisaema was mixed at a weight ratio of 1:1) of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1, respectively, and fermented in the same manner as above (Experimental Example 2), and the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of bound taurocholic acid in the fermentation broth were determined. The determination method was the same as that of Experimental Example 1, and the determination results are shown in Table 4.

[0039] Table 4: Effects of different bile-powder ratios on bile acid content during fermentation

[0040] The results show that when the bile: drug powder weight ratio is 4-6:1, the content of bile acid and deoxycholic acid produced by fermentation is high, while at a high bile-drug powder ratio, although the content of conjugated taurocholic acid is higher, the amount of its conversion products bile acid and deoxycholic acid is not high. Therefore, the optimal bile: drug powder weight ratio is determined to be 4-6:1.

[0041] Experimental Example 4: Investigation of stirring and fermentation time Take fresh ox bile and 80 mesh kudzu powder and arisaema powder, mix them evenly in a bile:powder (kudzu: arisaema is mixed in a weight ratio of 1:1) ratio, add the weight of bile and powder and 4% Proteus mirabilis ( Proteus mirabilis strain )、Vegetable Bacillus( Bacillus oleronii ), Saccharomyces cerevisiae ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. ) (the four bacteria are composed of a mass ratio of 1:1:1:1), fermented with stirring at 25°C and a stirring rate of 150 rpm. Samples were taken on the 0th, 3rd, 7th, 10th, 15th, 19th, 23rd, 27th and 30th days of fermentation to determine the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of conjugated taurocholic acid in the fermentation broth. The determination method was the same as that in Experimental Example 1. The results are shown in Table 5.

[0042] Table 5: Effect of different fermentation times on bile acid content

[0043] The results show that on the 15th day of fermentation, the content of bile acid and deoxycholic acid produced by fermentation is the highest. As the fermentation time increases, the content of conjugated taurocholic acid, bile acid and deoxycholic acid decreases, and may be further converted into other metabolites. Therefore, the fermentation time is determined to be 7-15 days, preferably 15 days.

[0044] Experimental Example 5: Investigation of stirring fermentation temperature Take fresh ox bile and ground into 80 mesh arisaema powder and kudzu powder, mix them evenly in the ratio of bile: powder (kudzu: arisaema in a weight ratio of 1:1) and add the weight of bile and powder and 4% Proteus mirabilis ( Proteus mirabilis strain )、Vegetable Bacillus( Bacillus oleronii ), Saccharomyces cerevisiae ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. ) (the four bacteria are composed of a mass ratio of 1:1:1:1), stirred and fermented at a stirring rate of 150 rpm, and fermented for 15 days at fermentation temperatures of 20°C, 23°C, 25°C, 28°C, and 30°C, respectively, to determine the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of bound taurocholic acid in the fermentation broth. The determination method is the same as that of Experimental Example 1, and the results are shown in Table 6.

[0045] Table 6: Effect of different fermentation temperatures on bile acid content

[0046] The results show that on the 15th day of fermentation at 25°C, the content of free bile acid and deoxycholic acid produced by fermentation is the highest; the fermentation results at 28°C are also good, slightly lower than 25°C. Therefore, the optimal fermentation temperature is determined to be 25°C-28°C.

[0047] Experimental Example 6: Sample 1: A method for preparing Guangdanxing decoction pieces The following steps are involved: (1) Fermentation in a fermentation tank: Take the medicinal materials of Pueraria lobata and Arisaema consanguineum, crush them through an 80-mesh sieve, then take fresh ox bile and mix them in a ratio of bile: medicinal powder (Pueraria lobata and Arisaema consanguineum are mixed in a mass ratio of 1:1) 4:1, add the weight of bile and medicinal powder and 4% Proteus mirabilis ( Proteus mirabilis strain )、Vegetable Bacillus( Bacillus oleronii ), Saccharomyces cerevisiae ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. ) (the four bacteria were composed of 1:1:1:1 by mass ratio), and fermented at 25°C with stirring and a stirring rate of 150 rpm for 15 days; (2) The mixed fermentation liquid obtained in the first stage is placed in the gallbladder skin of the cattle, and suspended for fermentation. The temperature is controlled to be lower than 28°C and the relative humidity is controlled to be lower than 80%. The gallbladder is hung and fermented for 30 days to complete the second stage of fermentation. (3) Molding process: Cut open the cow gallbladder skin, take out the fermented product, bake it at 70℃ for 20 hours, crush it into 80 mesh fine powder, wet granulate it, and press it into a cylindrical shape to obtain Guangdanxing decoction pieces.

[0048] According to the above method, 15 batches of pilot samples were prepared to determine the contents of two free bile acids, deoxycholic acid and cholic acid, and the content of conjugated taurocholic acid in the slices. The determination method was the same as that of Experimental Example 1, and the results are shown in Table 7.

[0049] Table 7: Content determination indicators of 15 batches of pilot products

[0050] As shown in Table 7, the bile acid content of the 15 batches of pilot products was between 6.43% and 7.26%, with an average content of 6.89%, and the deoxycholic acid content was between 2.70% and 2.97%, with an average content of 2.81%.

[0051] Sample 2: The only difference from sample 1 is that during the fermentation in the fermenter in step (1), Pueraria lobata powder is not added. The other steps are the same as sample 1. The contents of two free bile acids, deoxycholic acid and cholic acid, and the content of bound taurocholic acid in the prepared Arisaema confusa slices are determined. The determination method is the same as that of experimental example 1. The results of 6 batches are shown in Table 8. At the same time, the relevant test data of sample 1 and Arisaema confusa prepared by 9 fermentations using the traditional ancient method are added to Table 8.

[0052] Table 8: Content determination results of products prepared by different preparation methods

[0053] The results show that when fermented without adding kudzu vine, the average bile acid content is 3.80%, the deoxycholic acid content is 1.46%, and the conversion level of free bile acid is equivalent to the result of 9 fermentations in the ancient method, but the fermentation time is significantly shortened. The effect of fermentation with kudzu vine is significantly better than that without kudzu vine. It can be seen that kudzu vine is of great significance to the production of active bile acids.

[0054] In the Guangdanxing sample 1 obtained by the preferred method of the present invention, the content of bile acid and deoxycholic acid is higher than that of sample 2 and the traditional nine-turn gallbladder arisaema, which improves the activity of the medicinal slices. The preparation method of the present invention can promote the conversion of bound bile acid with low activity in bile to free bile acid with high activity, especially the addition of kudzu root. Compared with the traditional nine-time fermented gallbladder arisaema, the Guangdanxing obtained by the method of the present invention has a 97.4% increase in bile acid content and a 117.83% increase in deoxycholic acid content.

[0055] The production process conditions of the preparation method of the Arisaema ciliata obtained by the invention are reasonable and feasible, can meet the requirements of industrial production, and the obtained medicinal pieces have high contents of bile acid and deoxycholic acid.

[0056] Experimental Example 7: Identification of the fermentation strain of Guangdanxing (1) Isolation of bacterial strains: Take an appropriate amount of Guangdanxing decoction pieces (batch number 20220904), streak culture on a plate, and isolate a single bacterial strain.

[0057] (2) Classical identification: observe the characteristics of the colony, Gram stain (bacteria) and lactic acid cotton blue stain (fungi), and then observe under a microscope. Biochemical reaction identification includes glucose fermentation test, methyl red test, urea test, indole test, gelatin test and H 2 S experiment.

[0058] (3) Molecular biological identification The purified single strain was extracted and amplified using the TSINGKE plant DNA extraction kit, and the PCR bands were purified and recovered by 1% agarose gel electrophoresis and Sanger sequencing. The software ContigExpress was used for splicing, and the inaccurate parts at both ends were removed to obtain the gene sequence of the strain and construct a phylogenetic tree.

[0059] (4) Identification result: Identified as Proteus mirabilis ( Proteus mirabilis strain )、Vegetable Bacillus( Bacillus oleronii ), Saccharomyces cerevisiae ( Kazachstania servazzii ) and Penicillium ( Penicillium sp. .).

[0060] Experimental Example 8: Pharmacodynamics study of Guangdanxing against acute lung injury (1) Experimental methods: SPF BALB / c male mice were purchased from Guangdong Medical Experimental Animal Center, production license number: SCXK (Yue) 2018-0002. They were randomly divided into 9 groups: blank group (CON), model group (MODEL), dexamethasone positive control group (Positive), Guangdanxing (batch number: 20220902) low-dose group (TD, 1 g / kg), high-dose group (TG, 2 g / kg), Guangdanxing (batch number: 20220904) low-dose group (HD, 1 g / kg), high-dose group (HG, 2 g / kg), bile acid group (CA, 50 mg / kg), deoxycholic acid group (DCA, 50 mg / kg), with 10 mice in each group. Except for the blank group, mice in each group were intraperitoneally injected with LPS (5 mg / kg) to establish an acute lung injury model, with an intraperitoneal injection volume of 0.1 mL / 20g. The blank group was injected with an equal amount of normal saline. 24 hours after modeling, the corresponding drugs were administered by intragastric administration at a dose of 0.2 mL / 20 g, once a day for 7 consecutive days.

[0061] (2) Results: Pathological observation of lung tissue showed that the positive drug group and each drug-treated group were milder than the model group, with less inflammatory cell infiltration and mild capillary congestion. The low-dose group still had alveolar stenosis and alveolar wall thickening. The improvement effect of the monomeric bile acid group and the high-dose group was better than that of the low-dose group, indicating that cholic acid, CA and DCA can improve LPS-induced acute lung injury in mice. Figure 1 .

[0062] The levels of TNF-α, IL-6, IL-1β, and IL-18 in the lung tissues of mice in the model group were extremely significantly increased (P<0.01), indicating that the level of lung inflammation in mice was high; the low-dose group of Guangdanxing TD (batch number: 20220902) could significantly reduce the levels of TNF-α, IL-6, and IL-18 (P<0.05), and the level of IL-1β showed a decreasing trend but without statistical significance (P>0.05); the high-dose group of Guangdanxing TG (batch number: 20220902) could significantly reduce the level of IL-6 (P<0 .05), and significantly reduced the levels of TNF-α, IL-1β and IL-18 (P<0.01); the low-dose group of Guangdanxing (batch number: 20220904) could significantly reduce the levels of IL-1β and IL-18 (P<0.05), and significantly reduced the levels of TNF-α and IL-6 (P<0.01); the high-dose group of Guangdanxing (batch number: 20220904), CA and DCA groups could significantly reduce the levels of TNF-α, IL-6, IL-1β and IL-18 (P<0.01). See attached Figure 2 .

[0063] Conclusion: Guangdanxing can alleviate pulmonary edema, improve the pathological morphology of lung tissue, reduce the levels of inflammatory factors TNF-α, IL-6, IL-1β and IL-18, inhibit excessive inflammatory response in the body, and thus play an anti-inflammatory and anti-acute lung injury role.

[0064] Experimental Example 9: Antipyretic Effect of Guangdanxing (1) Experimental methods: SPF male SD rats were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd. (No.: SCXK (Beijing) 2021-00011). All animal experiments were approved by the Ethics Committee of Heilongjiang University of Chinese Medicine (Approval No. 2023092207).

[0065] Rats with stable body temperature were randomly divided into control group (Control), model group (Model), acetaminophen (200 mg / kg) group, and guandanxing (batch number: 20220904) group (1.4 g / kg), with 8 rats in each group. The rats in the control group were subcutaneously injected with normal saline (10 mL / kg) on ​​the back, and the other rats were subcutaneously injected with 20% dry yeast suspension on the back to establish a fever model. The rats in the acetaminophen group and guandanxing group were gavaged once 30 minutes before modeling and 3.5 hours after modeling, respectively. The control group and model group were gavaged with an equal volume of normal saline (10 mL / kg). Starting from the 4th hour after modeling, the body temperature of the rats was tested every 1 hour until the 8th hour, and the body temperature change value (AT, i.e., real-time body temperature value-basal body temperature value) of the rats in each group at each time point was calculated.

[0066] (2) Results: Effect on the body temperature of rats with dry yeast-induced fever: After the injection of dry yeast suspension at the 4th hour, the rectal temperature of the model rats increased significantly (P<0.01). After taking Guangdanxing, the rectal temperature of the rats decreased significantly (P<0.01) and lasted until 8 hours. The antipyretic time was similar to that of acetaminophen. The results are shown in the table below: Table 9: Changes in rectal temperature of rats in each group (`x, n=8)

[0067] Compared with the control group, ## P<0.01; **P<0.01 compared with the model group.

[0068] Effects on the levels of inflammatory factors in rats with dry yeast-induced fever: After dorsal injection of dry yeast suspension, the levels of TNF-α, IL-1β, IL-6 in rat serum and PGE2 and cAMP in the hypothalamus were significantly increased (P<0.01); compared with the model group, Guangdanxing could significantly reduce the levels of TNF-α, IL-1β, IL-6 in rat serum and PGE2 and cAMP in the hypothalamus (P<0.01). The results are shown in the table below: Table 10: Effects on the levels of inflammatory factors in rat serum (`x, n=8)

[0069] Compared with the control group, ## P<0.01; **P<0.01 compared with the model group.

[0070] Conclusion: Guangdanxing can significantly reduce the rectal temperature of febrile rats for a long time. The expression levels of pyrogenic factors (PGE2, cAMP, CRH, TNF-α, IL-1β and IL-6) in rats in the Guangdanxing group were significantly reduced.

[0071] Experimental Example 10: The therapeutic effect of Guangdanxing on mice with non-alcoholic fatty liver disease (1) Experimental methods: SPF male C57BL / 6 mice were purchased from Guangdong Medical Experimental Animal Center, and the animal production certificate number is SCXK (Yue) 2018-0002. The mice were randomly divided into 7 groups, including normal group (ControL), model group (ModeL), positive control group of EssentiaLe (0.178 g / kg), Guangdanxing (batch: 20220902) low-dose group (TD, 0.78 g / kg), high-dose group (TG, 2.34 g / kg), Guangdanxing (batch: 20220904) low-dose group (HD, 0.78 g / kg), high-dose group (HG, 2.34 g / kg), with 10 mice in each group. The mice in the normal group were fed with MCS feed, and the mice in the model group and the drug-treated group were fed with MCD feed three times a day. The weight of the feed consumed by the mice and the body weight of the mice were recorded. Four weeks after modeling, the liver function index and blood lipid index were used to determine the success of the modeling. Starting from the fifth week, the normal group and the model group were gavaged with 0.5% sodium carboxymethyl cellulose solution, and the mice in the other groups were gavaged with the corresponding drug solution. The gavage volume was 10 mL / kg, and the drug was administered once a day for 21 consecutive days. The dosage of the low-dose and high-dose groups was calculated based on the clinical medicinal dose and 3 times the clinical medicinal dose.

[0072] (2) Results: Effect on liver pathological histology of NASH mice: Compared with the model group, the cytoplasmic fatty degeneration and nuclear pyknosis of hepatocytes in each Guangdanxing treatment group of mice were significantly reduced, especially in the high-dose group of Guangdanxing (batch: 20220902), the improvement was most obvious. Oil Red O results: It can be seen that there is no obvious fatty degeneration in the hepatocytes of mice in the normal group; there are more red-stained lipid droplets of different sizes in the liver lobules of mice in the model group, and typical fatty degeneration and vesicular degeneration appear; compared with the model group, the red-stained lipid droplets in the hepatocytes of mice in each Guangdanxing treatment group of mice are significantly reduced, especially in the high-dose group of TG Guangdanxing (batch: 20220902), the improvement is most obvious. See attached Figure 3 .

[0073] Serum liver function indexes and blood lipid indexes: ALT and AST levels in the serum of mice in the model group were significantly increased (P<0.01), and TC and TG levels were significantly increased (P<0.01); compared with the model group, ALT and AST levels in the serum of mice in the EssentiaLe group, TD group, TG group, HD group, and HG group were significantly decreased (P<0.01), and TC and TG levels were significantly decreased (P<0.01). The results are shown in the table below.

[0074] Table 11: Effects on serum liver function indexes and blood lipid indexes in NASH mice (`x±SD, n=10)

[0075] Compared with the Control group, ## P<0.01; **P<0.01 compared with the Model group.

[0076] Effects on serum inflammatory factors in NASH mice: The levels of IL-6, IL-1β, and TNF-α in the serum of mice in the model group were significantly increased (P<0.01); the levels of IL-6, IL-1β, and TNF-α in the serum of mice in the EssentiaLe group, TD group, TG group, HD group, and HG group were significantly decreased (P<0.01). See the table below for specific results.

[0077] Table 12: Effects of Guangdanxing on serum inflammatory factors in NASH mice (`x±SD, n=10)

[0078] Compared with the Control group, ## P<0.01; **P<0.01 compared with the Model group.

[0079] Effect on oxidative stress in liver tissue of NASH mice: The MDA level in liver tissue of mice in the model group was significantly increased, and the SOD level was significantly decreased (P<0.01); the MDA level in liver tissue of mice in the EssentiaLe group, TD group, TG group, HD group, and HG group was significantly decreased, and the SOD level was significantly increased (P<0.01). The results are shown in the table below.

[0080] Table 13: Effects of Guangdanxing on oxidative stress in liver tissue of NASH mice (`x±SD, n=10)

[0081] Compared with the Control group, ## P<0.01; **P<0.01 compared with the Model group.

[0082] Conclusion: Guangdanxing exerts significant anti-inflammatory, antioxidant, anti-fat degeneration effects in NASH, regulates glucose and lipid metabolism and bile acid metabolism, reduces liver parenchymal cell damage, hepatic lipid accumulation and inflammatory cell infiltration in MCD diet mice, and has the effect of treating non-alcoholic fatty liver disease.

[0083] Experimental Example 11: Study on the efficacy of Guangdanxing on MPTP Parkinson's mouse model (1) Experimental methods: SPF grade C57BL-6J male mice were purchased from Guangdong Medical Experimental Animal Center, animal license number: SCXK (Yue) 2018-0002. According to body weight, they were randomly divided into normal group (CON), model group (MODLE), levodopa tablet group (POSITIVE, 80 mg / kg), Guangdanxing (batch number 20220902) low-dose group (TD, 0.39 g / kg), high-dose group (TG, 1.56 g / kg), bile acid group (CA, 10 mg / mL), deoxycholic acid group (DCA, 10 mg / mL), 10 mice in each group. Except for the normal group, the mice in the other groups were intraperitoneally injected with methyl-4-phenyl-1, 2, 3, 6 tetrahydropyridine (MPTP) solution (35 mg / kg) once a day for 5 consecutive days to establish the subacute PD model; the mice in the normal group were simultaneously intraperitoneally injected with normal saline (10 g / kg). Starting from the sixth day of the experiment, the mice in the normal group and the model group were gavaged with 0.5% sodium carboxymethylcellulose solution (10 g / kg) every day, and the mice in the other groups were gavaged with the corresponding drug solution once a day for 7 consecutive days.

[0084] (2) Results: Weight changes and behavioral observations: After intraperitoneal injection of MPTP, mice showed symptoms such as slow movement, crawling disorders, erect hair, and tail stiffness within half an hour. It can be seen that during the modeling period from the 1st day to the 5th day, except for the blank group mice, the weight of the mice in the other groups was decreasing; from the 6th day, the modeling was stopped and the corresponding therapeutic drugs were given, and it was found that the weight of the mice began to increase, and when the modeling was given again on the 12th day, the weight of the mice decreased again; This shows that MPTP intraperitoneal injection modeling can induce behavioral disorders and physical stress in mice, affecting their eating function, thereby leading to weight loss.

[0085] Results of the pole climbing test: After 5 days of intraperitoneal injection of MPTP, the climbing time of mice was significantly higher than that of CON (P<0.0001), indicating that the climbing ability was seriously reduced and the modeling effect was stable; after one week of treatment and the last modeling, the climbing time of MODEL mice was significantly prolonged (P<0.0001), indicating that the behavioral ability of mice was difficult to recover; although POSITIVE and TD could shorten the climbing time of mice compared with MODEL, there was no significant difference; TG significantly shortened the climbing time of mice compared with MODEL (P<0.001); CA significantly shortened the climbing time of mice compared with MODEL (P<0.05); and deoxy DCA significantly shortened the climbing time of mice compared with MODEL (P<0.01). The results of the pole climbing experiment showed that TG, CA and DCA could effectively shorten the climbing time of MPTP mice and improve the motor ability of MPTP mice.

[0086] The results of TH protein immunofluorescence in substantia nigra: After MPTP modeling, the number of TH-positive cells in the substantia nigra of MODEL decreased significantly compared with CON; compared with MODEL, the number of TH-positive cells in the substantia nigra of TD, TG, CA and DCA increased, among which the increase in TG and CA was particularly obvious.

[0087] ELISA was used to detect the inflammation level in the serum and substantia nigra of mice: the serum IL-1β level of MODEL was significantly increased (P<0.001); TD, TG, CA, and DCA all significantly reduced the serum IL-1β level of mice (P<0.001). The serum TNF-α level of MODEL was significantly increased (P<0.01); TG and CA significantly reduced the TNF-α level (P<0.05). The IL-1β level in the substantia nigra of MODEL was significantly high (P<0.001); TD and TG could significantly reduce the IL-1β level in the substantia nigra (P<0.01). The TNF-α level in the substantia nigra of MODEL was significantly increased (P<0.001); TD and TG significantly reduced the TNF-α level in the substantia nigra (P<0.001), CA significantly reduced the TNF-α level in the substantia nigra (P<0.01), and DCA significantly reduced the TNF-α level in the substantia nigra (P<0.05). The level of iNOS in the substantia nigra of MODEL was significantly increased (P<0.05); the level of iNOS in the substantia nigra of TD and DCA was significantly decreased (P<0.05), and TG significantly reduced the level of iNOS in the substantia nigra (P<0.01). The level of COX-2 in the substantia nigra of MODEL was significantly increased (P<0.05); TD significantly reduced the level of COX-2 in the substantia nigra (P<0.05), and TG significantly reduced the level of COX-2 in the substantia nigra (P<0.01). See Appendix Figure 4, where A is the serum IL-1β level of each group, B is the serum TNF-α level of each group, C is the IL-1β level of each group, D is the TNF-α level of each group, E is the INOS level of each group, and F is the COX-2 level of each group. Under the same indicators, compared with the CON group: # P<0.05, ## P<0.01, ### P<0.001; compared with the MODEL group: *P<0.05; **P<0.01, ***P<0.001; ns means P>0.05.

[0088] WB was used to detect the levels of PKA, GPX4, and FTH1 in the substantia nigra of mice: after treatment with TG, CA, and DCA, the PKA level in the TG group increased significantly (P<0.05), and the PKA levels in the CA and DCA groups also increased extremely significantly (P<0.01); the GPX4 level in the TG and CA groups increased significantly (P<0.05), and the GPX4 level in the DCA group increased extremely significantly (P<0.01); the FTH1 level in the MODEL group decreased significantly (P<0.05), and the FTH1 level in the TG, CA, and DCA groups increased significantly (P<0.05). See attached Figure 5 , where A is the protein expression level of PKA, GPX4 and FTH1, B is the relative expression of PKA, C is the relative expression of GPX4, and D is the relative expression of FTH1; under the same indicators, compared with the CON group: # P<0.05; compared with the MODEL group: *P<0.05, **P<0.01.

[0089] Conclusion: Guangdanxing can significantly shorten the climbing time of MPTP model mice, indicating that it can significantly improve the behavioral disorders of MPTP model mice. From the perspective of the number of TH-positive neurons, TG can significantly protect the number of DA neurons and has the effect of protecting nerve cells. From the perspective of anti-inflammatory factors, TG significantly reduced the levels of IL-1β, TNF-α, iNOS and COX-2 in MPTP model mice. Western blotting results showed that MPTP induced a decrease in the levels of GPX4 and FTH1 proteins in mice, while TG could inhibit the ferroptosis of substantia nigra neurons by increasing the levels of PKA, GPX4 and FTH1.

Claims

1. A method for preparing Guangdanxing decoction pieces, characterized in that: The preparation method comprises the following preparation steps: (1) Grinding Puerariae lobata and / or Arisaema confusa to obtain medicinal powder; (2) Mixing bile and drug powder in a mass ratio of 4-6:1, adding fermentation bacteria for fermentation, and obtaining fermentation liquid; (3) placing the fermentation liquid in a cow gallbladder and suspending it for fermentation to obtain a fermentation product; (4) The fermented product is dried at low temperature, crushed, and pressed into tablets or pellets to obtain Guangdanxing decoction pieces; The fermentation bacteria is selected from at least one of Proteus mirabilis, Bacillus vegetativeus, Saccharomyces cerevisiae and Penicillium.

2. The preparation method according to claim 1, characterized in that: The medicinal powder in step (1) is prepared by crushing Pueraria lobata medicinal material and Arisaema consanguineum medicinal material into 80 meshes, and the mass ratio of Arisaema consanguineum medicinal powder to Pueraria lobata medicinal powder is 0-1:0.25-1.

3. The preparation method according to claim 1, characterized in that: The amount of fermentation bacteria added in step (2) accounts for 3-6% of the mass of the medicine powder.

4. The preparation method according to claim 1, characterized in that: The fermentation temperature in step (2) is 25-28° C. and the fermentation time is 7-15 days.

5. The preparation method according to claim 1, characterized in that: The temperature of the suspended fermentation in step (3) is lower than 28° C., the relative humidity is lower than 80%, and the fermentation time is 30 days.

6. The preparation method according to claim 1, characterized in that: The temperature of low-temperature drying in step (4) is not higher than 80°C, the drying time is 12-24 hours, and the powder is crushed into 80-100 mesh fine powder.

7. A Guangdanxing decoction piece, characterized in that The method is prepared by any one of claims 1 to 6.

8. Use of the Guangdanxing decoction piece prepared by the preparation method according to any one of claims 1 to 6 or the Guangdanxing decoction piece according to claim 7 in preparing drugs for clearing heat phlegm in the lung meridian, respiratory tract infection, sore throat, phlegm-heat cough, lung injury, high fever convulsion, clearing phlegm and blood stasis in the blood, protecting the liver and lowering lipids, promoting metabolism, protecting nerves, preventing and treating liver injury, stroke, dizziness, Parkinson's disease, and epilepsy.

9. According to the use described in claim 8, the Guangdanxing can be used directly or as a raw material for preparing other compound preparations.

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